JP2009242345A - Scca-1 production inhibitor having 1-piperidinepropionic acid and/or its salt as active ingredient - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel drug for preventing and/or treating a disease caused by cell abnormal multiplication and a disease caused by SCCA-1 (squamous cell carcinoma antigen 1) production acceleration based on the SCCA-1 production inhibition. <P>SOLUTION: The SCCA-1 production inhibitor comprises 1-piperidinepropionic acid of formula (I) and/or its salt as an active ingredient. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a squamous cell carcinoma associated antigen-1 (hereinafter referred to as “SCCA-1”) production inhibitor comprising 1-piperidinepropionic acid and / or a salt thereof as an active ingredient.

  Squamous cell carcinoma-associated antigen (SCCA) is an antigen found in squamous cell carcinoma cells and has a high blood concentration in squamous cell carcinomas of the cervix, lungs, esophagus, and skin. It is often used (Non-patent document 1: H. Kato et al. Cancer 40: 1621-1628 (1977), Non-patent document 2: N. Mino et al. Cancer 62: 730-734 (1988)).

  SCCA is also known not only to be squamous cell carcinoma, but also from the inventors' research, that the expression is increased in the upper layer of the psoriatic epidermis (Non-patent Document 3: Takeda A. et al, J. Invest Dermatol. (2002) 118 (1), 147-154). Psoriasis is one of the skin diseases with a high morbidity and is a chronic, recurrent inflammatory keratosis characterized by abnormal proliferation / differentiation of epidermal cells and infiltration of inflammatory cells. Psoriasis is thought to develop by adding various environmental factors to a genetic predisposition (Non-patent Document 4: Hopso-Havu et al. British Journal of Dermatology (1983) 109, 77-85).

  SCCA is encoded by two genes, SCCA-1 and SCCA-2, arranged in tandem on chromosome 18q21.3. The proteins encoded by them, SCCA-1 and SCCA-2, are both proteins with a molecular weight of about 45,000 and are highly homologous, but the amino acid sequences of the reaction sites are different and are considered to have different functions. (Non-Patent Document 5: Schick et al. J. Biol. Chem. (1997) 27213, 1849-55).

  The present inventor conducted research aimed at elucidating the physiological mechanism of the epidermis in which SCCA-1 and SCCA-2 are involved, and both SCCA are both anti-apoptotic factors having an action of suppressing cell apoptosis. Knowledge has been obtained (Patent Document 1: JP-A-2005-281140).

  In addition, the present inventor conducted research aimed at evaluating the sensitivity of the skin with the expression of SCCA-1 as an index, in particular, 16 times for atopic dry skin, 90 times for exposed area skin, It has also been found that SCCA-1 expression is increased 232 times in hay fever allergic skin and 466 times in psoriatic skin (Japanese Patent Application No. 2006-075024).

Furthermore, the present inventor conducted various studies on the relationship between cell proliferation and SCCA, particularly SCCA-1,
-Cell proliferation is activated in SCCA high expressing mice,
-Thickening of the epidermis is observed in SCCA high-expressing mice,
The present inventors have found that there is a correlation between the cell growth and the SCCA-1 expression level in the -SCCA-1 high-expression cell line, and that the cell proliferation activity is reduced in the -SCCA knockdown cell line.

  From the above, if there is a substance that effectively suppresses the production of SCCA, particularly SCCA-1, it is considered useful for the prevention and / or treatment of diseases caused by abnormal cell growth and diseases caused by increased production of SCCA-1. It is done.

JP-A-2005-281140

Cancer 40: 1621-1628 (1977) Cancer 62: 730-734 (1988) J. Invest. Dermatol. 118 (1), 147-154 (2002) British Journal of Dermatology 109, 77-85 (1983) J. Biol. Chem. 27213, 1849-55 (1997)

  As a result of screening a wide variety of drugs by focusing on the relationship between cell proliferation and SCCA-1, the present inventors have found that 1-piperidinepropionic acid and / or a salt thereof significantly suppresses the production of SCCA-1. I found it. Therefore, the present inventor has proposed that 1-piperidinepropionic acid and / or a salt thereof prevent and prevent diseases caused by abnormal cell proliferation and diseases caused by increased production of SCCA-1 based on its SCCA-1 production inhibitory action. The present invention has been completed because it is considered extremely useful for treatment.

This application includes the following inventions:
[1] 1-piperidinepropionic acid of general formula (I)
And / or a SCCA-1 production inhibitor containing a salt thereof as an active ingredient.
[2] A pharmaceutical composition for preventing and / or treating a disease caused by abnormal cell proliferation, comprising 1-piperidinepropionic acid and / or a salt thereof according to [1] as an active ingredient.
[3] A pharmaceutical composition for preventing and / or treating epidermis thickening, comprising 1-piperidinepropionic acid and / or a salt thereof according to [1] as an active ingredient.
[4] Basal cell carcinoma (BCC), squamous cell carcinoma (SCC), Bowen containing 1-piperidinepropionic acid and / or a salt thereof according to [1] as an active ingredient Malignant tumors (carcinoma) such as Bowen's disease, pilomatricoma, sebarrheic keratosis, actinic keratosis, actinic keratosis, solar keratosis ) And precancerous conditions, lichen planus-like keratosis, benign lichenoid keratosis, acrochordon, cutaneous tag, pustular psoriasis Psoriasis (psoriasis vulgaris), psoriasis (psoriasis), xeroderma pigmentatosum (XP), atopic dermatitis, systemic lupus erythematosus (SLE), circular systemic Luthematode (Discoid lupus erythematosus: DLE) and other nevus and wrinkles such as erythematosus, porokeratosis, inflammatory linear verrucous epidermal naevus (ILVEN), hyperplasia A pharmaceutical composition for treating a disease selected from the group consisting of benign keratosis.

  The 1-piperidinepropionic acid and / or salt thereof according to the present invention is extremely useful for suppressing the production of SCCA-1. Specifically, it was found that when 1-piperidinepropionic acid was added to the medium during the cultivation of human keratinocytes, the SCCA1 production amount was significantly suppressed. It was also confirmed that epidermal thickening can be prevented and improved by culturing a three-dimensional skin model that has abnormal growth of epidermal cells and SCCA1 expression is significantly enhanced in a medium containing 1-piperidinepropionic acid. Furthermore, it was also confirmed that the amount of SCCA1 in the application part significantly decreased by continuously applying 1-piperidinepropionic acid to human skin.

  Further, when the effect of 1-piperidinepropionic acid and / or a salt thereof according to the present invention on human skin was examined, it was confirmed that it was useful for suppressing the increase in the thickening of the epidermis of human skin.

  Therefore, 1-piperidinepropionic acid and / or a salt thereof according to the present invention inhibits the production of SCCA-1 by inhibiting the production of cells, such as basal cell carcinoma, squamous cell carcinoma, Bowen's disease. , Malignant tumors and pre-cancerous conditions such as pilomatricoma (calcifying epithelioma), seborrheic keratosis, actinic keratosis (sun keratosis), lichen planus-like keratosis, benign lichen-like horn Psoriasis such as keratosis, soft adenoma, pustular psoriasis, psoriasis vulgaris, xeroderma pigmentosum, atopic dermatitis, systemic lupus erythematosus, and lupus erythematosus such as circular systemic lupus erythematosus It is useful for preventing and / or treating nevus such as epidermal nevus, wrinkles, and benign keratosis with thickening.

1-piperidinepropionic acid according to the present invention is a compound of the following general formula (I).

  1-piperidinepropionic acid represented by the general formula (I) according to the present invention is a known substance, and can be easily synthesized by a known method, or a commercially available product can be easily purchased.

  The 1-piperidinepropionic acid represented by the general formula (I) according to the present invention can be converted into an inorganic salt or an organic salt by a known method. Although it does not specifically limit as a salt used in this invention, For example, as an inorganic salt, hydrochloride, sulfate, phosphate, hydrobromide, sodium salt, potassium salt, magnesium salt, calcium salt, ammonium Examples include salts. Organic salts include acetate, lactate, maleate, fumarate, tartrate, citrate, methanesulfonate, p-toluenesulfonate, triethanolamine salt, diethanolamine salt, amino acid salt, etc. Can be mentioned.

  The SCCA-1 production inhibitor and pharmaceutical composition of the present invention comprise 1-piperidinepropionic acid and / or a salt thereof, and a pharmaceutically acceptable excipient and / or carrier. The pharmaceutical composition contains an effective amount of 1-piperidinepropionic acid and / or a salt thereof to exert its function, and the content may vary depending on the use of the pharmaceutical composition. For example, in the total amount of the pharmaceutical composition, 0.001 to 20.0% by mass is preferable, more preferably 0.01 to 10.0% by mass, and particularly preferably 0.2 to 10.0% by mass. Let's go. In addition, when 1-piperidinepropionic acid and / or its salt are mixed and used, it is preferable that the upper limit of those total content shall be 20.0 mass% or less, More preferably, it is 10.0 mass% or less. Will do.

  The SCCA-1 production inhibitor and pharmaceutical composition of the present invention may take various dosage forms depending on the use, for example, oral, for example, tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions or suspensions. It can also be administered in the form of a product or enterally, for example in the form of suppositories, or parenterally, for example in the form of injections, or externally, for example in patches, ointments, creams or emulsions.

  The SCCA-1 production inhibitor and pharmaceutical composition of the present invention may contain, for example, an inorganic or organic solid or liquid pharmaceutically acceptable carrier as appropriate, together with the active ingredient, if desired. For example, a diluent (lactose, dextrose, saccharose, mannitol, sorbitol, cellulose, etc.), a lubricant (silica, talc, stearic acid or a salt thereof such as magnesium stearate or calcium stearate), and / or polyethylene glycol, etc. May be included. Tablets may also contain binders (such as magnesium aluminum silicate, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone) and optionally disintegrants (starch, agar, alginic acid or salts thereof, and / or foaming. Sex mixtures, etc.), absorbents, colorants, flavors and / or sweeteners and the like.

  Furthermore, the SCCA-1 production inhibitor and pharmaceutical composition of the present invention are a preservative, a solubilizer, a stabilizer, a wetting agent, an emulsifier, a sweetener, a coloring agent, a flavoring agent, a salt for changing osmotic pressure, Buffers, coatings, or antioxidants can also be included. Moreover, the SCCA-1 production inhibitor and pharmaceutical composition of the present invention may further contain a therapeutically valuable substance such as an additional active ingredient other than 1-piperidinepropionic acid and / or a salt thereof according to the present invention.

  The dosage of SCCA-1 production inhibitors and pharmaceutical compositions of the present invention can vary within wide limits and can be determined by methods known in the art. Such dosage is tailored to individual requirements in each particular case involving the route of administration, the condition being treated, and the patient being treated. The dosage may vary depending on the use and dosage form of the pharmaceutical composition or the weight and body surface area of the patient, but a daily dosage of 0.1 μg to 10,000 mg is preferred, more preferably 100 μg to 1000 mg. Is preferred. These may be administered once or divided, and the dosage may be administered orally or by injection, or by application.

Examination using SCCA high expression transgenic mice:
SCCA1 is highly expressed in the upper layer of the epidermis in abnormally proliferating human skin, but almost no expression is observed in normal skin. Since involucrin is also a differentiation marker expressed in the final differentiation process of the epidermis, SCCA1 transgenic mouse mice were prepared using the involucrin promoter. SCCA-1 cDNA was fused to the involucrin promoter (LB Taichman, State University of New York, Stony Brook, NY; Carroll et al., 1993) and BDF1 mice were used to generate transgenic mice. In order to obtain a hairless phenotype, the involucrin-SCCA-1 transgenic mice were bred with HR-1 mice and SCCA-1 + / + mice were obtained after 3 passages. The transilluminator was irradiated with UVB for 2 days at 200 mJ / cm ² / day. Thereafter, a skin sample was collected, and in order to confirm that SCCA1 was highly expressed in the upper epidermis as intended in a transgenic mouse mouse, using a MoMap kit (Ventana Medical Systems, Inc.), anti-SCCA- As a result of immunostaining with a monoclonal antibody (Santa Cruz Biotechnology, CA, USA), strong expression of SCCA-1 was observed in the upper epidermis of the transgenic mouse. It was not recognized (FIG. 1). Furthermore, when the skin of the transgenic mouse was collected and fixed with PFA and stained with hematoxylin and eosin, remarkable epidermal thickening was observed, but in the similarly stained wild type mouse, such epidermal thickening was not observed. (FIG. 2).

  In order to examine the activity of cell proliferation in the transgenic mice prepared as described above, immunostaining was performed with an anti-Ki-67 antibody (sc-7846: Santa Cruz Biotechnology, CA, USA) and an anti-keratin 14 antibody. Keratin 14 as a layer marker and Ki-67 as a proliferative cell marker were enhanced, and Ki-67 cells proliferated remarkably in the basal cell layer of SCCA1 transgenic mice. Such enhancement was not observed in wild-type mice (FIG. 3).

Examination using SCCA highly expressing cells:
SCCA-1 and SCCA-2 cDNA (Takeda et al., 2002) cloned from a psoriatic cDNA library were subcloned into a p-target vector and transfected into 3T3 / J2 cells using Lipofectamine Plus (Invitrogen). After 4 weeks of culture in 500 μg / ml G418, 20 colonies for SCCA-1 were isolated and established as an SCCA-expressing cell line (FIG. 4).

When the SCCA expression level of the SCCA high-expressing cells prepared as described above was measured by Taqman PCR, the expression level of 20 colonies varied by 1 to 2772 times. In the SCCA high-expressing cells, an increase in cell proliferation activity was observed as the SCCA-1 expression level increased (FIG. 5). PCR analysis was performed as follows. RNA was prepared using Isogen (Nippon Gene), and an equivalent amount of complementary DNA was subjected to quantitative PCR analysis with an ABI PRISM 7900HT sequence detector (Taqman PE) (Applied Biosystems) using the following primers / probes.
・ Human SCCA1
Forward primer 5'-GTGCTATCTGGAGTCCT-3 '(SEQ ID NO: 1)
Reverse primer 5'-CTGTTGTTGCCAGCAA-3 '(SEQ ID NO: 2)
Probe 5'-CATCACCTACTTCAACT-3 '(SEQ ID NO: 3)
・ Human SCCA2
Forward primer 5'-CTCTGCTTCCTCTAGGAACACAG-3 '(SEQ ID NO: 4)
Reverse primer 5'-TGTTGGCGATCTTCAGCTCA-3 '(SEQ ID NO: 5)
Probe 5'-AGTTCCAGATCACATCGAGTT-3 (SEQ ID NO: 6)
・ Human GAPDH
Forward primer 5'-GAAGGTGAAGGTCGGAGTC-3 '(SEQ ID NO: 7)
Reverse primer 5'-GAAGATGGTGATGGGATTTC-3 '(SEQ ID NO: 8)
Probe 5'-AGGCTGAGAACGGGAAGCTTGT-3 '(SEQ ID NO: 9)
GAPDH was used as an internal control. Semi-quantitative RT-PCR analysis was performed using the following primers.
・ Mouse SCCA (Serpinb 3a)
Forward primer 5'-CTATCCTTCCTGCCCACTTC-3 '(SEQ ID NO: 10)
Reverse primer 5'-CTTTGCTCCATAGATACTGTTGG-3 '(SEQ ID NO: 11)
・ Mouse GAPDH
Forward primer 5'-CCCATCACCATCTTCCAG-3 '(SEQ ID NO: 12)
Reverse primer 5'-CCTGCTTCACCACCTTCT-3 (SEQ ID NO: 13)

Study using SCCA knockdown cells:
A double-stranded oligonucleotide is designed to correspond to the consensus sequence of human SCCA (5′-AAGCCAACACCAAGTTCATGT-3 ′: SEQ ID NO: 14) that forms a hairpin structure in the expressed oligo RNA, and a p silencer vector (Ambion) Cloned in and transfected into keratinocyte cell line HaCat cells. SiRNA against GFP mRNA (Ambion) was used as a control. Transfection was performed with Lipofectamine 2000 (Invitrogen), and hygromycin-B was added to the medium and cultured for 4-6 weeks to establish a cell line in which SCCA expression was constantly knocked down (Fig. 6).

  When the SCCA expression level of the SCCA knockdown cells prepared as described above was measured by Taqman PCR as described above, both SCCA-1 and SCCA-2 expression levels were suppressed by 90% or more (FIG. 7). In the SCCA knockdown cells, a significant decrease in keratinocyte proliferation activity was observed compared to the control (FIG. 8).

SCCA-1 expression inhibitory effect by 1-piperidinepropionic acid in cell lines:
Human keratinocytes were cultured and 1-piperidinepropionic acid at various concentrations was added in a 60-70% confluent state. After 24 hours, RNA was collected and the SCCA gene expression level was measured using quantitative PCR (G3PDH was used as an internal standard). The result is shown in FIG. As shown in this figure, a significant decrease in the dose-dependent SCCA-1 gene expression level was observed.

result:
A significant decrease in the dose-dependent expression of SCCA-1 was observed (FIG. 10).

Effect of 1-piperidinepropionic acid on human skin:
A 1% or 3% 1-piperidinepropionic acid-containing drug was applied continuously using the half-face method. That is, 1-piperidinepropionic acid-containing drug was applied to the half face, and 1-piperidinepropionic acid removal control was applied to the half face. Whether to apply the 1-piperidinepropionic acid-containing drug to the left or right was blind for both the subject and the measurer (double blind test). Before the continuous use, the amount of epidermal SCCA1, the amount of transdermal moisture transpiration (TEWL), the amount of stratum corneum, and the skin thickness (OCT) were measured after 2 months.

The expression level of SCCA-1 on both cheeks was measured by the following ELISA method.
ELISA method:
The skin stratum corneum sample was collected by tape stripping which peeled off after applying a transparent adhesive tape (Cellotape (registered trademark) (NICHIBAN)) to the skin surface. The tape with the skin horny layer attached was cut, immersed in an extraction buffer (0.1 M Tris-HCl (pH 8.0), 0.14 M NaCl, 0.1% Tween-20, 1 ml), and sonicated (20 sec. A sample extract was prepared by applying x4). 100 μl of a polyclonal anti-SCCA antibody (diluted 1: 1000) diluted in PBS was dispensed into each well of a 96-well ELISA plate, allowed to stand overnight at room temperature, and allowed to bind to the solid phase of the plate. Thereafter, in order to inhibit non-specific binding to the plate, the plate was incubated for 1 hour in a blocking solution (a solution obtained by diluting Block Ace with PBS-Tween 20; 300 μl / well). 50 μl of the sample extract was added to each well of the ELISA plate and reacted at 37 ° C. for 2 hours. Monoclonal anti-SCCA-1 antibody (diluted 1: 1000) was added and reacted at 37 ° C. for 1 hour. Next, a secondary antibody, horseradish peroxidase-labeled anti-mouse was added and reacted at 37 ° C. for 1 hour. After washing with 0.1% Tween-20 PBS, substrates 3 ′, 3 ′, 5 ′, 5'-tetramethylbenzidine (TMB) was added, and color was developed using TMB Peroxidase EIA substrate kit (BIO-RAD), and measurement was performed at 630 nm. By applying 3% 1-piperidinepropionic acid continuously for 2 months, the amount of SCCA-1 in the epidermis was significantly reduced as compared with 15% ethanol as a control (FIG. 10A). Moreover, it became clear from the difference (degree of improvement) result from 0 month data of each subject that SCCA-1 significantly decreased by continuous application (FIG. 10B).

  The transepidermal water evaporation (TEWL) of both cheeks was measured 5 times with a Vapometer (Delfin Technologies Ltd, Finland), and the average value was calculated. The higher the TEWL value, the higher the roughness of the skin. The result is shown in FIG. As shown in this figure, in the cheeks to which 1% or 3% 1-piperidinepropionic acid was applied, significant suppression of TEWL increase was observed 2 months after application.

  The average value was calculated by measuring the horny layer moisture content of both cheeks at five locations for each site using Corneometer CM825C (Courage + Khazaka electric, Germany). The lower the value, the more lost the stratum corneum is. The result is shown in FIG. As shown in this figure, in the cheeks to which 1% or 3% 1-piperidinepropionic acid was applied, a significant suppression of decrease in stratum corneum water content was observed 2 months after application.

Effect of 1-piperidinepropionic acid on skin thickening:
A drug containing 3% 1-piperidinepropionic acid was applied to one cheek, and the control was applied to the other cheek (for 2 months), using optical coherence tomography (OCT; ISIS optronics, Germany) The skin thickness was then measured. The result is shown in FIG. As shown in this figure, significant epidermal thickening suppression by 1-piperidinepropionic acid was observed.

A method for producing an SCCA-1 transgenic mouse will be described. Skin staining is shown for epidermal thickening in SCCA high expressing mice compared to aged wild type mice. The activation of cell proliferation in SCCA high expressing mice shows enhanced keratin 14 and Ki-67 compared to wild type mice. The establishment method of the SCCA-1 highly expressing cell is shown. The correlation between SCCA-1 expression level and cell proliferation activity is shown. Figure 3 shows a method for establishing SCCA knockdown cells using RNA interference. The expression level of SCCA-1 and SCCA-2 in the SCCA knockdown cell line is shown. FIG. 5 shows reduced cell proliferative activity in SCCA knockdown cell lines. The SCCA-1 expression inhibitory effect by 1-piperidine propionic acid in a cell system is shown. The effect of improving the amount of SCCA-1 after application of 1-piperidinepropionic acid to human cheeks is shown. The TEWL improvement effect after 1-piperidine propionic acid application to a human cheek is shown. The improvement effect of the stratum corneum water content after 1-piperidine propionic acid application to a human cheek is shown. The thickness of the epidermis after application of 1-piperidinepropionic acid to the human cheek is shown.

Claims (4)

1-piperidinepropionic acid of general formula (I)
And / or SCCA-1 (Squamous Cell Carcinoma Antigen 1) production inhibitor containing the salt as an active ingredient.   A pharmaceutical composition for preventing and / or treating a disease caused by abnormal cell proliferation, comprising 1-piperidinepropionic acid and / or a salt thereof according to claim 1 as an active ingredient.   A pharmaceutical composition for preventing and / or treating epidermis thickening, comprising 1-piperidinepropionic acid and / or a salt thereof according to claim 1 as an active ingredient.   A basal cell carcinoma (BCC), squamous cell carcinoma (SCC), Bowen's disease (Bowen's) containing 1-piperidinepropionic acid and / or a salt thereof according to claim 1 as an active ingredient. disease), pilomatricoma, seborrheic keratosis, actinic keratosis, solar keratosis, and the like Cancer state, lichen planus-like keratosis, benign lichenoid keratosis, acrochordon, cutaneous tag, pustular psoriasis, vulgaris Psoriasis (psoriasis vulgaris), psoriasis (psoriasis), xeroderma pigmentatosum (XP), atopic dermatitis, systemic lupus erythematosus (SLE), circular systemic lupus erythematosus (d) nevus and wrinkles such as iscoid lupus erythematosus (DLE), erythematosus, porokeratosis, inflammatory linear verrucous epidermal naevus (ILVEN), hyperplasia A pharmaceutical composition for treating a disease selected from the group consisting of benign keratosis with