JPH0579645B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a therapeutic agent for diseases accompanied by abnormal accumulation of collagen. For more details, please refer to the general formula ()

The present invention relates to a therapeutic agent for diseases accompanied by abnormal accumulation of collagen, which contains a compound represented by the following formula (wherein R ¹ and R ² each represent a hydrogen atom or a methyl group) as an active ingredient. [Prior Art] Diseases accompanied by abnormal accumulation of collagen include liver fibrosis (meaning liver fibrosis in a broad sense including liver cirrhosis and chronic hepatitis), pulmonary fibrosis, keloids, hypertrophic scars, and scleroderma. diseases (latest medicine,
42, No. 10, pp. 2075-2139, 1987). These diseases are difficult to treat, and it can be said that no therapeutic agents have been established for these diseases. In diseases accompanied by abnormal accumulation of collagen,
It has been suggested that the balance between collagen synthesis and degradation is lost; for example, liver fibrosis associated with cirrhosis is associated with increased collagen biosynthesis (Science, 176
Vol. 795, 1972) and decreased collagen decomposition ability (Biochemical Journal, Vol. 118, p. 229;
1970 and Life Sciences, Volume 30, Issue 16, 1379
p., 1982). Among these, the decrease in collagen decomposition ability is thought to be due to a decrease in collagenase activity, which is the rate-limiting enzyme for collagen decomposition. For example, skin fibroblasts from mice with bleomycin-induced fibrosis (see Skin, Vol. 14, No. 4, p. 217, 1972), skin from scleroderma patients (Journal of Clinical
Investigation, Vol. 56, p. 1175, 1975) and in the liver tissues of patients with alcoholic cirrhosis (Life
Sciences, Vol. 30, No. 16, p. 1379, 1982) says that collagenase activity is reduced. Therefore, in the treatment of diseases accompanied by abnormal accumulation of collagen, it is necessary to decompose the abnormally accumulated collagen, and for this purpose it is necessary to promote the production of collagenase. This has already been pointed out, for example, in the treatment of liver cirrhosis (the history of medicine,
136, No. 13, p. 1027, 1986). Collagenase is secreted from cells as a precursor, procollagenase, and is then activated to collagenase in vivo by proteolytic enzymes such as plasmin and stromelysin (Biochemical Journal, Vol. 166, p. 21, 1977 and Proceedings of the National Academy of
Sciences of the USA, Vol. 86, p. 2632, 1989), therefore, compounds having pro-collagenase production-promoting effects are considered effective in promoting collagenase production. [Problems to be Solved by the Invention] The present inventor conducted various studies in order to obtain a therapeutic agent for diseases accompanied by abnormal accumulation of collagen, which has a procollagenase production promoting effect. An object of the present invention is to provide an excellent therapeutic agent for diseases accompanied by abnormal accumulation of collagen. [Means for Solving the Problem] As a result of screening various compounds, the present inventors discovered that the compound represented by the above general formula () promotes procollagenase production, and based on this knowledge, the present inventors have developed the present invention. Completed the invention. Specific examples of the compound represented by the general formula () include L-serine, DL-serine, N-methyl-L-serine, N-methyl-DL-serine,
N,N-dimethyl-L-serine and the like can be mentioned. The therapeutic agent of the present invention is administered to humans orally or parenterally, such as by injection or transdermally. Dosage forms for oral administration include solid preparations such as tablets, granules, powders, fine granules, and hard capsules;
Includes liquid preparations such as syrups, elixirs, and soft capsules. Such preparations are manufactured by conventional methods, and tablets, granules, powders, and fine granules are prepared by combining the compound of general formula () with, for example, lactose, starch, crystalline cellulose, magnesium stearate, hydroxypropylcellulose, talc, etc. hard capsules are prepared by appropriately filling capsules with the above-mentioned fine granules and powders. In addition, syrups are manufactured by dissolving or suspending the compound of general formula () in an aqueous solution containing sucrose, D-sorbitol, carboxymethyl cellulose, etc. together with preservatives such as methyl parahydroxybenzoate and propyl parahydroxybenzoate. is produced by mixing glycerin, orange oil, lemon oil, corianter oil, anise oil, talc, etc. with an ethanol solution of the compound of general formula (). Soft capsules are produced by dissolving or suspending the compound of general formula () in a lipid excipient such as vegetable oil, oil emulsion, glycols, etc., and filling the solution into soft capsules. Injectables are produced by dissolving or emulsifying the compound of general formula () in physiological saline or a lipid excipient such as vegetable oil, oily emulsion, or glycol, and aseptically sealing the solution in ampoules or vials. . Transdermal preparations include ointments, lotions, poultices, gels, creams, liquids, sprays, patches, and the like. Such preparations contain the compound of general formula () and conventional pharmaceutical additives, such as hydrocarbons such as vaseline, squalane, and liquid paraffin, higher alcohols such as stearyl alcohol and cetanol, and higher fatty acids such as isopropyl myristate and isopropyl palmitate. lower alkyl esters, animal fats and oils such as lanolin, polyhydric alcohols such as glycerin and propylene glycol,
Polyethylene glycols such as Macrogol 400 and Macrogol 4000, glycerin fatty acid esters such as glyceryl monostearate, sodium lauryl sulfate, polyethylene glycol monostearate, polyoxyethylene alkyl ether phosphoric acid (product name, NIKKOL DDP-2, Nippon Surf) Surfactants such as Actant Industries Co., Ltd.)
It can be produced by a conventional method by mixing wax, resin, water and, if necessary, a preservative such as butyl paraoxybenzoate or methyl paraoxybenzoate. The therapeutic agent of the present invention is administered orally or parenterally. For example, for diseases such as liver fibrosis and pulmonary fibrosis, in which collagen is abnormally accumulated in organs, it can be administered orally or by injection, and for diseases in which collagen is abnormally accumulated in the epithelium, such as keloids and hypertrophic scars, it can be administered percutaneously or locally. Preferably, an inventive therapeutic agent is administered. The dosage varies depending on the patient's age, weight, symptoms, administration method, etc., but when administered to adults, the compound () is generally administered in an amount of 0.5 to 1000 mg one to three times a day. For example, when administering systemically orally or by injection to treat diseases such as liver fibrosis and pulmonary fibrosis, where collagen is abnormally accumulated in organs, a dose of 30 to 1000 mg per dose is appropriate. When locally administered transdermally for diseases such as sexual scars and other diseases in which collagen is abnormally accumulated in the epithelium, a dose of 1 to 50 mg per dose is appropriate. [Effects of the Invention] The compound of general formula () promoted the production of procollagenase (see Test Example 1 below). In tests using animals, it was also confirmed that it is effective in treating diseases associated with abnormal collagen accumulation, such as liver fibrosis (see Test Example 2 below).
reference). On the other hand, the compound of general formula () has low toxicity (see Test Example 3 below) and low skin irritation (see Test Example 4 below). The above facts indicate that the compound of general formula () is useful as a therapeutic and preventive agent for various diseases associated with abnormal accumulation of collagen. Hereinafter, the present invention will be explained in detail by giving test examples. In addition, the following abbreviations used in the test examples have the following meanings. HF medium: A medium prepared by dissolving 10.6 g of Ham's F-12 powder medium (manufactured by Nissui Pharmaceutical Co., Ltd.) in 11 parts of distilled water. HF-AV medium: 1.76 g of powdered Eagle amino acid vitamin medium (manufactured by Nissui Pharmaceutical Co., Ltd.) per 11 HF medium,
After adding 1.6 g of sodium bicarbonate, 50 mg of streptomycin sulfate and 60 mg of kanamycin sulfate;
A medium whose pH has been adjusted to approximately 7 by blowing carbon dioxide gas into it. Tris: Tris(hydroxymethyl)aminomethane MES buffer: Contains 0.5M NaCl, 1mM _CaCl2 , and 0.05v/v% Brij-35 (trade name for polyoxyethylene lauryl alcohol ether)
50mM 2-(N-morpholino)ethanesulfonic acid monohydrate aqueous solution was added to Tris aqueous solution at 4℃.
Buffer solution adjusted to PH6.5. Acetate buffer: 0.5M NaCl, 1mM CaCl _and
A buffer solution in which a 25mM acetic acid aqueous solution containing 0.05v/v% Brij-35 was adjusted to pH 4.5 at 4°C with a Tris aqueous solution. MES-A buffer: A buffer prepared by adjusting the MES buffer to pH 7 at 4°C with an aqueous Tris solution. MES-B buffer: A buffer solution in which an MES buffer solution and an acetate buffer solution were mixed and the pH was adjusted to 6.2 at 4°C. MES-C buffer: A buffer solution in which an MES buffer solution and an acetate buffer solution were mixed and the pH was adjusted to 5.2 at 4°C. Measurement buffer: 0.2MNaCl, _5mMCaCl2 ,
A 50mM Tris aqueous solution containing 0.05v/v% Brij-35 and 0.02w/v% _NaN3 was PHed with hydrochloric acid at room temperature.
Buffer adjusted to 7.5. (Test Example 1) Procollagenase production promoting effect 1 Test compound/L-serine/N-methyl-L-serine 2 Cells used Human fibrosarcoma cells HT1080 (ATCC CCL121)
Anchorage-independent cells (referred to as human fibrosarcoma HT-P11) that can grow in serum-free and protein-free media due to
Kanebo Co., Ltd., owned by the Biochemical Research Institute). The cells were precultured and cell suspensions were prepared and tested as follows. Human fibrosarcoma HT-P11 was suspended in HF-AV medium at a density of 1 x 10 ⁵ /ml, and 20 ml of this suspension was added to flasks (bottom area 75 cm ² each), and the mixture was heated with 95% air and 5% carbon dioxide. The cells were statically cultured at 37°C for 3 days in an atmosphere of After 3 days of culture, centrifuge (600 rpm, 10 minutes)
Cells were collected by The obtained cells were HF-
The cells were suspended in AV medium to prepare a cell suspension with a density of 7×10 ⁴ /ml. 3 Test method 2 ml of the above cell suspension was added to a 6-well plate (bottom area: 9.4 cm ² ) and cultured at 37° C. for 1 day in an atmosphere of 95% air and 5% carbon dioxide. Then 10mM
Aqueous solutions of each test compound at various concentrations were added to HF-AV medium.
Dilute to a concentration of 600 μM, add 0.4 ml of this solution to the culture solution, and add in an atmosphere of 95% air - 5% carbon dioxide.
Cultured for 13 days. After culturing, add 1/20 to the culture solution.
0 volume of 10 v/v% Brij-35 aqueous solution was added, and cells were removed by centrifugation (600 rpm, 10 minutes) to obtain a culture supernatant. Next, add 0.5 ml of MES-A buffer to 0.5 ml of culture supernatant.
Zinc chelating Sepharose 6B (manufactured by Pharmacia) equilibrated with MES-A buffer
It was applied to a column packed with 0.5 ml. MES on column
The collagenase inhibitor was eluted and removed from the column by flowing 1 ml of -B buffer four times. Next, 1 ml each of MES-C buffer was poured twice.
The eluate was collected to obtain 2 ml of procollagenase solution. After adding 1N NaOH to the procollagenase solution and adjusting the pH of the solution to about 7, add MES-A buffer to make 2.5ml, and then prepare a solution of about 0.1 to 0.7 units/ml of procollagenase with measurement buffer. This was used as the test solution. Next, 20 μl of trypsin solution (manufactured by Sigma, Type 12 adjusted to a concentration of 1 mg/ml with measurement buffer) was added to 50 μl of the test solution, and after incubating at 35°C for 5 minutes, soybean trypsin inhibitor solution ( Merck No. 24020 at a concentration of 3 in measurement buffer
Trypsin was inactivated by adding 30 μl (adjusted to mg/ml) to obtain a collagenase solution. 0.01N acetic acid solution (concentration 1 mg/ml) of type I collagen labeled with fluorescein isothiocyanate (FITC-collagen, manufactured by Cosmo Bio)
using the method of Nagai et al. (inflammation, 4) as a substrate solution.
Vol. 2, p. 123, 1984), the activity (units/ml) of the collagenase solution was measured.
The amount of collagenase generated from procollagenase by the above trypsin treatment decomposing 1 μg of type I collagen (FITC-collagen) per minute at 35°C is defined as one unit of procollagenase, and the amount of procollagenase produced (unit/ culture solution ml)
was calculated (this value is set as X). On the other hand, as a control test, distilled water was added instead of the aqueous solution of the test compound, and the amount of procollagenase produced (unit/mL of culture solution) when the test compound was not added was determined by the same procedure as above (this value is designated as Y). ). Next, the procollagenase production promotion rate (%) was calculated from these values using the formula below. Procollagenase production promotion rate (%)=X-Y/Y×100 4 Test results are shown in Table 1.

[Table] As shown above, the compound of general formula () promotes procollagenase production in cells. (Test Example 2) Therapeutic effect on liver fibrosis A liver fibrosis model animal was created and tested. 1 Test compound N-methyl-L-serine 2 Test animal Wistar male rat (5 weeks old, weight 122 g)
~134g, 5 animals per group). 3 Test method Carbon tetrachloride (hereinafter abbreviated as CCl ₄ ) and olive oil were tested according to the drug screening method for liver function improvement effect (supervised by Hikaru Ozawa, drug efficacy screening method for new drug development, p. 75, published by Maruzen, 1984). 2ml/Kg each time, twice a week (Monday,
(Thursday) subcutaneously on the back of rats for 10 weeks to create liver fibrosis rats. Aqueous solution of N-methyl-L-serine (concentration 66.6
mg/ml) was orally administered to rats at a rate of 3 ml/Kg once a day for 7 weeks (excluding Sundays) from the 4th week after the start of CCl ₄ administration. Thereafter, blood was collected from the abdominal aorta and placed in a heparin-treated test tube. This was centrifuged to obtain plasma, which was stored frozen at -80°C until the biochemical tests described below were performed. In addition, the rats were killed by whole blood sampling, the liver was removed, its weight was measured, and the liver was divided into two parts under ice-cooling. One half was frozen at -80°C for the determination of hydroxyproline in the liver, and the other half was fixed in 10% formalin for histological examination. On the other hand, as an untreated group, a group was established in which distilled water was administered in place of the test compound aqueous solution at a rate of 3 ml/Kg to liver fibrosis rats according to the same schedule as above, and blood sampling and liver removal were performed in the same manner. In addition, a group of normal rats to which neither CCl ₄ nor the test compound aqueous solution was administered was established, and blood sampling and liver extraction were performed in the same manner. Hydroxyproline in the liver and blood was then quantified using each of the above livers and plasma. Furthermore, hydroxyproline is an indicator of collagen content (Methods of Biochemical
Analysis, vol. 15, p. 25, 1967). In addition, the following plasma biochemical tests [see (b) to (g) below] and liver histological tests (h) are indicators of liver damage.
I also went. The respective inspection items and inspection methods are as follows. (a) Amount of hydroxyproline (hereinafter abbreviated as Hyp) in the liver and blood (a-1) Method for quantifying Hyp in the liver Take approximately 100 mg of liver, weigh it accurately, and add physiological After adding 250 μl of saline, the mixture was pulverized by vigorous stirring (28,000 rpm, 30 seconds) using a Hiscotron homogenizer (manufactured by Nippon Medical Science Instruments Manufacturing Co., Ltd.). The resulting suspension was centrifuged (15,000 rpm, 10 minutes) and divided into a supernatant sample and a precipitate sample. 6N hydrochloric acid was added to each sample to make 2 ml, and the mixture was hydrolyzed at 110°C for 24 hours. Hydrochloric acid was removed from the hydrolyzed solution by centrifugal concentration, and the concentrate was reduced to 0.01N.
Dissolved in hydrochloric acid. This solution was analyzed using a post-column amino acid analysis method using high-performance liquid chromatography (Proceedings of the National Academy of Sciences).
Sciences of the USA, Volume 72, Issue 2, Page 619,
The amount of Hyp in the supernatant sample and precipitate sample was determined by quantifying the amount of Hyp in the supernatant sample and the precipitate sample. and,
The amounts of Hyp in the supernatant sample and the precipitate sample were totaled, and the amount of Hyp per unit liver weight (μmol/g liver) was determined from this value and the liver weight. (a-2) Quantification method of Hyp in blood Add 5w/v% sulfosalicylic acid aqueous solution to 100 μl of plasma
Add 100 μl under ice-cooling and centrifuge (10000 rpm,
3 minutes) to obtain a supernatant, and remove Hyp in the supernatant (a-
It was quantified by post-column amino acid analysis using high-performance liquid chromatography in the same manner as in 1). (b) Glutamate pyruvate transaminase (hereinafter abbreviated as GPT) amount Karmen method (see Izumi Kanai, Summary of Clinical Testing Methods, revised 29th edition, p. 514, Kanehara Publishing Co., Ltd., 1983)
Measured at (c) Amount of glutamate oxaloacetate transaminase (hereinafter abbreviated as GOT) Karmen method (see Izumi Kanai, Summary of Clinical Testing Methods, revised 29th edition, p. 514, Kanehara Publishing Co., Ltd., 1983)
Measured at (d) Amount of γ-glutamyl transpeptidase (hereinafter abbreviated as γ-GTP) Method using L-γ-glutamyl-p-nitroanilide as a substrate (Izumi Kanai, Summary of Clinical Testing Methods, revised 29th edition, p. 532, Kanehara) Publishing Co., Ltd., 1983)
Measured at (e) Total bilirubin amount Malloy-Evelyn method (Izumi Kanai, Summary of Clinical Testing Methods, revised 29th edition, p. 724, Kanehara Publishing Co., Ltd.)
(see 1983). (f) Amount of alkaline phosphatase Bessey-Lowry method (Izumi Kanai, Summary of Clinical Testing Methods, revised 29th edition, p. 496, Kanehara Publishing Co., Ltd.)
(see 1983). (g) Zinc sulfate turbidity test value (hereinafter abbreviated as ZTT) Standard operating method of the Liver Function Research Group of the Japanese Society of Gastroenterology (Izumi Kanai, Summary of Clinical Testing Methods, revised 29th edition, p. 710, Kanehara Publishing Co., Ltd., 1983) Measured in 2008). (h) Histological examination of the liver The liver was sliced in a conventional manner and observed under a microscope after staining with hematoxin and eosin. 4 Test Results Table 2 shows the amounts of Hyp in the liver and blood, and the biochemical test values of plasma, which are indicators of liver damage.

[Table] As described above, the amount of Hyp in the liver of the test compound administered group was significantly lower and the amount of Hyp in blood was significantly higher than that of the untreated group. This means that the therapeutic agent of the present invention promotes the production of procollagenase, and as a result, promotes the degradation of collagen in fibrotic liver tissue and releases it into the blood.
This shows that Hyp was released. In addition, in the test compound administration group, compared to the untreated group,
Plasma biochemical test values (GPT, GOT, γ-
Since the levels of GTP, total bilirubin, alkaline phosphatase, and ZTT were significantly lower, it can be said that the test compound improved liver damage associated with liver fibrosis. On the other hand, in histological examination of the liver, the test compound-administered group showed more liver damage than the untreated group.
It was observed that diffuse hepatocyte vacuole formation, bile canalicular hyperplasia, and common bile duct epithelial cell hyperplasia were reduced. This also indicates that the test compound is effective in improving liver damage associated with liver fibrosis. The above test results indicate that the therapeutic agent of the present invention containing the above test compound as an active ingredient is effective in improving liver damage associated with liver fibrosis. (Experiment Example 3) Acute toxicity test 1 Test compound Same as Test Example 1. 2 Test method and test results Dissolve each test compound aqueous solution in purified water.
It was orally administered to ICR male mice (5 weeks old, weight 24-28 g, 5 mice per group) at a rate of 0.2 ml/Kg body weight (5 g/Kg body weight as a specimen). Thereafter, the mice were observed for 7 days, but no deaths were observed in any of the sample administration groups. (Test Example 4) Skin irritation test 1 Test compound Same as Test Example 1. 2 Test method: Using Japanese native male domestic rabbits (weighing approximately 3 kg), the Drez method (APPRAISAL OF THE SAFETY) was used.
OF CHEMICALS IN FOODS, DRAGS AND
COSMETICS, p.46, 1959, Edited and
Published by THE EDITORIAL
COMMITTEE, ASSOCIATION OF FOOD
& DRUG OFFICIALS OF USA). Specifically, an abrasion site (damaged skin) is created on the back of a rabbit whose hair has been shaved, and 0.1 ml of a 1 w/v % aqueous solution of the test compound is applied to each of the damaged skin and normal skin using a patch test bandage (1.2 x 1.6 cm, Ribbon Aid (manufactured by River Tape Pharmaceutical Co., Ltd.) was infiltrated and applied. After 24 hours, the bandage was removed and the skin was observed for erythema and edema, and the same observation was made 48 hours after removal of the bandage. Then, erythema score and edema score were given respectively according to the following evaluation criteria.

[Table] Based on this score and the formula below, the primary irritation score was calculated. Primary stimulation score calculation formula Primary stimulation score = 1/2 (A ₂₄ +A ₄₈ ) + 1/2
(X ₂₄ +X ₄₈ ) + 1/2 (B ₂₄ + B ₄₈ ) + 1/2 (Y ₂₄
+Y ₄₈ ) Here, each symbol has the following meaning. A ₂₄ ... Erythema score 24 hours after applying a bandage on normal skin A ₄₈ ... Erythema score 48 hours after removing the bandage from normal skin B ₂₄ ... Erythema score 24 hours after applying a bandage on damaged skin B ₄₈ ... Injury Erythema score 48 hours after the bandage is removed from the skin X ₂₄ ... Edema score 24 hours after the bandage is applied to normal skin X ₄₈ ... Edema score 48 hours after the bandage is removed from normal skin Y ₂₄ ... Bandage applied to damaged skin 24 Edema score after ₄₈ hours... Edema score 48 hours after the bandage is removed from the injured skin Next, based on the primary irritation score and the following criteria,
The degree of irritation of the test compound was determined. Test Compound Irritation Level Judgment Criteria Mild irritation...Primary irritation score 0 to less than 2 Moderate irritation...Primary irritation score 2 to less than 5 Strong stimulation...Primary irritation score 5 or more 3 Test results Primary irritation score and irritation for each test compound The degree is shown in Table 3.

[Table] [Examples] The present invention will be explained below with reference to Examples. Example 1 Tablet Tablets each containing 100 mg of L-serine as an active ingredient were prepared as follows. (Formulation) Ingredients Amount (g) L-serine 50 Lactose 10 Corn starch 30 Crystalline cellulose 8 Hydroxypropyl cellulose 1 Magnesium stearate 1 (Operation) Add hydroxypropyl cellulose to a mixture of L-serine, lactose, corn starch and crystalline cellulose. was dissolved in 30 g of water, added, and thoroughly kneaded. The mixture was passed through a 20-mesh sieve to form granules and dried. Magnesium stearate was mixed with the resulting granules and the mixture was compressed into 200 mg tablets. Example 2 Capsule N-methyl-L as an active ingredient in one capsule
- Capsules containing 100 mg of serine were prepared as follows. (Formulation) Ingredients Amount (g) N-methyl-L-serine 100 Lactose 100 Corn starch 50 Crystalline cellulose 47 Magnesium stearate 3 (Procedure) Thoroughly mix each of the above ingredients, and put 300 mg of the mixture into No. 2 capsules. Capsules were obtained by filling. Example 3 Granules Granules containing 100 mg of L-serine as an active ingredient in 1 g were prepared as follows. (Formulation) Ingredients Amount (g) L-serine 100 Lactose 470 Corn starch 400 Hydroxypropyl cellulose 30 (Procedure) Add hydroxypropyl cellulose dissolved in 1000 g of water to the mixture of L-serine, lactose and corn starch and mix thoroughly. did. This kneaded product was passed through a 20 mesh sieve, granulated, dried, and sized to obtain granules. Example 4 Syrup A syrup containing 100 mg of L-serine as an active ingredient in 1 ml was prepared as follows. (Formulation) Component Amount (g) (a) L-serine 100 (b) Methyl paraoxybenzoate 0.3 (c) Propyl paraoxybenzoate 0.15 (d) White sugar 300 (e) 70W/V% D-sorbitol aqueous solution 250 ( f) Sodium citrate 10 (g) Citric acid 1.5 (h) Add purified water to make a total volume of 1000 ml (operation) Add the ingredients (b) to (d) above to 400 ml of purified water at 90°C, dissolve, and then The above component (e) was added at the same temperature, thoroughly mixed, and then cooled to 30°C. L-serine dissolved in 100 ml of purified water was added to this mixture, and the mixture was stirred at 30°C for 30 minutes. Next, the above components (f) and (g) were added and stirred for 20 minutes, purified water was added to make a total volume of 1000 ml, and sterile filtration was performed to obtain a syrup. Example 5 Injection 10 g of N-methyl-L-serine was dissolved in physiological saline for injection to make a 200 ml solution, and the solution was sterilized by sterile filtration. The sterilized solution was dispensed in 2 ml portions into 3 ml brown ampoules. The inside of the ampoule was aseptically purged with nitrogen, and the ampoule was melt-sealed to obtain an injection containing 100 mg of N-methyl-L-serine as an active ingredient in one ampoule. Example 6 Ointment An ointment containing 100 mg of L-serine as an active ingredient in 100 g of ointment was prepared as follows. (Formulation) Component Amount (g) (a) L-serine 0.1 (b) Methyl paraoxybenzoate 0.1 (c) Propylene glycol 6.7 (d) Purified water 44.0 (e) Squalane 4.7 (f) White petrolatum 24.0 (g) Stearyl alcohol 8.7 (h) Isopropyl myristate 6.0 (i) Polyethylene glycol monostearate (product name NIKKOL MYS-45, manufactured by Nippon Surf Actant Industry Co., Ltd.) 1.3 (j) Polyoxyethylene alkyl ether phosphoric acid (product name NIKKOL) DDP-2, manufactured by Nippon Surf Actant Industry Co., Ltd.) 2.3 (k) Glyceryl monostearate 2.0 (l) Butyl paraoxybenzoate 0.1 (Procedure) Heat the components (a) to (d) above to 80°C in a hot water bath. The mixture was heated and mixed, and this mixture was gradually added to the mixture of components (e) to (1) above, which had been heated to 80° C., with stirring. Next, use a homogenizer (TOKUSHUKIKA
Stir vigorously for 2.5 minutes (2500 rpm) using a KOGYO product.
After sufficiently emulsifying and dispersing each component, the mixture was gradually cooled while stirring to obtain an ointment. Example 7 A lotion containing 100 mg of N-methyl-L-serine as an active ingredient in 100 g of lotion was prepared as follows. Ingredients Amount (g) (a) N-methyl-L-serine 0.1 (b) Sodium lauryl sulfate 0.5 (c) Purified water 92.8 (d) White beeswax 0.1 (e) Setanol (trade name Vinasol NAA 48,
(manufactured by NOF Corporation) 1.5 (f) Concentrated glycerin 5.0 (Operation) The components (a) to (c) above were heated to 80°C in a hot water bath and mixed. Meanwhile, mix ingredients (d) to (f) in the same way, and gradually add mixtures (a) to (c) to this mixture while stirring, using a homogenizer (TOKUSHUKIKA
Stir vigorously for 2.5 minutes (2500 rpm) using a KOGYO product.
did. The mixture was gradually cooled to room temperature while stirring to obtain a lotion.

Claims (1)

[Scope of Claims] 1. Diseases accompanied by abnormal accumulation of collagen containing a compound represented by the general formula () [Chemical formula] (wherein R ¹ and R ² each represent a hydrogen atom or a methyl group) as an active ingredient therapeutic agent. 2. The therapeutic agent according to claim 1, which contains L-serine as an active ingredient. 3. The therapeutic agent according to claim 1, which contains N-methyl-L-serine as an active ingredient.