JPH041130A - Remedy for disease accompanying abnormal accumulation of collagen - Google Patents

Abstract

PURPOSE:To obtain a remedy for diseases accompanying abnormal accumulation of collagen, having procollagenase-production promoting action and effective in ameliorating hepatic disorder caused by hepatic fibrosis by using a specific compound as an active component. CONSTITUTION:The objective agent contains a compound of formula (R<1> and R<2> are H or methyl) as an active component. The compound of formula is e.g. L-serine, DL-serine, N-methyl-L-serine, N-methyl-DL-serine or N,N-dimethyl- L-serine. The remedy of formula can be administered to person by oral adminis tration or parenteral administration such as injection or transcutaneous adminis tration. It has low toxicity and low skin-stimulation. The administration amount of the remedy is preferably 30-1,000mg/dose for diseases characterized by abnor mal accumulation of collagen to organs (e.g. hepatic fibrosis or pulmonary fibrosis) in the case of systemic administration by oral administration or injec tion and is 1-50mg/dose for diseases characterized by abnormal accumulation of collagen to epithelium (e.g. keloid or hypertrophic cicatrix) by transcutaneous administration to the diseased part.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention is directed to the treatment of diseases accompanied by abnormal accumulation of collagen (in the formula:
R1 and R2 each represent a hydrogen atom or a methyl group. ) The present invention relates to a therapeutic agent for diseases accompanied by abnormal accumulation of collagen, which contains a compound represented by the following 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. (Modern Medicine, Vol. 42, No. 10, 2075-
2139, 1987). These diseases are difficult to treat, and it can be said that no therapeutic agents have been established for these diseases.

It has been suggested that in diseases accompanied by abnormal accumulation of collagen, the balance between collagen synthesis and degradation is lost; for example, liver fibrosis associated with liver cirrhosis is caused by increased collagen biosynthesis (Science, Vol. 176, 795 pages, 1
972) and decreased collagen decomposition ability (Bioch
Chemical Journal, Volume 118, Page 229,
1970 and Life 5 Sciences, Vol. 30, No. 16, p. 1379, 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 fibrosis induced by pleomycin (Skin, Vol. 14, No. 4, 2
17, 1972), the skin of scleroderma patients (Jou
rnal of C11nical Invest-ti
56, p. 1175, 1975) and in the liver tissues of patients with alcoholic cirrhosis (Lif
eSc 1ences, Volume 30, No. 16, Page 1379,
1982), collagenase activity is said to be reduced.

Therefore, in the treatment of diseases accompanied by abnormal accumulation of collagenase, 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 (Igaku no Ayumi, Vol. 136, No. 13, p. 1027, 1
986).

Collagenase is secreted from cells as a precursor, procollagenase, and is then activated into collagenase by proteolytic enzymes such as plasmin and stromelysin in vivo (Biochemi-cal Jour
nal, vol. 166, p. 21, 1977 and Proc.
-eedings of the National
Academy of 5 sciences of the
U.S.A., vol. 86, p. 2632, 1989), therefore, compounds having an effect of promoting procollagenase production 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 Problems] As a result of screening various compounds, the present inventors discovered that the compound represented by the above general formula (I) promotes procollagenase production, and based on this knowledge, The invention has been completed.

Specific examples of the compound represented by general formula (I) include L-serine, DL-serine, N-methyl-L-serine, N-methyl-DL-serine, N,N-dimethyl-serine, etc. 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, as well as 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 (I) with lactose, starch, crystalline cellulose, magnesium stearate, hydroxypropyl cellulose, talc, etc. Hard capsules are produced by mixing with ordinary pharmaceutical additives, and hard capsules are produced by appropriately filling capsules with the above-mentioned fine granules and powders.

Syrups are also produced by dissolving or suspending the compound of general formula (I) in an aqueous solution containing white sugar, D-sorbitol, carboxymethyl cellulose, etc. together with preservatives such as methyl roseoxybenzoate and propyl roseoxybenzoate. The agent is prepared by mixing an ethanol solution of the compound of the general formula (I) with nigiri serine, orange oil, lemon oil, coriander oil, anise oil, talc, etc.

Soft capsules are produced by dissolving or suspending the compound of general formula (I) in a lipid excipient such as vegetable oil, oily emulsion, or molasses, and filling the solution into soft capsules.

Injections are produced by dissolving or emulsifying the compound of general formula (I) in physiological saline or a lipid excipient such as vegetable oil, oily emulsion, or glycol, and aseptically sealing the solution in an ampoule or vial.

Transdermal preparations include ointments, lotions, poultices, gels, creams, liquids, sprays, patches, and the like. Such preparations contain the compound of general formula (I) and conventional pharmaceutical additives, such as hydrocarbons such as vaseline, squalane and liquid paraffin, higher alcohols such as stearyl alcohol and cetanol, and higher alcohols such as isopropyl myristate and isopropyl palmitate. Lower alkyl esters of fatty acids, animal fats and oils such as lanolin, polyhydric alcohols such as glycerin and propylene glycol, macrogol 40
0, polyethylene glycol such as Macrogol 4000, glycerin fatty acid ester such as glyceryl monostearate, sodium lauryl sulfate, polyethylene glycol monostearate, polyoxyethylene alkyl ether phosphoric acid (trade name, NIKKOL DDP-2, Nippon Surfactant Kogyo Co., Ltd.) surfactants such as
It can be produced by a conventional method by mixing wax, resin, water and, if necessary, a preservative such as butyl roseoxybenzoate or methyl roseoxybenzoate.

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, oral or injection therapy is administered, and for diseases in which collagen is abnormally accumulated in the epithelium, such as keloids and hypertrophic scars, transdermal or local injections are administered. Preferably, the therapeutic agent of the invention is administered. The dosage varies depending on the patient's age, weight, symptoms, administration method, etc., but when administered to adults, generally 0.5 to 1000 mg of compound (I) is administered 1 to 3 times a day. Administer. For example, when administering systemically by oral or 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 administered locally through the skin for diseases in which collagen is abnormally accumulated in the epithelium, such as genital migraine, 1 dose per dose is administered locally.
A dosage of ˜50 mg is suitable.

[Effects of the Invention] The compound of general formula (I) 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 accumulation of collagen, such as liver fibrosis (see Test Example 2 below).

On the other hand, the compound of general formula (I) 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 (I) is useful as a therapeutic and preventive agent for various diseases associated with abnormal accumulation of collagen.

The present invention will be described in detail below with reference to test examples. In addition,
The following abbreviations used in the test examples have the following meanings.

HF medium: Ham F-12 powder medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) 1
A medium prepared by dissolving 0.6 g in distilled water 11.

Tsuji Danji Δy forgery: per 11 HF medium, 1.76 g of powdered Eagle amino acid vitamin medium (manufactured by Hinaga Pharmaceutical Co., Ltd.), 1.6 g of sodium bicarbonate, 5 ml of streptomycin sulfate.
After adding 0 mg of kanamycin sulfate and 60 mg of kanamycin sulfate, the pH was adjusted to about 7 by blowing carbon dioxide gas into the medium.

Tris: Tris(hydroxymethyl)aminomethane MES buffer: 0.5M NaCl, 1mM CaC
l2, and 0.05v/v% Br1j-35 (trade name of polyoxyethylene lauryl alcohol ether)
A 50mM aqueous solution of 2(N-morpholino)ethanesulfonic acid monohydrate containing
Buffer adjusted to H6.5.

Acetate buffer: 0.5M NaCl, 1mM CaCl2
and 25m containing 0.05v/v% Br1j-35
A buffer solution prepared by adjusting M acetic acid aqueous solution to pH 4.5 at 4°C with Tris aqueous solution.

MES-A buffer: MES buffer in Tris aqueous solution
Buffer adjusted to pH 7 at °C.

MES-B buffer: A buffer solution in which MES buffer solution and acetate buffer solution were mixed and adjusted to pH 6.2 at 4°C.

MES-C buffer: A buffer solution that was prepared by mixing an MES buffer solution and an acetate buffer solution and adjusting the pH to 5.2 at 4°C.

Measurement buffer: 0.2M NaC1, 5mMCaC1□
, 0.05v/v1% Br1j-35 and 0.02w
A buffer solution prepared by adjusting a 50 mM Tris aqueous solution containing /v% NaN to pH 7.5 at room temperature with hydrochloric acid.

(Test Example 1) Procollagenase production promoting effect 1) Test compound/L-serine/N-methyl-L-serine 2) Cells used Human fibrosarcoma cells HT1080 (ATCCCCCL12
The test was conducted using anchorage-independent cells (referred to as human fibrosarcoma HT-pH, owned by Kanebo Co., Ltd., Seikagaku Institute) that can grow in a serum-free and protein-free medium derived from 1). The cells were precultured and cell suspensions were prepared and tested as follows.

Human fibrosarcoma HT-pH was placed in HF-AV medium at a density of 1.
xlO'/ml, and 20 ml of this suspension was added to each flask (bottom area: 75 cm2) and cultured stationary at 37°C for 3 days in an atmosphere of 95% air-5% carbon dioxide.

After culturing for 3 days, cells were collected by centrifugation (600 rpm, 10 minutes). The obtained cells were suspended in HF-AV medium to prepare a cell suspension with a density of 7 x lO'/ml.

3) Test method Pour 2 ml of the above cell suspension into a 6-well plate (bottom area: 9
．． 4 cm2) and cultured at 37°C for 1 day in an atmosphere of 95% air-5% carbon dioxide. Thereafter, each test compound aqueous solution with a concentration of 10mM was diluted with a HF-AV medium to a concentration of 600kM, and 0.4ml of this solution was added to the culture medium.
The cells were cultured for 13 days in an atmosphere of 95% air and 5% carbon dioxide. After culturing, add 1/200 volume of 10v/ml to the culture solution.
Add v$Br1j-35 aqueous solution and centrifuge (600r
pm for 10 minutes) to remove the cells and obtain a culture supernatant.

Next, 0.5 ml of MES-A buffer was added to 0.5 ml of the culture supernatant.
ml and equilibrated with MES-A buffer.
Ting Sepharose 6B @ (manufactured by Pharmacia) 0
．． It was applied to a column packed with 5 ml. MES- on the column
1 ml of B buffer solution was passed four times, and the collagenase inhibitor was eluted and removed from the column.

Next, 1 ml each of MES-C buffer was poured twice, and the eluate was collected to obtain 2 ml of procollagenase solution.

Add lNNa0f to the procollagenase solution and
) After adjusting I to about 7, add MES-A buffer and 2.
5 ml, and then a solution with a collagenase activity of about 0.1 to 0.7 units/ml was prepared using a measurement buffer, and this was used as a test solution.

Next, trypsin solution (manufactured by Sigma,
After adding 20 μQ of Type 12 (adjusted to a concentration of 1 mg/ml with measurement buffer) and incubating at 35°C for 5 minutes, add soybean trypsin inhibitor solution (Merck No. 24020 with measurement buffer). Concentration 3m
Trypsin was inactivated by adding 30 μQ (adjusted to g/ml) to obtain a J collagenase solution.

Type 1 collagen labeled with fluoreracene isothiocyanate (FITC-collagen, manufactured by Cosmo Bio)
A 0.01N acetic acid solution (concentration 1 mg/ml) of
The activity (units/ml) of the above collagenase solution was measured according to the method described in Phys. The amount of collagenase generated from procollagenase by the above trypsin treatment decomposing 1 μg of Hz collagen (FITC-collagen) per minute at 35°C is defined as one unit of procollagenase, and the amount of procollagenase produced (unit/ (ml of culture solution) was determined (this value is designated as X).

On the other hand, as a control test, distilled water was added instead of the test compound aqueous solution, and the procollagenase production amount (unit/ml of culture solution) was performed in the same manner as above.
) was calculated (this value is designated as Y).

Next, the procollagenase production promotion rate (%) was calculated from these values using the formula below.

4) Test results Table 1 a) Standard error above the mean (n = 3) b) Significant difference in PcO, 01 compared to no additive (control) (Duncan
Method) As described above, the compound of general formula (I) 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 - 13
4g, 5 animals per group).

3) Test method Drug screening method for liver function improvement effect (supervised by Hikaru Ozawa,
According to the drug efficacy screening method for new drug development, p. 75, Marukubi, published in 1984), an isostatic compound of carbon tetrachloride (hereinafter abbreviated as CCL) and olive oil was
The drug was administered subcutaneously to the back of rats at a dose of ml/kg twice a week (Monday and Thursday) for 10 weeks to create liver fibrosis rats.

Aqueous solution of N-methyl-L-serine (concentration 66.6 mg/
m1) was orally administered to rats at a rate of 3 ml/kg once a day for 7 weeks (excluding Sundays) from 4 weeks after the start of CCL 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 and the liver was removed.
The weight was measured, and the product was divided into two parts under water cooling. Half of the sample was frozen at -80°C for quantitative 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 to liver fibrosis rats at a rate of 3 ml/kg in place of the test compound aqueous solution according to the same schedule as above, and blood sampling and liver extraction were performed in the same manner.

In addition, a group of normal rats to which neither CCL nor the test compound aqueous solution was administered was established, and blood sampling and liver removal were performed in the same manner.

Hydroxyproline in the liver and blood was then quantified using each of the above livers and plasma. Note that hydroxyproline is an indicator of collagen content (Metho
ds of Biochemical Analysis
s, vol. 15, p. 25, 1967). In addition, the following plasma biochemical tests that are indicators of liver damage [see (b) below]
~(g)] and histological examination of the liver (h) were also performed.

The respective inspection items and inspection methods are as follows.

(a) Hydroxyproline in the liver and blood (hereinafter referred to as
(abbreviated as Hyp) amount (a-1) Quantitative method of H3'l) in the liver Take 100 mg of the liver, weigh it accurately, add 250 μQ of physiological saline, and then Stir vigorously (280
00 rpm for 30 seconds). 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 dissolved in 0.01N hydrochloric acid. This solution was analyzed using the Bost column amino acid analysis method (P) using high performance liquid chromatography.
roceedings of the Nationa
lAcademy of 5sciences of t
he U,S,A,, vol. 72, no. 2, p. 619, 197
The amount of Hyp in the supernatant sample and the precipitate sample was determined by quantifying the amount of Hyp in the supernatant sample and the precipitate sample. Then, the amounts of Hyp in the supernatant sample and the precipitate sample were totaled, and the amount of Hyp per unit liver weight (ILmo 17 g liver) was determined from this value and the liver weight.

(a-2) Assay method for blood Hn 5 w/v% sulfosalicylic acid aqueous solution 1 to 100 μQ of plasma
00 μQ was added under water cooling, and centrifuged (110,000 r
p, 3 minutes) to obtain a supernatant, and Hyp in the supernatant was diluted with (a-1
) was quantified by Bost column amino acid analysis method using high performance liquid chromatography.

(b) Glutamate pyruvate transaminase (hereinafter abbreviated as GPT) amount Karmen method (Izumi Kanai, Summary of Clinical Testing Methods, Revised 2nd
(9th edition, 514 pages, Kanehara Publishing Co., Ltd., 1983)
Measured at

(C) Glutamate oxaloacetate transaminase (
Hereinafter abbreviated as GOT) quantity Karmen method (Izumi Kanai, clinical test method summary, revised 2nd
(9th edition, 514 pages, Kanehara Publishing Co., Ltd., 1983)
Measured at

(d) γ-glutamyl transpeptidase (hereinafter γ-
GTP) Method using γ-glutamyl-p-nitroanilide as a substrate (Izumi Kanai, Summary of Clinical Testing Methods, Revised 29th Edition, 53
2, Kanehara Publishing Co., Ltd., 1983).

(e) Total bilirubin amount Malloy-Evelyn method (Izumi Kanai, Summary of Clinical Testing Methods, revised 29th edition, p. 724, Kanehara Publishing Co., Ltd., 1
983).

(f) Alkaline phosphatase amount Bessey-Lowry method (Izumi Kanai, Summary of Clinical Testing Methods, revised 29th edition, p. 496, Kanehara Publishing Co., Ltd., 19
(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 liver The liver was sliced in a conventional manner and observed under a microscope after staining with hematoxin and eosin.

4) Test results The amount of I(yp) in the liver and blood is an indicator of liver damage.The biochemical test values of plasma are shown in Table 2.

Thus, compared to the untreated group, the amount of Hyp in the liver of the group administered with the test compound was significantly lower, and the amount of Hyp in blood was significantly higher. This indicates that the therapeutic agent of the present invention promoted procollagenase production and, as a result, promoted the degradation of collagen in fibrotic liver tissue and released Hyp into the blood.

In addition, plasma biochemical test values (GPT, GOT, γ-GTP, total bilirubin, alkaline phosphatase, and ZTT levels) in the test compound administration group compared to the untreated group.
It can be said that the test compound improved liver damage associated with liver fibrosis, since it was significantly lower.

On the other hand, in the histological examination of the liver, the test compound-administered group had significantly reduced liver damage, diffuse hepatocyte vacuolation, bile canalicular hyperplasia, and common bile duct epithelial cell formation compared to the untreated group. It was observed that there were. 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.

(Test Example 3) Acute toxicity test 1) Test compound Same as Test Example 1.

2) Test method and test results Each test compound aqueous solution was dissolved in purified water and 0.2 m
l/kg body weight (5 g/kg body weight as specimen)
CR male mice (5 weeks old, weight 24-28 g, -group 5
was administered orally to 100,000 animals. 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 (3 kg in weight), the tray method (APPRAISAL OF THE 5AFET) was conducted.
Y OF CHEMIC-ALS IN FOODS,
DRAGS AND CO8METIC3, p, 46,
1959゜Edited and Published
by THE EDITORIAL COMMI-T
TEE, ASSOCIATION OF FOOD &
DRUG 0FFICIALS 0FL1. S,A,
). Specifically, an abrasion site (damaged skin) is created on the back of a rabbit whose hair has been shaved, and 0.1% of a 1w/v% aqueous solution of the test compound is applied to each of the damaged skin and normal skin.
ml of patch test bandage (1.2 x 1.6 cm,
It was infiltrated with Ribbon Eight (manufactured by River Tape Pharmaceutical Co., Ltd.) and applied. After 24 hours, the bandage was removed and the skin was observed for erythema and edema.
The same observation was made after some time. Then, erythema score and edema score were given respectively according to the following evaluation criteria.

Based on this score and the formula below, the -order stimulation score was calculated.

Child station (B24+B48) Child station (Y24+Y4a) Here, each symbol has the following meaning.

Table 3 Next, the degree of irritation of the test compound was determined based on the -order irritation score and the following criteria.

3) Test results Primary irritation score and irritation degree of each test compound [Example] The present invention will be described below with reference to Examples.

Example 1 Tablets each containing 100 mg of L-serine as an active ingredient were prepared as follows.

(Formulation) Ingredients Amount I [L-serine 50 lactose
10 corn starch
30 Crystalline Cellulose 8 Hydroxypropyl Cellulose 1 Magnesium Stearate 1 (Operation) Hydroxypropyl cellulose dissolved in 30 g of water was added to a mixture of serine, lactose, corn starch and crystalline cellulose, and thoroughly kneaded. After passing this mixture through a 20-mesh sieve and granulating it into granules and drying, magnesium stearate is mixed with the obtained granules,
Each tablet weighed 200 mg.

Example 2 Capsules Capsules each containing 100 mg of N-methyl-L-serine as an active ingredient were prepared as follows.

(Formulation) Ingredients Dosage IJυN-Methyl-L-Serine 100 Lactose
100 corn starch 50 crystalline cellulose
47 Magnesium stearate
3 (Operation) The above components were thoroughly mixed, and 300 mg each of the mixture was filled into No. 2 capsules to obtain capsules.

Example 3 Granules containing 100 mg of monoserine as an active ingredient in 1 g of granules were prepared as follows.

(Formulation) Ingredients Amount (g) L-serine 100 Lactose
470 Corn starch 400 Hydroxypropyl cellulose 30 (Operation) Hydroxypropyl cellulose dissolved in 1000 g of water was added to a mixture of L-serine, lactose and corn starch and thoroughly kneaded. This kneaded product was passed through a 20-mesh sieve, granulated, dried, and sized to obtain granules.

Example 4 A syrup containing 100 mg of L-serine as an active ingredient in 1 ml of syrup was prepared as follows.

(Prescription) Ingredients (Button L1(a) L
-Serine 100 (b) Methyl paraoxybenzoate 0.3 (C) Propyl paraoxybenzoate 0.15 (d) White sugar
300(e) 70W/V$D -'
) Rubitol aqueous solution 250 (f) Sodium citrate 10 (g) Citric acid
1.5 (h) Add purified water to make total volume 10100O
(Procedure) Components (b) to (d) above were added to 400 ml of purified water at 90°C and dissolved, then component (e) was added at the same temperature, mixed thoroughly, and cooled to 30°C. . L to this mixture
- Dissolve serine in 100ml of purified water and add to it at 30°C.
The mixture was stirred for 30 minutes. Next, the above ingredients (f) and (g)
Add and stir for 20 minutes, then add purified water to bring the total volume to 10,100O
A syrup was obtained by sterile filtration.

Example 5 Injectable N-methyl-L-serine Log was dissolved in physiological saline for injection to make a 200 ml solution, and sterilized by sterile filtration. Pour the sterilized solution into a 3ml brown ampoule.
Dispensed in portions. 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-thi-serine as an active ingredient in one ampoule.

Example 6 An ointment containing 100 mg of L-serine as an active ingredient in 100 g of ointment was prepared as follows.

(Prescription) Ingredients [Plan 14) (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 (manufactured by Qutant Industries Co., Ltd.) 1.3 (k) Glyceryl monostearate 2.0 (1
) Butyl roseoxybenzoate 0.1 (Operation) The components (a) to (d) above were heated to 80°C in a hot water bath and mixed, and this mixture was mixed with the above (e) heated to 80°C. )
It was gradually added to the component mixture of ~(1) while stirring.

Next, use a homogenizer (TOKUSHUKIKA KO)
(manufactured by GYO) for 2.5 minutes (2500 rpm)
L/After thoroughly emulsifying and dispersing each component, the mixture was gradually cooled while stirring to obtain an ointment.

Example 7 N-methyl-L-serine 1 as an active ingredient in 100g of lotion
A lotion containing 00 mg was prepared as follows.

Ingredients 1 Gold 1 (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) Cetanol (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, the components (d) to (f) were mixed in the same manner, and the mixtures (a) to (C) were gradually added to this mixture while stirring, and the mixture was heated using a homogenizer (TOKUSHUKIK).
(made by AKOGYO) for 2.5 minutes with vigorous stirring (2500r
pm) L/ta. A lotion was obtained by gradually cooling to room temperature while stirring.

Procedural amendment (indication of own ship July 17, 1990 case 1o 1990 Patent Application No. 99579 2, title of invention: therapeutic agent for diseases accompanied by abnormal accumulation of collagen 3, relationship with the person making the amendment case) Patent Applicant Address: 5-17-4-6 Sumida, Sumida-ku, Tokyo, "Detailed Description of the Invention" column 7 of the specification to be amended, Contents of the amendment (1) Lines 3 to 4, page 13 of the specification Correct "collagenase activity" in line 1 to "rprocollagenase".

Kanebo Co., Ltd. Patent Department 5, 1-5-90 Tomobuchi-cho, Bejima-ku, Osaka 534, No number of claims increased due to amendment Teitomeri Neho Seisho (self-proposal) December 26, 1990, 1990 Patent Application No. 99579 2, Name of the invention: A therapeutic agent for diseases accompanied by abnormal accumulation of collagen 3, Relationship with the case of the person making the amendment Patent applicant address: 5-17-4 Sumida, Rota-ku, Tokyo; Column 7 of "Detailed Description of the Invention" Contents of Amendment (1) "Collagenase" in the fifth line from the bottom of page 3 of the specification is corrected to "r-collagen".

1-chome, Tomobuchi-cho, Bejima-ku, Osaka 534 Kanebo Co., Ltd. Patent Department Telephone: (06) 921-1251 4. Date of amendment order (voluntary) 5. Number of claims increased by amendment No. 5 No. 90

Claims (3)

[Claims] (1) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼...(I) (In the formula, R^1 and R^2 each represent a hydrogen atom or a methyl group.) Compounds represented by A therapeutic agent for diseases accompanied by abnormal accumulation of collagen, containing as an active ingredient. (2) Claim No. (2) which contains L-serine as an active ingredient
The therapeutic agent described in section 1). (3) The therapeutic agent according to claim (1), which contains N-methyl-L-serine as an active ingredient.