JPH0377898A - Muscular dystrophy-treating agent comprising sialosyl cholesterol - Google Patents

Abstract

PURPOSE:To provide the subject treating agent useful for the treatment of muscular dystrophy in a form of ophthalmic agent, inhalation agent, oral medicine, intravenous injection agent, muscular injection agent, hypodermic injection agent, etc., by containing a specific sialosyl cholesterol as an active ingredient. CONSTITUTION:A compound of formula I (Ac is acetyl) is reacted with approximately 2-5 times moles of cholesterol in the presence of a Kenigs-Knorr reaction catalyst at the ordinary pressure and at approximately 20-25 deg.C for approximately 2-7 days, and the reaction product is isolated and purified by a conventional method such as column chromatography. The prepared reaction product is hydrolyzed to convert the methoxycarbonyl group into sodium carboxyl group and also the acetyl group into hydrogen to prepare a sialosyl cholesterol of formula II (M is Na or K) or III, which is dissolved in distrilled water for injection or compounded with an excipient, etc., to provide the objective injection agent, oral medicine, etc.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a therapeutic agent comprising sialosylcholesterol useful for the treatment of muscular dystrophy, which is attracting attention as one of the intractable diseases worldwide. Muscular dystrophy is one of the incurable diseases, and includes Duehe+>ne type, Becker type, scapulohumeral type, myotonic dystrophy, congenital, distal type,
It is classified into various types such as ocular muscle type.

Currently, several therapeutic agents are being developed, but no effective therapeutic agent has yet been developed.

Therefore, there has been a strong demand for a drug that is effective in treating muscular dystrophy.

Therefore, an object of the present invention is to provide a drug that effectively acts on muscular dystrophy.

[Means to solve the problem]

As a result of intensive studies to achieve the above object, the present inventors have found a therapeutic agent for muscular dystrophy characterized by containing sialosylcholesterol represented by the following general formula:

(2) The therapeutic agent for muscular dystrophy according to claim (1), which is in the form of eye drops, inhalants, internal medicines, intravenous injections, intramuscular injections, and subcutaneous injections.

The present invention was achieved by discovering that muscular dystrophy can be effectively treated by using sialosylcholesterol represented by ().

By the way, sialic acid forms sialocomplexes (11 proteins, glycolipids, oligosaccharides, polysaccharides, etc.) on the cell surface of animals or bacteria.
It exists as a specific active molecule, and has recently been used in immunology, cancer,
Sialosylcholesterol is attracting medical and pharmacological attention as a substance involved in inflammation, viral infection, cell differentiation, hormone receptors, etc., but to date, there is no evidence that sialosylcholesterol is useful as a treatment for muscular dystrophy. It wasn't known.

The present invention will be explained in detail below.

The Na salt of sialosylcholesterol used in the present invention is a known compound, for example, in JP-A-1-935
It is described in Publication No. 29. As described in the publication, sialosylcholesterol represented by general formula (I) or (II) is generally produced as follows.

Compound (III) represented by formula (11 [): is reacted with cholesterol in the presence of a Koenigs-Knorr reaction catalyst at normal pressure at a temperature of about 20 to 25°C to react with about 2 to 7 Let it react for a day. At that time, cholesterol
Compound (III) is used in an amount of about 2 to 5 times the mole per mole. The obtained reaction product is isolated and purified by conventional methods such as column chromatography (Japanese Unexamined Patent Publication No. 61-24
(See Publication No. 3096).

Incidentally, the above compound (III) is a known compound that is easily available.

Furthermore, the above product is hydrolyzed to convert the methoxycarbonyl group to a sodium carboxyl group and the acetyl group to hydrogen to obtain a compound (■) (α form) and a metal compound (■) (β form). In addition, the novel salt of sialosylcholesterol can be obtained by first hydrolyzing the above product, converting its methoxycarbonyl group into a free carboxyl group, and simultaneously converting its acetyl group into hydrogen, and then neutralizing it with potassium hydroxide. or by direct treatment with potassium hydroxide.

The compound of the present invention can be administered either orally or parenterally, but eye drops, inhalants, double doses, intramuscular injection, subcutaneous injection, and intravenous injection are preferred. The dosage depends on the severity of the disease and the weight of the patient, but is preferably between 0.001 and 10 cm.

In order to prepare the compounds of the present invention for oral administration, for example, excipients, binders, disintegrants, lubricants, film-forming agents, etc. are generally used.

Examples of excipients include glucose, starch, lactose, mannitol, sorbitol, white sugar, kaolin, dextrin, cyclodextrin, titanium oxide, anhydrous calcium phosphate, light anhydrous silicic acid, talc, natural hydrated magnesium silicate, and precipitated R calcium carbonate. , magnesium aluminate metasilicate, crystalline cellulose, calcium carboxymethylcellulose, etc., or a combination of two or more thereof can be added.

Examples of binders include starch, dextrin, 1
- Lagant Cam, Gelatin, Polyvinyl Vinyl: I Ridone, Polyvinyl Alcohol, Methyl Cellulose, Ethyl Cellulose, Hydroxyethyl Cellulose, Hydroxypropyl Cellulose, Hydroxypropyl Methyl Cellulose, Hydroxypropyl Starch, Crystalline Cellulose,
Pectin, polypectic acid, polypectic acid I/Ryufu, polyacrylic acid, 6-polystorium acrylate, polyacrylic acid copolymer, polymethacrylic acid, sodium borimecacrylate, polyhydroxyethyl methacrylate, One type or a combination of two or more types of carboxymethylcellulose, sodium carboxymethylcellulose, gum arabic, sodium alginate, etc. can be added.

Examples of disintegrants include starch, crystalline cellulose 1,
Carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl starch 1, droxypropyl starch 5, agar powder, mannan, etc. 1
A species or a combination of two or more species can be added.

As the lubricant, for example, one type or a combination of two or more of talc, stearic acid, magnesium and calcium stearate, hydrogenated oil, sesame oil, and paraffin can be added.

Examples of film-forming substances include ethyl cellulose (EC), carboxymethylethyl cellulose (CMEC), and acetic acid cell r+-su derivatives.
CA), Fjt4E cellulose phthalate (CAP)
, hydroxypropylmethylcellulose (HPMC)
), Hydroxyb V: 12-pyl methyl cellulose phthalate -) (HPMCP) +, Methyl cellulose course (MC
), hydroxyprocellulose (RPC), also polyvinyl derivative; - Trimethylammonium ethyl methacrylate chloride copolymer, dimethylaminoethyl methacrylate/methyl methacrylate copolymer, and the like.

In addition, when coating the above-mentioned film-forming substance, in addition to commonly used coating aids such as plasticizers, various additives to prevent mutual adhesion of chemicals during coating operations can be added to the film-forming agent. When used in inhalation or injectable dosage forms, the following additives are generally used, which can improve the properties of the drug or make the coating operation easier.

Calcium chloride Storium chloride Citrate Succinate Potassium acetate Tartrate Potassium chloride Hydrochloride Sodium citrate Sodium acetate Sodium acetate Potassium hydroxide Thorium hydroxide Physiological saline Sodium bicarbonate Sodium sulfate Phosphate Potassium phosphate Sodium phosphate Dipotassium phosphate Sodium dihydrogen phosphate, aqueous sulfite, thorium, sodium pyrogrite, purified egg yolk lecithin, gelatin, large serum albumin, polyethylene glycol 400, polyethylene glycol 1500, polyethylene glycol 4000, polyethylene glycol 6000, polyoxyethylene 1/one, hydrogenated castor oil 60, sodium edetate ,Such.

water for injection carbonate/thorium lactic acid maleic acid phosphoric acid sodium phosphate Calcium phosphate hydrate Sodium hydrogen phosphate J, sodium sulfite Potassium pyrosulfite I-arginine purified gelatin lactose (Example) The drugs of the present invention will be shown in Examples below.

The light is not limited to these.

Reference Example 1 The following formula (a): 10.00 g of the compound represented by the present invention was added to a solution consisting of 8.188 g of potassium hydroxide and 600 taβ of ethanol with stirring under water cooling. Stirred. A 50% aqueous formic acid solution was added to this reaction solution to adjust the pH to acidic (approximately 2), then ethanol was removed under reduced pressure, and the residue was added to water, stirred, and then passed through the mouth. The obtained crystals were dried under reduced pressure to give 7.482 g (94.9%) of the following formula (b
) A white crystal was obtained.

The melting point is 175.5-177.4°C (decomposed).

υ (Reference Example 2) 601■ of the compound represented by the above formula (bl) was mixed with 20I of water.
After stirring and mixing with i#, 5% aqueous potassium hydroxide solution was added little by little to adjust the pH of the liquid to about 8.8, and the solution was sifted, freeze-dried, and prepared using the following formula. 609 cm (96%) of a white powder represented by (C) was obtained.

The physical property values of the compound represented by formula (C) are shown below.

Owner: 229.0-230.6℃ (decomposition) IR: 33
66.2938.286B, 1622.1122.10
7'6,1033em-'NMR: 0°705ppmn(3H,,,s,18'-
C143) 0.878ppm (3H,,d,26'or
27' CHs) 0.875 ppIl (3H, d,
2B'or27'-CH5)0,936ppm(3
H,d,21'-CH5)C0993ppm(3H
,s, [9' CH2)2゜005ppim(3H
,sS CH3C0NH-) 2.832 ppm (I H
, d, d, 3eq-H) Reference N3 After dissolving 9°138 g of the compound represented by the above formula (a) in 200 sJ of ethanol, 7.4 g of potassium hydroxide was added to the solution while stirring and cooling with water. A solution consisting of 255 mJ of ethanol and 255 mJ of ethanol was added, and the mixture was allowed to react for 2 days under water cooling. The precipitated crystals were collected, washed with ethanol, and then dried under reduced pressure to obtain 5.565 g (73.2%) of a white powder of the compound represented by the above formula (C). Its physical property values were the same as those of Reference Example 2.

[Acute toxicity test]

The acute toxicity test was confirmed by intravenous injection into ddy male mice at 4.5 weeks of age, and the results were as follows:
-93q/kg (compound (1)), LD,, = 29
1 q/kg (compound (II)').

[Pharmacological effect test]

We investigated the effects of the compound on this disease using hereditary muscular dystrophy mice, which are pathological model animals for muscular dystrophy. The efficacy of the drug was evaluated based on its effect on locomotor activity and survival time.

16 For experimental animal experiments, commercially available C57BL/6J-dy strain male mice with genetic muscular dystrophy (dy/4y) and male and female normal mice of the same strain (10/+, →-/dy) were used. did. CMC (Oriental Yeast Co., Ltd.) was used as bait, and the mice were given free access to feed and water.

2. Drug compound (I) was dissolved in distilled water immediately before administration and weighed 10
0.1-g/g was orally administered to 6-week-old mice once a day, 5 times a week, for 5 consecutive weeks to measure locomotor activity, and continuously until death for survival. Distilled water was orally administered to the control group.

3. Measurement of locomotor activity Measurement of locomotor activity was carried out every week from 6 weeks of age at 111 weeks of age using a rotating cage. The mouse was placed in a rotating cage, allowed to acclimate to the rotating cage for 5 minutes, and then its locomotor activity was measured for 15 minutes. Note that measurements were performed at fixed times (between 1:00 p.m. and 3:00 p.m.) taking into account diurnal fluctuations.

4. Measurement of survival period The survival rate was measured every week from 6 weeks of age to 200 weeks of age. Eight males and one female were used in the control group, and three males and one female were used in the treatment group. The survival rate was evaluated in 9 animals of both sexes in the control group, and in 4 animals of both sexes in the administration group. In the locomotor activity measurement test, the average body weight of muscular dystrophic mice was approximately 12 g, so the above compound (1
) was administered.

Result 1: Effect on locomotor activity Changes in exercise over time were examined in terms of relative exercise, with the exercise at 6 weeks of age as 100%. The amount of exercise of a normal mouse is
The amount of exercise increased significantly for both sexes from 6 to 8 weeks of age, but after that the increase became more gradual and remained almost constant (Figure 1); the maximum amount of exercise for normal mice was 232% for males.
, it was 289% in females. On the other hand, the amount of exercise of muscular dystrophic mice showed a slight increase in both sexes until they were 7 weeks old, but after this point, the amount of exercise gradually decreased (the first
Figure), the maximum amount of exercise in male mice with muscular dystrophy is 13
3%, and 132% in females.

When the compound ([) was administered orally at 2 kg/kg to mice with muscular dystrophy, there was a statistically significant difference (Student
's t-text), but the amount of exercise of muscular dystrophic mice in both sexes increased compared to that of control muscular dystrophic mice (Figure 2).

In other words, the maximum amount of exercise in mice with muscular dystrophy at 8 weeks of age was 158% in the treated group compared to 133% in male controls.
%, and in females, it was 149% in the treated group compared to 132% in controls. The amount of exercise of muscular dystrophic mice at 9 weeks of age is
For males, 153% in the treated group compared to 120% in the control group;
In females, 138% in the treated group compared to 121% in the control group.
%, Compound (1) was observed to have an inhibitory effect on the decrease in locomotor activity over time in mice with muscular dystrophy.

On the other hand, when normal mice were orally administered a dose of 2 kg/kg, no increase in locomotor activity was observed (Fig. 3); in fact, a decrease in locomotor activity was observed compared to the control group (Fig. 3).
.

2. Effect on survival period Compounds (1) on survival period of muscular dystrophic mice
) was examined at a dose of 2 lb/kg, which was found to have an effect on increasing locomotor activity and inhibiting decrease in locomotor activity. In the control group, the first mortality was observed at approximately 10 weeks of age, and all mice tested at 199 weeks of age died (Figure 4), with an average survival time of 144 weeks of age in the control group. On the other hand, in the administration group, no mice died at 188 weeks of age. 199
The first death at the age of 300 weeks was observed (Figure 4), and the longest surviving mouse among the treated groups was a female mouse that survived at 300 weeks of age. In the terminal stages of muscular dystrophic mice, the hind limbs become atrophied and the hind limbs remain stretched out in a rod-like manner, or the knees remain bent, and similar symptoms were observed in the treated group. However, the severity appeared to be milder than in the control group of the same age. Also, as the symptoms progress, a large amount of IN! may appear around the eyelids. Fat was resistant to adhesion, but it was not observed in the treated group until death.

From the above results, compound (I) showed a tendency to suppress the decrease in locomotor activity of mice with muscular dystrophy, and exerted a significant survival effect on the survival period.

Compound (1) has the potential to become a therapeutic agent for muscular dystrophy, which has a different mechanism of action from drugs that have been variously studied as therapeutic agents for muscular dystrophy.

Dormitory Example-Z-6 Enteric Coated Granule Compound (■) 2°Og, Cornstarch 60, Og,
After mixing 95.0 g of lactose using a mixer, 3.0 g of hydroxypropyl cellulose dissolved in ethanol C was added and kneaded well.The resulting mixture was formed into granules according to a conventional method. After drying, it was sieved to produce suitable granules.Next, 60°Og of hydroxypropyl methylcellulose phthalate ()4PMCP) was mixed with 19ml of water.
0s6 and ethanol 750m! Prepare a coating liquid dissolved in a solvent consisting of
g Put it in a mini flow coater and spray the coating liquid inside it to coat the granules, about 200
Film-coated enteric granules of g were obtained.

1 Ampoule of Injection: 2.5 ■ of Compound (X), 0.25 ■9 of sodium dihydrogen phosphate dihydrate, 3 ■ of disodium hydrogen phosphate dodecahydrate, and distilled water for injection. The total amount is 2
．． An injection of 5sβ was prepared.

1 fin, 0.5 I of compound (1) in 1 vial of injection solution before use! g and menstrual p1
: Freeze-dried injection containing 1 ml of salt water,
Dissolution Z7 was used using 1 mβ of distilled water for injection.

1 H.M5 Eye drops 1 vial contains 1■ of compound (■), 52.5■ of boric acid,
An eye drop containing 14.5+ g of powder sand, an appropriate amount of benzobenium chloride, and a solution for eye drops, and having a total amount of 5 FlN was prepared.

Fruit 1st grade Inhalant compound (1) was placed in an agate mortar and thoroughly ground to form a fine powder with a particle size of 1 to 20 μm. Lactose was added to the powder, pulverized and mixed, and lactose was added little by little to this fine powder and mixed thoroughly to give a 20-40 fold loss. This 20 to 40 μm was encapsulated or packaged in a conventional manner to form a preparation.

Kabuidol was used for powder aerosols, and sachets were used for liquid aerosols.

Even in the cases according to Examples 2 to 6 above, Example 1
A similar effect was obtained.

[Brief explanation of drawings]

Figure 1 shows changes in locomotor activity in normal mice and mice with muscular dystrophy. Figures 2 and 3 show changes in locomotor activity of normal mice and mice with muscular dystrophy.
) is a diagram showing the effect of

Claims (2)

[Claims] (1) General formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ ▲There are mathematical formulas, chemical formulas, tables, etc.▼ A therapeutic agent for muscular dystrophy characterized by containing sialosylcholesterol. (2) The therapeutic agent for muscular dystrophy according to claim (1), which is in the form of eye drops, inhalants, internal medicines, intravenous injections, intramuscular injections, and subcutaneous injections.