JPS5838219A - Novel leupeptin preparation - Google Patents

Abstract

PURPOSE:The titled preparation that is made by making leupeptin included in a liposome composed of lecithin, cholesterol and brain sulfatide, thus showing remarkable curing effect for demyelinating diseases. CONSTITUTION:Lecithin such as yolk lecithin, cholesterol and brain sulfatide (galactocerebroside-3'-sulfate) such as bovine brain sulfatide are dissolved in an appropriate solvent such as chloroform, then the solvent is removed, and an aqueous solution of leupeptin (acetyl-L-leucyl-L-leucyl-L-argininal), preferably of 0.05- 5% concentration is added. Then, when necessary, the mixture is subjected to ultrasonic treatment to give an aqueous suspension of liposome including leupetin. The amounts of the cholesterol and brain sulfatide are 10-25pts.wt. and 10-30pts.wt. per 100pts. of the lecithine, respectively.

Description

[Detailed description of the invention] Leupeptin (acetyl-leucyl-■,-leucyl-L-argininal) Id, % Kosho 45-1715
4 and Japanese Patent Publication No. 46-22012, etc., it is a compound known to have an anti-plasmin effect, and its toxicity is relatively weak, for example, LD3. 12 by intravenous injection
5/kg, subcutaneous volume, pages 283-286 and volume 22, pages 558-568 (19, 69)], and is a compound expected to have various medicinal uses.

The present inventors have conducted various studies on the use of leupeptin for demyelinating diseases, and have found that leupeptin alone rarely shows any effects.

It was discovered that the preparation of the present invention exhibits a remarkable therapeutic effect, and the present invention was completed.

Lecithin, cholesterol and galactoserebroside of the present invention
A novel leupeptin preparation in which leupeptin is incorporated into a liposome consisting of (-3'-5ulfate) is produced as follows.

First, lecithin, cholesterol, and brain sulfatide are mixed and dissolved in a suitable solvent such as chloroform.
After removing the solvent, an aqueous leupeptin solution is added, sufficiently stirred and mixed, and, if necessary, a conventional sonication treatment and/or ultrasonication treatment is performed.

Since the liposome containing leupeptin is suspended in an aqueous solution, it can be used as it is as an injection solution. However, depending on the case, the liposomes obtained from this suspension can be separated by a conventional method such as centrifugation and used in any desired formulation form.

The proportions of lecithin, cholesterol and cerebral sulfatide to be used can be appropriately determined through preliminary tests, etc., but usually 100 parts of lecithin (parts by weight: hereinafter, parts by weight are shown unless otherwise specified) to 5 parts of cholesterol. ~40 parts, preferably 10-2 s parts - brain sulfatide 5-5
0 copies.

Preferably it is used in a proportion of 10 to 30 parts.

The concentration of the aqueous leupegtin solution used to incorporate leupeptin is not particularly limited, but it is used at a concentration of 0.01 to 10 parts, preferably about 0.05 to 5%.

Egg yolk lecithin is usually used as the lecithin, and bovine brain sulfatide or the like is used as the brain sulfatide.

When the noroupeptin preparation of the present invention was intraperitoneally administered to guinea pigs with experimental allergic encephalitis on consecutive days, a remarkable effect of suppressing the onset of allergic encephalitis was observed. That is, only 10% of the patients who received Leuvep alone survived for 40 days, whereas 40% of the patients who received 5 preparations survived.

Therefore, the preparation of the present invention is particularly useful as a therapeutic agent for demyelinating diseases such as multiple sclerosis.

The dosage of the preparation of the present invention varies depending on the patient's symptoms, etc., but the amount of leupeptin is thought to be about 5 m9 to 300 m9/day/6.

Next, the present invention will be explained using specific examples.

Example 1゜Brain sulfuric acid 201■, cholesterol] 7.4
m9, egg yolk lecithin 123.9 m9 to chloroform 1
The solution was dissolved in a volume of 0 ml, placed in an eggplant-shaped flask, dried under reduced pressure using a rotary evaporator, and further vacuum-dried for 3 to 5 hours. A solution of Leupeptin 00m9 dissolved in 10 ml of phosphate buffer-physiological saline (PBS) was added to the flask, mixed for 3 minutes using a Vortex mixer, and then mixed using a sonicator (Branson, USA, 200KC8). 5
The mixture was treated for 1 minute to prepare a liposome suspension. Transfer this suspension to a small test tube and use an ultrasonic device (80KC8, Labo
Ratory 5uplies Co., USA) at 1
After treatment for 5 minutes, a liposome suspension containing leupeptin was obtained.

This can be used directly as an injection solution.

In addition, egg yolk lecithin was obtained by the method of Mizuben et al. (Jap, J.

Exp, Medicine, 44. p451-46
1 (] 974)) and purified by alumina chromatography and silicic acid chromatography. Brain sulfatide was obtained from the acetone-insoluble and chloroform:methanol (2:1) soluble fraction obtained from bovine brain using the method of Mizuben et al.
d Function of Gangliosides
” by L, Svennerholm, 1-1.D
reyfus and P, P, 1Jrban, p
13-21 (Plenum publishing
(1980)) and purified by DEAE-Sephadex chromatography.

Experimental Example (11) Method for Inducing Experimental Allergic Encephalitis 1000 killed Mycobacterium tuberculosis strain B strains prepared by heat treatment tt9-, Ar 1acel and Bayol
miniphosphorus basic protein [Neuroprogress and proo
1-1014 (1979)] 300 Rumonoto males, 5 weeks old (body weight 300-500 Ljf), were inoculated in front of the right leg.

(2) Treatment test method and results Thirty IJ Hartly guinea pigs (bars inoculated with the above miniphosphorus basic protein-containing adjuvant) were divided into groups with 10 dead in each group, and each group was divided into (1) a control group (no drug administration group), (210 ipebutin alone administration group, (3) leupeptin-containing liposome administration group.

The administration of the drug was calculated by taking the mouth inoculated with the above adjuvant as day 0, and administering the above preparation at 0.5 m/! Once a day from 6 sips until the 20th day, then 22.24°26.28,30
, 33, 36 and 39 days] was injected intraperitoneally once a day.

10 Ipep preparation for single administration A solution of 100 mL of dileupeptin dissolved in 10 mL of phosphate buffer-6 physiological saline-water2. Leupeptin-containing liposome suspension prepared in [Example] The effect on experimental animals was determined by Coates.
The method of et al. (Ce11. Jmmunol, +12.
p 370-381 (198)]. The results are shown in Table 1.

Table 1 Effect of leupeptin-containing liposomes in guinea pigs with experimental allergic encephalitis 10.12. +3.13° Comparison 118 13
．． 13, 14, 14. . As is clear from the /96-F table, a delay in the onset date was observed even in the group administered with leubegutin alone.

Only one animal survived after 40 days, whereas in the group treated with leupeptin-containing liposomes, the onset of morbidity was further delayed compared to the group treated with leupeptin alone.
Even after 40 days, 4 mice were found to be alive. The weight change in the surviving guinea pigs after 40 days showed that even if weight loss was observed during the course of treatment, by continuing administration, the weight increased by 20 to 30%1 compared to the time of adipant inoculation, indicating that the disease was completely cured. Ta.

Claims (1)

[Claims] A novel leupeptin [lJ