JPH1067665A - Hemolysis resistant liposome preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hemolysis resistant liposome preparation useful for the treatment of a disease and a disorder accompanying the hemolysis of erythrocytes. SOLUTION: This hemolysis resistant liposome preparation contains 16-90wt.% sulfolipid, 5-70wt.% phosphatidyl choline, 5-40wt.% cerebroside and 0-30wt.% cholesterol. The sulfolipid in the preparation is one or more selected from a group consisting of cerebroside sulfate, sulfolactosylceramide, sulfogalactosyldiacylglycerol and a sulfogalactosylalkylacylglycerol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to anti-hemolytic liposome preparations, and more particularly to liposome preparations containing sulfolipid, phosphatidylcholine, and cerebroside for treating hemolysis, especially complement-induced hemolysis. About.

[0002]

BACKGROUND OF THE INVENTION Several diseases and disorders (eg, neonatal diseases, paroxysmal nocturnal hemoglobinuria) are associated with red blood cell hemolysis. Hemolysis can occur in transfusions when blood selection is not very accurate, or in spinal cord transplants in case of incorrect blood group incompatibility.

[0003] EP 0 394 035 A ₂ disclosed a soluble form of the glycoprotein MACIF in erythrocytes membrane to protect against hemolysis (membrane attack complexes inhibitor). Recently, human MACI
A protein having F activity was obtained from a transformed cell using an expression vector containing a gene encoding this protein. However, the use of this protein as a therapeutic is limited by its high price and potential side effects.

[0004]

An object of the present invention is to provide an anti-hemolytic liposome preparation for treating diseases and disorders associated with hemolysis of red blood cells.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide an image processing apparatus comprising:
The purpose of the present invention is to provide an anti-hemolytic liposome preparation containing 5% by weight of sulfolipid, 5 to 70% by weight of phosphatidylcholine, 5 to 40% by weight of cerebroside and 0 to 30% by weight of cholesterol. Here, the sulfolipid is one or more selected from the group consisting of cerebroside sulfate, sulfolactosylceramide, sulfogalactosyldiacylglycerol, and sulfogalactosylalkylacylglycerol. Among these, the preferred sulfolipid is cerebroside sulfate.

The preparations of the present invention can be obtained by two steps: i) using commercially available lipids or lipid extracts from mammalian brain or spinal cord to obtain a lipid mixture having the above composition. And ii) dispersing the lipid mixture in an isotonic solution.

The 50% active concentration (IC ₅₀ ) of the preparation is between 4 and 120, depending on the content of sulfolipids and other polar lipids.
μg / ml.

However, another object of the present invention is to provide a convenient method for preparing the above-mentioned anti-hemolytic liposome preparation according to a process comprising the steps of: i) preparing a mammalian brain or spinal cord; Homogenizing; ii) removing water and neutral lipids with acetone; iii) extracting polar lipids with lower alcohols (methanol, ethanol, isopropanol); iv) evaporating the extract under reduced pressure; v) dissolving the residue with chloroform or methylene dichloride; vi) precipitating polar lipids with acetone; vii) repeating steps v) and vi) several times; viii) removing the organic solvent and removing the lipid mixture The step of obtaining

[0009] The mixture of lipids obtained in such a manner comprises 18-40% cerebroside sulfate, 10-30% phosphatidylcholine, 15-30% cerebroside, 10-25% phosphatidylethanolamine, and other Contains phosphatidylserine and sphingomyelin and the like. The 50% active concentration (IC ₅₀ ) of this preparation is between 4 and 15 μg /
ml.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The liposome preparation of the present invention can be obtained by the following method.

[0011] The anti-hemolytic lipid composition is obtained by dissolving a mixture of polar lipids, including sulfolipids, in chloroform, methanol, and / or ethanol. After removal of the solvent, an isotonic solution is added. The lipid preparation is then homogenized and dispersed by thorough shaking and sonication. Finally, the dispersion is 0.
Filtered through a 7μ or 0.4μ size membrane filter. The size of the liposome of the present invention is smaller than 1μ, preferably 0.5μ or less. This is because it is required for intravenous administration.

The characteristics of the anti-hemolytic liposome preparation of the present invention are as follows.

1) Inhibition of complement-induced hemolysis The preparations of the present invention can inhibit complement-induced hemolysis, for example, neonatal hemolytic disease, paroxysmal nocturnal hemoglobinuria. Effective concentrations for such hemolysis are from about 0.5 μg / ml to 20 mg / ml.

2) Inhibition of hypotonic hemolysis It is well known that mammalian cells rupture in hypotonic solutions due to differences in intracellular and extracellular permeability. Some infections can be associated with hypotonic hemolysis. This preparation is
Due to the strong acid groups in the sulfolipid, it has a strong negative charge. Thus, this preparation can be used for protection against hypotonic hemolysis. The effective concentration in this case is 0.01
It is between mg / ml and 0.1 mg / ml.

3) Utility for Preservation of Products Containing Blood and Red Blood Cells The preparations of the present invention are useful for storage and handling of blood and blood-containing products, tissues or organs. Such blood products may further contain blood preservative additives such as, for example, anticoagulants and nutrients. Typical additives are sodium chloride, citric acid, sodium citrate,
Glucose, sodium dihydrogen phosphate, glycerol,
Includes folate, adenine, and L-ascorbic acid. The preparations of the present invention are also useful as blood preservative additives.

The present invention relates to anti-hemolytic liposome preparations, more particularly 16-90% by weight of sulfolipid,
A liposome preparation for treating hemolysis, especially complement induced hemolysis, containing 70% by weight phosphatidylcholine, 5-40% by weight cerebroside, and 0-30% by weight cholesterol.

The present invention can be more specifically explained by the following examples. However, the scope of the present invention is not limited to these examples.

[0018]

【Example】

Example 1 This example illustrates the preparation method of the present invention from bovine spinal cord. The method is suitable for any other source: the spinal cord of any mammal, the brain of any mammal,
And any mammalian tissue containing sulfolipids.

First stage. Lipid blends containing sulfolipids
Acquisition of fresh or rapidly frozen bovine spinal cord (1 kg)
Homogenize in a tissue disruptor with ~ 1 L acetone.
The crushed spinal cord was placed in a reactor and chilled (−10 ° C.) acetone was added (2.5 L). Mix 5
Stir for a minute and then filter on a Nutsch filter. Remove the acetone solution. Transfer the residue into the reactor. Add cooled (-10 ° C) acetone (3 L) to the reactor and stir vigorously for 2 hours. The residue is filtered on a Nutsch filter (acetone extraction 2). This operation is further repeated once (acetone extraction 3). The residue is washed on a Nutsch filter with 0.2 L of ethanol. Acetone extractions 2 and 3 can be used for cholesterol separation. Add the residue into the reactor. Add ethanol (3 L). The mixture is stirred for 2 hours at 20-22 ° C. The residue is filtered on a Nutsch filter. The ethanol extract (extract 1) is placed in a refrigerator (−10 to −20 ° C.). Add the residue into the reactor and add petroleum ether (4L)
Is added. The mixture is stirred for 2 hours at 20-22 ° C. The residue is filtered on a Nutsch filter. Put the petroleum ether extract (extract 2) in the refrigerator (−10 to −20 ° C.). Add the residue into the reactor and add petroleum ether (3 L). The mixture is stirred vigorously for 2 hours (20-22 ° C). The residue is filtered on a Nutsch filter. Put the petroleum ether extract (extract 3) in the refrigerator (−10 to −20 ° C.).

Extracts 1, 2, and 3 are combined under reduced pressure with 35-
Evaporate at 40 ° C and dry. The residue is dissolved in 0.5 L of chloroform with stirring. Acetone (2.5L)
Is added to the lipid solution with stirring. The mixture is placed in a refrigerated case (0-4 ° C.) for 18 hours (eg, overnight). The precipitate is centrifuged. Remove the supernatant. The precipitate is dissolved in chloroform (0.3 L) with stirring. Acetone (1.5 L) is added to the lipid solution. Place the mixture in a refrigerated case (0-4 ° C)
Leave for 18 hours. The precipitate is centrifuged. Remove the supernatant. Dissolve the precipitate in chloroform (0.3 L). Ethanol (1.2 L) is added to the lipid solution with stirring. Evaporate the mixture to one third volume.

The mixture is placed in a refrigerated case (0-4 ° C.) for 18 hours. The precipitate is centrifuged. Remove the supernatant. Precipitation
Dissolve in 0.3 L of chloroform. Ethanol (1.5L)
Is added to the lipid solution with stirring. The mixture is placed in a refrigerated case (0-4 ° C) for 18 hours. The precipitate is centrifuged.
Remove the supernatant. Dissolve the precipitate in 0.3 L of chloroform. Physiological saline solution (0.3L) and ethanol (0.1L)
Is added to the lipid solution. Mix the mixture for 5 minutes. Separate the chloroform solution with a separatory funnel. Remove the aqueous layer.
The chloroform solution is dried by Na ₂ SO ₄ (anhydrous) in 0.1～0.2Kg. This is then placed in a refrigerated case (0-4 ° C) for 2 hours. The chloroform solution is separated by filtration.
Na ₂ SO ₄ is removed. The chloroform lipid solution is placed in a refrigerator (−10 to −20 ° C.) for storage. Determine the concentration of lipids.

Second stage. Evaporate the obtained chloroform solution of the preparation of the invention and disperse it in saline solution (or other isotonic solution) until the lipid concentration is 2.6%. Dispersion of the liposomes is performed 3-4 times (until the OD becomes constant) through the Microfluidizer. Sterilize the dispersion. Filter the dispersion through a filter (0.4 μm). Determine the final concentration of lipid. The dispersion is diluted to 2% with a sterile isotonic solution. The preparation is stored in a bottle under aseptic conditions.

Example 2 This example illustrates a method for preparing an anti-hemolytic composition in the form of a fat emulsion from bovine spinal cord and soybean oil. This method is suitable for any other source: any mammalian spinal cord, any mammalian brain, and any mammalian tissue containing sulfolipids and also related to triglycerides: any known Fats and oils of the type described above may be used in the present invention, but the use of edible oils such as soybean oil, corn oil, coconut oil, rapeseed oil, sesame oil, or peanut oil is preferred.

All operations are performed under a nitrogen atmosphere.

First stage. Lipid blends containing sulfolipids
Acquisition of compound This stage is the same as in the first embodiment.

Second stage. Capture of anti-hemolytic fat emulsion
The obtained soybean oil (12-16 parts of the preparation of Example 1 and 25 parts of glycerol for 100 parts oil) is added to the chloroform solution, then evaporated and a saline solution (2.6%) until the polar lipid concentration is 2.6%. Or other isotonic solution). Disperse 3-4 times (until OD is constant) with Microflu
Pass through idizer. Sterilize the dispersion. Filter the dispersion through a filter (0.4 μm). Determine the final concentration of lipid. The dispersion is diluted with a sterile isotonic solution to 2% polar lipids. The preparation is stored in a bottle under aseptic conditions.

Examples 3-6 These examples illustrate the immunohemolysis, ie the inhibition of neonatal hemolytic disease (in vitro) by the preparations according to the invention.

Procedure We use erythrocytes from children with hemolytic disease of newborns and serum from their mothers. Fibers are removed from the blood (1 ml) from the child's frontal vein, then saline solution is added to the red blood cells (9 ml + 1 ml) and the mixture is gently agitated for 0.5 minutes. The sample is then centrifuged (3 min, 3000 rpm)
And decant the supernatant. Repeat this operation twice.
Add 9 ml of saline solution to 0.3 ml of red blood cells.

The mother's blood (3 ml) is placed at 20 ° C. for 2 hours and then centrifuged (3 minutes, 300 rpm). The serum is diluted 10-fold with saline solution.

In this experiment: 0.5 ml of red blood cells is mixed with 0.5 ml of a preparation according to the invention, 0.5 ml of serum and 0.5 ml of saline solution.

In a blank experiment: 0.5 ml of red blood cells was
Mix with ml serum and 1.0 ml saline solution.

For light scattering control: 0.5 ml of the preparation is mixed with 0.5 ml of serum and 1.0 ml of saline solution. To calculate the percentage of hemolysis, we performed a total hemolysis. Measure OD ₄₁₈ for all samples. The level of hemolysis is calculated by the following formula: H = (OD _ex −OD _Ls −OD _bl ) × 100 / OD ₁₀₀ where: OD _ex −OD in this study, OD _bl −OD in blank experiment, OD O at ₁₀₀ -100% red blood cells
D; OD _Ls- optical density of the preparation diluted to a concentration equivalent to the concentration of the preparation in the experiment (liposome scattering control).

These experiments were performed with a final preparation concentration of 0.04 mg.
/ ml, 0.2 mg / ml, and 1 mg / ml. Table 1 shows the results.

[0034]

[Table 1]

In the absence of the preparation, it is shown that haemolysis has occurred from about 20% to about 50%, depending on the severity of the disease. In all cases, substantially complete delay of hemolysis occurred at concentrations of the preparation of 0.2 mg / ml and higher.

Examples 7-14 These examples demonstrate the inhibition of hemolysis by the preparations of the present invention in the case of immunohemolysis in mammals (sheep and rabbit) and also demonstrate the complement induced hemolysis. 2 shows a method for treating associated diseases and disorders. A lethal amount of anti-hemolytic serum was introduced into the animals. The preparation of the present invention (20 mg / ml) was introduced into experimental animals by intravenous injection for 10 minutes. Physiological saline solution was introduced into control animals. Table 2 shows the results.

[0037]

[Table 2]

Animals that have not received the preparations of the present invention
He died 6-36 hours after administration of a lethal dose of hemolytic serum.
Blood bilirubin levels in control animals are 29-36μ
M and the erythrocyte level of control animals is 4-6
Reduced by half in time.

Animals dosed with the preparation at a dose of 14-16 mg / kg had several disorders (weakness, frequent br
eathing) and malaise), which recovered within 24 hours (Examples 7, 11). All animals were healthy at the administration of the preparations ≧ 21 mg / kg.

The blood bilirubin level of the experimental animals was equal to 0, and the red blood cell levels of the experimental animals did not change.

In these examples, the therapeutic dose was about 20 mg / k
g indicates weight.

[0042]

According to the present invention, there is provided an anti-hemolytic liposome preparation for treating diseases and disorders associated with erythrocyte hemolysis.

Continuation of the front page (72) Inventor Kwon Kim South Korea Kungguid, Anyang-shi, Donggang-ku, Kyuin-dong, Hanshin Apty 707-804 Ngil-Don 54, Han Yang Apty 2-307 (72) Nina N. Inventor. Ivanova Ukraine Karkov 310091, Tankoy Street 7/1, Apty 12 (72) Inventor Alexander P. Kaplan Russian Federation Moscow Prospect 117571, Bernadoskogo 86

Claims (4)

[Claims] 1. An anti-hemolytic liposome preparation, comprising 16
-90% by weight of sulfolipid, 5-70% by weight of phosphatidylcholine, 5-40% by weight of cerebroside, and
A hemolytic liposome preparation containing 30% by weight of cholesterol, wherein the sulfolipid is one or more selected from the group consisting of cerebroside sulfate, sulfolactosylceramide, sulfogalactosyldiacylglycerol, and sulfogalactosylalkylacylglycerol. . 2. The anti-hemolytic liposome preparation according to claim 1, wherein said sulfolipid is cerebroside sulfate. 3. A process for preparing an anti-hemolytic liposome preparation, comprising two steps: i) a commercially available lipid or extracted from mammalian brain or spinal cord. Using a lipid to obtain a lipid mixture having the composition of claim 1; and ii) dispersing the lipid mixture in an isotonic solution. 4. A process for preparing an anti-hemolytic liposome preparation according to claim 3, wherein the lipid mixture is obtained by following a step comprising: i) mammalian brain Or homogenizing the spinal cord; ii) removing water and neutral lipids with acetone; iii) extracting polar lipids with lower alcohols (methanol, ethanol, isopropanol); iv) evaporating the extract under reduced pressure V) dissolving the residue with chloroform or methylene dichloride; vi) precipitating polar lipids with acetone; vii) repeating steps v) and vi) several times; viii) removing the organic solvent Obtaining a lipid mixture.