GB2316869A

GB2316869A - An antihaemolytic liposomal preparation

Info

Publication number: GB2316869A
Application number: GB9714037A
Authority: GB
Inventors: Ho Moh Shon; Hee Sock Park; Kwon Kim; Jin Kyu Park; Nina N Ivanova; Alexander P Kaplun
Original assignee: Dong Kook Pharmaceutical Co Ltd
Current assignee: Dong Kook Pharmaceutical Co Ltd
Priority date: 1996-08-26
Filing date: 1997-07-02
Publication date: 1998-03-11
Anticipated expiration: 2017-07-02
Also published as: FR2752526A1; CN1174705A; DE19729641A1; KR100190826B1; KR19980016013A; JPH1067665A; GB9714037D0; GB2316869B; DE19729641C2

Abstract

This invention relates to an antihaemolytic liposomal preparation comprising 16#90 wt% of sulfolipids, 5#70 wt% of phosphatidyl choline, 5#40 wt% of cerebroside and 0#30 wt% of cholesterol for treating haemolysis, especially, complement-induced haemolysis.

Description

2316869

(Title of the Invention)

Am Antihaemolytie Liposomal Preparation (Background of the Invention)

Field of the Invention

This invention relates to an antihaemolytic liposomal preparation, more specifically, a liposomal preparation comprising sulfolipid, phosphatidyl, choline and cerebroside for treating haemolysis, especially, complementinduced haemolysis.

Description of Prior Art

Some diseases and disorders are accompanied by haemolysis of erythrocytes, for example, disease of new-born infants, paroxysmal nocturnal hemoglobinuria. Haemolysis is possible under blood transfusion, if a choice of blood is not quite accurate, or under bone marrow transplantation in case of wrong blood group incompatibility.

1 EP 0 394 035 A2 disclosed the soluble forms of glyoprotein MACIF (membrane attack complex inhibition factor) in erythrocyte membrane for defence from haemolysis. Recently, the protein having human MACIF activity are obtained from the transformed cells with the expression vectors, which contain the genes for coding this protein. However, the use of this protein as therapeutic agent is limited due to its high price and possible side effects.

(Summary of the Invention)

The object of the present invention is to provide an antihaemolytic liposomal preparation comprising 16-90 wt% of sulfolipids, 5-70 wC/6 of phosphatidyl choline, 5-40 wt% of cerebroside and 0-30 wt'/o of cholesterol, wherein sulfolipids are one or more selected from the group consisting of cerebroside sulfate, sulfolactosylcerarnide, suifogalactosyldiacylglycerol and suifogalactosylaikylacylgylcerol. Among them, preferable sulfolipid is cerebroside sulfate.

The preparation of the present invention can be obtained by following two steps:

i) obtaining the lipid mixture to have said composition using commercially marketed lipids or lipids extracted from brain or spinal cord of mammals and ii) dispersing the said lipid mixture in isotonic solution.

2 The concentration of 50% activity(ICw) of the preparation is 4120,gg/n-d depending on the content of sulfolipid and other polar lipid.

However, the other object of present invention is to provide a convenient method for preparing said antihaemolytic liposomal preparation with following process comprising the steps of: i) homogenizing the brain or spinal cord of mammals ii)removing the water and neutral lipids by acetone; iii) extracting the polar Epid by lower alcohol(methanol, ethanol, iso-propanol) iv) Evaporating the extracts in reduced pressure v) dissolving the residue with chloroform or methylene dichloride vi) precipating the polar Epids by acetone vii) repeating step v) and vi) several times vi) removing the organic solvent to obtain the lipid mixture.

The mixture of lipid obtaining such methods contains 18-40% of cerebroside sulfate, 10-30% of phosphatidyl choline, 15-30% of cerebroside, 1025% Of phosphatidyl ethanolazine and other phosphatidyl serine and sphingomyelin etc. Concentration of 50% activity(ICw) of this preparation is 4 15pg/n-d.

(Detailed Description of the Invention)

3 The liposomal preparation of the present invention can be obtained by following methods.

The antihaemolytic lipid composition is obtained by dissolving the mixture of polar lipids containing sulfolipids in chloroform, methanol and/or ethanol. After removal of solvent the isotonic solution is added. Then, the lipid preparation is homogenized and dispersed by sufficient shaking and ultrasonic treatment. Finally, the dispersion is filtered through membrane filter of 0.7p or 0.4p size under sterile condition. The size of liposome of this invention is less than 1P, prefarably, not more than 0.5p, because it is required for intravenous administration.

The indications of andhaemolytic liposomal preparation of the present invention are as follows.

1) Inhibition of complement-induced haemolysis The preparation of the present invention can inhibit the complementaryinduced haemolysis, such as, haemolytic disease of new-born, paroxysmal nocturnal haemoglobinuria. The effective concentration of such haemolysis is from about 0.5pg/n-d to 20mg/nil.

2) Inhibition of hypotonic haemolysis It is well known that mammalian cells are burst in hypotonic solution due to the difference of osmolality inside and outside of cell. Several infections can be accompanied hypotonic haemolysis. This preparation has strong negative charge because of strong acidic group in sulfolipid. Therefore, this 4 preparation can be used for the protection against hypotonic haemolysis. Effective concentration of this case is from O.Olmg/n-d to 0.lmg/ml.

3) Useful for the storage of blood and products containing erythrocytes The present preparation is useful for the storage and handling of blood and blood-containing products, tissues or organs. Such blood products may further comprise the blood storage additives, for example, anti-coagulant and nutritive ingredient. Typical additives include sodium chloride, citric ac'id, sodium citrate, glucose, sodium dihydrogenephosphate, glycerol, folic acid, adenine and L-ascorbic acid. The preparation of present invention is also useful for blood storage additive.

The present invention can be explained more concretly by following examples. But the scope of this invention is not limited to these examples.

(Example 1)

This example demonstrates a preparation method of present invention from cattle spinal cord. This method is suitable for any other source: spinal cord of any mammals, brain of any mammals and any marrimal tissue containing sulfolipid.

Stage 1. Obtainin-e of lidd mixture comprising sulfolipid Fresh or quickfrozen spinal cord of cattle(lkg) is homogenized with 05-1 L acetone in tissue disintegrator. Crushed spinal cord is placed in a reactor, and cooled(-10IC) acetone is added(2.5 U A mixture is stirred for 5 min, then it is filtered on a Nutsch-filter. Acetone solution is removed. The residue is transferred in a reactor. A cooled(-10r-) acetone(3 L) is added in a reactor, and it is intensively stirred for 2 h. The residue is filtered on a Nutsch-filter(acetone extract 2). This working is repeated once more(acetone extract 3). The residue is washed with 0.2 L ethanol on a Nutsch-filter. The acetone extracts 2 and 3 can be used for separation of cholesterol. The residue is loaded in a reactor. EthanolQ L) is added. The mixture is stirred for 2 h at20-22C. The residue is filtered on a Nutsch-filter. Ethanol extract(extract 1) is placed in a refrigerator(- -20 IC). The residue' is loaded in a reactor, -petroleum ether(4 L) is added. The mixture is stirred for 2 h (20-22t). The residue is filtered on a Nutsch-filter. Petroleum ether extract(extract 2) is placed in a refiigerator(-10-20C). The residue is loaded in reactor, petroleum ether(3 L) is added. The mixture is stirred intensively for 2 h (20-221C). The residue is filtered on a Nutsch-filter. Petroleum ether extract(extract 3) is placed in a refrigerator(- 10 - - 20 IC).

The extracts 1, 2, and 3 are evaporately dried at 35-401C in vacuum. The residue is dissolved with 0.5 L chloroform with stirring. Acetone(2.5 L) is added to the lipid solution with stirring. The mixture is placed in- a refrigerator case(04C) for 18 hrs (for example, over night). The precipitate is centrifuged. The supernatant is removed. The precipitate under stirring is dissolved with(O.3 L) chloroform. Acetone(L5 L) is added to the lipid solution. The mixture is placed in a refrigerator case(0-4r,) for 18 h. The precipitate is centrifuged. The supernatant is removed. The precipitate is dissolved with(O.3 L) chloroform. Ethanol (1. 2 L) is added to the lipid solution with stir-ring. The mixture is evaporated into 1/3 volume.

6 The mixture is placed in a refrigerator case(O4'C) for 18 h. The precipitate is centrifuged. The supernatant is removed. The precipitate is dissolved with 0.3 L chloroform. Ethanol(L5 L) is added to the lipid solution with stirring. The mixture is placed in a refrigerator case(O410 for 18 h. The precipitate is centrifuged. The supernatant is removed. The precipitate is dissolved with 0.3 L chloroform. Saline(O.3 L) and ethanol(O.1 L) are added to the lipid solution. The mixture is mixed during 5 min. Chloroform solution is separated in a separating funnel. Water layer is removed. Chloroform solution is dehydrated by 01-0.2kg Na2S04(free of water). Then it is placed in a refrigerator case(O-41C) for 2 h. Chloroform solution is separated by filtration. Na2S04 is removed. Chloroform lipid solution is placed in refrigerator(- 10 - 20 t) for storage. The concentration of lipids is determined.

Staize 2. Obtaining of the Perwaration of present invention Chloroform solution is evaporated, dispersed in saline(or other isotonic solution) up to concentration of lipids 2.6%. The dispersion of liposome is passed through Nficrofluidizer 3-4 times (up to constant OD). The dispersion is steri. The dispersion is filtered through filter(0.4pm). The final concentration of lipis is determined. The dispersion is diluted up to 2% with sterile isotonic solution. The preparation is bottled in sterile condition.

(Example 2)

This example demonstrates a preparation method for antihaemolytic 7 composition in the form of fat emulsion from cattle spinal cord and soybean oil. This method is suitable for any other source: spinal cord of any mammals, brain of any mammals, and any mammal tissue containing sulfolipid, and also as concerned with triglycerides: any known type of fats and oils may be used in this invention, but it is desirable to use an edible oil such as soybean oil, corn oil, coconut oil, rape seed oil, sesarn oil or peanut oil.

All operations are carried out under nitrogen atmosphere.

Stage 1. Obtainina of lipid mixure comprisiniz sulfolipid This stage is the same as in example 1.

Staize 2. Obtaining of antihaemolytic fat emulsion.

Soybean oilGOO parts oil to 12-16 parts of the preparation in example 1 and 25 parts of glycerol) is added in chloroform solution, then it is evaporated, dispersed in saline(or other isotonic solution) up to concentration of polar hpids 2.6%. The dispersion is passed through Microfluidizer 3-4 times(up to constant OD). The dispersion is sterilized. The dispersion is filtered through filter(O.4pm). The final concentration of lipids is determined. The dispersion is diluted up to 2% polar lipids with sterile isotonic solution. The preparation is bottled in sterile condition.

(Example 3-6)

8 These examples illustrate inhibition of immune haernolysis by the preparation of present invention haernolytic disease of new-born infants(in vitro).

(Procedure) We used erythrocytes from children with haemolytic disease of new-bom infants and serum from their mothers. The blood from child forehead venue(lmi) is defibrinated, then saline is added to erythrocytes (9M1 + ln-d), the mixture is stirred gently for 0.5 n-dn. Then the sample is centiifuged(3min, 300Orpm), supernatant is decanted. This working is repeated twice, 0.9m1 saline is added to 0.3nd erythrocytes.

The blood from mother(3mD settle at 20C for 2 h, then is centrifuged (3 rnin, 300 rpm). The serum is diluted in 10 times with saline.

In the experiment: 0.5rrd erythrocytes is rnixed with 0.5nd of the preparation of present invention, 0.5n---d serum and 0.5mI saline.

In the blank experiment: 0.5m1 erythrocytes is n-dxed with 0.5mI serum and 1.0n-d saline.

In the control of ligh scattering: 0.5m1 of preparation is n-dxed with 0. 5rnl serum and 1.0m1 saline. In order to account percentage of haemolysis we carried out total haemolysis. For all samples OD418 is measured. Level of 9 haemolysis is calculated according to equation:

H = (0De, - ODLs - MA) x 100/0Dloo wherein: 0De.-OD in experiment under study, ODti-01) in blank experiment, ODloo-OD under 100% haemolysis; 0Dis-optical density of a preparation, diluted up to concentration, equal to concentration of a preparation in experiment(Control of liposome scattering).

These experiments were carried out at the final concentration of preparation 0.04mg/fid, 0.2mg/n-d and lmg/ml. Results are represented in Table 1.

[Table 11 HaemolysisM at the different concentrations of the preparation o Dwsent invention Example Concentration of preparation of present invention (mg/ml) number 0 0.04 0.2 1 3 43 15 3 3 4 21 13 2 <1 48 18 3 2 6 29 14 2 2 It is shown that in the absence of preparation, haemolysis occurred from about 20% to about 50% in depends on a disease heaviness. In all cases, practically full retard of haemolysis occurred at the concentration of preparation 0.2mg/rrd and more.

(Example 7-14) These examples illustrate the inhibition of haemolysis by the preparation of present invention in case the immune haemolysis in mammal (in sheep and in rabbits) and also the method for treating diseases and disorders accompanied with complement-induced haemolysis. The lethal dose of the antihaemolytic serum was introduced to animals. Preparation of present invention (20mgAnD was introduced to the experimental animals by i. v. in 10 min. Saline was introduced to the control animals. The results is represented in table 2.

[Table 21 Antihaemolytic activity of preparation in vivo. Inhibition o complement-induced haemolysis in animals.

Sample Animals Weight Number Volume of Amount of Volume No of haemolytic lipids per 1 Of Obeervatiens animals serum kg animal saline kg (titer) weight mi mg nd 7 Rabbits 3.5 2 4(1:100) 15.7 5.0 Weakness, frequent breathing, in 24 h recocered Sample Animals Weight Number Volume of Amount of Volume No of haemolytic lipids per 1 of Observatiens animals serum kg animal saline kg (titer) weight M.1 M9 rrd 8 Rabbits 3.2 2 4(1:100) 21.2 5.0 Healthy 9 Rabbits 4.2 2 4(1100) 32.2 6.0 healthy Rabbits 4.1-3.8 4 4(1:100) 0 6.0 Dead in 6-16 hours 11 Sheep 60 2 15(1:1200) 14.2 75 Languor, frequent breathing, in 24 h recovered 12 Sheep 63 2 15(1:1200) 25.1 75 Healthy 13 Sheep 57 2 150:1200) 36.8 70 Healthy 14 Sheep 62 2 15(11200) 0 70 Dead in 36 h The animals who did not receive preparation of present invention were dead in 6-36 h after administration of the lethal dose of haemolytic serum. Level of blood bilirubin of the control animals increased to 29- 36PM, level of erythrocytes of the control animals decreased twice in 46 hours.

The animals, to whom preparation was administrated in dose 14-16mgAg, showed some disorders(weakness, frequent breathing, languor), but they were recovered within 24 h(examples 7, 11). Under administration 21mgAg of preparation or more, all animal were healthy.

12 Level of blood bibrubin of the experimental animals was equal zero, level of erythocytes of the experimental animals did not change.

This examples demonstrate that therapeutic dose is about 20rngAg of weight.

13

Claims

we claim

1. An andhaemolytic liposomal preparation comprising 16-90 wt% of sulfolipids, 5-70 wtO/o of phosphatidyl choline, 540 wto/o of cerebroside and 0-30 wtO/6 of cholesterol, wherein sulfolipids are one or more selected from the group consisting of cerebroside sulfate, sulfolactosylceramide, suifogalactosyldiacylglycerol and sulfogalactosylaikylacylgylcerol -

2. The antihaemolytic liposomal preparation according to claim 1, wherein sulfolipid is cerebroside sulfate.

3. A process for preparing an antihaemolytic liposomal preparation by following two steps comprising: i) obtaining the lipid mixture to have the composition disclosed in claim 1 using commercially marketed lipids or lipids extracted from brain or spinal cord of mammals; and ii) dispersing the said lipid mixture in isotonic solution.

4. The process for preparing an antibaemolytic liposomal preparation according to claim 3, wherein said lipid mixture is obtained by following steps comprising: i) homogenizing the brain or spinal cord of mammals ii) removing the water and neutral lipids by acetone 14 iii) extracting the polar lipid by lower alcohol(methanol, ethanol, isopropanol); iv) evaporating the extracts in reduced pressure v) dissolving the residue with chloroform or methylene dichloride vi) precipating the polar lipids by acetone; vii) repeating step v) and vi) several times vffi) removing the organic solvent to obtain the lipid mixture.