DE3428736A1 - PHARMACEUTICAL COMPOSITION ON THE BASIS OF LIPOSOMES AND METHOD FOR MANUFACTURING LIPOSOMES - Google Patents

Description

Case 15394

description

The invention relates to a therapeutic composition using liposomes, the fructose-1,6-diphosphate (FDP) contain, to introduce this compound into the interior of cells, whose membrane is normally is impermeable to this substance.

Fructose-1,6-diphosphate can work in this way directly act on the cell, which explains a greater therapeutic effect.

The invention also provides the process for the preparation of these liposomes and of related liposomes pharmaceutical compositions described.

The fructose-1,6-diphosphate (FDP) is a well-known compound, which is widely used for the therapy of trauma, of intestinal and uterine atony as well as in therapy of intoxication or hypercatabolic states is used.

Recent research has confirmed that fructose 1,6-diphosphate cannot penetrate into the interior of cells whose membrane is impermeable to this substance. the The therapeutic effect of fructose-1,6-diphosphate is based on its ability to stimulate the flow of ions through membranes

QQ vary and in this way across the intracellular Metabolism to influence the aforementioned pharmaceutical effects.

In order to deny the effectiveness of the above compound strengthen a therapeutic method is proposed by the invention, the direct introduction of Allowing fructose-1,6-diphosphate into the cell, which allows the drug to be more active.

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The procedure of the invention envisions the use of liposomes whose composition is suitable for taking up fructose-1,6-diphosphate and / or its isomers.

It is known that liposomes made up of lipid bilayers of different Shapes and sizes are built up and can contain other substances inside. You know them Possibility of fusion of the lipid part of this vesicle with the plasma membrane (cf. G. Poste and D. Papahadjopoulos: Les liposomes et leurs usages en biologie et en medecine, Pp. 164-184, Am. N.Y. Acad. May be. 1978 and M. Giomini and on. Giuliani, Il Farmaco (Prat.) 34, 3-14, 1979).

However, there are hypotheses, after which the liposomes may function as transport elements for other substances in that they allow for the introduction of molecules into the cellular cytoplasm, which are in the aqueous portion of the liposomes _n included. Further research has confirmed the correctness of these hypotheses, which can be clearly seen from the following.

Therefore, the therapeutic method according to the invention consists of following stages:

a) inclusion of the drug substance inside liposomes;

b) Introduction of the substance into the interior of the cell cytoplasm 30

mas, utilizing the method of fusion of the lipid part of the liposomes with the plasma membrane.

The invention also relates to the process for the production of liposomes, which consists of the following stages:

a) Preparation of liposomes from the phospholipid compound, which is purified by column chromatography, dissolved

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3A28736

in an organic solvent, and in contact with an aqueous solution of fructose-1,6-diphosphate Ultrasound is treated;

b) concentrating through a membrane in the ultrafiltration cell;

c) Purification of the non-included fructose-1,6-diphosphate by gel filtration.

It should be noted that as to the amount of the principle of action included in the obtained particles, that indicated Order of phases is critical.

The pharmacological effect of the shape thus obtained is assessed by incubation with human blood at 17 ⁰ C with stirring, and multiple control of the stabilities of the ATP-erythrocyte levels. In fact, this parameter has so far proven to be the most effective for studying the pharmacological effects of fructose-1,6-diphosphate and its isomers.

The following production example for liposomes serves to further illustrate the invention:

example 1

5 g of raw lecithin (e.g. from eggs) are used in QQ about 10 ml of chloroform and dissolved over a silica gel filled column. Elute with a continuous gradient of chloroform-methanol.

The fraction (n-5) containing 3.5 g of phosphatidylgg choline is collected, eluting with methanol. The purity of this fraction is confirmed by thin layer chromatography (glass plates with silicon dioxide _{gel), eluting with chloroform-methanol-H 2} O (65-35-5). Rf = 0.24 '

-6-1

One conserved in a freezer at -20 ⁰ C under a nitrogen atmosphere 500 mg of this product that has become solubilized in 150 ml of ethyl ether, ml in a sealed with ground glass stoppers 500 container. A clear solution is obtained, mixed with 150 ml of chloroform and successively 50 ml of fructose-1,6-diphosphate solution (with a concentration of 1 mg / ml) at a pH of 7.4.

The operations described are carried out under a nitrogen atmosphere carried out to reduce the risk of undesirable formation Eliminate products through possible oxidation of lipids.

The mixture thus obtained is a treatment in one

An ultrasonic bath, is subjected for 15 minutes (keeping in every 5 minutes to pause of 1 minute), while avoiding that the bath temperature exceeds 15 ⁰ C. A single stable phase is obtained that lasts for an hour

lets stand. 20th

The organic solvents are then slowly evaporated off under vacuum at a temperature of 45.degree. You get first a gelatinous mass, which in the course of the complete removal of all remaining solution

^ΔΌ by means of liquefied. The large vesicles with a lamella obtained in this way are filtered through a membrane of 0.45 μm in order to make the mass of the liposomes uniform (particle size about 30 nm or 300 Å). After this filtration, about 45 ml of suspension remain, which is treated with ultrasound

^^ is in an Amicon filtration unit with a Diaflo YM 30 membrane under nitrogen pressure (Amicon and Diaflo are registered trademarks). Through this previous concentration about 60 to 65% of the outer fructose-1,6-diphosphate is removed. The remaining volume (about 20 ml) is transferred to a column

35 _au fgetragen containing dextran polymers, commercially available as "Sepharose 4B" (registered trademark) are known and were previously saturated with a standard liposome suspension.

It is eluted with a Tris-HCl-NaCl-0.1 m buffer (pH = 7.5), the first fractions being collected according to the "empty volume" (which was previously determined using dextran blue ° ). The volume of the liposomes obtained is subjected to a final concentration in an ultrafiltration cell (Amicon) with a Diaflo YM 30 membrane under nitrogen pressure. A suspension is obtained which contains 1.3-1.5 mg of fructose-1,6-diphosphate per ml in total, which is free from external medicament which is not enclosed in the vesicles.

The liposomes containing the fructose-1,6-diphosphate, the so obtained were in vitro with analog not included drug dosages and with an analog Amount of liposomes containing 0.9% NaCl, as described below, examined:

Example 2

2.25 ml of human blood were mixed with 0.25 ml of liposomes, which were obtained as described above and contained 1.35 mg / ml of fructose-1,6-diphosphate, and were placed in a thermostatically controlled bath at 37 ^° C. with continuous stirring held. Two other samples of human blood (from the same person) were incubated at the same time, 2.25 ml each, the 0.25 ml fructose-1,6-diphosphate solution (1.35 mg / ml) and 0.25 ml, respectively Liposomes containing 0.9% NaCl contained.

After incubation, the erythrocyte levels of ATP were measured at various times. _:

The results obtained are in the table below I listed.

40 ' 160 ' Fructose-1,6-diphosphate in the liposomes 550 γ / ml 664 γ / ml free fructose-1,6-di phosphate 552 γ / ml 601 γ / ml Liposomes as such 524 γ / ml 532 γ / ml

Note: The base value for the ATP is 530 // ml.

Each base value is the mean of several (6) tests. Analysis of these results is convincing that the in the liposome vesicles enclosed fructose-1,6-diphosphate the erythrocyte metabolism in clearly different ways Way to fructose-1,6-diphosphate not entrapped in liposomes. This shows the possibility a fusion of the liposomes with the cell membrane with a corresponding release of the entrapped drug in the cell.

To confirm these hypotheses, the human blood incubation test using liposomes was carried out performed using the technique described

and fructose-1,6-diphosphate marked with C include. The parallel with an analogous amount of free fructose-1,6-diphosphate, which also was marked, showed that the fructose-1,6-diphosphate entrapped in the liposomes in vesicles can cross the erythrocyte membrane.

The percentage of fuctose-1,6-diphosphate ( in the liposomes) compared to that of free fructose-1,6-diphosphate, recorded:

Fructose-1,6-diphosphate in the liposomes -% in the cell
free fructose-1,6-diphosphate -% in the cell

Claims (3)

Dr. F. Zumstein Sr. - Dr. E. Assmann ^ Dipl.-Ing. F. Klingseisen - Dr. F. Zumstein jun. 3428736 PATENT LAWYERS APPROVED REPRESENTATIVES AT THE EUROPEAN PATENT OFFICE REPRESENTATIVES BEFORE THE EUROPEAN PATENT OFFICE D SOOO MUNICH 2, BRÄUHAUSSTRASSE 4 TELEPHONE: (O89) 225341 TELEGRAMMA. Rome / Italy Pharmaceutical composition based on liposomes and process for the production of liposomes Patent claims 1. Pharmaceutical composition containing fructose-1,6-diphosphate and / or its isomers incorporated into the interior of liposomes. 2. Process for the preparation of fructose-1,6- * containing diphosphate Liposomes, characterized by the following stages: a) Preparation of a phospholipid component purified by column chromatography, dissolved in an organic one Solvent and treated with ultrasound in contact with an aqueous solution of fructose-1,6-diphosphate. POST CHECK ACCOUNT: MUNICH 91139-809. BLZ 70010080 BANK ACCOUNT: BANKHAUS H. AUFHÄUSER ACCOUNT NO. 3Θ7997. BLZ 7003060 -2- 3428735 b) Concentration with a membrane in an ultrafiltration cell , 5 c) Purification of the non-entrapped fructose-1,6-diphosphate by gel filtration, and d) final concentration with a membrane in the Ultrafiltration cells. 10 3. A pharmaceutical composition obtainable by the method of claim