CH662946A5 - PROCEDURE FOR THE PREPARATION OF INCORPORATING LIPOSOMES FRUCTOSE 1,6 DIPHOSPHATE. - Google Patents

Description

The present invention proposes a process for the preparation of liposomes incorporating fructose 1,6 diphosphate, where the following steps are envisaged:

a) preparation of the phospholipid component purified by column chromatography, dissolved in organic solvent and sonicated in contact with the aqueous solution of fructose 1,6 diphosphate;

b) concentration for membranes in ultrafiltration cell;

c) purification by gel filtration of fructose 1,6 diphosphate not incorporated;

d) final concentration for membranes in ultrafiltration cell.

The liposomes thus produced can be used to introduce FDP into cells, whose membrane is normally impermeable to this substance.

Fructose 1,6 diphosphate can thus act directly on the cells, exhibiting greater therapeutic activity.

Fructose 1,6 diphosphate (FDP) is a well-known compound, widely used in the therapy of shocks, intestinal and uterine atony, in the therapy of toxic and hypercatabolic states.

FDP, as recent studies have confirmed, is unable to penetrate inside the cells, whose membrane is impermeable to this substance. The therapeutic activity of FDP, therefore, is due to its ability to vary transmembrane ionic flows, thus influencing the intracellular metabolism to determine the aforementioned pharmacological effects. To increase the effectiveness of the action performed by the described compound, FDP can be introduced directly into the cell, thus allowing the drug to exert greater activity. To introduce FDP into cells, the use of liposomes is envisaged, the composition of which is suitable for containing FDP and / or its isomers.

It is known that liposomes are made up of double lipid layers of different shapes and sizes, capable of enclosing other substances inside them. The possibility of fusion of the lipid part of these vesicles with the plasma membrane is known (see POSTE G., PAPAHADJOPOULOS D .: Lyposom and their uses in biology and medicine, pp. 164-184 Am. NY Acad. Sci 1978, and GIOMINI M „GIULIANI AM: Il Farmaco (Prat.) 34; 3-14, 1979).

It has therefore been hypothesized that the liposomes could function as transporters of other substances, so as to make it possible to introduce into the cell cytoplasm the molecules incorporated in the aqueous part of these liposomes.

Subsequent experiments have confirmed the accuracy of this hypothesis, as will become clear from the data reported below.

The present invention relates to the process for the production of these liposomes, a process which consists of the following steps:

a) preparation of liposomes from the phospholipid component, purified by column chromatography, dissolved in organic solvent and sonicated in contact with the aqueous solution of fructose 1,6 diphosphate;

b) concentration for membranes in ultrafiltration cell;

c) purification by gel filtration of fructose 1,6 diphosphate not incorporated;

d) final concentration for membranes in ultrafiltration cell.

It should be noted that, both as regards the quantity of active ingredient incorporated and as regards the quality of the particles obtained, the sequence of the listed phases is critical.

The pharmacological efficacy of the form thus obtained was evaluated by incubating blood at 37 °, under stirring, checking the erythrocyte levels of ATP at set times; in fact, this parameter has so far proved to be the most effective for the study of the pharmacological effects of both FDP and its isomers.

For further clarification, a detailed example relating to the preparation of liposomes will now be provided.

Example 1

5 g of crude lecithin (ex ovo) are dissolved in about 10 ml of chloroform and passed through a column filled with silica gel. It elutes with a continuous chloroform-methanol gradient.

The fraction (n. 5) eluent methanol is collected which contains 3.5 g of phosphatidyl-choline. The purity of this fraction is confirmed by thin layer chromatography (silica gel glass plates) eluent: chloroform-methanol-H ^ O (65: 35: 5).

Rf = 0.24.

It can be kept in the freezer at -20 ° C under a nitrogen atmosphere.

500 mg of this product are dissolved in 150 ml of ethyl ether in a 500 ml emery neck flask; obtained a clear solution, 150 ml of chloroform and subsequently 50 ml of fructose-1,6-diphosphate solution (at the concentration of 1 mg / ml) are added at a pH = 7.4.

The operations described must be carried out in a nitrogen atmosphere in order to eliminate the risks of formation of any unwanted lipid oxidation products.

The mixture thus obtained is subjected to sonication in an ultrasonic bath for the duration of 15 '(interspersing, every 5', one minute break), avoiding that the bath temperature exceeds 15 ° C. The result is a single stable phase which is left to rest for about an hour.

The organic solvents are then evaporated under vacuum slowly at a temperature of 45 ° C. A gelatinous mass is obtained first, which subsequently fluidizes by total elimination of all the residual solvent. The L.U.V. (Large Unila-mellar Vescicles) thus obtained are filtered through 0.45 um membranes in order to uniform the population of liposomes (particle size ~ 300 À).

Once this filtration is completed, about 45 ml of suspension remain which is subjected to ultrafiltration in a filter unit of Amicon, with Diaflo YM 30 membranes, under nitrogen pressure (Amicon and Diaflo registered trademarks).

This preliminary concentration operation frees us of about 60-65% of the external fructose-1,6-diphosphate.

The residual volume (-20 ml) is loaded on a column with 5

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662 946

containing commercially known dextran polymers under the name "Sepharose 4 B" (registered trademark) previously saturated with a suspension of standard liposomes. It is eluted with tris buffer (0.1 M HCl • NaCl; pH = 7.5) collecting the first fractions after the "void volume" (previously determined with the use of bleu dextran). The volume of the liposomes obtained is subjected to final concentration in the Amicon ultrafiltration cell with Diaflo YM 30 membranes, under nitrogen pressure.

A suspension containing 1.3-s-1.5 mg of fructose-1.6-diphosphate per ml is obtained, completely free of drug not incorporated outside the vesicles.

The FDP-containing liposomes thus obtained have been tested in vitro in comparison with similar doses of non-incorporated drug, and with similar quantities of liposomes containing 0.9% NaCl, as described in the following:

Example 2

2.25 ml of human blood were added with 0.25 ml of liposomes, obtained as described, containing 1.35 mg / ml of fructose-1,6-diphosphate, and incubated in a thermostat bath at 37 ° C , under constant stirring. ' At the same time two other human blood samples (of the same individual) were incubated, each of 2.25 ml, containing 0.25 ml of FDP solution (1.35 mg / ml) and 0.25 ml of liposomes containing NaCl respectively 0.9%.

After incubation, ATP erythrocyte values were checked at various times.

The data obtained are inserted in the attached table I:

TABLE I

40 '

160 '

FDP in liposomes

550 y / ml

664 y / ml

Free FDP

552 y / ml

601 y / ml

Liposomes t.q.

524 y / ml

532 y / ml

10 N.B. The basic value of the ATP is 530 y / mi.

Each value is the average of multiple observations (6). The analysis of these data shows that the FDP conveyed in the liposomes influences the erythrocyte metabolism in a significantly different way from the FDP with incorporated in the liposomes, thus demonstrating the possibility of a fusion of the liposomes with the cell membrane with consequent release in the cell of the drug incorporated. In order to confirm this hypothesis, the incubation test with human blood was repeated using liposomes, prepared according to the described technique, which incorporate fructose-1,6 - labeled diphosphate (C14). The test, performed in parallel with similar quantities of free fructose-1,6-diphosphate, also on the market, has shown that the FDP conveyed in the liposomes is able to overcome the erythrocyte membrane; the percentage of FDP (carried in the liposomes), entered into the cell over time, in comparison with the free FDP is reported below -25 to:

5 '

40 '

160 '

- FDP in liposomes

-%

in cells:

0

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20

30 - Free FDP

-%

in cells:

0

0

0

v

Claims (3)

662 946 1. Procedure for the preparation of liposomes containing fructose 1,6 diphosphate, characterized in that it provides the following steps: a) preparation of the purified phospholipid component by column chromatography, dissolved in organic solvent and sonicated in contact with the aqueous solution of fructose 1,6 diphosphate; b) concentration for membranes in ultrafiltration cell; c) purification by gel filtration of fructose 1,6 diphosphate not incorporated; d) final concentration for membranes in ultrafiltration cell. 2. Pharmaceutical preparation obtained by the process according to claim 1. 2 3. Pharmaceutical preparation characterized in that it provides fructose 1.6 diphosphate and / or its isomers incorporated within liposomes, obtained with the process according to claim 1.