SE459782B - PHARMACOLOGICAL PREPARATION OF FRUCTOS-1,6-DIPHOSPHATE AND SATISFIED FOR PREPARATION - Google Patents

Description

20 25 'JJ C) we Ul go. 459 782 Recent experiments have confirmed the correctness of these hypotheses, as is clear from the data reported below.

Consequently, horses the therapeutic procedure according to the finding of the following steps: (a) entrapment of the medicinal substance in the interior of the posomer; including the introduction of said substance into the interior of the cellular cytoplasm, using the fusion procedure the lipid portion of the liposomes with the plasma membrane.

The invention further relates to a sweet to produce lipo- which consists of the following steps: a) preparation of liposomes of the phospholipidic composition sion, purified by column chromatography, dissolved in an organic solvent and treated with ultrasound in contact with a aqueous solution of fructose-1,6-diphosphate; b) membrane concentration in an ultrafiltration cell; and c) purification by gel filtration of non-entrapped fructose 1,6-diphosphate.

It should be noted that in terms of the amount of the the active ingredient and in terms of the particles obtained the order between the specified steps is critical.

The pharmacological effect of the form thus obtained has been studied by incubating human blood at 17 ° C below movement under control from time to time of the stability and erythrocyte levels of ATP. In practice, this parameter If' until proved to be the more effective for the study of the pharmacological effects of fructose isomers. 1,0-diphosphate than before For a closer understanding, an example is given here, regarding the preparation of the liposomes.

Exemgel l 5 g of crude lecithin (eg from eggs) are dissolved in about 10 ml of chloroform and passed through a column, filled with silica gel. Elution with a continuous gradient of chloroform and methanol.

The fraction (n-5) eluted with methanol is collected, and contains 3.5 g of phosphatidylcholine. The purity of this fraction confirmed by thin layer chromatography (glass plates with silica kagol) at elucring with Rx = n, 2l. chlorofnrm-methanol-water (OS-35-Sl.

SUN mg of this product, dissolved E l3 = wl vlyletvr stored 15 20 25 -1 ll 5,459,782 in a freezer at ZOOC under a nitrogen atmosphere in a 500 ml container with slipnd plug. When a clear solution is obtained, 150 ml are added chloroform and successively SU ml solution of fructose-1,6-diphosphate with a content of 1 mg / ml and a pH of 7.4.

The specified processes are performed in a nitrogen atmosphere to eliminate the risks of possible, unwanted oxidation products of the lipids.

The mixture thus obtained is subjected to a in an ultrasound hand for a period of IS minutes (with input a break of one minute every five minutes), thereby preventing that the temperature of the bid exceeds ISOC. In this way a single, permanent phase is obtained, which is left to stand under In an hour.

The organic solvents are then driven slowly under vacuum at a temperature of 45oC. You first get one gelatinous mass, which in the following becomes liquid through it complete elimination of any remaining solvent.

The large and unilamellar blisters thus obtained are filtered. through a 0.45 nm membrane to make the population of liposomes uniform (particle size about 300 Å). When this filtration is complete, about 45 ml of suspension remains, which is set for the ultrasonic filtration unit from Amicon, with one membrane Diaflo YM 30 under nitrogen pressure (Amicon and Diaflo registered trademarks). This pre-concentration process releases about 60-65% of external fructose-1,6-diphosphate. The remaining the volume (about 20 ml) is charged to a column containing dextran polymers, commercially known as "Sepharose 4 B" ®, which first saturated with a suspension of standard liposomes.

The elution is performed with a buffer tri-HCl-NuCl-0.1 M (pH = 7.5) with recovery of the first fractions after "dead volume" (predetermined using dextran bluel.

The volume of liposomes obtained is subjected to a final centering in an Amicon ultrafiltration cell with a membrane Diaflo YM 30 under nitrogen pressure. A suspension is obtained which per ml of the whole contains 1.3-1.5 mg of fructose-1,6-diphosphate, and which is free from non-enveloped drug outside the bladder.

They thus contain liposomes, containing fructose 1, o-diphosphine, hnr experimentally examined in vitro in comparison see with nnnlogn doses nx not inneslntet_lükemedvl and with one unnlog amount nr liposomvl, innehnllnndt H, U 1 Nu fi l, snsum he- (II 20 459 782 l H is written in the following: Examples 2 2.25 ml of human blood is added with 0.25 ml of liposomes. containing and set for incubation prepared as described above, 1.35 mg / ml of fructose 1,6-diphosphate, in a thermostat bath at 37 ° C with continuous stirring. Sam- early incubate two other samples of 2.25 ml each of human blood (from the same individual), containing 0.25 ml of solution of fructose-1,6-diphosphate (1.35 mg / ml), resp. 0.25 ml liposomes, containing 0.9% NaCl.

After incubation during different times. table 1: Table 1 the erythrocyte values for ATP are sought at The results obtained are given in the following 160 ' Fructose-1,6-diphosphate i i 40 'i liposomes I 5550 Y / ml 664 Y / ml Free fructose-1,6-diphosphate Åssz Y / ml 601 v / m1 ; Liposomes as such š524 Y / ml 532 Y / ml I š I Note. The baseline value for ATP is 530 Y / ml.

Each basic value is the mean of a plurality (6) observations. A study of these results shows that fructose 1,6-diphosphate transported in the liposomes affects erythrocyte metabolism in a way that is clearly different from fructose-1,6-diphosphate not entrapped in the liposomes and thereby demonstrating the possibility of a fusion of liposomes cells with the cell membrane, with a consequent release of the enclosed drug in the cell.

To confirm these hypotheses, the incubation attempt repeated with human blood and using liposomes, which can be prepared by the specified method and enveloped labeled fructose-1,6-diphosphate (C14). A completed parallel experiment with an analogous amount of free, likewise labeled fructose-1,6-diphosphate has shown that it fructose f, 6-diphosphate as transports posomcrna is as i li- capable of penetrating the erythrocyte membrane. In it the following indicates the proportion of Fructose 1.6 -diphosphate (transport- tcrnd i liposomcrnu) som intrñngt i the cell at different times in ratio to free fructose l, n-diphosphate | Fructose-1,6-diphosphate i íliposomer ~% in cells : Free fructose-1,6-diphosphate - 'å in the cell

Claims (5)

459 782 k Patent claims 1. Pharmacological preparation of fructose-1,6-diphosphate and / or its isomers as active compound, t e c k n a d thereof, knowing that the active compound is incorporated into the interior of liposomes. A process for preparing a pharmacological preparation of fructose-1,8-diphosphate and / or its isomers incorporated into liposomes, characterized in that the following process steps are carried out: a) a phospholipid component is purified by chromatography, and the purified product is dissolved in a organic solvent; b) the resulting solution is mixed with an aqueous solution of fructose-1,6-diphosphate, and the resulting mixture is sonicated until a stable emulsion is obtained; c) the organic solvent is evaporated off in vacuo, unincorporated fructose 1,6 and diphosphate are removed by gel filtration, and d) the product obtained under c) is membrane concentrated in an ultrafiltration cell. 3. A method according to claim 2, characterized in that the aqueous solution of fructose-1,6-diphosphate used in b) has a content of about img / ml and a pH of about 7.4. 4. A method according to claim 2 or 3, characterized in that the mixture under b) is treated with ultrasound for 15 minutes, with pauses of 1 minute every 5 minutes, return not exceeding 15 ° C. hence, at a tempe- 5. A method according to any one of claims 2-4, characterized in that the vacuum stripping under c) is carried out at a temperature of not more than 4S ° C.