GB2144332A - Pharmaceutical preparations containing fructose-1,6-diphosphate - Google Patents

Abstract

The invention provides a pharmaceutical preparation wherein fructose-1,6-diphosphate and/or one or more isomers thereof are incorporated into the interior of a liposome.

Description

SPECIFICATION Pharníaceutical Preparations Containing Fructose-l ,6-diphosphate The present invention relates to pharmaceutical preparations containing fructose-1,6diphosphate (FDP) and processes for their preparation.

Fructose-l ,6-diphosphate (FDP) is a known compound, which is widely used in therapy in the treatment of traumatisms, intestinal and uterine atony, and toxic or hypercatabolic states.

As recent studies have confirmed, fructose-l ,6-diphosphate is not capable of penetrating into the interior of the cell, the membrane of which is impermeable to that substance. The therapeutic activity of fructose-l ,6-diphosphate is believed to be due to its capacity for varying transmembranal ionic movement, thereby influencing intercellular metabolism and resulting in above-mentioned pharmaceutical effects.

In order to increase the therapeutic effectiveness of FDP we have now discovered a therapeutic process which makes it possible for fructose-l ,6-diphosphate to be introduced directly into the cell, thus enabling the compound to exhibit a higher degree of activity.

Thus, in one aspect the invention provides a pharmaceutical composition wherein fructose-1,6diphosphate and/or one or more isomers thereof are encapsulated by or incorporated into the interior of a liposome.

The invention provides for the use of liposomes, the structure of which is capable of containing fructose-l ,6-diphosphate and/or isomers thereof.

It is known that liposomes comprise double layers of lipids of various forms and sizes, capable of containing other substances in their interior. The possibility of fusion of the lipidic part of the liposomes with plasmatic membranes is known (see G. Poste and D. Papahadjopoulos: Liposomes and uses thereof in biology and medicine, pages 1 64-i 84, Am. N. Y. Acad. Sci. 1978, and M. Giomini and A.

M. Giuliani, II Framaco (Prat.) 34, 3-14 1979).

However, it is proposed that liposomes could be used as elements for transporting other substances, thus making it possible to introduce molecules which are encased by the aqueous part of the liposomes into the cellular cytoplasm. Further experiments have confirmed the correctness of this proposal, as is clearly apparent from the data set out hereinafter.

In consequence, the therapeutic process according to the invention comprises the following steps: a) enclosing the medicament substance within the liposomes; and b) introducing said substance into the interior of the cellular cytoplasm, by making use of the process of fusion of the lipidic part of the liposomes with the plasmatic membrane.

The invention also provides a process for the production of liposomes for use according to the invention which comprises the following steps a) preparation of a phospholipidic liposome, purified by column chromatography, dissolved in an organic solvent and treated with ultrasound in contact with an aqueous solution of fructose-1,6diphosphate and/or isomers thereof; b) concentration using an ultra-filtration cell membrane; and c) purification by gel filtration of fructose-l ,6-diphosphate which is not encased by the liposome.

It should be noted that, both as regards the quantity of active ingredient encapsulated and as regards the particles produced, the sequence of the listed phases is generally critical.

The pharmacological activity of the compositions produced according to the invention has been evaluated by incubating human blood at 1 70C, while stirring, and periodically monitoring the stabilities and the erythrocyte levels of ATP. That parameter in fact has been found hitherto to be the most effective for studying the pharmacological activity both of fructose-l ,6-diphosphate and of isomers thereof.

The following non-limiting Examples serve to illustrate the invention.

EXAMPLE 1 5 g of crude or untreated lecithin (for example egg) is dissolved in about 10 ml of chloroform and passed down a silicagel column. A continuous gradient of chloroform-methanol is used as eluent.

The fraction (n-5) eluted by methanol is collected, that fraction containing 3.5 g of phosphatidylcholine. The purity of that fraction is confirmed by thin-layer chromatography (silica gel glass plates), eluting with chloroform-methanol-H20 (65-35-5); Rf=0.24.

500 mg of the substance, which may be preserved in a freezer at --200C under a nitrogen atmosphere, is dissolved in 1 50 ml of diethyl ether in a 500 ml stoppered container. Aclear solution is obtained, 1 50 ml of chloroform is added, and subsequently 50 ml of a solution of fructose-i ,6- diphosphate (at a concentration of 1 mg/ml), at a pH-value of 7.4, is added.

The operations described are carried out under a nitrogen atmosphere in order to avoid the risk of the formation of possible undesirable products from oxidation of the lipids.

The resulting mixture is subjected to treatment in an ultrasound bath for a period of 1 5 minutes (with a pause of 1 minute every 5 minutes), ensuring that the bath temperature does not exceed 1 50C.

This results in a single stable phase which is left to stand for 1 hour.

The organic solvents are then slowly evaporated under vacuum at a temperature of 450C, first giving a gelatinous mass which is subsequently liquified with total elimination of all the residual solvent. The large unilamellar vesicles produced in this way are filtered through a 0.45 um membrane in order to obtain liposomes of uniform size (particle size about 300 A). After the filtration operation about 45 ml of suspension is obtained which is subjected to ultrafiltration in an Amicon filtering unit using a Diaflo YM 30 membrane, under nitrogen pressure (Amicon and Diaflo are registered trade marks). This preliminary concentration operation yields about 60 to 65% of the unbound fructose-1 ,6- diphosphate.The remaining volume (about 20 ml) is charged to a column containing dextran polymers commercially known as 'Sepharose 4 B' (registered trade mark) which has been previously saturated with a standard liposome suspension.

Elution is effected with a buffertri-HCI-NaCI-0.1 M (pH=7.5), collecting the first fractions after the 'empty volume' (previously determined by using dextran blue). The liposomes fractions which are obtained are subjected to final concentration in an Amicon ultrafiltration cell with a Diaflo YM 30 membrane, under nitrogen pressure. A suspension containing 1 .3-1.5 mg of fructose-1,6- diphosphate per ml of the whole, devoid of non-encased active compound which is external to the vesicles, is obtained.

The liposomes containing fructose-i ,6-diphosphate which are produced in this way were subjected to experiment in vitro in comparison with similar doses of non-encased medicament, and with a similar amount of liposomes, containing 0.9% of NaCI, as described hereinafter: EXAMPLE 2 To 2.25 ml of human blood was added 0.25 ml of liposomes, produced as described, containing 1.35 mg/ml of fructose-1 ,6-diphosphate, and the mixture was incubated in a thermostatically controlled bath at 370 C, with continuous agitation. Two other samples of human blood (from the same person) were incubated at the same time, each being of 2.25 ml and respectively containing 0.25 ml of fructose-1 ,6-disphosphate solution (1.35 mg/ml) and 0.25 ml of liposomes containing 0.9% of NaCI.

After incubation, the ATP erythrocyte values were monitored at various times.

The data obtained are set out in Table 1 below: TABLE 1 40 Mins 160 minus Fructose-1,6-diphosphate in 550 j/ml 664 11ml liposomes Freed frustose-l ,6-diphosphate 552y/ml 601 y/ml Liposomes t.q 524 y/ml 532 y!ml N.B.-the base value of ATP is 530 y/ml.

Each base value is the average of several (6) observations. From analysis of the data obtained, it is found that fructose-I ,6-diphosphate carried in the liposomes influences the erythrocyte metabolism in a markedly different manner from fructose-1,6-diphosphate which is not contained in the liposomes, consequently showing the possibility of fusion of the liposomes with the cellular membrane, with consequential liberation in the cell of the encased medicament.

For the purposes of confirming the above-hypotheses, the incubation test with human blood was repeated, using liposomes prepared in accordance with the procedure described, and which contain C14 tagged fructose-1 ,6-diphosphate. The test which was carried out in parallel with a similar amount of free fructose-1 6-diphosphate, which was also tagged, showed that fructose-i 6-diphosphate carried in the liposomes is capable of passing through the erythrocyte membrane. A comparison is made below to show the percentage of fructose-i 6-diphosphate (carried in the liposomes) which passed into the cell in time in relation to free fructose-l ,6-diphosphate: 5 Mins 40Mins 160Mins Fructose-i ,6-diphosphate in 0 5 20 liposomes--o/o in the cell Freed fructose-i 6-diphosphate 0 0 0 % in the cell

Claims (6)

1. CLAIMS 1. A pharmaceutical preparation wherein fructose-l ,6-diphosphate and/or one or more isomers thereof are incorporated into the interior of a liposome.
2. 2. A liposome encapsulating fructose-i ,6-diphosphate and/or one or more isomers thereof.
3. 3. A process for the preparation of liposomes containing fructose-l ,6-diphosphate and/or one or more isomers thereof which comprises the following steps: a) preparation of a phospholipidic compound, which is purified by column chromatography, dissolved in an organic solvent and treated with ultrasound in contact with an aqueous solution of fructose-i 6-diphosphate and/or isomers thereof, b) concentration using an ultrafiltration cell membrane, c) purification by gel filtration of fructose-i 6-diphosphate which is not encased by the liposome, and d) final concentration using an ultrafiltration cell membrane.
4. 4. A pharmaceutical preparation produced with the process according to claim 3.
5. 5. A pharmaceutical preparation substantially as herein described.
6. 6. Each and every novel process, method, compound and composition substantially as herein described.