Title:  Use of complexes among cationic liposomes and polydeoxyribonucleotides and medicaments

United States Patent:  6,767,554

Issued:  July 27, 2004

Inventors:  Porta; Roberto (Como, IT); Ferro; Laura (Milan, IT); Trento; Fabio (Como, IT); Nastruzzi; Claudio (Ferrara, IT); Esposito; Elisabetta (Ferrara, IT); Menegatti; Enea (Ferrara, IT)

Assignee:  Gentum S.p.A. (Como, IT)

Appl. No.:  330215

Filed:  June 11, 1999

Abstract

The invention relates to the use as medicaments, specifically as antiinflammatories, of complexes formed by cationic liposomes and polydeoxyribonucleotides having a molecular weight in the range 7,000-60,000 Da obtainable by depolymerization of nucleic acids, wherein in said complexes the polydeoxyribonucletoides are located on the outer surface of the liposome.

Description of the Invention

The present invention relates to the use as medicaments of complexes formed by cationic liposomes and polydeoxyribonucleotides. More specifically the present invention relates to the use of the above mentioned complexes which possess a remarkable stability in time as medicaments having anti-inflammatory activity.

It is well known that the liposomes can be used as carriers for drug systemic administration. They are administered by intravenous, subcutaneous, intramuscular injection, or by infusion.

As far as the structure of the complexes between liposomes and DNAS is concerned, it is known that oligodeoxyribonucleotides and plasmid DNAs can bind by means of an ionic bond to the external surface of cationic liposomes (C. F. Bennet et Al. Mol. Pharmacol. 41,1023-1033,1992; Xiang Gao et Al. Biochem. Biophys. Res. Comm. 179,280-285, 1991). However no indication is given on the stability in the time of said complexes and on their use as anti-inflammatory drugs. It is also known by the patent application WO 97/04787 that when oligonucleotides have a chain length between 8 and 50 nucleotides, they can be entrapped into liposomes. Also in this reference no information is given on the stability of the complexes in the time.

Complexes with liposomes and polydeoxyribonucleotides having molecular weight of 16000 Da, obtained by depolymerization of nucleic acids, wherein these polymers are contained inside the lipidic vesicle (Gursoy et Alii, Pharmazie 48, (1993)H. 7, 559-560 ), have been described. The same as above said for WO 97/04787 can be repeated.

It is also known that liposome complexes with oligonucleotides and polydeoxyribonucleotides have the property to remarkably increase the pharmacologic activities of the latter substances (Bennet et Al, Gursoy et Al., see above; A. Colige, Biochemistry 1993, 32, 7-11). However tests carried out by the Applicant have shown that these complexes of the prior art cannot be used as therapeutical agents because, when suspended in aqueous media as requested for their administration, they loose very quickly their activity in time. Besides this, in said complexes the cationic components of the liposome, such as for example stearylamine and quaternary ammonium surfactants, can be potentially toxic agents and can cause toxic side effects. The complex degradation is also evident since the physical appearance of the aqueous phase changes in time, turning from opalescent (initial emulsion) to final limpid, with formation of a precipitate.

The polydeoxyribonucleotides, and specifically that know as defibrotide, are well known as medicaments having profibrinolytic activity (R. Pescador et al., Thromb. Res. 30: 1-11, 1983), antithrombotic-thrombolytic (R. Niada et Al., Pharmacol. Res. Commun. 14 (10), 949-957 1982) antihypertensive (F. Trento et Al., XXVII Congr. Naz. Soc. It. Farmacol. Torino 25-29 September 1994, Abstract Book pag. 703), antiischaemic, cytoprotective (G. Rossoni et Al. J. Cardiovasc. Pharmacol. 27, 680-685 1986) and anti-inflammatory activity (R. Scalia, Meth. Find. Exp. Clin. Pharmacol. 18(10) 669-676 1996). The daily doses range from 600 to 1200 mg. All these pharmacologic activities of the substance are essentially referable to their property to locally release therapeutically effective amounts of endogenous prostacyline from the vascular endothelium (ref. R. Niada et alii, above, C. Thiemermann et Alii, Am. J. Cardiol. 56 978-982 1985).

It has been now surprisingly and unexpectedly found by the Applicant that it is possible to prepare complexes from liposomes and polydeoxyribonucleotides having an high activity lasting in time, devoid of any toxic side effect.

This affords to use the aqueous emulsions containing the complexes of the invention for subsequent treatments, for one or more days, and also also for long lasting administrations, such as infusions.

Therefore it is an object of the invention the use as medicaments, specifically as anti-inflammatories, of complexes formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range 7,000-60,000, preferably 10,000-60,000, most preferably 15,000-60,000 Da, obtainable by depolymerization of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome.

Said liposome complexes are characterized in that their solutions, by addition of aliquots of at ceytlpyridinium chloride solution, form a quantity of a precipitate with said quaternary ammonium ion that is different from that obtainable by treating in the same conditions a solution of the liposome complexes of the same polydeoxyribonucleotides and cationic liposomes wherein the polydeoxyribonucleotides are instead located inside the liposome.

In a preferred embodiment of the invention the polydeoxyribonucleotide is defibrotide.

Therefore according to the present invention it is also possible to reduce the daily dose to be administered to the patient, without affecting the therapy effectiveness.

The liposomes are lipidic vesicles, which are formed in aqueous phase, and are generally constituted of phospholipides. Said compounds in the presence of water and an insoluble organic solvent form a spherical shell which wall is a double layer, wherein the molecule polar portion (hydrophilic) is on the outer side of the liposome and the lipidic portion (hydrophobic) is inside the double layer. The vesicle in this case is called monolamellar. There are also multilamellar liposomes, which are composed of more lipidic layers.

The polydeoxyribonucleotides having a molecular weight in the range 15,000-60,000 which are used in the complexes with liposomes according to the present invention are obtainable by extraction and subsequent depolymerization of high molecular weight nucleic acids.

The extraction of high molecular weight nucleic acids can be carried out as disclosed in the U.S. Pat. No. 3,770,720, herein incorporated by reference. It is possible to obtain polydeoxyribonucleotides with molecular weight in the range 15,000-30,000 by carrying out the depolymerization of nucleic acids as described in U.S. Pat. No. 4,985,552 herein incorporated by reference. The Applicant has ascertained that it is possible to obtain also polymers having a molecular weight in the range 30,000-60,000, using the same conditions of the process of U.S. Pat. No. 4,985,552 stopping depolymerization when the value of reversible hyperchromicity, as defined in Methods in Enzymol. vol. III pag. 708-712, is comprised between 20 and 40% (with reference to the absorbance value of reversible hyperchromicity the non denatured sample), or, stopping depolymerization when the value of reversible hyperchromicity is above or equal to 3 for obtaining polydeoxyribcnucleotides having molecular weight above or equal to 7,000. Reversible hyperchromicity is the parameter by which depolymerization progress is followed,

The preferred polydeoxyribonucleotides to form the complex with the cationic liposome are the ones known as defibrotides (D.C.I.) having a molecular weight in the range 15,000-30,000 (Informations Pharmaceutiques O.M.S. n. 4, vol. 1/1987 pag. 272).

The main lipidic components of the liposomes of the invention are phosphatidylcoline or phosphatidylethanolamine, which can be combined in the liposome with other lipids as disclosed in the R.R.C. New volume "Liposomes, a practical approach" IRL Press 1994, herein incorporated by reference. The preferred associated lipids are ergosterol and cholesterol.

One or more antioxidants, selected from the known ones and which are listed in the same reference previously mentioned, can be added to the composition. The preferred antioxidant is alpha-tocopherol.

To the liposomes of the invention are added cationic surfactants, containing one or more mono-, di-substituted amminic groups, or quaternary ammonium groups. Said quaternary ammonium groups contain one or more aliphatic chains with a number of carbon atoms ranging from 8 to 22.

The quaternary ammonium surfactants having aliphatic chains with 18 carbon atoms, are preferred.

The molar ratio between the total amount of the liposome lipid/s and cationic surfactant ranges from 10:0.05 to 10:3, preferably is 10:1. When together with the phosphatidylcoline (or phosphatidylethanolamine) there is a second and different lipid, the internal molar ratios between each of the two lipids and the surfactant (phosphatidylcoline (or phosphatidylethanolamine): second lipid: surfactant) range from 9:1:0.05 to 7:3:3, preferably 8:2:1.

The weight ratio between the liposome amount and that of the active principle (polydeoxyribonucleotides) ranges from 10:2 to 10:0,1, preferably is 10:1.

The preparation of the cationic liposome complexes used in the present invention can be carried out as described by D. C. Litzinger, Biochim. Biophys. Acta 1281 139-149, 1996, or in the above mentioned R. R. C. New's volume. In particular a process usable for preparing the present invention complex comprises the following steps:

a. liposome preparation by means of the solvent reverse phase evaporation method, ref. Szoka P. et Alii. Proc. Natl. Acad. Sci. USA 75 4194 1978): 4 parts of organic phase, which can be polar (ex. linear or branched C₁ -C₄ lower aliphatic alcohols) or apolar (ex. linear or branched C₁ -C₄ dialkylethers, such as for example diethylether, partially chlorinated C₁ -C₂ hydrocarbons, preferably chloroform), wherein are solubilized the lipids, the cationic surfactant and the antioxidant, with one part of water, the thus obtained biphasic system is subjected to sonication at 0^oC. for 5-20 minutes, the organic phase is then evaporated at room temperature at a reduced pressure, thus obtaining an emulsion,

flowing said emulsion, according to the technique described at pages 52-54 of the R. R. C. New volume, through a polycarbonate membrane having a pore diameter ranging from 100 to 600 nm, preferably 400 nm; the step is repeated for at least three times, so to obtain a vesicle average diameter comparable with that of the membrane pores,

lyophilizing the aqueous emulsion, after addition of an aqueous solution of a lyophilizing coadjuvant, for example monosaccharides such as saccharose, sorbitol, mannitol, fructose, or polysaccharides such as dextranes, maltodextrines having different molecular weight, so that the coadjuvant is in excess of at least 7 times with respect to the lipids. Preferably the excess is comprised between 10 and 15 times,

preparation of the final emulsion for pharmaceutical use by adding in a sterile environment, under stirring, a diluted sterile isotonic aqueous solution of polydeoxyribonucleotides to the vessel containing the lyophilized emulsion. An emulsion is formed containing a liposome complex wherein the polydeoxyribonucleotides are linked with an ionic bond to the liposome outer wall. Alternatively, a sterile isotonic solution is added to the vessel containing the lyophilized liposomes and the so obtained emulsion is mixed in a sterile environment with the solution containing the active principle.

The stability of the liposomes of the invention has been evaluated by assaying the pharmacologic activity immediately after the emulsion preparation and then at the 30th day of conditioning under sterile conditions at 25^oC. in the dark.

The emulsion containing the entrapped polydeoxyribonucleotides liposome complex (Gursoy et al, see above) underwent the same test and was used as comparative formulation.

The Applicant has also found that the present invention complexes can be used also as antihypertensive and antithrombotic agents having an high activity in the time, without side toxic effects.

The pharmacologic activity has been determined in the following experimental models.

Anti-inflammatory activity (Miyasaka et al., Eur. J. Pharmacol. 77 229-236 1982).

Arterial hypertension (F. Trento et Al., see above).

Antithrombotic activity (R. Niada et alii, Thromb. Res. 23 233-246. 1981).

In the experiment relating to the anti-inflammatory activity the myeloperoxidase amount present in the obtained polymorphonucleates of the animal pleural exudate, has been assayed. The enzyme amount is directly proportional to the produced inflammation. The results are expressed as % variation of the myeloperoxidase (MPO) amount with respect to that of the controls, determined with the formula: ##EQU1##

In the arterial hypertension model the parameter used to determine the activity was blood-pressure which was monitored up to 30 minutes from the treatment with L-NAME, the inhibitor of the release of endogenous nitric oxide. In the antithrombotic activity model the carotid temperature has ben monitored up to 60 minutes after induction of the local endothelial lesion. The results have been expressed as % variation of the area under the curve (.DELTA.AUC %) obtained with the tested sample with respect to that of the controls, by means of the following ratio: ##EQU2##

The obtained results, reported respectively in Tables I, II and III, show that the complex among liposomes and polydeoxyribonucleotides according to the present invention is stable in time, differently from the comparative formulation.

According to the present invention it is therefore possible to administer to the patient a lower amount of active principle maintaining the therapeutic effect unchanged.

It is also possible to use a same complex emulsion, suitably formulated and with a suitable active principle concentration, for a whole therapy cycle as requested in the above mentioned pathologies.

It is also known that polydeoxyribonucleotides known as defibrotide have an antithrombotic activity (R. Niada, Pharmacol. res. Comm., see above), anti-ischaemic, cytoprotective (C. Thiermemann, see above), anti-inflammatory activity (G. Rossoni, J. Cardiovasc. Pharmacol., see above) and in the atherosclerosis (P. Lobel et Al. Atherosclerosis 80, 69-79 1989). Said actitivites are referable to the local release of endothelial prostacyclin in the blood flood in therapeutically effective amounts.

It has been found by the Applicant that the liposome-polydeoxyribonucleotide complexes described in the present invention can be used for the therapy of pathologies which treatment requires a sustained release of endothelial prostacyclin.

The pharmaceutical formulations containing the cationic liposome-polydeoxyribonucleotides include the usual carriers and excipients. Said formulations can be in the form of sterile and apyrogenic emulsions, or of lyophilisates, stored in sterile containers, to be extemporaneously dissolved in sterile aqueous solvents. In the latter case it is preferred that the liposome lyophilisate is separately stored and that the polydeoxyribonucleotides are already dissolved in the aqueous sterile solvent to be added to the liposomes.

As aqueous sterile solvents, sterile isotonic solutions containing conventional buffers (citrates, phosphates) can be used together with known preservatives.

The administration routes of the emulsion containing the complex of the invention are those parenteral, i.e. by intravenous, intramuscular, subcutaneous injection, and by infusion.

The active principle amount contained in the preparation ranges from 1 to 20 mg/ml of polydeoxyribonucleotide.

The polydeoxyribonucleotide daily doses administered with the liposome complexes range from 10 to 200 mg, preferably from 20 to 120 mg.

Claim 1 of 15 Claims

What is claimed is:

1. A pharmaceutical formulation containing complexes of cationic liposomes constituted of phospholipids and polydeoxyribonucleotides having a molecular weight in the range 15,000-50,000, said polydeoxyribonucleotides obtained by depolymerization of nucleic acids, in the complexes the polydeoxyribonucleotides being located on the outer surface of the liposome wherein the weight ratio between the liposome amount and the polydeoxyribo-nucleotides ranges from 10:2 to 10:0.1.

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