Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1999-06-11
2004-07-27
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C514S04400A
Reexamination Certificate
active
06767554
ABSTRACT:
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 o
Esposito Elisabetta
Ferro Laura
Menegatti Enea
Nastruzzi Claudio
Porta Roberto
Arent & Fox PLLC
Gentum S.p.A.
Kishore Gollamudi S.
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