AU776014B2 - Use of complexes among cationic liposomes and polydeoxyribonucleotides as medicaments - Google Patents

Description

j AUSTRAL IA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant eR-iNOs iNDUSTRIA rAPMACODIOLOCIGA S.P. \ST.1 Invention Title: 7 USE OF COMPLEXES AMONG CATIONIC LIPOSOMES AND POLYDEOXYRIBONUCLEOTIDES AS MEDICAMENTS The following statement is a full description of this invention, including the best method of performing it known to me/us:

T

2 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 antiinflammatory 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 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 9808/1!;: E:;T oligonucleotides have a chain length between 8 and 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 chese 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 o 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, *V 9808/151.EST 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 Pescador et al., Thromb. Res. 1-11, 1983), antithrombotic-thrombolytic Niada et Al., Pharmacol. Res. Commun. 14 949-957 1982) antihypertensive 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 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 CV 9808/151.EST or more days, and also for long lasting administrations, such as infusions.

In a first aspect, the present invention provides complexes formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range 7,000-60,000 Da, preferably 10,000-60,000, most preferably 15,000-60,000 Da, obtained by depolymerisation of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome.

In a second aspect, the present invention provides a pharmaceutical composition comprising a complex according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a 15 method of treating inflammation, thrombosis or hypertension in a patient comprising administering to the patient a therapeutically effective amount of a complex formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range of 7,000-60,000 Da obtained S 20 by depolymerisation of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome.

Sgo In a further aspect, the present invention provides a method of treating a condition which requires a sustained release of the endothelial prostacyclin in a patient comprising administering to the patient a therapeutically effective amount of a complex formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range of 7,000-60,000 Da obtained by depolymerisation 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 a 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 o possible to reduce the daily dose to be administered to the patient, without affecting the therapy effectiveness.

o The liposomes are lipidic vesicles, which are formed in a .o *oo aqueous phase, and are generally constituted of phospholipides. Said compunds 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 o weight nucleic acids.

The extraction of high molecular weight nucleic acids can be carried out as disclosed in the USP 3,770,720, herein incorporated by reference. It is possible to obtain polydeoxyribonucleotides with molecular weight in the range e* 15,000-30,000 by carrying out the depolymerization of nucleic acids as described in USP 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 USP 4,985,552 stopping depolymerization when the value of CV 9808/151.EST 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 polydeoxyribonucleotides 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 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 liposcmes 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 CV 9808/151.EST 8 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 liposcme 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 CV 9808/151.BST 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 Ci-C, lower aliphatic alcohols) or apolar (ex. linear or branched dialkylethers, such as for example diethylether, partially chlorinated C 1 hydrocarbons, preferaly 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°C for 5-20 minutes, the organic phase is then evaporated at rcom temperature at a reduced pressure, thus obtaining an emulsion, b. 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 a repeated for at least three times, so to obtain a vesicle average diameter comparable with that of the membrane pores, c. lyophilizing the aqueous emulsion, after addition of an aqueous solution of a lyophilizing coadjuvant, for example monosaccharides such as saccharose, sorbitol, CV 9808/151.BST I mannitol, fructose, or polysaccharides such as dexcranes, 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, d. 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 concaining 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 liposcmes 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 30t: day of conditioning under sterile conditions at 25CC 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.

CV 9808/151.EST 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 Trento et Al., see above).

Antithrombotic activity 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 0' 2 0 produced inflammation. The results are expressed as 0* variation of the myeloperoxidase (MPO) amount with respect to that of the controls, determined with the formula: MPOTreated MPOcontrol, MPOcontrolo 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 CV 9808/1S1.BST 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 (AAUC obtained with the tested sample with respect to that of the controls, by means of the following ratio: AREATreated Areacontrolo Areacontrolo 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 concentrtion, for a whole therapy cyle as requested in the above mentioned pathologies.

It is also known that polydeoxyribonucleotides known as defibrotide have an antithrombotic activity Niada, Pharmacol. res. Comm., see above), anti-ischaemic, cytoprotective Thiermemann, see above), anti-inflammatory activity (G.

Rossoni, J. Cardiovasc. Pharmacol., see above) and in the CV 9808/151.ST 13 atherosclerosis 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 liposomepolydeoxyribonucleotide 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 0* 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, ad by intravenous, intramuscular, subcutaneous injection, and by CV 9808/151.EST 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.

The following examples have the purpose to clarify the content of the present invention and are not to be considered as a limitation of the scope of the same.

EXAMPLE 1 Preparation of the polydeoxyribonucleotide liposomes.

The preparation of the liposomes used in the present invention is carried out according to the solvent reverse phase evaporation method.

100 mg of soya phosphatidylcoline (Phospholipon® Natterman Phospholipid GmbH), dioctadecyldimethyl ammonium bromide (abbr. DIDAB -Fluka Chemie AG) and 0.1% w/w of alphatocopherol (Fluka Chemie AG) are dissolved in diethylether.

Phosphatidylcoline and cationic surfactant are mixed in a 10:1 molar ratio.

To the organic phase, bidistilled water is added in a ratio of 4 parts of organic phase/1 part of water, thus obtaining an emulsion W/O.

On the emulsion a sonication at 0°C is carried out for CV 9808/151.EST minutes by using a Branson 2200 sonicator batch. The ether is then removed by evaporation at a reduced pressure until an aqueous liposomal system is obtained, which is then made to flow through polycarbonate membranes (Nucleopore) having a pore diameter of 0.4 pm. Said step through the membrane is repeated three more times A mannitol amount equal to 10 times the lipid weight is added and the suspension is lyophilized.

mg of a polydeoxyribonucleotide having a molecular weight of 28,000, obtained by depolymerization according to USP 4,985,552, are dissolved in 5 ml of isotonic physiologic solution. The above obtained lyophilizate is dissolved in 5 ml of bidistilled water. The two aqueous phases are mixed and stirred. In the so obtained emulsion the concentration of phosphatidylcoline is of 10 mg/ml and that of the polydeoxy- S• ribonucleotide is of 5 mg/ml.

EXAMPLE 2 (comparative Preparation of polydeoxyribonucleotide liposomes according to the prior art (Gursoy et alii, Pharmazie 48 (19- 93) H 7 559-560) having the polydeoxyribonucleotide entrapped in the liposome.

The same organic phase of Example 1, with the same com- •ponents mentioned above, is separately dried in a vessel. An aqueous solution of a polydeoxyribonucleotide having molecular CV 9808/151.EST weight 16,000, prepared as the polydeoxyribonucleotide solution of the previous example is added. The liposomal vesicles englobing the active principle are obtained by sonication. The phosphatidylcoline and polydeoxyribonucleotide concentrations are the same as those in the complex of example 1.

EXAMPLE 3 Demonstration of the formation of the polydeoxyribonucleotide-liposome complex of Example 1 by electrophoretic method.

The electrophoresis is carried out in a 3% agarose gel containing 0.5 Ag/ml of ethidium bromide as fluorescence agent. The electrophoretic system is constituted by a small electrophoretic chamber containing a layer of gel of thickness comprised between 1-3 mm, to which a 50 mV electric field is applied.

In the gel are respectively seeded, in 6 separate zones near the negative pole, 20 ~l of the solution of Example 1 (polydeoxyribonucleotide concentration 5 mg/ml), and of solutions containing the polydeoxyribonucleotide .lone at concentrations of 4, 3, 2, 1, 0.5 mg/ml.

The electric field is applied for 40 minutes. The polydeoxyribonucleotide moves from the seeding zone towards the positive pole. At the end of the electrophoretic run the CV 9808/151.EST agarose gel is stained with ethidium bromide. The liposome complex does not show any coloration. In the gel are evidenced the bands corresponding to the seeds of the polydeoxyribonucleotide solutions, which intensity is proportional to the amount seeded.

EXAMPLE 4 Comparison of the stability of the liposome-polydeoxyribonucleotide complex obtained according to Example 1 with that of the complex wherein the polydeoxyribonucleocide is contained inside the liposome (Example 2 comparative), by evaluating the polydeoxyribonucleotide anti-inflammatory activity in rats treated with samples of solutions of said complexes freshly prepared and with samples of said solutions conditioned for 30 days at 25 0 C in closed vessels, in the **dark.

S"Sprague Dawley male rats weighing 250-270 g were used.

3 groups, each group of 18 animals, were formed and to each of the groups were respectively administered intravenously one of the following solutions at the stated doses: 1. Control group: physiologic solution, at 2 ml/Kg.

2. Group treated with the liposome-polydeoxyribonucleotide complex (ref. Ex. 1) physiologic solution containing the complex in amounts equal to a polydeoxyribonucleotide CV 9808/151.EST concentration of 1 mg/ml, at 2 mg/Kg.

3. Group treated with the liposome-polydeoxyribonucleotide complex according to A. Gursoy et alii (see above): physiologic solution containing the complex in amounts equal to a polydeoxyribonucleotide concentration of 1 mg/ml, at a 2 mg/Kg dose.

minutes after the treatment, under a mild ether anaesthesia, pleuritis was caused in the animals by administering by intrapleural route 0.5 ml of a 1% w/v carragenine physiologic solution and of 5 ml of water per os.

After 6 hours the animals were sacrificed. By means of a syringe the pleural exudate was recovered, and the content in polymorphonucleate neutrophil leucocytes (PNK) was determined by asssaying the myeloperoxidase (MPO) enzyme, which is the characteristic enzyme of these cells.

The assay was carried out as described-Schierwagen C. et Al. J. Pharmacol. Methods 23 179 1990.

The exudate samples were stirred and then 0.2 ml were added to 4.8 ml of a 0.5% w/v HTAB (hexadecyltrimethylammonium bromide) buffered solution. The samples were then frozen at 80 0 C so as to cause cell breaking, unfrozen and then subjected to 80 watt sonication for 1 minute. The preparations were then heated to 60 0 C for two hours in order to degrade the myeloperoxidase inhibitors and subsequently centrifuged at 11,800 g CV 9808/151.EST for 5 minutes at Before proceeding to the enzyme spectrophotometric assay (wavelength 650 nm), the samples were diluted with the HTAB solution in order to bring the reading values in the range of the standard curve obtained by using the pure MPO enzyme.

The results are expressed as per cent variation with respect to the MPO amount found in the controls and are reported in the following Table I.

The Table evidences that the anti-inflammatcry activity of the two preparations at time zero is substantially the same and after 30 days the activity of the preparation acccrding to the invention does not significantly differ from the initial value, while the preparation containing the liposomes according to the comparative example shows a 70% activity decrease with respect to the initial value. At the same time it was noticed that said latter preparation was degraded, since the aqueous phase appeared limpid and a precipitate was present that could not be resuspended The animals treated with this preparation showed evident signs of pain and pronounced dyspnoea.

.EXAMPLE Comparison of the stability of the liposome-polydeoxyribonucleotide complex obtained according to Example 1 with that of the complex wherein the polydeoxyribonucleozide is CV 9808/151.BST contained inside the liposome (Example 2 comparative), by evaluating the polydeoxyribonucleotide anti-hypertensive activity in rats, with hypertension induced by inhibition of endogenous nitric oxide (NO) release, administered with the samples of solutions containing the above complexes freshly prepared and with samples of the same solutions conditioned at 0 C for 30 days in closed vessels in the dark.

Sprague Dawley male rats weighing 250 20 g, not fasted are anaesthetized with ethylurethane. Two catheters were respectively inserted in the left carotid artery, for recording the mean arterial blood pressure (MABP), and in the right jugular vein, for administering the tested compositions.

The trachea was cannulated and the animal body temperature was maintained at 37 0 C. The MABP was continuously recorded throughout the experiment. Heparin (500 U.I./Kg was administered to avoid blood coagulation in the recording system.

Ater 30 minutes the rats were randomized in homogeneous groups.

The treatment with the compositions or the placebo was carried out by bolus, immediately followed by perfusicn. After one hour from the start of the perfusion all the animals receive an intravenous bolus of L-NAME (10 mg/Kg). The perfusion lasted 30 minutes after the injection of L-NAME.

CV 9808/151.EST The pressure modifications induced by the compositions are expressed as area under the curve (AUC) in the 30 minutes interval following L-NAME treatment.

In the experimental model under consideration the animals were divided in four groups (6 animals for group), each of them was intravenously treated with a bolus of 1 ml/Kg, immediately followed by a a perfusion of 2 ml/Kg/h, as explained hereinafter: 1. Control group (CTR): physiologic solution 1 ml/Kg bolus 2 ml/Kg/hour in perfusion.

2. Group treated with a bolus of a polydeoxyribonucleotide mol. wt. 28,000 in a physiologic solution at concentration of 10 mg/ml, at the dose of 10 mg/Kg (bolus) 20 mg/Kg/h in perfusion.

3. Group treated with a bolus of the liposomepolydeoxyribonucleotide complex of the invention in a physiologic solution at a polydeoxyribonucleotide Sconcentration of 5 mg/ml, at the dose of 5 mg/Kg (bolus) 10 mg/Kg/h in perfusion.

4. Group treated with a bolus of the liposomepolydeoxyribonucleotide complex according the comparative example 2 in a physiologic solution at a polydeoxyribo nucleotide concentration of 5 mg/ml, at the dose of mg/Kg (bolus) 10 mg/Kg/h in perfusion.

CV 9808/151.EST The pharmacologic activity determined by using samples of freshly prepared solutions containing the liposome complexes above referred, and samples of the same solutions stored in closed vessels at 25 0 C in the dark, is reported in Table II.

The results show that whereas at time zero the two preparations have nearly the same antihypertensive activity, after 30 days the activity of the preparation according to the prior art is lowered of about 70% in the confront with the corresponding starting value.

The animals treated with said preparation showed evident signs of pain with marked dyspnoea.

EXAMPLE 6 Comparison of the stability of the lipcsome-polydeoxyribonucleotide complex obtained according to Example 1 with that of the complex wherein the polydeoxyribonucleo ide is •contained inside the liposome (Example 2 comparative) by evaluating the polydeoxyribonucleotide anti-thrombotic activity in rats, treated with samples of solutions containing the above complexes freshly prepared and with samplesof the same solutions conditioned at 25 0 C for 30 days in closed vessels in the dark.

Sprague Dawley male rats weighing 200-230 g, fasted for 16 hours, were anaesthetized with urethane (1.25 g/Kg ***eeA After the right carotid artery and the left jugular vein CV 9808/151.EST 23 of the animals were isolated. A bipolar electrode (Lesion Producing Device 3500 Ugo Basile Comerio, Varese) was positioned on the right artery and, at a 0.5 cm distance, a thermosensitive probe connected to a polygraph. A catheter was inserted in the vein for administering the preparations.

After 15 minutes of stabilization, the carotid temperature was continuously recorded from 5 minutes before to minutes after the endothelial lesion induction by means of the electrode. This allowed to indirectly determine the formation of endoluminal thrombi formation by correlation between the decreasing temperature of the vessel and the blood flow reduction. The endothelial lesion was caused by a series of 5 electric stimuli. The stimuli at intervals of one minute one from the other, were such that the impedance measured on the lesioned artery was of 10 mA. The impedance was measured with a tester and was regulated for each animal during the first 30 seconds of stimulation, and required applied voltage of about 30 Volt.

The carotid temperature was determined immediately before the electric stimulation (basal value) and az constant intervals of time 10, 15, 30, 45 and 60 minutes) afte the o stimulation.

•The groups were each formed by 10-12 rats.

All the treatments were carried out as intravenous bolus All the treatments were carried out as intravenous bolus CV 9808/151.SST 24 which was administered 5 minutes before the beginning of the electric stimulation.

The groups were the following: 1. Control group SHAM, wherein the animals were operated and monitored as above described, but they were not subjected to the electric stimulation.

2. Control group, treated with physiologic solution ml/Kg 3. Group treated with the liposome-polydeoxyribonucleotide complex (ref. Ex. 1) physiologic solution containing the complex in an amount equal to a polydeoxyribonucleotide concentration of 5 mg/ml; administered dose: 7.5 mg/Kg.

4. Group treated with the liposome-polydeoxyribonucleotide complex according to A. Gursoy et alii (see above): physiologic solution containing the complex in amounts equal to a polydeoxyribonucleotide concentration of 5 mg/ml; administered dose: 7.5 mg/Kg.

The activity was determined at the time of the preparation of the solutions with the two complexes and after days of conditioning said solutions at 25 0 C in the dark.

The results are reported in Table III.

From the Table it is noticed that while the antithrombotic activity of the two preparations at time zero is sub- ******stantially comparable, after 30 days the activity of the stantially comparable, after 30 days the activity of the CV 9808/151.EST preparation according to the prior art is lowered of about in the confront with the corresponding starting value.

EXAMPLE 7 Pharmaceutical formulation, containing the liposomes used in the present invention, for single-dose administration.

3 ml sterile vial containing the lyophilized liposome: phospholipon 90 mg 100 DIDAB mg alpha-tocopherol mg 0.1 saccarose g 1 Before use add 1 ml of water for injection. Add then in a sterile way the following sterile solution, premanufactured in 1 ml disposable sterile syringe is added to the above: polydeoxyribonucleotide (ref. Ex. 1) mg bihydrate trisodic citrate mg S- water for injections and preservatives, enough to ml 1 EXAMPLE 8 Extemporaneous pharmaceutical formulation to be used for a whole therapeutic cycle.

30 ml sterile bottle containing the lyophilized liposome: phospholipon 90 g 1 DIDAB g 00 DIDAB mg 100 CV 9808/151.EST 26 alpha-tocopherol mg 1 saccarose g Before use add 10 ml of water for injection in a sterile way in the bottle. Add to the so prepared emulsion the following sterile solution contained in a 15 ml bottle or in a 10 ml disposable premanufactured syringe: polydeoxyribonucleotide (ref. Ex. 1) mg 100 dihydrate trisodic citrate mg water for injections and preservatives enough to ml The preparation supply a number of 20 mg/die doses for 5 days lasting therapy.

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CV 9808/151.EST TABLE I Stability at 30 days of the complex according to Example 1 (group 2 in the Table) in comparison with that of the lioosome-polydeoxyribonucleotide complex according to Gursoy et Al. (group 3 in the Table), evaluated by means of the polydeoxyribonucleotide anti-inflammatory activity (reduction of the myeloperoxydase activity in the pleural exudate of rats with pleuritis induced by carragenine).

group No. MPO vs. CTR) MPO vs. CTR) (zero time) (30 days) 2 -86 3 -85 -18 CV 9808/151.EST 1. 1 28 TABLE II Stability at 30 days of the complex according to Example 1 (group 3 in the Table) in comparison with that of the liposome-polydeoxyribonucleotide complex according to Gursoy et Al. (group 4 in the Table), evaluated by means of the polydeoxyribonucleotide anti-hypertensive activity.

group No. AUC vs. CTR) AUC vs. CTR) (zero time) (30 days) 2 0* 3 -32 -29 4 -30 -9 The polydeoxyribonucleotide administered dose (10 mg/Kg bolus 20 mg/Kg/h) is too low to give rise to a meaningful anti-hypertensive activity with respect to the controls.

eoeo oo*ee CV 9808/151.EST 29 TABLE III Stability at 30 days of the complex according to Example 1 (group 3) in comparison with that of the liposomepolydeoxyribonucleotide complex according to Gursoy et Al., (group 4) evaluated through the anti-thrombotic activity of the polydeoxyribonucleotides.

A reference herein to a prior art document is not an admission that the document forms part of the common general knowledge in the art in Australia.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Claims (38)

1. Complexes formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range 7,000-60,000 Da obtained by depolymerization of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome, for use as a medicament.

2. Complexes according to claim 1 wherein the polydeoxyribonucleotide has a molecular weight in the range 10,000-60,000 Da.

3. Complexes according to claim 1 or 2 for preparing medicaments having an anti-inflammatory activity.

4. Complexes according to claim 1 or 2 for preparing medicaments having an anti-thrombotic activity.

5. Complexes according to claim 1 or 2 for preparing p •medicaments having an anti-hypertensive activity.

6. Complexes according to claim 1 or 2 for preparing medicaments for the therapy of pathologies the treatment of which requires a sustained release of the endothelial prostacyclin.

7. Complexes according to any one of claims 1 to 6 ;o *wherein the polydeoxyribonucleotide is defibrotide. S: 8. Complexes according to claim 7 wherein the polydeoxyribonucleotide has a molecular weight in the range 15,000-30,000.

9. Complexes according to any one of claims 1 to 8 wherein one or more antioxidants are added. 31 Complexes according to claim 9 wherein the antioxidant is alphatocopherol.

11. Complexes according to any one of claims 1 to wherein cationic surfactants containing one or more mono-, di-substituted amminic groups, or quaternary ammonium groups, are present, said quaternary ammonium groups containing one or more aliphatic chains with a number of carbon atoms ranging from 8 to 22.

12. Complexes according to claim 11 wherein the cationic surfactants are quaternary ammonium surfactants having aliphatic chains with 18 carbon atoms.

13. Complexes according to any one of claims 1 to 12 wherein the molar ratio between the total amount of the liposome lipid/s and cationic surfactant ranges from 10:0.05 to 10:3.

14. Complexes according to claim 13 wherein the molar ratio between the total amount of the liposome lipid/s i* and cationic surfactant is 10:1. Complexes according to claim 13 or 14 wherein, together with the phosphatidylcoline (or a phosphatidylethanolamine) there is a second and different lipid and the molar ratio phosphatidylcoline (or phosphatidylethanolamine): second lipid: a surfactant ranges from 9:1:0.05 to 7:3:3.

16. Complexes according to claim 15 wherein the molar ratio phosphatidylcoline (or 32 phosphatidylethanolamine): second lipid: surfactant is 8:2:1.

17. Complexes according to any one of claims 1 to 16 wherein the weight ratio between the liposome amount and the active principle ranges from 10:2 to 10:0.1.

18. Complexes according to claim 17 wherein the weight ratio between the liposome amount and the active principle is 10:1.

19. Complexes according to any one of claims 1 to 18 obtainable by a process comprising the following steps: *O*O 33 a. liposome preparation by mixing 4 parts of polar cr apolar organic phase, wherein are solubilized the lipids, the cationic surfactant and the antioxidant, with 1 part of water, then subjecting the obtained biphasic system to sonication at 0 0 C for 5-20 minutes and evaporating the organic phase at room temperature at a reduced pressure, thus forming an emulsion; b. flowing said emulsions through a polycarbonate membrane having a pore diameter ranging from 100 to 600 nm, said step repeated for at least three times, c. lyophilizing the emulsion after addition of an aqueous o solution of a lyophilizing coadjuvant, so that the amount of said coadjuvant is in excess of at least 7 times with respect to that of the lipids, d. preparing the emulsion for pharmaceutical use by adding i :in a sterile environment under stirring a diluted sterile isotonic aqueous solution of polydeoxyribonucleotides to the vessel containing the lyophilizate, or alternatively by adding a sterile isotonic solution to the vessel containing the lyophilized liposome and the thus obtained emulsion mixed in a sterile environment with the solution containing the active principle. 34 Complexes according to claim 19 wherein the pore diameter of the polycarbonate membrane is 400 nm.

21. Complexes according to claim 19 or 20 wherein the amount of said coadjuvant is in excess of from 10 to times with respect to that of the lipids.

22. Complexes according to any one of claims 1 to 18 contained in pharmaceutical formulations for parenteral administration.

23. Use of the complexes according to any one of claims 1 to 22 for the preparation of medicament having antiinflammatory activity.

24. Use of the complexes according to any one of claims 1 Sto 22 for the preparation of medicament having antithrombotic activity.

25. Use of the complexes according to any one of claims 1 o to 22 for the preparation of medicament having antihypertensive activity.

26. Use of the complexes according to any one of claims 1 Sto 22 for the preparation of medicament for treating pathologies which require a sustained release of the

27. Complexes formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range 7,000-60,000 Da obtained by depolymerisation of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome.

28. Complexes according to claim 27 wherein the polydeoxyribonucleotide has a molecular weight in the range 15,000-60,000 Da.

29. Complexes according to claim 27 or 28 wherein the polydeoxyribonucleotide is defibrotide. Complexes according to any one of claims 27 to 29 wherein cationic surfactants containing one or more mono-, di-substituted amminic groups, or quaternary ammonium groups, are present, said quaternary ammonium groups containing one or more aliphatic chains with a number .of carbon atoms ranging from 8 to 22.

31. Complexes according to claim 30 wherein the molar ratio between the total amount of the liposome lipid/s *o and cationic surfactant ranges from 10:0.05 to 10:3.

32. Complexes according to claim 31 wherein, together with the phosphatidylcoline (or phosphatidylethanolamine) there is a second and different lipid and the molar ratio of phosphatidylcoline (or *o :phosphatidylethanolamine): second lipid: surfactant ranges from 9:1:0.05 to 7:3:3. o

33. Complexes according to any one of claims 27 to 32 wherein the weight ratio between liposome amount and the active principle ranges from 10:2 to 10:0.1.

34. A pharmaceutical composition comprising a complex as claimed in any one of claims 27 to 33 and a pharmaceutically acceptable carrier. 36 A composition according to claim 34 further comprising one or more antioxidants.

36. A pharmaceutical composition according to claim wherein the antioxidant is alphatocopherol.

37. A method of treating inflammation in a patient comprising administering to the patient a therapeutically effective amount of a complex formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range of 7,000-60,000 Da obtained by depolymerisation of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome.

38. A method of treating thrombosis in a patient comprising administering to the patient a therapeutically effective amount of a complex formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range of 7,000-60,000 Da obtained by depolymerisation of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome.

39. A method of treating hypertension in a patient comprising administering to the patient a therapeutically effective amount of a complex formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range of 7,000-60,000 Da obtained by depolymerisation of nucleic acids, 37 wherein the polydeoxyribonucleotides are located on the outer surface of the liposome. A method of treating a condition which requires a sustained release of the endothelial prostacyclin in a patient comprising administering to the patient a therapeutically effective amount of a complex formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range of 7,000-60,000 Da obtained by depolymerisation of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome.

41. A method according to any one of claims 37 to :o wherein the complex is a complex as claimed in any one of claims 28 to 33.

42. A method according to any one of claims 37 to 41, wherein the patient is a human.

43. Complexes according to any one of claims 27 to 33 when used in the treatment of inflammation, thrombosis, or hypertension.

44. Complexes according to any one of claims 27 to 33 when used in the treatment of a condition which requires a sustained release of the endothelial prostacyclin. Complexes formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range 7,000-60,000 Da obtained by depolymerization of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome, 38 substantially as herein described with reference to Example 1.

46. A pharmaceutical composition comprising a complex formed by cationic liposomes and by polydeoxyribonucleotides having a molecular weight in the range 7,000-60,000 Da obtained by depolymerisation of nucleic acids, wherein the polydeoxyribonucleotides are located on the outer surface of the liposome, substantially as herein described with reference to Example 7 or Example 8. Dated this 8th day of June 2004 GENTIUM S.p.A. By its Patent Attorneys GRIFFITH HACK 0* B go o go*