CN102028702A - Application of compound of cation liposome and polydeoxyribonucleotide as medicament - Google Patents
Application of compound of cation liposome and polydeoxyribonucleotide as medicament Download PDFInfo
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- CN102028702A CN102028702A CN2010105427202A CN201010542720A CN102028702A CN 102028702 A CN102028702 A CN 102028702A CN 2010105427202 A CN2010105427202 A CN 2010105427202A CN 201010542720 A CN201010542720 A CN 201010542720A CN 102028702 A CN102028702 A CN 102028702A
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Abstract
The invention relates to application of a compound as a medicament, particularly an inflammatory agent, wherein the compound is prepared from cation liposome and polydeoxyribonucleotide with the molecular weight of 7000-60000; the polydeoxyribonucleotide is obtained by depolymerizing nucleic acid; and the polydeoxyribonucleotide in the compound is located on the outer surface of the liposome.
Description
The application be that June 11, application number in 1999 are 99109491.3 the applying date, denomination of invention divides an application for the application for a patent for invention of " complex of cationic-liposome and polydeoxyribonucleotide is as the purposes of medicament ".
Technical field
The present invention relates to the complex that forms by cationic-liposome and polydeoxyribonucleotide purposes as medicament.More particularly, the present invention relates to the above-mentioned purposes that significant temporal stability can be used as the complex with anti-inflammatory activity medicament that has.
Background technology
As everyone knows, liposome can be as the pharmaceutical carrier of whole body administration.They are by intravenous, subcutaneous, intramuscular injection or by the infusion administration.
As for the structure that relates to complex between liposome and the DNAs, known oligodeoxynucleotide of people and plasmid DNA s can combine (C.F.Bennet etc., Mol.Pharmacol.41,1023-1033,1992 with the outer surface of cationic liposome by ionic bond; Xiang Gao etc., Biochem.Biophys.Res.Comm.179,280-285,1991).But, do not provide the hint of any relevant temporal stability when the antiinflammatory medicine with them at this complex.People are also known from patent application WO 97/04787 has the chain long time of 8 to 50 nucleotide when oligonucleotide, and they can be loaded in the liposome by bag.In these documents, do not provide relevant this class complex any information of stability in time yet.
Described now that to contain liposome and molecular weight be that the complex of the polydeoxyribonucleotide of 16000Da can obtain by separating polynucleic acid, wherein can be included in (Gursoy etc. in the lipid capsule to these polymer, Pharmazie 48, (1993) H.7,559-560).WO 97/04787 has repeated above-mentioned identical description.
The also known liposome complex that contains oligonucleotide and polydeoxyribonucleotide of people have the remarkable characteristic that increases the pharmacologically active of back one material (Bennet etc., Gursoy etc. are on seeing; A.Colige, Biochemistry 1993,32,7-11).However, the test that the applicant carries out has shown that these complex of the prior art can not be used as medicament, because in they being suspended in the required water-bearing media of administration the time, they will lose its activity rapidly.In addition, in this complex the cation constituent of liposome, be potential toxic agents and can cause toxic and side effects as stearmide and quaternary ammonium type surfactant.Because contain the variation of the physical appearance of water, promptly final limpid from there being opalescence (colostrum) to change into, be accompanied by sedimentary formation, the degraded of complex also is tangible.
As everyone knows; the polydeoxyribonucleotide class; the polydeoxyribonucleotide that particularly is called Defibrotide can be used as (profibrinolytic) active (R.Pescador etc. that have plasmin; Thromb.Res.30:1-11; 1983); antithrombotic-thromboclastic (thrombolytic) (R.Niada etc.; Pharmacol.Res.Commun.14 (10); 949-957; 1982); antihypertensive (F.Trento etc.; XXVII Congr.Naz.Soc.It.Farmacol.Torino 25-29; in JIUYUE, 1994; the 703rd page of Abstract Book); ischemia; cytoprotective (G.Rossoni etc.; J.Cardiovasc.Pharmacol.27; 680-685 1986) and anti-inflammatory activity (R.Scalia; Meth.Find.Exp.Clin.Pharmacol.18 (10) 669-679,1996) medicament.Every day dosage range from 600 to 1200mg.The pharmacologically active of all these materials all mainly refers to from the local characteristic that discharges the interior living prostacyclin of treatment effective dose of their blood vessel endotheliums (referring to R.Niada etc., above-mentioned, C.Thiemermann etc., Am.J.Cardiol.56 987-9821985).
Summary of the invention
Now the applicant unexpectedly and be surprisingly found out that from liposome and polydeoxyribonucleotide and can prepare the complex that has lasting high activity of time and remove toxic and side effects.
This aqueous emulsions that will make that use contains complex of the present invention is used for treating continuously being achieved and also making to continue medication for a long time in one day or many days and is achieved such as infusion.
So the objective of the invention is having 7 by cationic liposome with by what separate that polynucleic acid obtains, 000-60,000 dalton, preferred 10,000-60,000 dalton, most preferably be 15,000-60, the complex that the polydeoxyribonucleotide of 000 Dalton molecular weight forms is as medicament, especially antiinflammatory, wherein polydeoxyribonucleotide is positioned at the outer surface of liposome.
This liposome complex is characterised in that by adding the cetylpyridinium chloride of equal portions
Solution, their solution and the quantitative precipitate that this quaternary ammonium ion forms be different under the same conditions by handle the liposome complex formed by identical polydeoxyribonucleotide and cationic liposome, that wherein polydeoxyribonucleotide is positioned at liposome interior is resulting.
In the preferred embodiment of the invention, polydeoxyribonucleotide is Defibrotide.
So according to the present invention, under the condition that does not influence the treatment effectiveness, dosage every day that reduces to patient's administration also is possible.
Liposome is a lipid vesicle, and it is containing aqueous phase formation, and generally is made up of phospholipid.In the presence of water and a kind of insoluble organic solvent, described chemical compound can form a kind of spherical shell, and its shell wall is double-deck, wherein the polarity of molecule part (hydrophilic) in the outside of liposome and lipid part (hydrophobic) in bilayer.In this case, this vesicle is referred to as the monolayer shape.Multilamellar shape liposome is also arranged, and they are made up of multiple lipid layer.
Being used for the molecular weight that the present invention contains the complex of liposome is 15,000-60,000 daltonian polydeoxyribonucleotide can by extract and then the high-molecular weight nucleic acid of depolymerization obtain.
As USP 3,770, carry out the extraction of high molecular nucleic acid described in 720, this paper is cited as list of references with the document.As USP 4,985,552 described can to obtain molecular weight by the depolymerization of carrying out nucleic acid be 15,000-30, and 000 daltonian polydeoxyribonucleotide, this paper is with USP 4,985, and 552 are cited as list of references.The applicant verified in as Methods in Enzymol. the 3rd volume 708-712 page or leaf defined reversibility dark color using USP 4 between 20 and 40% when (with regard to the absorption value of the reversible hyperchromicity of unmodified sample), 985, the same terms of 552 technologies stops depolymerization also can obtain molecular weight 30,000-60,000 polymer, perhaps when the value of reversible hyperchromicity was equal to or greater than 3, the termination depolymerization can obtain molecular weight and be equal to or greater than 7,000 polydeoxyribonucleotide.Reversible hyperchromicity is to follow a parameter of separating collecting process.
The polydeoxyribonucleotide that preferred and cationic liposome forms complex is that everybody known molecular weight is 15,000-30,000 defibrotide acids (D.C.I.) (Information Pharmaceutiques O.M.S.n.4, the/1987,272nd page of the 1st volume).
The main lipid components of liposome of the present invention is lecithin or PHOSPHATIDYL ETHANOLAMINE, they can with R.R.C.New Volume " liposome; a kind of actual approach " IRL publishing house, disclosed other lipid is combined in the liposome in 1994, this paper is cited as list of references with the document.Preferred relevant lipid is ergosterol and cholesterol.
Can add in the said composition and be selected from known and be listed in one or more antioxidants in the above-mentioned same document.Preferred anti-oxidants is an alpha-tocopherol.
Add cationic surface active agent in liposome of the present invention, this surfactant contains one or more single or disubstituted amino or quaternary ammonium group.This quaternary ammonium group comprises one or more aliphatic chain of carbon number between 8 to 22.
The quaternary ammonium type surfactant that preferably has 18 carbon atom aliphatic chains.
The scope of mol ratio between 10: 0.05 to 10: 3 between the lipid of liposome/lipid total amount and the cationic surface active agent is preferably 10: 1.When with lecithin (or PHOSPHATIDYL ETHANOLAMINE) when using, also have second kind of different lipid, inside mol ratio between two kinds of lipids and the surfactant (lecithin (or PHOSPHATIDYL ETHANOLAMINE): second kind of lipid: surfactant) in the scope between 9: 1: 0.05 to 7: 3: 3, preferred 8: 2: 1.
Liposome is used for and the weight ratio of active component (polydeoxyribonucleotide) in 10: 2 to 10: 0.1 scope, preferred 10: 1.
According to D.C.Litzinger, Biochim.Biophys.Acta1281,139-149,1996 or in the preparation that can carry out the used cation lipid nanocrystal composition of the present invention described in above-mentioned R.R.C.New ' the s Volume.Specifically, the technology that can be used for preparing complex of the present invention comprises the following steps:
A. prepare liposome (list of references is Szoka P.et Alii.Proc.Natl.Acad.Sci.USA 75 4,194 1978) by the solvent reverse phase evaporation: 4 parts of organic faciess and 1 part of water, wherein organic facies can be polar (for example, the C of straight or branched
1-C
4Lower aliphatic alcohols) or nonpolar (as the C of straight or branched
1-C
4Dialkyl ether, as diethyl ether, the chlorating C of part
1-C
2Hydrocarbon, preferred chloroform), wherein be dissolved with lipid, cationic surfactant and antioxidant, the binary system that obtains was like this carried out supersound process 5-20 minute at 0 ℃, reduction vaporization organic facies at room temperature obtains a kind of emulsion thus then,
B. the described technology of 52-54 page or leaf according to R.R.C.New volume makes this emulsion flow through bore dia between 100 to 600nm, the polycarbonate membrane of preferred 400nm; This step triplicate at least, thereby obtain the folliculus of the average diameter suitable with fenestra,
C. behind the aqueous solution that adds the lyophilizing adjuvant, containing the aqueous emulsion lyophilizing, lyophilizing adjuvant wherein such as monosaccharide such as sucrose, Sorbitol, mannitol, fructose or polysaccharide such as glucosan, have the maltodextrins of different molecular weight, thereby make this adjuvant Duo 7 times at least than lipid.Between preferred many 10 to 15 times,
D. the preparation of final medicinal Emulsion: under gnotobasis, stir on the limit, and the limit adds the sterile isotonic aqueous solution of the polydeoxyribonucleotide that diluted is equipped with in the container of lyophilizing emulsion.Formed the emulsion that contains liposome complex, wherein polydeoxyribonucleotide is connected with the outer wall of liposome with an ionic bond.Another kind method is equipped with a kind of sterile isotonic solution adding in the container of freeze-dried lipidosome, and mixes with the solution that contains active component at following emulsion that obtains like this of gnotobasis.
The stability of liposome of the present invention is by at Emulsion preparation back rapid test drug activity, places in 25 ℃ of dark and under the aseptic condition then to measure drug activity on the 30th day and assess.
To contain the Emulsion that is enclosed with polydeoxyribonucleotide liposome complex (Gursoy etc. are on seeing) carries out identical test and is used as control formulation.
The applicant has been found that also complex of the present invention can not have the hypotensive agent and the antithrombotic agent of toxic and side effects simultaneously as having greater activity yet.
In following test model after measured pharmacologically active.
-anti-inflammatory activity (Miyasaka etc., Eur.J.Pharmacol.77 229-236 1982).
-Arterial Hypertention (F.Trento etc. are on seeing).
-antithrombotic activity (R.Niada etc., Thromb.Res.23 233-246,1981).
In the test that relates to anti-inflammatory activity, after testing be present in the amount of myeloperoxidase of the polymorphonuclear leukocyte (polymorphonucleates) of the animal pleural exudate that obtains.The amount of this enzyme is directly proportional with the inflammation of generation.The result uses the percentage variable quantity of the myeloperoxidase (MPO) with respect to matched group to represent, calculates with following formula:
In the model of Arterial Hypertention, being used to measure active parameter is blood pressure, and monitoring uses the L-NAME that discharges the endogenous nitric oxide inhibitor to treat back 30 minutes blood pressure.In the anti-thrombosis activity model, after bringing out local endothelial injury, monitoring carotid artery temperature to 60 minute.Result's percentage rate of change (Δ AUC%) expression of comparing the area under a curve that obtains at test specimen with matched group obtains with following ratio:
The result who is obtained who reports in table I, II and III represents that the complex of liposome of the present invention and polydeoxyribonucleotide is stable always, is different from control formulation respectively.
So,, can take the therapeutical effect that the low active component of measuring remains unchanged to the patient according to the present invention.
Also can use identical complex Emulsion, suitably prepare and have the concentration of suitable active component, thereby be used for the required whole treatment cycle of above-mentioned pathologic condition.
People also known are that the polydeoxyribonucleotide class of Defibrotide has anti-thrombosis activity (R.Niada.Pharmacol.res.Comm.; on seeing), ischemia, cytoprotective (C.Thiermemann; on seeing), anti-inflammatory activity (G.Rossoni; J.Cardiovasc.Pharmacol.; on seeing) and treat atherosclerotic effect (P.Lobel etc.; Atherosclerosis 80, and 69-79 1989).Described activity is relevant with the endothelium prostacyclin of local release treatment effective dose in the blood.
The applicant has been found that liposome of the present invention-polydeoxyribonucleotide complex can be used in the pathology that treatment need continue to discharge the endothelium prostacyclin.
The medicament that contains cationic-liposome-polydeoxyribonucleotide comprises carrier commonly used and excipient.Described preparation can be aseptic and apyrogenic Emulsion form, or is dissolved in the lyophilized products form in the sterile chamber of being kept in the sterilized water solvent temporarily.At latter event, preferably the lyophilized powder of liposome is separated to deposit and polydeoxyribonucleotide to be dissolved in moisture aseptic solvent and add liposome again.
As moisture aseptic solvent, the sterile isotonic solution that contains conventional buffer (citrate, phosphate) can be used with known antiseptic.
The route of administration that contains the Emulsion of complex of the present invention is a parenterai administration, promptly passes through intravenous, intramuscular, subcutaneous injection and passes through the infusion administration.
The amount that is contained in the active component in the preparation is 1 to 20mg/ml polydeoxyribonucleotide.
Dosage every day with the polydeoxyribonucleotide of liposome complex administration is 10 to 200mg, preferred 20 to 120mg.
The specific embodiment
The following example has the clear purpose of illustrating content of the present invention, can not think to be used for to limit scope of the present invention.
Embodiment 1
The preparation of polydeoxyribonucleotide liposome.
Be used for the preparation of liposome of the present invention according to the solvent reverse phase evaporation.
In diethyl ether, dissolve in the soybean lecithin (Phospholipon of 100mg
90-NattermanPhospholipid GmbH), the alpha-tocopherol (Fluka Chemie AG) of two (octadecyl) dimethyl amine bromide (dioctadecyldimethyl ammonium bromide) (being abbreviated as DIDAB-Fluka Chemie AG) and 0.1%w/w.Mixed in molar ratio lecithin and cationic surfactant with 10: 1.
Ratio with 4 parts of organic facies/1 part water in organic facies adds double distilled water, obtains the Emulsion of a kind of w/o like this.
At 0 ℃, this Emulsion was carried out sonication 10 minutes with Branson 2200 sonicators in batches.Reduction vaporization is removed ether till obtaining aqueous liposome system then, makes it flow through the polycarbonate membrane that bore dia is 0.4 μ m (Nucleopore) then.Described step at least more than the triplicate.
Adding equals the mannitol of 10 times of lipid weight also the suspension lyophilizing.
Oozing the molecular weight that dissolves in 50mg in the physiological solution in the grade of 5ml is a kind of polydeoxyribonucleotide of 28,000, and it is by according to USP 4,985, and 552 depolymerization obtain.The above-mentioned lyophilized products that obtains is dissolved in the double distilled water of 5ml.Mixing is also stirred biphase.In the Emulsion that obtains like this, the concentration of lecithin is 10mg/ml and the concentration of polydeoxyribonucleotide is 5mg/ml.
Embodiment 2 (contrast)
According to prior art (Gursoy etc., Pharmazie 48 (19-93) H 7559-560), preparation is wrapped in polydeoxyribonucleotide liposome in the liposome to polydeoxyribonucleotide.
Drying has the identical organic facies of the embodiment 1 of above-mentioned identical component respectively in container.The used molecular weight of polydeoxyribonucleotide solution that adds the preparation previous embodiment is the aqueous solution of 16,000 polydeoxyribonucleotide.Obtain being surrounded by the lipid somatocyst of active component by supersound process.The complex of the concentration of lecithin and polydeoxyribonucleotide and embodiment 1 identical.
Embodiment 3
Confirm the formation of polydeoxyribonucleotide-liposome complex of embodiment 1 with electrophoresis method.
Electrophoresis is to carry out in the ethidium bromide that contains 0.5 μ g/ml is made 3% agarose gel of fluorescent agent.Electrophoresis system is that the little Electrophoresis Lab of the gel layer of 1-3mm is formed by containing thickness, applies the electric field of 50mV.
In gel, add the solution (concentration of polydeoxyribonucleotide is 5mg/ml) of the embodiment 1 of 20 μ l respectively near 6 different districts of negative electrode and respectively contain 4,3,2,1, the solution of the polydeoxyribonucleotide of 0.5mg/ml.
Applied electric field 40 minutes.Polydeoxyribonucleotide moves from adding district's anode.When electrophoresis motion finishes, dye to agarose gel with ethidium bromide.This liposome complex does not show any color.Band with the adding spot correlation of polydeoxyribonucleotide solution in gel is that significantly its intensity is directly proportional with the amount of adding.
Embodiment 4
By with the sample solution of freshly prepared described complex and be used in anti-inflammatory activity that this solution example treatment rat of preserving in 25 ℃ of hermetic container dark 30 days assesses polydeoxyribonucleotide contrast liposome-polydeoxyribonucleotide complex of being obtained according to embodiment 1 and complex wherein polydeoxyribonucleotide be included in the stability of liposome interior (embodiment 2, contrast).
Use the Sprague Dawley male rat of body weight as 250-270g.
Be divided into three groups, every group of 18 animals, give in every group of rat every respectively with given dose a kind of intravenous administration that carries out in the following solution:
1. matched group: physiological solution, 2ml/Kg.
2. with the treatment group of liposome-polydeoxyribonucleotide complex (referring to embodiment 1): contain consumption and be equivalent to the physiological solution that polydeoxyribonucleotide concentration is the complex of 1mg/ml, 2mg/Kg.
3. use treatment group: contain consumption and be equivalent to the physiological solution that polydeoxyribonucleotide concentration is the complex of 1mg/ml, 2mg/Kg according to the liposome-polydeoxyribonucleotide complex of (on seeing) such as A.Gursoy.
Treat after 30 minutes, under appropriate etherization, by oral and in pleura approach the water of the carrageenin physiological solution of the 1%w/v of 0.5ml and 5ml is carried out the pleuritis that administration causes animal.
Put to death animal after 6 hours.Collect pleural exudate with a syringe, determine the content of polymorph neutrophile leucocytes (PMN) by detecting myeloperoxidase (MPO), wherein myeloperoxidase is the feature enzyme of these cells.
Measure described in J.Pharmacol.Methods 23 179 1990 according to Schierwagen C. etc.
Stir the exudate sample, then 0.2ml is joined in HTAB (cetab) buffer solution of the 0.5%w/v of 4.8ml.Thereby then 80 ℃ of following samples freezing make cell cracked, thaw and then through 80 watts of sonications 1 minute.Thereby then this preparation liquid be heated to 60 ℃ of two hours degraded myeloperoxidase inhibitor and then 4 ℃ with 11, centrifugal 5 minutes of 800g.
In the spectrophotometry of carrying out enzyme (wavelength 650nm) before, thus make reading value in the scope of the standard curve that uses pure MPO enzyme to obtain with this sample of HTAB solution dilution.
The result uses the percentage ratio with respect to the amount of the MPO that measures in the matched group to change to represent and lists in down in the tabulation I.
This table demonstrates zero the time that the anti-inflammatory activity of two kinds of preparations is substantially the same, and after 30 days, the activity of preparation of the present invention can not significantly be different from initial value, and the preparation that contains the liposome of comparative examples has 70% activity to descend with respect to initial value.Notice that simultaneously a kind of preparation in back has been degraded because water occurred becoming limpid and exist can not resuspending precipitate.
Animal with the said preparation treatment has demonstrated tangible pain symptom and significant dyspnea.
Embodiment 5
Place the hypertensive rat administration of sample to bringing out of 30 days same solution with the release that suppresses endogenous nitrogen oxide (NO) the sample solution that contains above-mentioned freshly prepared complex with in 25 ℃ of hermetic container dark, to suffering from the antihypertensive active of hypertensive rat, come relatively the liposome-polydeoxyribonucleotide complex that obtains according to embodiment 1 and polydeoxyribonucleotide wherein to be included in the stability of the intermediary complex of liposome (contrast of embodiment 2) by the assessment polydeoxyribonucleotide.
With urethanes anesthesia body weight is the Sprague Dawley male mouse that does not have fasting of 250 ± 20g.Two conduits are inserted in respectively write down in the left neck artery in mean arterial blood pressure (MABP) and the right jugular vein, be used for a subject composition administration.In trachea, insert sleeve pipe and animal heat is remained on 37 ℃.Continuous record MABP in whole test.Use heparin (500U.I./Kg intravenous injection) to prevent that blood from solidifying in recording system.
After 30 minutes, in same group the rat randomization.
Then inculcate the treatment of carrying out compositions or placebo by using pill at once.From inculcating beginning after 1 hour, the vein medicine group (10mg/Kg) of all animals received L-NAME.Behind the injection L-NAME, will inculcate lasting 30 minutes.
Being improved by the inductive pressure of compositions is to treat area under a curve in the back 30 minutes interval (AUC) with L-NAME to represent.
In test model, under considering, animal is divided into four groups (every group of 6 animals), give every infusion intravenous therapy of immediately using 2ml/Kg/h with the medicine group of 1ml/Kg in them, following explanation is arranged:
1. matched group (CTR): the physiological solution+2ml/Kg/h infusion of 1ml/Kg medicine group.
2. be that the molecular weight of 10mg/ml is the treatment group of the physiological solution of 28,000 polydeoxyribonucleotide medicine group with concentration: the infusion of the dosage of 10mg/Kg (medicine group)+20mg/Kg/h.
3. with the deoxynucleoside acid concentration treatment group of physiological solution of the liposome of the present invention-polydeoxyribonucleotide complex medicine group of 5mg/ml: the infusion of the dosage of 5mg/Kg (medicine group)+10mg/Kg/h.
4. with the deoxynucleoside acid concentration treatment group of physiological solution of medicine group of the liposome-Deoxydization nucleotide complex of the comparative examples 2 of 5mg/ml: the infusion of the dosage of 5mg/Kg (medicine group)+10mg/Kg/h.
Reported the pharmacologically active that uses the sample determination that contains the freshly prepared sample solution of above-mentioned liposome complex and the same solution that the hermetic container in 25 ℃ of dark is deposited in the table II.
Two kinds of preparations almost had identical antihypertensive active when the result was presented at 0, and after 30 days, the activity of prior art formulations has descended about 70% with corresponding initial value ratio.
Animal with described preparation for treating shows with obvious dyspneic pain symptom.
Embodiment 6
The sample of same solution of placing 30 days the sample solution that contains above-mentioned freshly prepared complex with in 25 ℃ of hermetic container dark is to the rat administration, to the antithrombotic activity of rat, come relatively the liposome-polydeoxyribonucleotide complex that obtains according to embodiment 1 and polydeoxyribonucleotide wherein to be included in the stability of the complex (comparative examples 2) of liposome interior by the assessment polydeoxyribonucleotide.
With urethanes (1.25g/Kg peritoneal injection) anesthesia body weight is 16 hours the Sprague Dawley male mouse of fasting of 200-230g.
Behind the right carotid of separating animal's and the left jugular vein, on right tremulous pulse, place bipolar electrode (Lesion Producing Device 3500Ugo Basile-Comerio, Varese), and at the 0.5cm place connect a polygraph with a thermo-responsive probe.Inserting a conduit in vein comes the said preparation administration.
After stablizing 15 minutes, bring out preceding 5 minutes temperature to back 60 minutes tremulous pulsies by electrode continuous record endothelial injury.Relation between this slows down by reduction blood vessel temperature and blood flow is with indirect determination endothelium thrombosis.Cause endothelial injury by a series of 5 electricity irritation.Once make that to one minute the stimulation of being spaced apart between another time the resistance of being measured is 10mA on the tremulous pulse of damage.Measure resistance and between the 30th second stimulation period, every animal is all regulated with tester, and the voltage that need apply approximately is 30 volts.
(base value) measures the tremulous pulse temperature and Fixed Time Interval (5,10,15,30,45 and 60 minutes) mensuration tremulous pulse temperature after stimulation at once before electricity irritation.
Form by 10-12 rat for every group.
The vein medicine is rolled into a ball administration in preceding 5 minutes in the beginning electricity irritation and carry out whole treatments.
These groups are as follows:
1. matched group SHAM, wherein to animal surgery and as above-mentioned monitoring, but they do not pass through electricity irritation.
2. matched group is treated (1.5ml/Kg intravenous injection) with physiological solution.
3. with the treatment group of liposome-polydeoxyribonucleotide complex (referring to embodiment 1): contain consumption and be equivalent to the physiological solution that polydeoxyribonucleotide concentration is the complex of 5mg/ml; Dosage: 7.5mg/Kg.
4. with the treatment group of liposome-polydeoxyribonucleotide complex (seeing above-mentioned) of A.Gursoy etc.: contain consumption and be equivalent to the physiological solution that polydeoxyribonucleotide concentration is this complex of 5mg/ml; Dosage: 7.5mg/Kg.
When preparing solution and described solution, after depositing 30 days, 25 ℃ of dark places measure activity with two kinds of complex.
The table III has been reported the result.
From this table, can notice that the anti-thrombosis activity when two kinds of preparations when zero is suitable basically, after 30 days, the activity of prior art preparation is compared with corresponding initial value and has been reduced about 70%.
Embodiment 7
The pharmaceutical preparation that contains the used liposome of the present invention of single dose administration.The 3ml sterile vials that contains freeze-dried lipidosome:
—phospholipon?90 mg?100
—DIDAB mg?10
-alpha-tocopherol mg 0.1
-sucrose g 1
Add 1ml water for injection before the use.Add following sterile solution with sterile manner then, add above-mentioned with the accessible asepsis injector of 1ml before producing:
-polydeoxyribonucleotide (referring to embodiment 1) mg 10
-trisodium citrate two water platform thing mg 2.5
-water for injection and antiseptic, capacity is to ml 1
Embodiment 8
Be used for the interim pharmaceutical preparation of whole treatment circulation.
-contain the aseptic bottle of the 30ml of freeze-dried lipidosome:
—phospholipon?90 g1
—DIDAB mg?100
-alpha-tocopherol mg 1
-sucrose g10
Before the use, in bottle, add 10ml water for injection with sterile manner.The following sterile solution that in the Emulsion of such preparation, contains in the syringe before adding 15ml bottle or the accessible production of 10ml:
-polydeoxyribonucleotide (referring to embodiment 1) mg 100
-citrate trisodium dihydrate mg 25
-water for injection and antiseptic capacity are to ml 10
Said preparation provides a large amount of 20mg/ that are used for 5 days continued treatments dead dosage.
The table I
The complex (in the table the 2nd group) of comparing embodiment 1 in the anti-inflammatory activity process (reduction suffers from myeloperoxidase activity in the pleuritic mice pleural exudate that brings out with carrageenin) of this polydeoxyribonucleotide of assessment with liposome-polydeoxyribonucleotide complex of Gursoy etc. (in the table the 3rd group) was the 30th day stability.
The table II
The complex (in the table the 3rd group) of comparing embodiment 1 in the antihypertensive active process of this polydeoxyribonucleotide of assessment with liposome-polydeoxyribonucleotide complex of Gursoy etc. (in the table the 4th group) was the 30th day stability.
*The dosage of this polydeoxyribonucleotide (cross low and can not produce and compare significant antihypertensive active with matched group by 10mg/Kg medicine group+20mg/Kg/h).
The table III
In the anti-thrombosis activity process of this polydeoxyribonucleotide of assessment, compare the complex (the 3rd group) of embodiment 1 the 30th day stability with liposome-polydeoxyribonucleotide complex (the 4th group) of Gursoy etc.
Claims (12)
1. medicament, it contains by cationic liposome and has 15,000-60, the complex that the polydeoxyribonucleotide of 000 Dalton molecular weight constitutes, described polydeoxyribonucleotide is by obtaining the nucleic acid depolymerization, polydeoxyribonucleotide is positioned at the outer surface of liposome in complex, and the weight ratio between wherein said liposome and the polydeoxyribonucleotide was from 10: 2 to 10: 0.1.
2. the medicament of claim 1, it has anti-inflammatory activity.
3. the medicament of claim 1, it has anti-thrombosis activity.
4. the medicament of claim 1, it has antihypertensive active.
5. the medicament of claim 1 is used for the treatment that needs continue the pathology therapy of release endothelium prostacyclin.
6. according to the medicament of claim 1, wherein polydeoxyribonucleotide is Defibrotide.
7. according to the medicament of claim 6, wherein the molecular weight of polydeoxyribonucleotide is in 15,000 to 30,000 scope.
8. according to the medicament of claim 1, wherein add one or more antioxidants.
9. according to the medicament of claim 1, wherein exist contain one or more lists-, two-amino of replacing or the cationic surfactant of quaternary ammonium group, it is 8 to 22 aliphatic chain that described quaternary ammonium group contains one or more carbon numbers.
10. according to the medicament of claim 1, wherein the total amount of the lipid/lipid of liposome and the mol ratio between the cationic surfactant are 10: 0.05 to 10: 3.
11. medicament according to claim 10, wherein the phospholipid in the liposome comprises lecithin or PHOSPHATIDYL ETHANOLAMINE and second kind of different lipid, and lecithin or phosphatidyl ethamine: second kind of lipid: the molar ratio range of cationic surfactant was from 9: 1: 0.05 to 7: 3: 3.
12. cationic liposome and have 15,000-60, the complex that the polydeoxyribonucleotide of 000 Dalton molecular weight constitutes is used for the treatment of patient's inflammation, thrombosis or hypertension or is used to provide the lasting purposes that discharges the medicine of endothelium prostacyclin in preparation, described polydeoxyribonucleotide is by obtaining the nucleic acid depolymerization, polydeoxyribonucleotide is positioned at the outer surface of liposome in complex, and the weight ratio between wherein said liposome and the polydeoxyribonucleotide was from 10: 2 to 10: 0.1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105427202A CN102028702A (en) | 1999-06-11 | 1999-06-11 | Application of compound of cation liposome and polydeoxyribonucleotide as medicament |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010105427202A CN102028702A (en) | 1999-06-11 | 1999-06-11 | Application of compound of cation liposome and polydeoxyribonucleotide as medicament |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99109491 Division CN1277024A (en) | 1999-06-11 | 1999-06-11 | Application of cationic liposome and polydeoxyribonucleosides composite using as medicament |
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| CN102028702A true CN102028702A (en) | 2011-04-27 |
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| CN2010105427202A Pending CN102028702A (en) | 1999-06-11 | 1999-06-11 | Application of compound of cation liposome and polydeoxyribonucleotide as medicament |
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Application publication date: 20110427 |