Background technology
Anthracycline antibiotics is a kind of antimitotic and has Cytotoxic medicine.Anthracycline antibiotics can successfully suppress Several Kinds of Malignancy, comprises acute leukemia, lymphoma, soft tissue and osteosarcoma, children malignant tumors and becomes human solid tumor.Its mechanism of action is the penetrable cell that enters of medicine, is combined with karyomit(e).Thereby the planar rings in medicines structure is inserted between base pair and is combined with DNA and forms mixture, severe jamming DNA is synthetic, DNA dependent rna is synthetic and protein synthesis.But the drug level that produces tumor locus in the required Dx concentration ratio clinical treatment of anti-proliferative effect by this mechanism wants high.In addition anthracycline antibiotics is also relevant with redoxomorphism, can participate in reaction and obtain cytotoxic compound, as hydroxy and the hydrogen peroxide etc. of superoxide, activity.The site of action of anthracycline antibiotics may be at cytolemma, anthracycline antibiotics is not also reached common understanding in scientific circles for the pharmacodynamic action of various diseases at present, mechanism of action for every kind of disease does not all have general character, needs further scientific research to be explained.Anthracycline antibiotics is widely used at present, but often needs to be become salt, for example Dx at Point of View of Clinical, common medicinal form is doxorubicin hydrochloride, needs to become hydrochloride, because of the solubleness of Dx own very low, do not transform the form of salify, cannot apply.And become after hydrochloride, the pharmacodynamics function of Dx declines to a great extent, and therefore this brings very large difficulty to clinical application.
Another shortcoming of anthracycline antibiotics is due to strong cytotoxicity itself, therefore the toxicity of itself is very large, after using through being everlasting, within about 10 days, there is obvious bone marrow depression, use after one week, can show very significantly gastrointestinal side effect and cardiac toxic, therefore must after accurate calculation, could apply when medication, and the transformation period of this medicine is very short, brings limitation equally to application.
In the time that anthracene nucleus medicament is applied in to eye, dosage and medicine efficacy relation are very responsive, anthracycline antibiotics Grafting Star-shape segmented copolymer of the present invention, can make the nanometer formulation of the new texture that contains anthracycline antibiotics of preparation dissolve in water, well control the amount of drug delivery, and then reach treatment disease related with intraocular neovascularization or illness.
The end product of preparing at this patent far exceedes other drug result for the treatment of in the time for the treatment of eye disease.Preparation method of the present invention, can make the new texture polymkeric substance that contains anthracycline of preparation dissolve in water, prolong half-life, this preparation method has solved application limitation in the lump, and makes anthracycline antibiotics in age-related macular degeneration disease, bring into play extraordinary action effect.
Summary of the invention
Of the present invention theing contents are as follows:
The star multi-block polymer that the invention discloses the anthracycline antibiotics grafting shown in following formula I, its structure is as follows: StructureY is:
Wherein the molecular weight polyethylene glycol in polymkeric substance is 100-200000, the integer between W=1-500, the preferably integer between W=1-300, n=1-300, the preferably integer of 1-200.
It is characterized in that:
1) sebacic acid is refluxed in diacetyl oxide, form ethanoyl-sebacic acid (also can be purchased);
2) compound L 2 that contains polynary amino reacts with Boc-NH-PEG-COOH, that is:react with L2, obtain the star compd A 1 with the polyamino ending of Boc protection;
3) compd A 1 obtains with the sebacic acid reaction that the unit carboxylic acid L1 reaction that contains polyvalent alcohol obtains star compd A 2 ethanoyl that end up with polyvalent alcohol the star polymer B that ethanoyl ends up;
4) polymer B is reacted and is obtained final product with the bad class microbiotic of anthracene C;
Wherein anthracycline antibiotics C is selected from Dx, Zorubicin, pidorubicin, pirarubicin, daunorubicin, daunoblastin, idarubicin, aclarubicin, zhengdingmeisu, aclacinomycin or carminomycin.
Wherein anthracycline antibiotics and connector 1,2, and Boc-NH-PEG-COOH is purchased.The required solvent of described chemical step of synthetic final product is selected from: one or more in benzene, toluene, pyridine, tetrahydrofuran (THF), trifluoroacetic acid, chloroform, tetracol phenixin, methylene dichloride, methyl alcohol, ethanol, methylene dichloride, dimethyl sulfoxide (DMSO), DMF.
The compound of new system can be prepared into the nanometer formulation that is suitable for topical, microball preparation.Purposes is the medicine of preparation treatment age-related macular degeneration.Also be used for the treatment of Accretive Type diabetic retinopathy, proliferative vitreoretinopathy, retinopathy of prematurity, pathologic myopia, supposes ocular histoplasmosis's syndrome, branch retinal vein occlusion, central retinal vein occlusion, branch retinal obstruction of artery, central retinal artery occlusion, the retinal ischemia of new vessel and Tumor-assaciated.
Preparation method of the present invention is specific as follows:
1) sebacic acid is refluxed in diacetyl oxide, form ethanoyl-sebacic acid (also can be purchased);
2) compound L 2 that contains polynary amino reacts with Boc-NH-PEG-COOH, that is:react with L2, obtain the star compd A 1 with the polyamino ending of Boc protection;
3) compd A 1 obtains the star compd A 2 with polyvalent alcohol ending with the unit carboxylic acid L1 reaction that contains polyvalent alcohol under trifluoroacetic acid catalysis, ethanoyl-sebacic acid is mixed to reaction at 100-200 ℃, reaction times 20min to 2h with compd A 2; After question response mixture is cooling, washing, the dry star polymer B that obtains;
4) by anthracycline antibiotics C and polymer B as for 0.5-24 hour in solvent, in subzero 30 ℃ of 0-after ultrasonic reaction 2-20 minute, then homogenizer high-speed stirring 1-20 minute, rotation volatilization obtains crude product, and rear centrifugal collection and treatment obtains the nanometer formulation of finished product formula I.
The star polymer that what the present invention obtained contain anthracycline structure, is easy to dissolve in water, and its transformation period extend much than Dx, such new compound effect in the time for the treatment of eye disease is splendid.
The nanometer formulation that the end product of preparing at this patent is made treatment eye disease relatively in, the medication effect of this patent new compound is very good, surmounts the action effect by the nanometer formulation of the anthracycline antibiotics of other preparation ways completely.
Embodiment
Specific embodiment is described in further detail the present invention below, but the present invention not only limits to following examples.
Preparation Example is as follows:
Embodiment 1
The mixture of 1 sebacic acid 80g in 800ml diacetyl oxide refluxes, to form ethanoyl-sebacic acid;
2 compound Ls that contain polynary amino 2:200mg puts into flask with Boc-NH-PEG-COOH2g and reacts, that is: react with L2, puts into dicyclohexylcarbodiimide 160mg and tetrahydrofuran (THF) 8mg simultaneously, mixes and adds 15ml chloroform, at room temperature stirs and spends the night; Then wash with ether, and dry under vacuum, obtain the star compd A 1 with the polyamino ending of Boc protection;
3 compd As 1 are the same unit carboxylic acid L1 that contains polyvalent alcohol under trifluoroacetic acid catalysisafter 30mg reaction, obtain the star compd A 2 with polyvalent alcohol ending through cellulose acetate membrane filtration, ethanoyl-sebacic acid is mixed with compd A 2, at 175 ℃, decompression contains intermingle with reaction 1 hour; Treat that polymkeric substance is cooled to room temperature chloroform and dissolves, and with petroleum ether the dry star polymer B that obtains;
The 800mg polymkeric substance of 120mg Dx and step 3 is put into the dichloromethane solution 48 hours of 8ml dimethyl sulfoxide (DMSO) and 12ml by 4; Ultrasonic 3 minutes; Then insert in baking oven 1 hour; In subzero 10-20 degree, homogenizer ultra-high speed stirs 3 minutes, then puts into 1% polyvinyl alcohol solution 600 and turns and stir 2 hours; Freeze-drying after centrifugal collection, obtains the nanoparticle of end product U1.
Embodiment 2
The mixture of 1 sebacic acid 100g in 900ml diacetyl oxide refluxes, to form ethanoyl-sebacic acid;
2 compound Ls that contain polynary amino 2:50mg puts into flask with Boc-NH-PEG-COOH3g and reacts, that is: react with L2, puts into dicyclohexylcarbodiimide 160mg and tetrahydrofuran (THF) 6mg simultaneously, mixes and adds 18ml methylene dichloride, at room temperature stirs and spends the night; Then wash with ether, and dry under vacuum, obtain the star compd A 1 with the polyamino ending of Boc protection;
3 compd As 1 are the same unit carboxylic acid L1 that contains polyvalent alcohol under trifluoroacetic acid catalysisafter 45mg reaction, obtain the star compd A 2 with polyvalent alcohol ending through cellulose acetate membrane filtration, ethanoyl-sebacic acid is mixed with compd A 2, at 180 ℃, decompression contains intermingle with reaction 1 hour; Treat that polymkeric substance is cooled to room temperature chloroform and dissolves, and with petroleum ether the dry star polymer B that obtains;
Pirarubicin and this polymkeric substance are put into the solution being mixed by 5ml methyl alcohol and 5ml methylene dichloride by 4; Then insert in baking oven 4 hours; In subzero 10-20 degree, ultrasonic 20 minutes; Product is put into 3% cholic acid solution 400 and is turned and stir 2 hours; Freeze-drying after centrifugal collection, obtains the nanoparticle of end product U2.
Embodiment 3
The mixture of 1 sebacic acid 2g in diacetyl oxide 20ml refluxes, to form ethanoyl-sebacic acid;
2 compound Ls that contain polynary amino 2:300mg puts into flask with Boc-NH-PEG-COOH2.5g and reacts, that is: react with L2, puts into dicyclohexylcarbodiimide 120mg and tetrahydrofuran (THF) 5mg simultaneously, mixes and adds 25ml chloroform, at room temperature stirs and spends the night; Then wash with ether, and dry under vacuum, obtain the star compd A 1 with the polyamino ending of Boc protection;
3 compd As 1 are the same unit carboxylic acid L1 that contains polyvalent alcohol under trifluoroacetic acid catalysisafter 30mg reaction, obtain the star compd A 2 with polyvalent alcohol ending through cellulose acetate membrane filtration, ethanoyl-sebacic acid is mixed with compd A 2, at 175 ℃, decompression contains intermingle with reaction 1 hour; Treat that polymkeric substance is cooled to room temperature chloroform and dissolves, and with petroleum ether the dry star polymer B that obtains;
The polymkeric substance of aclarubicin and step 3 is put into the solution of mountain 10ml methylene dichloride and the mixing of 6ml dimethyl sulfoxide (DMSO) by 4; Then insert in baking oven 8 hours; In subzero 20-30 degree, ultrasonic 20 minutes; Product is put into 5% cholic acid solution 400 and is turned and stir 2 hours; Freeze-drying after centrifugal collection, obtains the nanometer formulation of end product.
Embodiment 4
The mixture of 1 sebacic acid 35g in diacetyl oxide 500ml refluxes, to form ethanoyl-sebacic acid;
2 compound Ls that contain polynary amino 2:50mg puts into flask with Boc-NH-PEG-COOH3g and reacts, that is: react with L2, puts into dicyclohexylcarbodiimide 160mg and tetrahydrofuran (THF) 6mg simultaneously, mixes and adds 18ml methylene dichloride, at room temperature stirs and spends the night; Then wash with ether, and dry under vacuum, obtain the star compd A 1 with the polyamino ending of Boc protection;
3 compd As 1 are the same unit carboxylic acid L1 that contains polyvalent alcohol under trifluoroacetic acid catalysisafter 45mg reaction, obtain the star compd A 2 with polyvalent alcohol ending through cellulose acetate membrane filtration, ethanoyl-sebacic acid is mixed with compd A 2, at 180 ℃, decompression contains intermingle with reaction 1 hour; Treat that polymkeric substance is cooled to room temperature chloroform and dissolves, and with petroleum ether the dry star polymer B that obtains;
The polymkeric substance of daunorubicin and step 3 is put into the solution of mountain 10ml methylene dichloride and 6mlDMF mixing by 4; Then insert in baking oven 24 hours; Subzero 10 in 0-, ultrasonic 10 minutes; Product is put into 3% cholic acid solution 400 and is turned and stir 2 hours; Freeze-drying after centrifugal collection, obtains the nanometer formulation of end product.
Effect experiment is as follows:
Sample prepared by embodiment 1-4, the polymkeric substance that anthracycline-poly-sebacic acid-connector-polyoxyethylene glycol forms after connecting (are that structure isthe nanometer formulation of making (is anthracycline and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymerthere is the polymkeric substance of the non-star of containing anthracycline antibiotics structure of combination reaction gainedthe nanoparticle of preparation), star polymer B in each embodiment:(there is not combination reaction in the nanoparticle medicine group of the anthracycline antibiotics of parcel directly, anthracycline antibiotics does not have structural changes), each anthracycline antibiotics ordinary preparation (powder injection) carries out respectively the drug action test of stability test, drug release in vitro test and age-related macular degeneration.
Stability test:
By 1 group of embodiment, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing Dx structure of combination reaction gained occurs for Dx and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that Dx-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting, (there is not combination reaction in the star polymer B directly nanoparticle medicine group of the Dx of parcel, Dx does not have structural changes), and Dx ordinary preparation component have another name called get equivalent medicine (in Dx, every group containing 50mg Dx) measure respectively absorbance.Then put into 20 degree incubator 3 months for three groups, take out subsequently and measure 480 nanometer absorbances, before and after the absorbance of 1 group of the visible embodiment of result and the Dx of ordinary preparation group without changing, and the nanoparticle group absorbancy of parcel declines 20%, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing Dx structure of combination reaction gained occurs for Dx and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that Dx-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting declines 15%.
Drug release in vitro test:
By 1 group of embodiment, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing Dx structure of combination reaction gained occurs for Dx and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that Dx-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting, (there is not combination reaction in the star polymer B directly nanoparticle medicine group of the Dx of parcel, Dx does not have structural changes), and Dx ordinary preparation component have another name called get equivalent medicine (in Dx, every group containing 10mg Dx), then by each group of medicine as in test tube, after soaking with PBS damping fluid, 37 degrees Celsius of lower joltings in shaking table, after timing sampling, under ultraviolet spectrophotometer, measure the content of medicine, and the medicament contg per-cent that after record, calculating discharges, do releasing curve diagram, X-coordinate be the time (my god), ordinate zou is the per-cent discharging.See Fig. 2, the medicine that visible embodiment discharges is more even, more permanent, makes drug half-life longer.
Solubility test in water:
By embodiment 1-4 group, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing anthracycline antibiotics structure of combination reaction gained occurs for anthracycline antibiotics and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that anthracycline antibiotics-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting, (there is not combination reaction in the star polymer B directly nanoparticle medicine group of the anthracycline antibiotics of parcel, anthracycline antibiotics does not have structural changes), and anthracycline antibiotics ordinary preparation component have another name called get equivalent medicine (in anthracycline antibiotics, every group containing 100mg anthracycline antibiotics), put into respectively test tube, with the dissolving of 10mlPBS damping fluid jolting, situation is dissolved in static rear observation.The dissolved state that records 3 minutes and 20 minutes, result is as follows.
The comparison of table 1 solubleness
The restraining effect of medicine to tela chorioidea's hyperplasia:
Get 340 of male rats, be divided at random 17 groups, it is control group, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing Dx structure of combination reaction gained occurs for Dx and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that Dx-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting, synthetic sebacic acid-ethylene glycol star polymer the B of embodiment 1 directly wraps up nanoparticle (not reacting with Dx) prepared by Dx, synthetic sebacic acid-ethylene glycol star polymer the B of nanometer formulation embodiment 3 that the synthetic sebacic acid-ethylene glycol star polymer B of embodiment 2 directly wraps up the polymkeric substance that nanoparticle (not reacting with pirarubicin) pirarubicin prepared by pirarubicin-poly-sebacic acid-connector-polyoxyethylene glycol is made after being connected (polymkeric substance of the non-star of containing pirarubicin structure of pirarubicin and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer) generation combination reaction gained) directly wraps up nanoparticle (not reacting with aclarubicin) prepared by aclarubicin, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing aclarubicin structure of combination reaction gained occurs for aclarubicin and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that aclarubicin-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting, synthetic sebacic acid-ethylene glycol star polymer the B of embodiment 4 directly wraps up nanoparticle (not reacting with daunorubicin) prepared by daunorubicin, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing daunorubicin structure of combination reaction gained occurs for daunorubicin and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that daunorubicin-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting, embodiment 1-4 group, Dx general formulation group, aclarubicin ordinary preparation, pirarubicin ordinary preparation, daunorubicin ordinary preparation.Each treated animal number is 20.Every rat is chosen a glance at random for experimental eye, and another eye is contrast eye.The equal intraocular injection 10 μ g anthracycline antibiotics medicines of each group or the medicine carrying prolonged action preparation containing 10 μ g medicines except control group, control group is given isopyknic PBS solution.
Use laser radiation rat eye, after light is solidifying, has Bubble formation or break up Bruch film with hyporrhea (sometimes with light sound) mark, be designated as available point.After laser photocoagulation, inject each group of medicine to rat right eye eyeball.14d after light is solidifying, tissues observed hyperplasia area also carries out histological examination.Result is as follows:
Table 2 retina and hyperplasia Area comparison (unit: mm of tela chorioidea2)
With the comparison of control group group*p < 0.05,*p < 0.01, with the comparison of ordinary preparation group#p < 0.05,##p < 0.01
The result of upper table shows, control group retina and choroid generation hyperplasia area are larger, each treatment group respectively different limits dwindled hyperplasia area.Experiment to rat part tissue of eye hyperplasia area shows, each embodiment group can reduce pathology retina and choroidal hamartoplasia area simultaneously, but effect difference to some extent, and the embodiment 1-4 group effect that combination reaction wherein occurs is better.
The result of histological examination is that under light microscopic, control group light coagulates the visible outer retina in spot place and choroid structure disturbance, retinal pigment epithelium, choroid cambium hyperplasia, free companion's inflammatory cell infiltration.Medicine group compared with control group, the rarely found and less companion's limitation of cambium serous detachment of retina; Embodiment group compare the simple parcel of superpolymer and there is not the nanoparticle medicine group of combination reaction and the effect of the non-star-like compound coupling of poly-acid anhydrides-polyoxyethylene glycol anthracycline antibiotics more remarkable, the compound occurring after combination reaction can significantly reduce tela chorioidea's hyperplasia.Histological examination shows, embodiment group is more thorough to the treatment of age related maculopathy than other nanoparticle medicine groups of any one group, and retina and choroid to pathology play a role simultaneously.
Separately get the 5-6 female mice in age in week, mean body weight 25g, mouse is divided into 17 groups at random, it is control group, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing Dx structure of combination reaction gained occurs for Dx and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that Dx-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting, synthetic sebacic acid-ethylene glycol star polymer the B of embodiment 1 directly wraps up nanoparticle (not reacting with Dx) prepared by Dx, synthetic sebacic acid-ethylene glycol star polymer the B of nanometer formulation embodiment 3 that the synthetic sebacic acid-ethylene glycol star polymer B of embodiment 2 directly wraps up the polymkeric substance that nanoparticle (not reacting with pirarubicin) pirarubicin prepared by pirarubicin-poly-sebacic acid-connector-polyoxyethylene glycol is made after being connected (polymkeric substance of the non-star of containing pirarubicin structure of pirarubicin and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer) generation combination reaction gained) directly wraps up nanoparticle (not reacting with aclarubicin) prepared by aclarubicin, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing aclarubicin structure of combination reaction gained occurs for aclarubicin and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that aclarubicin-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting, synthetic sebacic acid-ethylene glycol star polymer the B of embodiment 4 directly wraps up nanoparticle (not reacting with daunorubicin) prepared by daunorubicin, the nanometer formulation of the polymkeric substance (polymkeric substance of the non-star of containing daunorubicin structure of combination reaction gained occurs for daunorubicin and poly-sebacic acid-connector-polyethylene glycol polymer (non-star polymer)) that daunorubicin-poly-sebacic acid-connector-polyoxyethylene glycol is made after connecting, embodiment 1-4 group, Dx general formulation group, aclarubicin ordinary preparation, pirarubicin ordinary preparation, daunorubicin ordinary preparation.Except control group, in the equal ball of each group, inject 3 μ g anthracycline antibiotics medicines or the preparation containing 3 μ g anthracycline antibiotics medicines, control group is given isopyknic PBS solution.In every group of mouse, get at random 12 eyes and carry out induced with laser CNV disposal, after laser photocoagulation, inject above-mentioned each group of medicine (inject 3ug medicine or the preparation containing 3ug medicine except control group in the equal ball of each group, control group is given isopyknic PBS solution) to eyeball of mouse more subsequently.After 7 days, mouse is carried out to fundus fluorescein angiography (being called for short FFA), the formation of CNV has or not fluorescence leakage as basis for estimation according to FFA.Result is as follows:
The incidence of 7d CNV after table 3 light is solidifying
The capable FFA of 7d after laser photocoagulation, in every eye, the situation of 6 the solidifying spot formation of light CNV, sees the above table respectively.