A kind of stable adriablastina albumin lipid drug-loading system and preparation method thereof
Technical field
A kind of stable adriablastina albumin lipid drug-loading system and preparation method thereof, by ultrasound wave, high pressure homogenize or Micro Fluid technology with albumin bound on amycin lipid cyst, can significantly improve the stability of lipid cyst and improve the envelop rate of medicine in the lipid cyst, especially can improve the therapeutic effect of amycin.
Background technology
Malignant tumor (cancer) is that China is only second to the cardiovascular disease mortality rate and accounts for second disease, and chemotherapy is one of important means of treatment malignant tumor, and amycin class medicine has important use to be worth in the treatment of malignant tumor.Because the sickness rate of malignant tumor is high, be that the new doxorubicin formulations of representative is subjected to new affirming with the pirarubicin on treatment of diseases such as tumor, amycin class medicine still will occupy one seat in a few years from now in anti-tumor medicine.Because new and effective antineoplaston medicine difficulty in a very long time has breakthrough, and amycin class medicine is wide owing to the antitumor spectrum, and curative effect is clear and definite, all will continue to be seized of critical role on consumption still is the amount of money.Because whole tumour medicine field competing phase is to relaxing, the market growing space is big, and profit is higher, and this has created good development space for new amycin class preparation dominates the market.
Commercially available amycin class medicine has doxorubicin, epirubicin, aclarubicin, pirarubicin and idarubicin etc. at present, wherein epirubicin and pirarubicin occupy critical role as the representative of adriablastina antineoplastic medicine in antitumor drug market.React according to the oncology doctor, amycin class hydrochlorate antitumor drug has very strong zest when clinical use, if can improve the zest of such medicine from preparation, have better market prospect as this kind of a line medication, must bring good economic benefit.
The lipid cyst is to utilize phospholipid bilayer to be embedded in cholesterol formed vesicle packaging medicine molecule and the preparation that forms, and phospholipid is the main chemical compositions that constitutes the lipid cyst, and wherein the most representative is lecithin.Mainly from egg yolk and Semen sojae atricolor, preparation cost is low for lecithin, and stable in properties belongs to neutral phospholipid.Phosphatidylcholine is the main component that forms many cell membrane, it also is the primary raw material of preparation lipid cyst, cholesterol also is another important composition composition of lipid cyst, it is the important component of many natural biological films, itself does not form membrane structure, but can insert in the immobilized artificial membrane with the mol ratio of 1:1 even 2:1.Add cholesterol and can change the phase transition temperature of adipose membrane, thereby influence the permeability and the flowability of film.Therefore cholesterol has the effect of stable phospholipid bimolecular film.
Press the structure and the particle size classification of lipid cyst, can be divided into: single chamber lipid cyst, multicell lipid cyst and heterogeneous lipid cyst can be divided into neutral lipid cyst, elecrtonegativity lipid cyst, electropositive lipid cyst by lipid cyst electric charge.
Pharmaceutical pack is wrapped in the lipid cyst, can be reduced in the tissue diffusion and slowly in blood, discharge medicine, thus prolong drug action time, and the lipid cyst can optionally be distributed in some tissue and organ, increase medicine to lymphoid directionality, improve the treatment concentration of medicine at target site.Especially to cancer therapy drug, can make it optionally to kill and wound cancerous cell or anticancer, to the obviously reduction or the harmless effect of toxicity of normal structure, cell.Lipid capsule cellular surface character is changed,, can improve the selectivity of medicine, thereby also reduce toxicity, reduced untoward reaction the target area as size, surface charge, tissue specificity antibody etc.
Owing on the cell membrane albumin receptor GP60 is arranged, after Gp60 and albumin bound, activate after birth cave sample indent, carry out cell traffic, be rich in cysteine acidic secretion albumen by tumor cell secretion, be present between multiple malignant tumor tissue in the matter, with patient's prognosis direct relation is arranged, it and GP60 homology, can and albumin bound, utilize the characteristic of itself and albumin bound, make the surface be inlaid with albuminous lipid cyst relative rich collection in mesenchyma stroma of tumors, discharge medicine in tumor cell by film fusion and transhipment, the medicine local concentration in the tumor cell is increased, thereby increase the curative effect of tumor cell.
By the preparation method and relevant research of literature search to albumin microsphere and liposome, and the lipid cyst is inlayed the also not discovery of research that albumin improves amycin lipid cyst curative effect and drug loading and envelop rate.
Adriablastina albumin lipid drug-loading system of the present invention has not only effectively been realized the enrichment of tumour medicine around tumor cell, improve the antineoplastic effect, it is low more meaningfully can effectively to overcome lipid cyst envelop rate, the defective of easy seepage, the technology that the present invention adopts can realize that suitability for industrialized production makes the product of clinical use, has very important realistic price.
Summary of the invention
The invention discloses a kind of stable adriablastina albumin lipid drug-loading system and preparation method thereof, it is characterized in that albumin bound on amycin lipid cyst, can significantly improve the stability of lipid cyst and improve the envelop rate of medicine in the lipid cyst, wherein comprise in the albumin lipid drug-loading system and have amycin, phospholipid, sterol and albumin, its preparation method is rolled in pharmaceutical pack in the lipid cyst of phospholipid and sterol formation for adopting film dispersion method in conjunction with the PH gradient method, in system, add albumin solution again, make the injectable albuminous lipid drug-loading system that combines with press filtration method or high pressure homogenize method, also albumin and medicine can be added the phospholipid that dissolves in organic solvent simultaneously, form precursor lipid cyst in the sterol, make the injectable albuminous lipid drug-loading system that combines with press filtration method or high pressure homogenize method after the reuse buffer salt solution aquation.
The albumin lipid drug-loading system that the present invention is stable, wherein each constituent mass percentage ratio is as follows:
Amycin medicine 0.01-10%, phosphatidase 10 .1-60%, sterol 0.01-20%, albumin 0.1-90%
Preferred each constituent mass percentage ratio is as follows:
Amycin medicine 0.01-10%, phosphatidase 13-60%, sterol 1-20%, albumin 0.1-90%
The amycin medicine albumin lipid drug-loading system that the present invention is stable, can also comprise frozen-dried supporting agent, be selected from micromolecule aminoacid, polysaccharide or polyhydric alcohol, specifically comprise a kind of or combination in any in 20 kinds of natural amino acids, trehalose, sucrose, lactose, maltose, mannitol, the sorbitol, described consumption is 2%-50% by weight.
The amycin medicine albumin lipid drug-loading system that the present invention is stable, also comprise the PH regulator, be selected from hydrochloric acid, phosphoric acid, citric acid or its alkali metal sodium salt, oxalic acid or its metal and receive salt, sodium hydroxide, potassium hydroxide, sodium carbonate, glacial acetic acid or its alkali metal salt.
The amycin medicine albumin lipid drug-loading system that the present invention is stable, can also further comprise stabilizing agent, be selected from micromolecule aminoacid or PEG200,400,600, a kind of or its any mixture in Tween 80, poloxamer (poloxamer), the NaTDC, described consumption is 0.2%-5% by weight, wherein aminoacid specifically is selected from 20 kinds of aminoacid commonly used, such as 20 kinds of a-amino acids:
Nonpolar amino acid: alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine (8 kinds);
Polarity, neutral aminoacid: serine, glycine, threonine, cysteine, agedoite, glutamine, tyrosine (7 kinds);
Acidic amino acid: aspartic acid, glutamic acid (2 kinds);
Basic amino acid: arginine, lysine, histidine (3 kinds)
The amycin medicine albumin lipid drug-loading system that the present invention is stable can also comprise antioxidant, is selected from one or more the mixture in vitamin E, vitamin C, sulfites, ethylenediaminetetraacetic acid and derivant thereof, the cysteine hydrochloride.
The amycin medicine albumin lipid drug-loading system that the present invention is stable can also comprise osmotic pressure adjustment agent, is selected from a kind of or combination in any in glucose, sodium chloride, glycerol, the sorbitol, and described consumption is 0.2%-5% by weight.
The amycin medicine albumin lipid drug-loading system that the present invention is stable, organic solvent is selected from low boiling point solvents such as dichloromethane, chloroform, ethanol, ethyl acetate, ether, acetone.
The amycin medicine albumin lipid drug-loading system that the present invention is stable, phospholipid is selected from natural or synthetic phospholipid in each component, such as lecithin (soybean lecithin, Ovum Gallus domesticus Flavus lecithin, two lauroyl phospholipid, two myristoyl phospholipid, two palmityl phospholipid or distearyl phospholipid etc.), PHOSPHATIDYL ETHANOLAMINE (two lauroyl PHOSPHATIDYL ETHANOLAMINE, two myristoyl PHOSPHATIDYL ETHANOLAMINE, two palmityl PHOSPHATIDYL ETHANOLAMINE or DSPE etc.) Phosphatidylserine (two lauroyl Phosphatidylserine, two myristoyl Phosphatidylserine, two palmityl Phosphatidylserine or distearyl Phosphatidylserine etc.), phosphatidic acid, phosphatidyl glycerol (two lauroyl phosphatidyl glycerols, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, two palmityl phosphatidyl glycerols or distearyl phosphatidyl glycerol etc.), phosphatidylinositols (two lauroyl phosphatidylinositols, two myristoyl phosphatidylinositols, two palmityl phosphatidylinositols or distearyl phosphatidylinositols etc.), LYSOLECITHIN SUNLECITHIN A, sphingomyelins, Ovum Gallus domesticus Flavus lecithin, soybean lecithin, hydroxylated lecithin, the mixture of one or more in the phospholipid of hydrogenated phospholipid and process PEG modified; Sterol is selected from cholesterol and derivant thereof, specifically comprises one or more the mixture in cholesterol, Polyethylene Glycol butanediol cholesterol ethers (preferred short chain polyalkylene glycol), Cholesteryl hemisuccinate, ergosterol, the lanosterol; Albumin is selected from animal albumin (such as ovalbumin, serum albumin, lactalbumin or muscle albumin) and phytalbumin, a kind of among preferred animal serum albumin, human serum albumin and the human serum albumin that produces by genetic engineering.
The amycin medicine albumin lipid drug-loading system that the present invention is stable gets preparation method, and concrete preparation method has two kinds, and first kind may further comprise the steps:
The first step: select the rotary evaporation method with medicine, phospholipid and sterol organic solvent dissolution, the lipid capsule cell space that forms by film dispersion method is, use the buffer salt solution ultrasonic dissolution, make the lipid cyst form relatively homogeneous granules by ultrasound wave, high pressure homogenize or Micro Fluid technology, size is at 50nm-1000nm;
Second step: at the lipid capsule cell space is to add albumin solution in the system, forms the injectable albuminous lipid drug-loading system that combines by ultrasound wave, high pressure homogenize or Micro Fluid technology, and size is filtered promptly canned at 20nm-1000nm;
The 3rd step: if the preparation freeze-dried preparation adds an amount of frozen-dried supporting agent in system, lyophilizing promptly.
Second kind of preparation method may further comprise the steps:
The first step: albumin and medicine adding are dissolved in the phospholipid and sterol of organic solvent, reduction vaporization adds buffer salt solution after removing organic solvent, make the lipid cyst form relatively homogeneous granules by ultrasound wave, high pressure homogenize or Micro Fluid technology, size is filtered promptly canned at 20nm-1000nm;
Second step: if the preparation freeze-dried preparation adds an amount of frozen-dried supporting agent in system, lyophilizing promptly;
If preparing product, environment, thermal source, aseptic etc. need finishing according to the requirement of big production.
The amycin medicine albumin lipid drug-loading system that the present invention is stable, the PH scope of solution was after injection or lyophilizing were redissolved: 5.0-7.0.
Specific embodiment
Embodiment 1: pirarubicin albumin lipid conjugate
Get pirarubicin 30mg, Ovum Gallus domesticus Flavus lecithin 2.5g, hydrolecithin 0.5g, Polyethylene Glycol butanediol cholesterol ethers 0.1g, tryptophan 0.2g, albumin 0.5g, add the stirring of 50ml chloroform/ethanol and make pirarubicin, Ovum Gallus domesticus Flavus lecithin, hydrolecithin, cholesterol dissolving, remove chloroform/ethanol with the rotary evaporator distilling under reduced pressure, add sucrose 3g, glucose 2g, PH4 citric acid buffer salt to cumulative volume is 50ml, and ultrasonic dissolution is used Na
2CO
3Regulator solution PH is 7.5,40 ℃ of insulations 30 minutes, is 50-200nm with press filtration method or high-pressure uniform machine homogenizing to granularity, and 0.22 filter membrane canning sealing promptly excessively.
If the preparation freeze-dried preparation adds the lyophilizing of 5% (percentage by weight) mannitol promptly in aforesaid liquid.
If preparing product, environment, thermal source, aseptic etc. need finishing according to the requirement of big production.
Embodiment 2: epirubicin albumin lipid conjugate
Get epirubicin 50mg, Ovum Gallus domesticus Flavus lecithin 1g, Polyethylene Glycol butanediol cholesterol ethers 0.3g, albumin 3.0g, add to stir in the 50ml dehydrated alcohol and make epirubicin, Ovum Gallus domesticus Flavus lecithin, cholesterol dissolving, remove ethanol with the rotary evaporator distilling under reduced pressure, add sucrose 5g, cysteine hydrochloride 0.02g, PH6-7 citric acid buffer salt to cumulative volume are 100ml, and high-pressure uniform machine homogenizing to granularity is 50-200nm, and 0.22 filter membrane canning sealing promptly excessively.
If the preparation freeze-dried preparation, as freeze drying protectant, lyophilizing promptly than the mannitol of 2-3% for the adding system weight in aforesaid liquid.
If preparing product, environment, thermal source, aseptic etc. need finishing according to the requirement of big production.
Embodiment 3: aclarubicin albumin lipid conjugate
Get aclarubicin 150mg, Ovum Gallus domesticus Flavus lecithin 3g, cholesterol 1g, albumin 0.7g, add the stirring of 50ml dichloromethane and make aclarubicin, Ovum Gallus domesticus Flavus lecithin, cholesterol dissolving, remove dichloromethane with the rotary evaporator distilling under reduced pressure, add sucrose 5 grams, PH6-7 citric acid buffer salt to cumulative volume is 100ml, and high-pressure uniform machine homogenizing to granularity is 30-200nm, and 0.22 filter membrane canning sealing promptly excessively.
If the preparation freeze-dried preparation, the technology lyophilizing is promptly routinely than the sucrose of 2-3% to add system weight in aforesaid liquid.
If preparing product, environment, thermal source, aseptic etc. need finishing according to the requirement of big production.
Embodiment 4: idarubicin albumin lipid conjugate
Get idarubicin 100mg, Ovum Gallus domesticus Flavus lecithin 2.5g, cholesterol 0.8g, albumin 0.6g, add the stirring of 50ml ethanol and make idarubicin, Ovum Gallus domesticus Flavus lecithin, cholesterol dissolving, remove chloroform with the rotary evaporator distilling under reduced pressure, add sucrose 5 grams, PH6-7 citric acid buffer salt to cumulative volume is 100ml, and high-pressure uniform machine homogenizing to granularity is 30-200nm, and 0.22 filter membrane canning sealing promptly excessively.
If the preparation freeze-dried preparation, the technology lyophilizing is promptly routinely than the mannitol of 2-3% to add system weight in aforesaid liquid.
If preparing product, environment, thermal source, aseptic etc. need finishing according to the requirement of big production.
Embodiment 5: doxorubicin albumin lipid conjugate
Get doxorubicin 50mg, Ovum Gallus domesticus Flavus lecithin 2.5g, hydrolecithin 0.5g, cholesterol 0.8g, albumin 0.5g, add the stirring of 50ml ethanol and make doxorubicin, Ovum Gallus domesticus Flavus lecithin, hydrolecithin, cholesterol dissolving, remove ethanol with the rotary evaporator distilling under reduced pressure, add sucrose 3g, glucose 2g, PH4 citric acid buffer salt to cumulative volume is 50ml, and ultrasonic dissolution is used Na
2CO
3Regulator solution PH is 7.5,40 ℃ of insulations 30 minutes, is 50-200nm with press filtration method or high-pressure uniform machine homogenizing to granularity, and 0.22 filter membrane canning sealing promptly excessively.
If the preparation freeze-dried preparation adds the lyophilizing of 5% (percentage by weight) mannitol promptly in aforesaid liquid.
If preparing product, environment, thermal source, aseptic etc. need finishing according to the requirement of big production.