CN102755292A - Vesicle-type medication system containing metformin, and application thereof - Google Patents

Abstract

The invention belongs to the field of pharmaceutical preparation, and specifically discloses a vesicle-type medication system containing metformin, and an application thereof. According to the invention, metformin is used for replacing part of ammonium in a classic ammonium ion gradient, and an ammonium gradient and/or metformin gradient in water phase inside and outside liposome is established. A novel vesicle-type medication system (comprising various liposomes) is prepared. With the trans-membrane gradient, an anti-tumor drug is actively loaded into liposome. The metformin is still retained in water phase in liposome. Therefore, the two are both encapsulated in liposome. According to the invention, a surface active substance is adopted as a basic membrane material; a solution of a guanidine-containing cationic compound is adopted as a hydration medium, and blank vesicles are prepared; and trans-membrane gradient is established upon the blank vesicles, so that the medication system is prepared. The vesicle-type medication system is characterized by wide drug loading range, high drug encapsulation efficiency, and good stability. With the vesicle-type medication system, drug toxicity can be reduced, and tumor reoccurrence can be inhibited.

Description

A kind ofly contain metformin vesicle class drug-supplying system and application thereof

Technical field

The invention belongs to field of pharmaceutical preparations, be specifically related to a kind of vesicle class drug-supplying system and application thereof that contains metformin and can utilize gradient realization active medicine carrying.

Background technology

Tumor recurrence and transfer are to be difficult to thoroughly two hang-ups of solution in the treating malignant tumor process all the time.In tumor research process in the past, traditional theory thinks that the generation of tumor and development are the results of the common propagation of all tumor cells, and therefore research is devoted to how to kill a large amount of tumor cell colonies always.It not is that all tumor cells all have infinite multiplication potential that but Recent study is found, the cell colony that has only a small amount of stem cell character is the potential that tumor stem cell has self renewal and infinite multiplication.

Cancer stem-cell hypothesis thinks that existence is heterogeneous between the tumor cell, has only the sub-fraction tumor stem cell to have infinite multiplication and the ability that forms tumor.2006 AACR (American Association for Cancer Research) (Cancer stem cell/Tumor stem cell, the definition of CSC/TSC) is: have the self renewal ability in the tumor and can produce the cell of heterogeneous tumor cell to tumor stem cell.Discover at present between CSC and the common stem cell and exist very similar characteristic: (1) all has the characteristics of infinite multiplication and differentiation; (2) there is the signal transduction path of similar adjusting self renewal; (3) all have different phenotypes, heterogeneity; (4) all have telomerase activation, can damage by DNA plerosis; (5) can both transfer to various tissue and similar going back to the nest and route of metastasis arranged.Certainly, CSC also has the characteristics that are different from common stem cell:: the negative feedback mechanism of (1) self renewal signal transduction pathway is destroyed; (2) lack the differentiation and maturation ability; (3) tend to accumulate the mistake of duplicating, and stem cell can prevent the development of copy error etc.Think that at present the possible source of tumor stem cell has three kinds: (1) has the cancerous phenotype normal stem cell; (2) committed progenitor that has a Cancer-causing mutation regains the self renewal ability; (3) rare fusion event between stem cell and other cell.Up to the present; Except in granulocyte leukemia, breast carcinoma and the cerebral tumor, having identified the existence of tumor stem cell, scientists has obtained the evidence of tumor stem cell in succession in pulmonary carcinoma, malignant melanoma, carcinoma of prostate, ovarian tumor, colon cancer, hepatocarcinoma.Selectively targeted tumor stem cell, remove the tumor stem cell storehouse, for long-term stability, the alleviation of malignant tumor and even cure most important.

The medicine of targeting tumor stem cells commonly used mainly contains following three types at present: small-molecule substance, albumen and virus.

The research worker of Harvard University's medical college shows that in the research of Cancer Research issue on JIUYUE 14th, 2009 metformin can improve breast cancer development.Discover that metformin can selectivity kills the tumor stem cell in the different breast carcinoma of four kinds of genotype; Tumor stem cell and the non-tumor stem cell that has killed cultivation used in uniting of amycin and metformin, effectively suppressed the growth of xenotransplantation mouse tumor.

Some amphipathic molecule; Like many natural or synthetic surfactants and can not simply associate into the phospholipid of micelle; Can spontaneous formation have the double-deck molecular assembly assembly of sealing for one type when being scattered in the water, be called vesicle (vesicle), be also referred to as liposome (liposome).The meaning of these two terms of vesicle and liposome some ambiguity in document.It is generally acknowledged that if these amphiphile, amphiphilic molecules are natural surfactant lecithin, the structure that then forms just is called liposome; If form, then be called vesicle by synthetic surfactant.Therefore, liposome refers in particular to the one type of special vesicle that is formed by phospholipid, and it is the human vesicle system of finding at first.

Liposome wraps up hydrophilic kernel by single or a plurality of double-layers of lipoid to be formed, and the main component of lipid layer is a phospholipid, in most of the cases also contains cholesterol.Lipophilic drugs can be wrapping in the double-layer of lipoid, and hydrophilic medicament then can enter into water layer or the aqueous kernel between the lipid layer.The bilayer similar cell membrane of liposome; Has excellent biological compatibility; Compare with the other medicines carrier, liposome has special advantages: the main component of (1) liposome is phospholipid and cholesterol, is the natural component of mammalian cell membrane; Has biocompatibility, biodegradable, avirulence and non-immunogenicity; (2) size of liposome, phospholipid composition, surface charge etc. have very big selection space; (3) lipid physical ability parcel hydrophilic and lipophilic drugs are enclosed pharmaceutical pack in the liposome, can protect medicine not to be degraded in vivo, avoid the interference of medicine to the receptor recognition ligand simultaneously; (4) be prone to obtain appropriate drug-carrier ratio, preparation is simple; (5) different with solid polymer support system (like microgranule, nanoparticle); The liposome bilayer lipid membrane is in liquid crystal state more than phase transition temperature; Allow the targeted molecular of surface combination that bigger degree of freedom is arranged, so targeted molecular can be with preferred configuration and target site receptors bind; (6) liposome comprises that also for the targeting property of institute's packaging medicine provides new possibility the extracellular discharges, cell membrane merges and endocytosis, has improved the targeting scope of medicine; (7) no covalent bond is connected between liposome and medicine, is beneficial to medicine and in lysosome, discharges; (8) can deliver drugs into particular target tissue and target cell at drug-loaded liposome surface combination different aglucon such as antibody, sugar ester etc.

In recent years, liposome at numerous disease, especially demonstrates obvious superiority in the treatment for cancer as pharmaceutical carrier.The toxic and side effects (after one's own heart dysentery property, Toxicity of Kidney) of system and specific part can be obviously reduced after antitumor drug is prepared into liposome, and drug targeting property, medicament curative effect enhancement can be improved.Think at present that it is because medicine slowly discharges from the lipid carrier composition and liposome is caused to the effect of tumor locus passive target that the liposome curative effect strengthens, the reason that toxicity reduces then is to have reduced contacting of medicine and the interior normal structure of body.

Liposome as the pharmaceutical carrier clinical practice early develops comparatively perfect.The liposome product that has gone on the market at present has Evacet (Doxil, Caelyx and Myocet), daunorubicin liposome (DaunoXome) and cytosine arabinoside liposome (DepoCyt) etc.

As pharmaceutical carrier; The method of liposome drug loading mainly comprises passive medicine carrying method and active loading method; But the common envelop rate of liposome that traditional passive medicine carrying method makes is low, particularly seals some amphipathic weak acid or weak base drug, because its profit partition coefficient receives the pH value of medium and ionic strength affect bigger; The condition of sealing is difficult for grasping, and the liposome encapsulation of preparation differs greatly.Therefore, envelop rate low and be prone to infiltration problem to a great extent limit the popularization of liposome as pharmaceutical carrier.Active loading method (also claiming the ion gradient method) can be loaded into some amphipathic medicine in the liposome efficiently, has overcome that early stage medicine is prominent to be released and leak, and has solved the large-scale industrial production difficult problem of these drug liposome preparations to a certain extent.The foundation of suitable ion gradient difference is depended in the realization of ion gradient method; Like the hydrion gradient; Promptly through forming " proton pond ", tart internal medium makes the neutral amphipathic medicine generation protonation that diffuses into liposome interior and lotus positive electricity stops it to stride film once more.

The ion gradient method is subdivided into: (1) pH gradient method; (2) ammonium sulphate gradient; (3) calcium acetate gradient method; (4) ion gradient-cell plasma support methods.Wherein back three kinds of methods all can be summed up as the pH gradient with the motive power that medicine initiatively is loaded into liposome, promptly induce through suitable ion gradient to produce pH gradient, these three kinds of specific form that method is the pH gradient method in fact.Load weakly basic drugs and can adopt pH gradient method or ammonium sulphate gradient, load weak acidic drug and then can adopt the calcium acetate gradient method.

The ultimate principle that the ion gradient legal system is equipped with liposome is: (1) liposome phospholipid bilayer film can comprise H optionally with the water-soluble substances inside and outside the liposome ⁺, K ⁺, Na ⁺, Ca ²⁺, Mn ²⁺Etc. various cationes, SO ₄ ^2-, various aniones such as citric acid radical, acetate separate effectively, form inside and outside two the relatively independent environment of water of liposome; (2) weak base or weak acidic drug are fat-soluble at the nonionic state, can see through immobilized artificial membrane according to gradient difference and get into water in the liposome.In living things system, many material turnover cells are also realized transporting for power through ion gradient (being also referred to as ion channel or ionic pump).It is generally acknowledged that ion gradient method efficiently drug loading mainly relies on following two kinds of mechanism: directly produce the pH gradient and produce the pH gradient indirectly.

Directly produce the pH gradient: pH gradient method (pH gradient method) is that the pH of water forms certain pH gradient difference inside and outside the liposome through regulating; Utilize the difference of weak acid or weakly basic drugs dissociated state in different pH solution; Make medicine outside aqueous phase exist with molecularity, see through behind the liposome bimolecular film at interior aqueous phase and form ionic drug again and sealed.Blank liposome is in liquid crystal state being higher than when phase transition temperature is hatched, bimolecular film is mobile to be increased, and permeability increases, and helps molecule-type medicine and sees through the liposome bimolecular film.Under the effect of pH gradient, the medicine of outer aqueous phase constantly diffuses into interior aqueous phase through bimolecular lipid membrane, in this process lasts till always water and outer aqueous phase drug level than with hydrogen ion concentration than equating ([D] _i/ [D] _o=[H ⁺] _i/ [H ⁺] _o) kinetics equation sealed of pH gradient method Chinese medicine sees formula (1):

[D(t)] _i=[D(eq)] _i(1-e ^-kt) （1）

Wherein, k=PA _mK _d/ V _o[H ⁺] _o, [D (t)] _iBe the constantly interior water drug level of t, [D (eq)] _iBe interior water drug level after the medicine carrying balance, k is a speed constant, and P is the film transmission coefficient of neutral drug molecule, A _mBe film surface area, K _dBe the dissociation constant of medicine, V _oBe outer water volume, [H ⁺] _oBe outer water hydrogen ion concentration.

The pH gradient that produces indirectly: although successfully multiple medicine is loaded into liposome through interior water for the pH gradient that the citric acid buffer salt forms, not all weakly basic drugs all is fit to load in this way, like ciprofloxacin.Ciprofloxacin is charged and solubilized in acidity or alkaline environment; But exactly be that the outer aqueous phase of drug delivery technologies is neutrality and dissolubility is low in the physiological pH scope; Therefore, be difficult to adopt the citric acid gradient method that it is written into liposome (envelop rate is usually less than 20%).But the transmembrane gradient that is to use ammonium sulfate to form just can reach very high envelop rate.

Have in the large unilamellar vesicle of ammonium sulphate gradient at inside and outside water, have a spot of neutral amino molecule and dissociate out from interior aqueous phase, water pH is 2.7 non-buffering system in forming, thereby produces the pH gradient of an inside and outside water indirectly.At this moment the neutral drug molecule of outer water will diffuse into water in the liposome under the effect of concentration difference, consumes a proton simultaneously, forms charged particle and is wrapped into liposome.Along with neutral molecule gets into water in the liposome, proton is consumed, and causes more neutral amino molecule to be diffused into outer water and produces more proton, as the power of medicine carrying.This process finishes when all medicines are sealed the proton full consumption of completion or interior water.This technology is suitable for the medicine that ciprofloxacin etc. is supplied with hydrochloride form, or is dissolved in the medicine that is faintly acid and solubility behind the water.In addition, the envelop rate of the Evacet that makes of ammonium gradient is also very high.This method is applied to the preparation of most medicinal liposomes all the time, like amycin, and epirubicin, ciprofloxacin, vincristine.This method also can use alkyl ammonium salt (like the sulphuric acid ammonium methyl) to replace ammonium salt formation gradient to reach the medicine carrying purpose.

Summary of the invention

The present invention is intended to substitute the part ammonium in the classical ammonium ion gradient with metformin; Set up the ammonium gradient and/or the metformin gradient of the inside and outside water of liposome; Prepare a kind of new vesicle class drug-supplying system (comprising various liposomees); Utilize transmembrane gradient that antineoplastic agent initiatively is loaded into liposome, metformin still is stranded in water in the liposome simultaneously, so that the two is encapsulated in the liposome jointly; Thereby realize heightening the effect of a treatment, reducing toxicity, the target tumor cell, kill tumor cell and tumor stem cell and suppress the effect of tumor recurrence.

For realizing the object of the invention, the inventor provides following technical scheme.

A kind of vesicle class drug-supplying system (comprising liposome), it adopts following method preparation:

At first be the underlying membrane material, be that the aquation medium prepares blank vesicle, then blank vesicle is set up transmembrane gradient and promptly get with the solution that contains the guanidine radicals cationic compound with the surfactant.

The present invention is the underlying membrane material with the surfactant; Comprise suitable ion-type and nonionic surfactant, macromolecular substances; Like various phospholipid and derivant thereof; Anhydro sorbitol fatty acid lipid, polyethenoxy sorbitan fatty acid lipid, also optional sterols and derivant thereof, and other necessary film material.

Above-mentioned transmembrane gradient is a notion in this professional field, i.e. composition, the Concentraton gradient of the inside and outside water of vesicle; Contain tradable various cation or anion or two kinds of ions of negative and positive in the blank vesicle aquation medium, ionic species can be a kind of or two kinds or two or more, with the blended any concentration of arbitrary proportion.The method of setting up transmembrane gradient can be according to the operation of existing conventional method, like dialysis, slipstream, polydextran gel sieve method, Amberlyst process or supercentrifugal process or the like.Also can set up transmembrane gradient according to ion exchange; So-called ion exchange promptly adopts suitable ion-exchanger to remove anion, the cation of the outer aqueous phase of vesicle; Perhaps zwitterion is removed simultaneously; And the zwitterion of interior aqueous phase is not removed or removes seldom, thereby sets up outside gradient in the film, realizes initiatively medicine carrying.

As preferred version, according to vesicle class drug-supplying system of the present invention, wherein, described to contain guanidine radicals cationic compound general structure following:

Be that the described guanidine radicals cationic compound that contains is a metformin.

As preferred version, according to vesicle class drug-supplying system of the present invention, wherein, the said solution concentration that contains the guanidine radicals cationic compound is 10-1000 mmolL ^-1As more preferably scheme, the described solution concentration 50-300 mmolL that contains the guanidine radicals cationic compound ^-1

As preferred version, according to vesicle class drug-supplying system of the present invention, wherein, the described pH value that contains guanidine radicals cationic compound solution is between 3.0-9.0.As more preferably scheme, the pH value of the described solution that contains the guanidine radicals cationic compound is between 5.0-8.0.

As preferred version; According to vesicle class drug-supplying system of the present invention; Wherein, The described guanidine radicals cationic compound solution that contains can adopt following method preparation: in conventional aquation medium (like ammonium sulfate, citric acid-liquor sodii citratis, ethylenediaminetetraacetic acid ammonium salt solution, calcium acetate solution etc.), add melbine salt, example hydrochloric acid metformin, sulphuric acid metformin, citric acid metformin etc.

As preferred version,, wherein, add inorganic or organic base regulator solution pH value such as ammonia, triethylamine, triethanolamine in the described solution that contains the guanidine radicals cationic compound between 3.0-9.0 according to vesicle class drug-supplying system of the present invention.As more preferably scheme, add inorganic or organic base regulator solution pH value such as ammonia, triethylamine, triethanolamine in the described solution that contains the guanidine radicals cationic compound between 5.0-8.0.

As preferred version, according to vesicle class drug-supplying system of the present invention, wherein, when it was used to load antitumor drug, the mass ratio of guanidine radicals cationic compound and antitumor drug was 0.05-5:1.As more preferably scheme, when it was used to load antitumor drug, the mass ratio of guanidine radicals cationic compound and antitumor drug was 0.1-2:1.

As preferred version, according to vesicle class drug-supplying system of the present invention, wherein, described aquation medium is for containing the formed saline solution of guanidine radicals cationic compound and monovalence, bivalence, trivalent or multivalent anions.

As preferred version, according to vesicle class drug-supplying system of the present invention, wherein, described anion is to contain following one or more anionic chemical compound: carboxylate radical, sulfate radical, sulfonate radical, phosphate radical, citric acid radical or phosphonate radical.As scheme most preferably, the solution concentration of wherein said anionic compound is 150-500mmolL ^-1

Anion of the present invention is selected from least a in the following material:

HP0 ₄ ^2-, H ₂P0 ₄ ^-, Cl ^-Malate; Tartrate anion; Acetate; The hexa metaphosphoric acid root; Tripolyphosphate; Pyrophosphate; The phosphoglycerol root; The fructose diphosphate root; The ATP root; The phytic acid root; Phthalate; The M-phthalic acid root; Terephthaldehyde's acid group; Sulfonic group; P-phthalic acid; Benzoic acid; M-phthalic acid; 1,3,5-Benzenetricarboxylic acid; Lactobionic acid; The 2-5 dihydroxy benzenes sulfonic acid; Hydroxy benzenesulfonic acid; DNA; RNA; SiRNA; RNAi; Protein; Succinylated gelatin; Enzyme; Charged polysaccharide or succinyl chitosan etc.Also can with the combination of various phosphonic acid based medicines (derivant) and its esters, as lythidathion, tiludronic acid, clodronic acid pamidronic acid two receive, fosfomycin amido butantriol, the acid of ethylenediamine tetraacetic methene, etidronic acid receive; Diethylenediamine pentamethylene phosphoric acid seven is received salt, 2-phosphate butane, 1; 2; 4-tricarboxylic acids four is received, ATMP, pamidronate disodium, (3-aminophenyl) phosphonic acids, hexamethylene diamine tetramethylene phosphonic acid potassium salt, sodium benzene phosphinate, Alendronic acid and its esters, methylphosphonic acid (5-ethyl-2-methyl-2-oxo-1; 3; 2 dioxy phospha hexamethylene-5 bases) ylmethyl fat, pamidronic acid, di 2 ethylhexyl phosphonic acid butane-1,2,4 tricarboxylic acids are received salt, hexamethylene diamine tetramethyl fork phosphonic acids six potassium salt, 2-phosphonic acids-l, 2; 4-tricarboxylic acids, hydroxy ethylene diphosphonic acid four are received, two hexene triamines, five methylenephosphonic acids, risedronic acid, aminomethyl phosphonic acids, ibandronic acid, sharp match phosphonic acids are received, azoles comes that Teng's acid, pamidronic acid are received, GS-504, tiludronic acid two are received, vinyl phosphonate, (3-((being methyl) amino)-3-carbonyl propyl group)-dimethyl phosphonate, 2-amino-ethyl phosphonic acids, ATMP are received, the smooth (i.e. [3-[[(2R of the husky pyrrole of good fortune; 3S)-2-[(1R)-1-[3, two (trifluoromethyl) phenyl of 5-] ethyoxyl]-3-(4-fluorophenyl)-4-morpholinyl] methyl]-2,5-dihydro-5-oxo-1H-1; 2, the 4-triazol-1-yl] phosphonic acids).

As more preferably, anionic compound of the present invention is selected from least a in the following material: sulfate ion, phosphate anion, citric acid radical ion, ethylenediaminetetraacetic acid radical ion, phosphoglycerol radical ion, phytic acid radical ion, fructose diphosphate radical ion or sucrose octasulfate radical ion.

Phospholipid can be selected from least a in the following material in the said film material: soybean lecithin; Ovum Gallus domesticus Flavus lecithin; EPG; Phosphatidic acid; Cardiolipin; Sphingomyelins; The phosphatidic acid serine; Phosphatidylinositols; PHOSPHATIDYL ETHANOLAMINE; Hydrogenated soy phosphatidyl choline; Hydrogenated yolk lecithin; DSPC; Dipalmitoyl phosphatidyl choline; The dioleoyl phospholipid phatidylcholine; Dimyristoyl phosphatidyl choline; Two Laurel phosphatidyl cholines; DDPC; Two decoyl phosphatidylcholines; Two hexanoyl phosphatidylcholines; Distearyl phosphatidyl glycerol and sodium salt thereof; Two palmityl phosphatidyl glycerol and sodium salts thereof; L-α-GLYCEROL,DIMYRISTOYL PHOSPHATIDYL and sodium salt thereof; Two lauroyl phosphatidyl glycerols; Two caprinoyl phosphatidyl glycerols; Two decoyl phosphatidyl glycerols; Two hexanoyl phosphatidyl glycerols; DSPE; Two palmityl PHOSPHATIDYL ETHANOLAMINEs; DOPE; Two myristoyl PHOSPHATIDYL ETHANOLAMINEs; Two lauroyl PHOSPHATIDYL ETHANOLAMINEs; Two distearyl phosphatidyl glycerols and sodium salt thereof; Two two palmityl phosphatidyl glycerol and sodium salts thereof; Two GLYCEROL,DIMYRISTOYL PHOSPHATIDYLs and sodium salt thereof; Two two lauroyl phosphatidyl glycerols; The distearyl phosphatidylinositols; Two palmityl phosphatidylinositols; Dioleoyl phospholipid acyl inositol; Two myristoyl phosphatidylinositols; Two lauroyl phosphatidylinositols; Palmityl oleoyl phosphatidylcholine; The inferior oleoyl phosphatidylcholine of palmityl; The inferior oleoyl phosphatidylcholine of stearoyl; Stearoyl oleoyl phosphatidylcholine; Stearoyl arachidonic phosphatidyl choline; DSPE-PEG; DPPE-PEG; DMPE-PEG; DLPE-PEG; Wherein said DSPE-PEG; DPPE-PEG; DMPE-PEG; The molecular weight of PEG is 100～10000 among the DLPE-PEG.

The underlying membrane material can add the PEG derivant, and its consumption is 0 ~ 50% of the total consumption of lipid.

As preferred version, add the 2-50% that the PEG derivant accounts for lipid phase raw material gross mass in the vesicle class drug-supplying system of the present invention.As more more preferably scheme, the PEG derivant that adds in the described underlying membrane material accounts for the 5-40% of lipid phase raw material gross mass.As scheme most preferably, the PEG derivant that adds in the described underlying membrane material accounts for the 5-20% of lipid phase raw material gross mass.Wherein, above-mentioned PEG derivant includes but not limited to various phospholipid PEG derivants, and like DSPE-PEG, DPPE-PEG, DMPE-PEG, DLPE-PEG, wherein the molecular weight of PEG is 100 – 100000; Also comprise other various PEG lipid derivates; Like PEG sterols derivant, PEG derivative of fatty acid, PEG aliphatic alcohols derivant; Specifically like cholesterol half amber nitric acid fat PEG derivant, Solulan C-24 (PEG-24 cholesterol ether), Tweens, Brij (Brij), the PEG derivant (like TPGS) of vitamins, the intoxicated derivant of PEG fatty acid (Myrij); Polyglycereol lipid derivant is like polyglycereol phospholipid derivative, polyglycereol list (two) oleic acid vinegar, stearic sour-sweet etc.; The polypropylene glycol lipid derivate; The polyamino acid lipid derivate; The sterols derivant; Oxygen ethylene-oxypropylene copolymerization is like F68, Polyethylene Glycol-two acid glyceride (perhaps monoester) or the like.

Ratio in the blank vesicle of the present invention between the various mould materials can be confirmed by the technical staff according to concrete experimental result and actual needs.Described film material also comprises acid-sensitive, temperature sensitive, photosensitive or targeting property material except underlying membrane material, PEG derivant.Specifically how to add and to confirm according to the kind and the needs of medicine carrying, operate getting final product with reference to the common technology means of this area.

The present invention also provides the above-mentioned application of vesicle class drug-supplying system on antitumor drug.

As preferred version, described antitumor drug is selected from a kind of in the following medicine: anthracene nucleus class/amerantrone class AGPM, catharanthus alkaloid class, camptothecine or Difluoronucleosides class antineoplastic agent.Include but not limited to following medicine or its salt specifically: doxorubicin, daunorubicin, epirubicin, a left side soften than star, aklavine, ametycin, mitoxantrone, bisantrene, hydroxycamptothecin, irinotecan, TPT, 9-aminocamptothecin, vinblastine, vincristine, vindesine, vinorelbine, 10; 11-methylene-dioxy camptothecine, 9-nitrocamptothecin, 7-(4-methyl-piperazinyl-methylene)-10,11-methylene-dioxy-20 (S) camptothecine, 7-(2-N-isopropylamine base) ethyl-20 (S) camptothecine, two fluorine deoxycytidine, methotrexate, alkaloid, Sutent, gefitinib.Be used for various tumor treatment.

Above-mentioned vesicle class drug-supplying system can carry out medicine carrying as follows: will set up the blank vesicle (comprising various liposomees) and drug solution mixing of transmembrane gradient, and under 20～70 ℃, hatch, and can make the medicine carrying vesicle.

Among the present invention, before vesicle is set up transmembrane gradient, can do the processing that homogenizes as required,,, more help medicine carrying such as the vesicle of particle diameter between 30-300nm to obtain the suitable vesicle of particle diameter.

The present invention has the following advantages:

1), can kill tumor stem cell and common tumor cell simultaneously the present invention adopts metformin to substitute the pH gradient medicine carrying that ammonium ion is set up indirectly, and the characteristics that the vesicle class drug-supplying system that contains metformin of preparation has comprise:; 2), the medicine carrying scope is wide, is applicable to the loading of a lot of antitumor drug; 3), tumor stops growing after the administration; 4), can reach high envelop rate, the toxicity of reduction antineoplastic agent; 5), can prolong patient's life span; 6), the preparation storage stability is high, leakage rate is few, can long term storage; 7), comprise the vesicle of metformin because the EPR effect gets into tumor, thereby improve the concentration of metformin in tumor tissues, performance is united the effect that presses down tumor with Farmorubine Hydrochloride; 8) metformin in the preparation both can have been killed tumor stem cell also can provide antineoplastic agent is loaded into the intravital power of lipid.

Description of drawings

Fig. 1 is a gross tumor volume change curve in time

Wherein on behalf of normal saline matched group (NS), " ▲ ", " ■ " represent Farmorubine Hydrochloride solution group, " ▼ " to represent (MetH) ₄EDTA-gradient liposome group, " ★ " represent (NH ₄) ₄EDTA-epirubicin hydrochloride liposome group, " ◆ " representative (MetH) ₄EDTA-epirubicin hydrochloride liposome group.

Fig. 2 is for connecing mice survival analysis after the tumor among the embodiment 9

Wherein on behalf of normal saline matched group (NS), " ▲ ", " ■ " represent Farmorubine Hydrochloride solution group, " ▼ " to represent (MetH) ₄EDTA-gradient liposome group, " ◆ " representative (NH ₄) ₄EDTA-epirubicin hydrochloride liposome group, " ★ " represent (MetH) ₄EDTA-epirubicin hydrochloride liposome group.

The specific embodiment

Below in conjunction with embodiment, content of the present invention is described more specifically.Should be appreciated that enforcement of the present invention is not limited to following embodiment, all will fall into protection domain of the present invention any pro forma accommodation and/or the change that the present invention made.

In the present invention, if not refer in particular to, all equipment and raw material etc. all can be buied from market or the industry is commonly used.Method among the following embodiment if no special instructions, is the conventional method of this area.

Embodiment 1The selection of phospholipid, cholesterol ratio

The stability and the medicine efficiency of loading of the ratio regular meeting appreciable impact liposome membrane of phospholipid and cholesterol.With 200 mmolL ^-1(MetH) ₄EDTA (being the ethylenediaminetetraacetic acid metformin) is as the aquation medium, and the preparation blank liposome is set up transmembrane gradient, loads Farmorubine Hydrochloride.Measure the mean diameter and the envelop rate of preparation, the result sees table 1.

The influence of the different phospholipid of table 1, cholesterol ratio

Annotate: metformin concentration is 600 mmolL in the aquation medium ^-1(HSPC is a hydrogenated soy phosphatidyl choline; CH is a cholesterol; PEG2000-CHS is poly glycol monomethyl ether and Cholesteryl hemisuccinate reaction products therefrom, i.e. the Cholesteryl hemisuccinate macrogol ester of molecular weight 2000).

Can know that by table 1 particle diameter of prescription 1 is slightly larger than prescription 2, but 1 envelop rate of writing out a prescription is higher, and the two is all greater than 95%.

Embodiment 2The pH value influence

Preparation pH is respectively 5.50,6.00,6.50,7.00,7.50,8.00,9.00 200 mmolL ^-1(MetH) ₄EDTA solution is equipped with epirubicin hydrochloride liposome as the aquation medium according to experimental example 1 below legal system, investigates envelop rate, and the result sees table 2.

Table 2 aquation PH values is to the influence of epirubicin hydrochloride liposome envelop rate

Can know that according to table 2 its envelop rate of rising along with the aquation PH values increases, the aquation PH values is 6 ~ 9 o'clock, and envelop rate is greater than 80%.

Embodiment 3The influence of aquation concentration of medium

Compound concentration is respectively 100,200,300,400 mmolL ^-1(MetH) ₄EDTA solution (pH7.0) prepares hydrochloric doxorubicin liposome as the aquation medium according to the prescription of optimizing, and investigates the variation of variable concentrations preparation different time medicine carrying envelop rate, and the result sees table 3.

Table 3 aquation concentration of medium is to the influence of epirubicin hydrochloride liposome envelop rate

Can know 100 mmolL by table 3 ^-1With 400 mmolL ^-1(MetH) ₄The EDTA liposome does not reach more than 90% at 120 min envelop rates, 300 mmolL ^-1(MetH) ₄EDTA liposome envelop rate when 80 min is maximum, and 200 mmolL ^-1(MetH) ₄EDTA envelop rate when 60 min is maximum.

Embodiment 4The medicine carrying temperature is to the influence of envelop rate

Set up 200 mmolL ^-1(MetH) ₄EDTA gradient preparation is pressed medicine fat than 1:10 (w/w) mixing with gradient preparation and Farmorubine Hydrochloride solution, respectively at hatching 60 min in 30,40,50,60 ℃ of water-baths, measures the envelop rate of epirubicin hydrochloride liposome, and experimental result is seen table 4.

Table 4 medicine carrying temperature is to the influence of envelop rate

Can know by table 4, (MetH) ₄EDTA-epirubicin hydrochloride liposome envelop rate raises with temperature to be increased, and envelop rate was greater than 90% when envelop rate was merely 17.2%, 50 ℃, 60 ℃ in the time of 30 ℃.

Embodiment 5The metformin addition is to the influence of envelop rate in the aquation medium

The preparation of aquation medium: precision takes by weighing each 7 parts of EDTA0.5845 g, places 25 mL beakers respectively, adds distilled water 6 mL; And add metformin 0,0.013,0.065,0.130 respectively; 0.260,0.390 and 0.764 g, using ammonia regulator solution pH value respectively is 7.00; Be transferred in the 10 mL volumetric flasks, thin up shakes up to scale.

The preparation of blank liposome

Prescription:

HSPC 200?mg

CH 66.6?mg

PEG2000-CHS 66.6?mg

Method for preparing: under 65 ℃,, get the lipid phase with 5% ethanol (v/v) dissolving recipe quantity film material; With the aquation medium that is preheated to uniform temp, inject the lipid phase with middling speed, hatch 20 min, make the liposome first product.Behind ultrasonic preliminary mixing 2 min of 200 W, 400 W ultra-sonic dispersion, 6 min (3 s that work, intermittently 3 s) through the microporous filter membrane of 0.8,0.45,0.22 μ m, promptly get blank liposome successively.

The preparation of gradient liposome and medicine carrying: adopt the mixture iron exchange resin method to set up the liposome transmembrane gradient, with Farmorubine Hydrochloride solution (medicine fat ratio, 1:4; W/w) with gradient liposome mixing; Hatch in 60 ℃, the different time 300 μ L that take a sample, ice bath stops medicine carrying; Measure envelop rate, the result sees table 5.

Table 5 metformin addition is to the influence of envelop rate

Annotate: "-" expression undetermined envelop rate in the table.

Can know by table 5, as metformin addition≤200 mmolL ^-1The time; It does not make significant difference to the epirubicin hydrochloride liposome envelop rate; The metformin of water has neither part nor lot in medicine carrying in promptly in the medicine carrying process, so both can improve medicine carrying speed, has also increased the delay of metformin water in liposome to a certain extent; (enhanced permeability and retention effect) can be transported to tumor locus with more metformin by the EPR effect, so that it better brings into play anti-tumor activity.

Embodiment 6Different melbine salt additions are to the influence of envelop rate in the aquation medium

The preparation of different aquation media: it is an amount of to take by weighing ammonium sulfate, ethylenediaminetetraacetic acid and citric acid respectively; Add a certain amount of metformin hydrochloride, sulphuric acid metformin and citric acid metformin; Add an amount of distilled water; And use ammonia to regulate ethylenediaminetetraacetic acid and the citric acid soln pH value is 7.00, make ammonium sulfate in the final aquation medium, ethylenediaminetetraacetic acid and citric acid concentration be respectively 200,200 and 300 mmolL ^-1, metformin concentration is respectively 200,400 and 600 mmolL ^-1

The preparation of blank liposome

Prescription:

HSPC 200?mg

CH 66.6?mg

PEG2000-CHS 66.6?mg

Method for preparing: under 65 ℃,, get the lipid phase with 5% ethanol (v/v) dissolving recipe quantity film material; With the aquation medium that is preheated to uniform temp, inject the lipid phase with middling speed, hatch 20 min, make the liposome first product.Behind ultrasonic preliminary mixing 2 min of 200 W, 400 W ultra-sonic dispersion, 6 min (3 s that work, intermittently 3 s) through the microporous filter membrane of 0.8,0.45,0.22 μ m, promptly get blank liposome successively.

The preparation of gradient liposome and medicine carrying: adopt the mixture iron exchange resin method to set up the liposome transmembrane gradient, with Farmorubine Hydrochloride solution (medicine fat ratio, 1:10; W/w), hatch 20 min, ice bath termination medicine carrying in 60 ℃ with gradient liposome mixing; Measure envelop rate, the result sees table 6.

Different melbine salt additions are to the influence of envelop rate in the table 6 aquation medium

Annotate: metformin concentration is 200,400,600 mmolL in " 200 ", " 400 ", the final aquation medium of " 600 " expression ^-1

Can know that by table 6 the metformin hydrochloride addition surpasses 200 mmolL in the aquation medium ^-1The time, because the existence of chloride ion can make the envelop rate decrease to some degree, but the addition of working as sulphuric acid metformin and citric acid metformin is no more than 600 mmolL ^-1The time can not produce appreciable impact to liposome encapsulation.

Embodiment 7(MetH) ₄The EDTA-hydrochloric doxorubicin liposome

The basic prescription of its raw material is seen table 7.

Preparation technology: in 65 ° of C water-baths, get the lipid phase with 10% ethanol (v/v) dissolving recipe quantity film material, the water that will be preheated to uniform temp then is (promptly with 200 mmolL ^-1The ethylenediaminetetraacetic acid metformin is the aquation medium), inject the lipid phase with method conventional in the prior art, hatch 30 min, make the liposome first product.Behind ultrasonic preliminary mixing 2 min of 200 W, 400 W ultra-sonic dispersion, 6 min (3 s that work, intermittently 3 s) through the microporous filter membrane of 0.8,0 .45,0.22 μ m, promptly get blank liposome successively.Adopt the mixture iron exchange resin method to set up the liposome transmembrane gradient, (w/w) mixing is hatched 20 min in 60 ° of C for medicine fat ratio, 1:8, promptly gets metformin hydrochloric acid Evacet (i.e. (MetH) for gradient liposome that obtains and drug solution ₄The EDTA-hydrochloric doxorubicin liposome, doxorubicin hydrochloride concentration is 1.67 mgmL in the final preparation ^-1I.e. 2.88 mmolL ^-1, metformin concentration is 0.83 mgmL ^-1I.e. 6.43 mmolL ^-1).

Table 7 liposome membrane material is formed

Embodiment 8(MetH) ₄The long-term shelf-stability of EDTA-hydrochloric doxorubicin liposome is investigated

According to the medicine stability test guideline, with reference to listing product amycin Doxil ^òCondition of storage, (MetH) that under 4 ± 2 ℃, embodiment 1 is obtained ₄The EDTA-hydrochloric doxorubicin liposome carries out long-term stable experiment.Get 3 batches (MetH) ₄EDTA-hydrochloric doxorubicin liposome solution is sub-packed in the cillin bottle, fills nitrogen, sealing, and under 4 ± 2 ℃ of conditions, lucifuge is placed, and respectively at sampling in 0,30,90,150 day, investigates preparation outward appearance, granularity, envelop rate variation, and the result sees table 8.

Table 8 long-term stable experiment result

Table 8 is the result show, the hydrochloric doxorubicin liposome of optimization is under 4 ± 2 ℃ of refrigerated conditions, and mean diameter slightly increases in 150 d, and envelop rate has reduced by 3.0% approximately.

Embodiment 9(MetH) ₄Pharmacodynamic study in the EDTA-epirubicin hydrochloride liposome body

With reference to " embodiment 7 " preparations (MetH) ₄The EDTA-epirubicin hydrochloride liposome.Envelop rate 98.3%, particle diameter 106nm.

The mice of 50 inoculation S-180 tumors is divided into 5 groups at random, i.e. normal saline matched group (NS), Farmorubine Hydrochloride solution group, (MetH) ₄EDTA-gradient liposome group, (NH ₄) ₄EDTA-epirubicin hydrochloride liposome group with (MetH) ₄EDTA-epirubicin hydrochloride liposome group, every group of 10 animals, inoculation in the 0th day, gross tumor volume is measured with slide gauge in the inoculation back, when gross tumor volume increases to 20 ~ 50 mm ³The time, the beginning administration, dosage is counted 5 mg/kg with Farmorubine Hydrochloride, counts 2.5 mg/kg with metformin, and dosing interval is 2 days, and administration number of times is 3 times.The administration continued is measured gross tumor volume, and draws gross tumor volume change curve in time, and the result sees Fig. 1.

Can know by Fig. 1, after the drug withdrawal 20 days, NS group, Farmorubine Hydrochloride solution group and (MetH) ₄The growth of EDTA-gradient liposome group mouse tumor is very fast, (NH ₄) ₄EDTA-epirubicin hydrochloride liposome group mouse tumor has begun recurrence, and (MetH) ₄The complete obiteration of EDTA-epirubicin hydrochloride liposome group mouse tumor is about to Farmorubine Hydrochloride and is loaded into (MetH) ₄Effectively suppressed the recurrence of tumor behind the EDTA-liposome; This pharmacodynamic result has proved the correctness of tumor stem cell hypothesis to a certain extent; Promptly pass through EPR effect (enhanced permeability and retention effect) liposome with Farmorubine Hydrochloride and the common target tumor tissue of metformin; By Farmorubine Hydrochloride to tumor cell and metformin to the killing and wounding of tumor stem cell, reached the treatment tumor and prevented the purpose of tumor recurrence.

In the present embodiment, the dosage of metformin is 2.5 mg/kg, is converted into oral dose and is about 10 mg/kg; Calculate document (Cancer Res, 2009 according to mice 5 mL that drink water every day; 69 (19): mice oral administration of metformin dosage is mg/kg every days 25 7507-7511), and it be successive administration every day, so present embodiment is when reaching the inhibition tumor recurrence; Further reduced the dosage of metformin; Improved the curative effect of metformin so on the one hand, reduced the side effect that metformin possibly bring on the other hand again, for clinical practice has from now on brought convenience at anti-tumor aspect.

Embodiment 10Survival analysis

As object of study, the mice life span is that index is carried out survival analysis after the tumor to connect with mice among the embodiment 9, and the result sees Fig. 2.

Can know by Fig. 2; The 12nd day physiology saline control group (NS) mice begins death to occur after connecing tumor, and all dead in the time of the 52nd day, Farmorubine Hydrochloride solution group mice began to occur dead on the 24th day; And Farmorubine Hydrochloride solution group mice is all dead in the time of the 46th day, (MetH) ₄EDTA-gradient liposome group mice began to occur dead on the 28th day, and is all dead during to the 54th day, (NH ₄) ₄Dead 2 of EDTA-epirubicin hydrochloride liposome group mice the 40th day, the 56th and the 60th day dead 1 and 2 respectively, (MetH) ₄EDTA-epirubicin hydrochloride liposome group mice did not all occur dead in 65 days.

Embodiment 11Drug withdrawal is the tumor-bearing mice survival condition after 20 days

As object of study, drug withdrawal was estimated the mice survival condition after 20 days with the mice among the embodiment 3.The atrophy of discovery: NS group mice right fore is observed in drug withdrawal after 20 days, the fur low in glossiness can't be walked, and survival condition is poor; Farmorubine Hydrochloride solution group mice right fore can't be movable, and health is become thin, the fur low in glossiness, and survival condition is poor; (MetH) ₄EDTA-gradient liposome group mice forelimb can't be movable, and the fur glossiness is relatively poor, and survival condition is relatively poor; (NH ₄) ₄EDTA-epirubicin hydrochloride liposome group mouse tumor has obtained certain inhibition, the mice freedom of movement, and the fur glossiness is better, and survival condition is better; (MetH) ₄EDTA-epirubicin hydrochloride liposome group mouse tumor is approaching to disappear, the mice freedom of movement, and the fur glossiness is good, and survival condition is good.

Embodiment 12(MetH) ₃CA (citric acid metformin) Evacet

Prescription HSPC 3.0 g

CH 1.0?g

PEG2000-DSPE 1.0?g

300 mmolL ^-1(MetH) ₃CA aquation medium adds to 50mL

Method for preparing lipidosome: take by weighing recipe quantity film material and dissolve with 10% ethanol (v/v), get the lipid phase, inject the aquation medium that is preheated to uniform temp with middling speed in 60 ℃; Hatch 20 min, make the liposome first product, handle (pressure is 14000 psi) through microjet; Particle diameter is decreased to 100 nm; Through the microporous filter membrane of 0.8,0.45 and 0.22 μ m, promptly get about 100 nm of mean diameter, the blank liposome of narrow particle size distribution successively.

Gradient is set up: get blank liposome, with composite fibre (ZB-1Na ⁺Type cation exchange fibre and ZB-2Cl ^-The mixed proportion of type anion-exchange fibre is wet apparent volume 1:2) mix place 5 min after, centrifugal 4 min under 2000 rpm set up gradient.

Medicine carrying: the blank liposome behind the desalination (gradient liposome) is mixed than 1:10 (w/w) according to medicine fat with 4 mg/ml doxorubicin hydrochloride solution, and 60 ℃ of following medicine carrying 30 min get (MetH) ₃The CA Evacet, envelop rate is greater than 95%.

Embodiment 13(MetH) ₃The CA Evacet compares with the preparation pharmacodynamics that other adds metformin

With 70 inoculation H ₂₂The mice of tumor carries out random packet (7 groups, 10/group); Be normal saline matched group (NS), amycin solution group (5 mg/kg; Intravenous injection), oral administration of metformin solution group (30 mg/kg), amycin-metformin mixed solution group (5 mg/kg; 2.5 mg/kg, intravenous injection), (MetH) ₃CA Evacet group (5 mg/kg, 2.5 mg/kg, intravenous injection), (NH ₄) ₃CA Evacet and metformin mixed solution group (5 mg/kg, 2.5 mg/kg, intravenous injection), (MetH) ₃CA gradient preparation group (2.5 mg/kg, intravenous injection).Inoculation in the 0th day, the inoculation back is irritated the stomach group and is administered once continuous 10 days every day; Contain the amycin group, respectively at tail vein injection administration in the 3rd, 6,9 day.Animal is normally raised after the administration, and weighing every day mice body weight is observed the growth conditions of mice simultaneously.Animal is put to death in inoculation back the 12nd day, completely peels off Subcutaneous tumor and weighs, and calculates tumour inhibiting rate, and average tumor heavily reaches tumour inhibiting rate and sees table 9.

The different Evacet tumor killing effects of table 9

Annotate: oral administration of metformin solution group dosage regimen is following: connect after the tumor the 3rd day and rise that to give metformin hydrochloride concentration every day be the drinking water of the distilled water of 60 μ g/mL as mice, connecing after the tumor the 3rd, 6,9 day simultaneously is the dosage tail vein injection (NH of 4 mg/kg by doxorubicin concentration ₄) ₃The CA Evacet.

Can know from table 9 result: with (MetH) ₃CA gradient preparation group is compared, the various dosage forms of amycin all have significant tumor killing effect ( P<0.05), wherein (MetH) ₃CA Evacet group have extremely significant tumor killing effect ( P<0.01).(MetH) ₃CA gradient liposome group tumor killing effect minimum (tumour inhibiting rate is 18.58%), (MetH) ₃CA Evacet group tumor killing effect the highest (tumour inhibiting rate is 75.25%); Relatively oral administration of metformin solution group with (MetH) ₃CA Evacet group can be found: oral administration of metformin can not effectively suppress tumor growth under the metformin dosage that equates; With amycin solution group, oral administration of metformin solution group, amycin-metformin mixed solution group and (NH ₄) ₃The CA Evacet is compared with metformin mixed solution group, (MetH) ₃CA Evacet group have extremely the tumor killing effect of significance ( P<0.01), the metformin that the performance drug effect is described is the metformin of water in the liposome.

Embodiment 14Vinorelbine lipoplast

Prescription HSPC 3.0 g

CH 1.0?g

PEG2000-DSPE 1.0?g

300 mmolL ^-1(MetH) ₃CA aquation medium adds to 50 mL

Method for preparing lipidosome: take by weighing recipe quantity film material and dissolve with 10% ethanol (v/v), get the lipid phase, inject the aquation medium that is preheated to uniform temp with middling speed in 60 ℃; Hatch 20 min, make the liposome first product, handle (pressure is 14000 psi) through microjet; Particle diameter is decreased to 100 nm; Through the microporous filter membrane of 0.8,0.45 and 0.22 μ m, promptly get about 100 nm of mean diameter, the blank liposome of narrow particle size distribution successively.

Gradient is set up: get blank liposome, with composite fibre (ZB-1Na ⁺Type cation exchange fibre and ZB-2Cl ^-The mixed proportion of type anion-exchange fibre is wet apparent volume 1:2) mix place 5 min after, centrifugal 4 min under 2000 rpm set up gradient.

Medicine carrying: the blank liposome behind the desalination (gradient liposome) is mixed than 1:10 (w/w) according to medicine fat with 5 mg/ml vinorelbine solution, and 60 ℃ of following medicine carrying 30 min get vinorelbine lipoplast, and envelop rate is greater than 95%.

Embodiment 15The vinorelbine lipoplast pharmacodynamic study

With 40 inoculation H ₂₂The mice of tumor is divided into 4 groups at random, i.e. normal saline matched group (NS), vinorelbine solution group, (NH ₄) ₃CA-vinorelbine lipoplast group with (MetH) ₃CA-vinorelbine lipoplast group, 10 every group.Inoculation in the 0th day, the inoculation back was respectively at the dosage tail vein injection administration by 4 mg/kg in the 3rd, 6,9 day.Animal is normally raised after the administration, and weighing every day mice body weight is observed the growth conditions of mice simultaneously.Animal is put to death in inoculation back the 14th day, completely peels off Subcutaneous tumor and weighs, and calculates tumour inhibiting rate, and the result sees table 10.

The tumor killing effect of the different vinorelbine preparations of table 10

Can know from table 10, (MetH) ₃The tumor killing effect of CA-vinorelbine lipoplast will be far above (NH ₄) ₃The tumor killing effect of CA-vinorelbine lipoplast, and tumour inhibiting rate can be up to 91.2%.

Embodiment 16Daunorubicin liposome

Prescription DPPC 3 g

300 mmolL ^-1(MetH) ₃CA (pH7.0) adds to 100 mL

Method for preparing is following: get the DPPC (two Palmic acid phosphatidylcholines) of recipe quantity, and with dissolved in chloroform, preparation DPPC chloroformic solution.Chloroform is removed in decompression, the preparation lipid film; Add aquation medium (MetH) in 60 ℃ ₃CA solution, dispersed with stirring 30 min make the liposome first product, and high pressure homogenize is handled, and successively through the microporous filter membrane of 0.8,0.45,0.22 μ m, promptly gets blank liposome.With 10% sucrose is the dialysis medium, adopts dialysis to remove to set up liposome transmembrane gradient (the EDTA concentration of interior water is 46 times of outer water).With gained gradient liposome and daunorubicin drug solution (10mg/ml) according to medicine fat than 1:10 (w/w) mixing, hatch 60 min in 60 ° of C, promptly get daunorubicin liposome.Envelop rate is greater than 90%.

Embodiment 17Miaow holder anthraquinone liposome

Prescription EPC 2.5 g

CH 1.0?g

300 mmolL ^-1(MetH) ₃CA (pH7.0) adds to 100 mL

Method for preparing is following: get the EPC (Ovum Gallus domesticus Flavus lecithin) of recipe quantity, CH (cholesterol) prepares EPC, the CH chloroformic solution with dissolved in chloroform.Chloroform is removed in decompression, the preparation lipid film; Add aquation medium (MetH) in 50 ℃ ₃CA solution, dispersed with stirring 30 min make the liposome first product, and high pressure homogenize is handled, and successively through the microporous filter membrane of 0.8,0.45,0.22 μ m, promptly gets blank liposome.Get Na ⁺Type cation exchange resin and Cl ^-The type anion exchange resin mixes than 1:2 with volume (looking humid volume) in right amount, and the preparation ion exchange resin column is handled liposome and eluting and promptly got the gradient liposome.With gained gradient liposome and miaow holder anthraquinone drug solution (10 mg/ml) according to medicine fat than 1:10 (w/w) mixing, hatch 60 min in 50 ° of C, promptly get miaow holder anthraquinone liposome, envelop rate is greater than 95%.

Sphingomyelins is changed EPC also can obtain equifinality.

Embodiment 18The TPT liposome

Prescription HSPC 1.0 g

CH 0.5?g

PEG1000-DSPE 0.1?g

PEG2000-DSPE 0.2?g

Aquation medium: 250 mmolL ^-1Sulphuric acid metformin solution 20 mL of pH7.5

The preparation of blank liposome: take by weighing recipe quantity film material, dissolve with 5% ethanol (v/v), inject the aquation medium that is preheated to uniform temp with middling speed in 65 ℃; Hatch 20 min; Make the liposome first product, after probe is ultrasonic (3 s that work, intermittently 3 s); Through the microporous filter membrane of 0.80,0.45,0.22 μ m, promptly get blank liposome successively.

The preparation of gradient liposome: get ZB-1 type Na ⁺Type cation exchange fibre and ZB-2 type Cl ^-The type anion-exchange fibre mixes than 1:2 with volume (looking humid volume) in right amount, and preparation ion-exchange fibre post is handled liposome and eluting and promptly got the gradient liposome.Medicine carrying: the gradient liposome that obtains is mixed (medicine fat is than 1:10) with topotecan hydrochloride solution, 60 ℃ of medicine carrying 40 min promptly get the TPT liposome, and envelop rate is greater than 90%.

Embodiment 19Freeze-dried type TPT liposome

Prescription HSPC 1.5 g

CH 0.5?g

PEG2000-DSPE 0.5?g

Trehalose 0.25 g

Maltose 0.25 g

Aquation medium: 200 mmolL ^-1Ethylenediaminetetraacetic acid metformin solution 30 mL

The preparation of blank liposome: take by weighing HSPC, CH, the PEG2000-DSPE of recipe quantity, dissolve with 5% ethanol (v/v), inject the aquation medium that is preheated to uniform temp with middling speed in 60 ℃; Hatch 20 min; Make the liposome first product, handle (pressure is 14000 psi), particle diameter is decreased to 100 nm through microjet; Through the microporous filter membrane of 0.8,0.45 and 0.22 μ m, promptly get blank liposome successively.

The preparation of gradient liposome: get ZB-1 type Na ⁺Type cation exchange fibre and ZB-2 type Cl ^-The type anion-exchange fibre mixes than 1:2 with volume (looking humid volume) in right amount; Preparation ion-exchange fibre post is handled liposome and eluting and is promptly got the gradient liposome, adds the trehalose and the maltose of recipe quantity in the gradient liposome; Carry out lyophilization, preparation lyophilizing gradient liposome.

Medicine carrying: the lyophilizing gradient liposome that obtains is redissolved with the glucose injection of sterilized water for injection or 5% or 0.9% sodium chloride injection; Mix (medicine fat mass ratio 1:5) with the cytosine arabinoside drug solution; 55 ℃ of medicine carrying 30 min promptly get the cytosine arabinoside liposome, and envelop rate is greater than 95%.

Explain: adopt present embodiment can prepare two bottled liposomal pharmaceutical prepns; Wherein one bottle is lyophilizing gradient liposome, and another bottle is a drug powder, faces the glucose injection of time spent employing sterilized water for injection or 5% or 0.9% sodium chloride injection etc. and respectively lyophilizing gradient liposome is redissolved; Drug powder is dissolved; With the two mixing, uniform temperature is hatched then, promptly gets the pastille liposome.Two bottled liposomees are that preparation and transportation have brought convenience on the one hand, and the what is more important liposome adopts lyophilized form, helps the long-time stability of preparation and keeping of gradient, and existing with join at present, reduces to the leakage of medicine minimum.

Embodiment 20The irinotecan liposome

Prescription HSPC 3.0 g

CH 1.0?g

PEG2000-DSPE 1.0?g,

200 mmolL ^-1(MetH) ₄EDTA (ethylenediaminetetraacetic acid melbine salt) (pH7.0) adds to 50 mL

Take by weighing HSPC (hydrogenated soya phosphatide), CH (cholesterol), the PEG2000-DSPE of recipe quantity, dissolve with 5% ethanol (v/v), inject the aquation medium that is preheated to uniform temp with middling speed in 60 ℃; Hatch 20 min; Make the liposome first product, handle (pressure is 14000 psi), particle diameter is decreased to 100 nm through microjet; Through the microporous filter membrane of 0.8,0.45 and 0.22 μ m, promptly get blank liposome successively.Adopt Sephadex G-50 to remove (MetH) of outer water ₄(the eluting medium is 10 mmolL to EDTA ^-1The PBS buffer), preparation gradient liposome is pressed medicine fat than 1:8 (w/w) mixing with gained gradient liposome and irinotecan drug solution (10 mg/ml), hatches 40 min in 60 ° of C, promptly gets the irinotecan liposome, envelop rate is greater than 90%.

Embodiment 21The Sutent liposome

Prescription DOPC 2.4 g

CH 0.8?g

PEG2000-DSPE 0.8?g,

The aquation medium adds to 50 mL

The preparation of aquation medium: 300 mmolL that get 20ml pH7.0 ^-1(NH ₄) ₃300 mmolL of CA solution (can also adopt the saline solution such as diethylamine, triethylamine or ethanolamine of citric acid to increase the delay of Met water in liposome) and 20 ml pH7.0 to shorten the medicine carrying time ^-1(MetH) ₃CA solution joins both in the 50 ml volumetric flasks, and to scale, mixing gets the aquation medium with distilled water diluting.

Method for preparing lipidosome: take by weighing recipe quantity film material and dissolve with 10% ethanol (v/v), get the lipid phase, inject the aquation medium that is preheated to uniform temp with middling speed in 60 ℃; Hatch 20 min, make the liposome first product, handle (pressure is 14000 psi) through microjet; Particle diameter is decreased to 100 nm; Through the microporous filter membrane of 0.8,0.45 and 0.22 μ m, promptly get about 100 nm of mean diameter, the blank liposome of narrow particle size distribution successively.

Gradient is set up: getting blank liposome, serves as the dialysis medium with 5% xylitol, adopts dialysis to set up the liposome transmembrane gradient.

Medicine carrying: the blank liposome behind the desalination (gradient liposome) is mixed than 1:10 (w/w) by medicine fat with 5 mg/ml Sutent solution, and 60 ℃ of following medicine carrying 15 min get the Sutent liposome, and envelop rate is greater than 98%.

Embodiment 22The Sutent liposome

Prescription DPPC 2.4 g

CH 0.8?g

PEG2000-DSPE 0.8?g,

The aquation medium adds to 50 mL

The preparation of aquation medium: take by weighing ammonium sulfate 2.6 g, metformin hydrochloride 3.3 g put in the 100 ml beakers, add distilled water 80 ml, and stirring and dissolving is transferred to above-mentioned solution in the 100 ml volumetric flasks,, shakes up to scale with distilled water diluting, promptly gets.

Method for preparing lipidosome: take by weighing recipe quantity film material and dissolve with 10% ethanol (v/v), get the lipid phase, inject the aquation medium that is preheated to uniform temp with middling speed in 60 ℃; Hatch 20 min, make the liposome first product, handle (pressure is 14000 psi) through microjet; Particle diameter is decreased to 100 nm; Through the microporous filter membrane of 0.8,0.45 and 0.22 μ m, promptly get about 100 nm of mean diameter, the blank liposome of narrow particle size distribution successively.

Gradient is set up: getting blank liposome, serves as the dialysis medium with 5% xylitol, adopts dialysis to set up the liposome transmembrane gradient.

Medicine carrying: the blank liposome behind the desalination (gradient liposome) is mixed than 1:10 (w/w) by medicine fat with 5 mg/ml Sutent solution, and 60 ℃ of following medicine carrying 15 min get the Sutent liposome, and envelop rate is greater than 96%.

Adopting uses the same method can prepare the gefitinib liposome.

Embodiment 23 Epirubicin liposome 1

Prescription HSPC 3.0 g

CH 0.05?g

PEG2000-CHS 1.0?g

The aquation medium adds to 50 mL

The preparation of aquation medium: 200 mmolL that get 20 ml pH7.0 ^-1(NH ₄) ₄300 mmolL of EDTA solution and 20 ml pH7.0 ^-1(MetH) ₄EDTA solution joins both in the 50 ml volumetric flasks, and distilled water diluting is to scale, and mixing obtains the aquation medium.

Method for preparing lipidosome: take by weighing recipe quantity film material and dissolve with 5% ethanol (v/v) in 60 ℃; Inject the aquation medium that is preheated to uniform temp with middling speed, hatch 20 min, make the liposome first product; First product is extruded through the continuous circulation of the microporous filter membrane of 0.8,0.45 and 0.22 μ m successively; Filter membrane through 0.1 μ m circulates 5-8 time continuously at last, promptly gets about 120 nm of mean diameter, the blank liposome of narrow particle size distribution.

Gradient is set up: get blank liposome, after hybrid resin (732 cation exchange resiies are wet apparent volume 1:2 with the mixed proportion of 717 anion exchange resin) mixed placement 5 min, centrifugal 4 min under 2000 rpm set up gradient.

Medicine carrying: add a certain amount of histidine in the blank liposome behind desalination (gradient liposome), the dissolving back adds epirubicin solution according to medicine fat than 1:10 (w/w), and 60 ℃ of following medicine carrying 20 min get the epirubicin liposome, and envelop rate is greater than 96%.

Embodiment 24Epirubicin liposome 2

Prescription DSPC 3.0 g

CH 0.5?g

PEG2000-DSPE 1.0?g

The aquation medium adds to 50 mL

The preparation of aquation medium: take by weighing EDTA5.8 g; Sulphuric acid metformin 3.5 g put in the 100 ml beakers, add distilled water 80 ml, and under stirring, adding the about 6 ml regulator solution pH value of ammonia is 7.0; Above-mentioned solution is transferred in the 100 ml volumetric flasks;, shake up to scale with distilled water diluting, promptly get.

Method for preparing lipidosome: take by weighing recipe quantity film material and dissolve with 5% ethanol (v/v) in 60 ℃; Inject the aquation medium that is preheated to uniform temp with middling speed, hatch 20 min, make the liposome first product; First product is extruded through the continuous circulation of the microporous filter membrane of 0.8,0.45 and 0.22 μ m successively; Filter membrane through 0.1 μ m circulates 5-8 time continuously at last, promptly gets about 120 nm of mean diameter, the blank liposome of narrow particle size distribution.

Gradient is set up: get blank liposome, after hybrid resin (732 cation exchange resiies are wet apparent volume 1:2 with the mixed proportion of 717 anion exchange resin) mixed placement 5 min, centrifugal 4 min under 2000 rpm set up gradient.

Medicine carrying: add a certain amount of histidine in the blank liposome behind desalination (gradient liposome), the dissolving back adds epirubicin solution according to medicine fat than 1:10 (w/w), and 60 ℃ of following medicine carrying 20 min get the epirubicin liposome, and envelop rate is greater than 96%.

 

Embodiment 25

The aquation medium is 10mmolL ^-1Metformin hydrochloride and 300 mmolL ^-1Ammonium sulfate (pH6.0) prepares blank liposome according to " embodiment 12 " film material, and gradient is loaded amycin, and envelop rate is greater than 95%.

Embodiment 26

The aquation medium is 50mmolL ^-1Metformin hydrochloride and 250 mmolL ^-1Ammonium citrate (pH 9.0) prepares blank liposome according to " embodiment 12 " film material, and gradient is loaded mitoxantrone hydrochloride, and envelop rate is greater than 95%.

Embodiment 27

The aquation medium is 100mmolL ^-1Metformin hydrochloride and 150 mmolL ^-1Ammonium sulfate and 150 mmolL ^-1Phytic acid ammonium (pH 8.0) prepares blank liposome according to " embodiment 12 " film material, and gradient is loaded a China fir alcohol, and envelop rate is greater than 95%.

Embodiment 28

The aquation medium is 50mmolL ^-1Metformin hydrochloride and 250 mmolL ^-1Ammonium citrate (pH 3.0) prepares blank liposome according to " embodiment 12 " film material, and gradient is loaded topotecan hydrochloride, and envelop rate is greater than 95%.

Embodiment 29

The aquation medium is 100mmolL ^-1Metformin hydrochloride and 100 mmolL ^-1Ammonium sulfate and 250 mmolL ^-1Ammonium citrate (pH 4.0) prepares blank liposome according to " embodiment 12 " film material, and gradient is loaded irinotecan hydrochloride, and envelop rate is greater than 95%.

Embodiment 30

The aquation medium is 100mmolL ^-1Sulphuric acid metformin and 300 mmolL ^-1Sucrose octasulfate triethylamine salt (pH6.6) prepares blank liposome according to " embodiment 12 " film material, and gradient is loaded vincristine or vindesine, and envelop rate is all greater than 90%.

Embodiment 31

The aquation medium is 500mmolL ^-1Metformin salicylic acid sulfonate (perhaps dihydroxy benzenes sulfonic acid salt) (pH5.0) prepares blank liposome according to " embodiment 12 " film material, and gradient is loaded vinorelbine, and envelop rate is greater than 90%.

Embodiment 32

The aquation medium is 1000mmolL ^-1Metformin sucrose octasulfate salt (pH7.0) prepares blank liposome according to " embodiment 12 " film material, and gradient is loaded vinorelbine, and envelop rate is greater than 90%.

Embodiment 33The compound recipe antitumor medicinal liposome

The aquation medium is 100mmolL ^-1Phosphoric acid metformin and 200 mmolL ^-1EDTA melbine salt (pH6.5); Prepare blank liposome according to " embodiment 12 " film material; Gradient is loaded vincristine and amycin (mass ratio 1:2) (perhaps daunorubicin, epirubicin, aklavine replace amycin), and envelop rate is all greater than 95%.

Although the inventor has done in more detail the present invention and has enumerated; But; The content that those skilled in the art is disclosed according to summary of the invention part and embodiment can be made various modifications or/and additional or to adopt similar mode to substitute be obvious to described specific embodiment, and can realize technique effect of the present invention; Therefore, give unnecessary details no longer one by one here.The term that occurs among the present invention is used for the elaboration of technical scheme of the present invention and understanding are not construed as limiting the invention.

Claims (11)

1. one kind contains metformin vesicle class drug-supplying system; It is characterized in that; Described drug-supplying system is is the underlying membrane material with the surfactant, be that the aquation medium prepares blank vesicle with the solution that contains the guanidine radicals cationic compound, then blank vesicle is set up transmembrane gradient and obtains.

2. vesicle class drug-supplying system as claimed in claim 1 is characterized in that, the described general structure that contains the guanidine radicals cationic compound is following:

。

3. vesicle class drug-supplying system as claimed in claim 1 is characterized in that, the said solution concentration that contains the guanidine radicals cationic compound is 10-1000 mmolL ^-1

4. vesicle class drug-supplying system as claimed in claim 2 is characterized in that, the described solution pH value that contains the guanidine radicals cationic compound is between 3.0-9.0.

5. vesicle class drug-supplying system as claimed in claim 1 is characterized in that, described aquation medium is the solution that contains guanidine radicals cationic compound and monovalence, bivalence, trivalent or the formed salt of multivalent anions.

6. vesicle class drug-supplying system as claimed in claim 5 is characterized in that described anion includes but not limited to SO ₄ ^2-, PO ₄ ^3-, HPO ₄ ^2-, H ₂PO ₄ ^-, Cl ^-, citric acid radical, acetate, EDTA ^4-, hexa metaphosphoric acid root, sucrose octasulfate root, tripolyphosphate, pyrophosphate, phosphoglycerol root, fructose diphosphate root, ATP root, phytic acid root, phthalate, M-phthalic acid root, terephthaldehyde's acid group, benzoate anion, M-phthalic acid root, 1,3,5-Benzenetricarboxylic acid root, lactose acid group, dimercaptosuccinic acid root, diethylenetriamine pentaacetic acid root, ethylene glycol bis (2-amino-ethyl ether) tetrem acid group or aminotriacetic acid root.

7. vesicle class drug-supplying system as claimed in claim 1 is characterized in that, adds inorganic or organic base regulator solution pH value such as ammonia, triethylamine, triethanolamine in the described solution that contains the guanidine radicals cationic compound between 3.0-9.0.

8. the application of the described vesicle class of claim 1 drug-supplying system on antitumor drug.

9. like claim 1 or 8 described application, it is characterized in that the mass ratio of guanidine radicals cationic compound and antitumor drug is 0.05-5:1.

10. vesicle class drug-supplying system as claimed in claim 1; It is characterized in that; The preferred phospholipid of described underlying membrane material is selected from least a in the following material: the molecular weight of PEG is 100～10000 among soybean lecithin, Ovum Gallus domesticus Flavus lecithin, EPG, phosphatidic acid, cardiolipin, sphingomyelins, phosphatidic acid serine, phosphatidylinositols, PHOSPHATIDYL ETHANOLAMINE, hydrogenated soy phosphatidyl choline, hydrogenated yolk lecithin, DSPC, dipalmitoyl phosphatidyl choline, dioleoyl phospholipid phatidylcholine, dimyristoyl phosphatidyl choline, two Laurel phosphatidyl cholines, DDPC, two decoyl phosphatidylcholines, two hexanoyl phosphatidylcholines, distearyl phosphatidyl glycerol and sodium salt thereof, two palmityl phosphatidyl glycerols and sodium salt, L-α-GLYCEROL,DIMYRISTOYL PHOSPHATIDYL and sodium salt thereof, two lauroyl phosphatidyl glycerols, two caprinoyl phosphatidyl glycerols, two decoyl phosphatidyl glycerols, two hexanoyl phosphatidyl glycerols, DSPE, two palmityl PHOSPHATIDYL ETHANOLAMINEs, DOPE, two myristoyl PHOSPHATIDYL ETHANOLAMINEs, two lauroyl PHOSPHATIDYL ETHANOLAMINEs, two distearyl phosphatidyl glycerol and sodium salt thereof, two two palmityl phosphatidyl glycerols and sodium salt, two GLYCEROL,DIMYRISTOYL PHOSPHATIDYL and sodium salt thereof, two two lauroyl phosphatidyl glycerols, distearyl phosphatidylinositols, two palmityl phosphatidylinositols, dioleoyl phospholipid acyl inositol, two myristoyl phosphatidylinositols, two lauroyl phosphatidylinositols, palmityl oleoyl phosphatidylcholine, the inferior oleoyl phosphatidylcholine of palmityl, the inferior oleoyl phosphatidylcholine of stearoyl, stearoyl oleoyl phosphatidylcholine, stearoyl arachidonic phosphatidyl choline, DSPE-PEG, DPPE-PEG, DMPE-PEG, DLPE-PEG, wherein said DSPE-PEG, DPPE-PEG, DMPE-PEG, the DLPE-PEG.

11. vesicle class drug-supplying system as claimed in claim 10 is characterized in that, adds the PEG derivant in the described underlying membrane material, its consumption is 0 ~ 50% of the total consumption of lipid.

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