CN103622909A - Cardiolipin-containing new liposome preparation, and its application in antitumor drugs - Google Patents

Abstract

A new mitoxantrone liposome preparation of components comprising cardiolipin and the like is internationally prepared in the invention for the first time, wherein a preparation method of a mitoxantrone liposome and the characterization characteristics of the preparation are described, and researches on the amplification of the technology of the preparation, the endozoic pharmacokinetics, the medicinal toxicity and the antitumor effect are also carried out. The novel preparation has the characteristics of high entrapment rate, strong stability and the like, and solves the defects of short half life, strong toxic side effects comprising marrow inhibition and the like, limited antitumor effect and the like of routine mitoxantrone preparations in the clinical application process. The researches in the invention prove that the above novel liposome dosage form has more advantages than other dosage forms.

Description

A kind of liposome novel preparation containing cuorin and the application in antitumor drug thereof

Technical field

The present invention relates to the research of the aspects such as the preparation method of mitoxantrone liposome and relevant drug safety, pharmacokinetics, therapeutic efficiency.

Research background

At present, tumor remains the principal disease of harm humans health, and its fatality rate is still in front three in all diseases, and its reason is mainly because tumor is difficult to be diagnosed in early days, once by after diagnosis often in the middle and advanced stage stage.At present, the Therapeutic Method of tumor remains surgical operation therapy, radiotherapy and chemotherapy.The patient who has shifted in middle and advanced stage stage tumor for those, the tumor patient after undergoing surgery and cannot perform the operation or the tumor patient of radiotherapy, chemotherapy is occupied very important status in the treatment of tumor, to delaying patient's life cycle, increase patient's therapy apparatus, can wait the very important effect that plays.But conventional chemotherapy medicine often exists many defects in tumor therapeutic procedure, because its molecular weight is little, in body, with causing the medicine peak concentration in important biorgan too high in plasma metabolism process, cause the toxic action of important biorgan and bring serious toxicity to patient; Because chemotherapeutics molecular weight is little, in metabolic process, by glomerular filtration, by kidney, disposed the metabolic half life making in its body very soon in vivo very short simultaneously, seriously reduced its therapeutic effect.Therefore, overcome chemotherapeutics existing major defect in oncotherapy, increase the half-life of medicine, change medicine metabolic pathway, reduce drug toxicity, improve curative effect of medication and become the primary problem solving in chemotherapeutics process of clinical application.

It is unique that liposome has advantages of as a kind of novel pharmaceutical carrier, by liposome system, carries medicine, can protect medicine not to be degraded in blood circulation, reduces the clearance rate of medicine in kidney, thus prolong drug half-life of metabolism in vivo; By this carrying system, can change the medicine distribution of each histoorgan in vivo simultaneously, reduce the peak concentration of medicine in important biorgan and alleviate the toxic and side effects that medicine produces body; By increasing self physical ability of the metabolic half life of medicine and body, improve the treatment curative effect of medicine; Moreover the applied preparation raw material of Liposomal formulation itself has biodegradability, can not produce immunoreation and various toxic and side effects to body, be therefore a kind of good medicament carrier system.

First mitoxantrone or mitoxantrone hydrochloride were synthesized by U.S. Murdock and Lederle laboratory in 1979 and prove its anti-tumor activity.Mitoxantrone molecular structure and doxorubicin are close, and there is plane aromatic rings and be easy in the base pair of intercalation of DNA double helix, be a kind of embedded type TopoII inhibitor, it by with DNA interconnection, suppress TopoII, cause DNA chain interruption, thus performance antitumor action.。Mitoxantrone also has inhibitory action to RNA synthetic, is cell cycle nonspecific agent (CCNSA).Mitoxantrone can with other drug coupling, also can be applied to separately therapeutic field of tumor.Mitoxantrone has antitumor curative effect to leukemia, lymphoma, breast carcinoma, carcinoma of prostate etc., especially to intractable, intractable leukemia is evident in efficacy, is recently used as again immunosuppressant and for the treatment of multiple sclerosis.But mitoxantrone is similar to other anthracycline antibiotics (as amycin), has more serious toxic and side effects, can cause bone marrow depression, and there are the myocardial toxicity of dose dependent, the serious toxic and side effects such as gastrointestinal toxicity and alopecia.Studies confirm that, mitoxantrone short term toxicity and long term toxicity are all low than amycin.Because free mitoxantrone medicine enters most of (more than 95%) after blood, be combined with plasma protein, efficiency is lower, but uses can cause toxic reaction again in a large number, thereby has limited greatly its clinical practice.The object of this project research is intended to solve the limitation of mitoxantrone in process of clinical application, utilize the liposome containing cuorin to wrap up mitoxantrone, prepare a kind of novel mitoxantrone Liposomal formulation, thereby reduce medicine clearance rate in vivo, change the metabolic pathway of medicine, reduce the toxic and side effects of medicine to body, and improve its antitumor curative effect.At present, for mitoxantrone or mitoxantrone hydrochloride, in the limitation of clinical use, both at home and abroad the dosage forms such as the nanoparticles of mitoxantrone or mitoxantrone hydrochloride, albumin microsphere and solid lipid nanoparticle are studied, but effect is all undesirable.Domestic at this area research also in the stage at the early-stage, both at home and abroad the report of relevant mitoxantrone liposome only has several pieces, the envelop rate that the deficiency of its result of study and defect part are to be wrapped medicine is lower; The pharmacokinetic parameter that is wrapped medicine do not have be improved significantly; Antitumous effect is unsatisfactory etc.Application yet there are no other reports containing the Liposomal formulation of the liposomal encapsulated mitoxantrone of cuorin.

Summary of the invention

The present invention has carried out the pilot scale of the prescription of mitoxantrone Liposomal formulation, preparation technology's optimization and preparation process to amplify research, solve the problems such as liposome encapsulation is low, poor stability, clinical practice complicated operation, can obtain high envelop rate, high stability, clinical administration is simple to operate and can repeat to amplify and produce the mitoxantrone Liposomal formulation that meets quality standard.

In mitoxantrone liposome of the present invention, can contain the compound that any various neutrality or charged liposome form material and be considered to like this such as cuorin to be combined with mitoxantrone.It can be amphipathic molecule that described liposome forms material, such as phospholipid, and phosphatidylcholine, two palmityl Nyingchi County choline, Phosphatidylserine, cholesterol etc., they form liposome in polar solvent.Although can use the mitoxantrone of wide concentration range in this preparation, the most useful concentration is in the scope of 0.5-2mg/ml.The mol ratio of mitoxantrone and lipid components also can extensively change, but the most useful scope is about 1: 10-1: 20.If necessary, the size of mitoxantrone liposome can be used the filter membrane of different size to control.

By resulting mitoxantrone liposome is applied in to the pharmacokinetic in beasle dog, analyze said preparation drug metabolism situation in vivo, confirmation mitoxantrone Liposomal formulation has good pharmacokinetics test parameters, has solved that the conventional mitoxantrone preparation half-life is short, kidney clearance rate fast, medicine the problem such as accumulates at cardiac muscular tissue's middle and high concentration.

The toxicity research of mitoxantrone Liposomal formulation in Mice Body, further confirm Liposomal formulation and can change medicine metabolic pathway in vivo, reduce peak concentration and the content of medicine in the vitals such as cardiac muscular tissue, thereby reduce the toxic and side effects that medicine produces body, solved conventional mitoxantrone preparation has the higher problems such as toxic and side effects in clinical practice.

The research of the antitumor action of mitoxantrone Liposomal formulation in nude mouse, by utilizing Mus source leukemia animal model and people source breast cancer animal model, confirm mitoxantrone Liposomal formulation and had better antitumor curative effect than conventional formulation, be expected to solve the undesirable problem of conventional mitoxantrone preparation for treating effect, can further improve clinical treatment index.

Detailed Description Of The Invention

The object of the invention is to prepare the novel mitoxantrone liposome nanometer formulation of high envelop rate, high stability.Said preparation should meet clinical administration simple to operate, can repeat to amplify the requirements such as productions, and can further reduce the drug toxicity of mitoxantrone, the therapeutic index of raising medicine.By correlational study of the present invention, can solve the limitation of mitoxantrone in process of clinical application, reach better antitumor curative effect.

Object of the present invention by the following method step realizes:

The preparation method of liposome can be selected conventional method for preparing lipidosome, any means all can, as long as related preparation method does not affect liposome the loading of Drug-Mitoxantrone is sealed, the present invention mainly adopts thin film aquation method and ammonium sulphate gradient to prepare mitoxantrone liposome, and by lyophilization, carries out pilot scale and amplify research.

Thin film aquation method, first phospholipid and cholesterol are mixed according to definite proportion row, be dissolved in chloroform-alcoholic solution, utilize Rotary Evaporators dry under vacuum condition in mixture, under 30 ℃ of conditions, form lipid membrane, dried residue continues time drying process process 10min again after occurring.Secondly certain density mitoxantrone saline solution is joined and on lipid film, make lipid film aquation 1～3h, carrying out afterwards vortex vibrates to suspension in homogeneous state, again sample is carried out to supersound process to obtain the mitoxantrone of liposome, i.e. mitoxantrone Liposomal formulation.

First ammonium sulphate gradient, after precise weighing be dissolved in phospholipid and cholesterol in organic solvent according to a certain percentage, under the effect of nitrogen current, forms thin film, and the vacuum of spending the night is except organic solvent.Utilize ammonium sulfate to carry out aquation 1～3h to lipid film.To mixture probe type ultrasonic, can obtain blank liposome.Next sets up ammonium sulphate gradient, and method, for blank liposome is loaded in bag filter, is placed in the conical flask that fills normal saline, in Mi Sai and water-bath Turning motion instrument, dialyses, and replaces the outer medium of bag filter at set intervals with normal saline.Finally carry out medicine loading, method, for add mitoxantrone sodium chloride solution in liposome after dialysis, is adjusted mitoxantrone concentration with normal saline.

Lyophilization refers to the cold drying technology that adopts, by repeatedly sealing, lyophilizing and again merge and realize higher envelop rate.Lyophilization provides good solution for improving the stability of liposome storage life, and it has changed the unstable and oxidizable shortcoming of liquid lipidosome, has process stabilizing, is suitable for suitability for industrialized production, and quality is easy to control and the feature such as product stability is good.

Phospholipid and cholesterol are dissolved in to chloroform with the ratio of 1: 1, and logical nitrogen rotary evaporation adds containing 2% polyvidon to the complete evaporate to dryness of chloroform, and the phosphate buffer of pH7.4 soaks 2h.Under turbine mixer, shake that 10min makes it to be distributed into bottle after complete miscibility, the how empty solid of a visible block white in bottle after freeze dryer Program lyophilization 24h.Take out bottle, dried lipid article is salable to be stored in room temperature or 4 ℃ of refrigerators.While preparing mitoxantrone liposome, by the mitoxantrone saline solution of 1mg/ml, mix with the lipid of lyophilizing, medicine fat mol ratio is controlled at 1: 15.Mixture is aquation 2h at room temperature, and supersound process is 10 minutes afterwards.Obtain mitoxantrone Liposomal formulation.

In the preparation process of mitoxantrone liposome, its liposome material that can Gong select has a variety of, and available material comprises natural, synthetic and semisynthetic biocompatible materials.Material can be hydrophilic or hydrophobic.Can select with positive charge or with negative charge and electroneutral material the material of preparing as liposome.Suitable liposome is prepared material and is comprised: phospholipid, fatty acid, sterols etc.If liposome is further optimized, optional with cuorin, phosphatidylcholine, cholesterol, Phosphatidylserine, phosphatidylinositols, tocopherol, even can introduce the stability that glycosyl chain improves liposome.

The preparation process of mitoxantrone liposome should be noted, the solvent of lipin dissolving body should be suitable, and the kind of solvent can be polarity, low pole, neutral or nonpolar.Such as selecting: acetic acid, propanoic acid, chloroform, acetone, triethylamine etc., also can comprise sterile water for injection.The choice criteria of solvent should be the easy evaporative removal of solvent and do not produce residually, or its residual quantity is within allowing limit.Can utilize several different methods by the removal of solvents of introducing in liposome preparation process, for example can select Filtration, centrifuging and the way of distillation to remove coordinative solvent, but best scheme is to select under vacuum condition, to take the method for distillation to remove solvent, also can adopt above several different methods to remove solvent under the prerequisite that does not affect mitoxantrone medicine and liposome stability.

Liposome of the present invention can be introduced cuorin, and cuorin can be any kind, comprises natural and synthetic.In the preparation process of liposome, can add suitable solvent that cuorin is dissolved, the solvent that dissolves cuorin should be easily to remove, and solvent residual amount should be in allowing limit.Can first utilize solvent that cuorin is dissolved, then add mitoxantrone, after also cuorin and mitoxantrone can being mixed, together be dissolved among solvent.Because cuorin is with negative charge, under electrostatic attraction effect, add cuorin can improve the envelop rate of mitoxantrone in liposome, the negative charge of plasma membrane surfaces can also increase the repulsive interaction between liposome, improves the spatial stability of liposome.Therefore under the prerequisite that does not affect medicine stability and therapeutic effect, can in mitoxantrone liposome, introduce a certain amount of cuorin and increase liposome to mitoxantrone envelop rate.Electronegative phospholipid similarly also has phosphatidylinositols, Phosphatidylserine, also can according to circumstances be applied in the middle of the preparation of liposome.

Liposome with electric charge can be the distinctive electric charge of liposome itself, also can be the electric charge that liposome shows under certain pH condition, for example under pH neutrallty condition, can prepare the liposome with negative charge, the selection formula of liposome can comprise lecithin, cholesterol, cuorin, phosphatidylinositols, Phosphatidylserine etc.Under this condition, also can prepare the liposome with positive charge, the selection formula of liposome can comprise phosphatidylcholine, stearylamine and cholesterol etc. simultaneously.The liposome of more optimizing is prepared the material of scheme and is selected to comprise cuorin, phosphoric acid lecithin, cholesterol.

In above-mentioned liposome, the relative molal weight of mitoxantrone and lipid is than there being a suitable scope, and preferred scope is 1: the more preferred scope of 1-30 is 1: 1-25, preferred scope are 1: 1-20, most preferred ratio were at 1: 15.

The composition of the preferred Liposomal formulation of above-mentioned liposome comprises cuorin, phosphatidylcholine and cholesterol.Three's preferred proportion scope can be controlled in 0.1-5: 1-25: 5-10.Also can further optimize proportioning, proportion is controlled to 0.3-3: 5-20: 6-9.Preferred scheme also can be by proportion control at 0.5-2: 7-15: 6-8.Most preferred scheme can be by three's proportion control at 0.8-1.5: 9-11: 6.5-7.The Lipidosome of optimizing also can comprise the related substances such as the antioxidants such as a-tocopherol and raising transfection efficiencies such as arginine, polymine and raising liposome stability.Wherein the concentration range of a-tocopherol can be controlled in more than 0.1% or under 6%.Arginic concentration proportioning is within the scope of 1%-20%, and the concentration proportioning of polymine is in 2%-25% concentration range.

In liposome preparation process, can carry out the formation of the operation induction liposomees such as aquation of adipose membrane, the solution of aquation can be selected polar solvent, specifically can comprise normal saline solution and PBS solution, and solution can further be dissolved with mitoxantrone.After adding, solution can select multiple hybrid mode to carry out the parcel of adipose membrane to medicine.The formation that DL oscillatory process should be liposome provides enough shearing forces, for example, can adopt magnetic agitation, and the modes such as vortex vibration, supersound process are carried out the preparation of liposome.Preferred method can adopt the mode of vortex vibration, and the liposome form obtaining is multilamellar liposome, if expect, unilamellar liposome can be by membrane filtration or the further mode such as ultrasonic.

In pilot scale amplification process, can carry out lyophilizing subpackage to the liposome making, in freeze-drying process, can add the protective agent that lyophilizing is relevant, protective agent can be monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyhydric alcohol and other water-soluble high-molecular substances.Wherein disaccharide be use be at most also the most effective freeze drying protectant, conventional disaccharide has sucrose or trehalose, wherein sucrose can be controlled in 5 with the ratio of lipid: 1-10: in 1 scope; Trehalose can be controlled in 3.5 with the ratio of lipid: 1-12.5: in 1 scope.But highly preferred scheme should be by sucrose with the proportion control of lipid 8.5: 1-10.5: in 1 scope interval; Trehalose can be controlled in 9.0 with the ratio of lipid: 1-10.5: in 1 scope interval.The liposome that lyophilizing obtains should be powder type or agglomerate thing form.The liposome of freeze-dried type should comparatively easily dissolve again.

The normal saline solution of the mitoxantron solutions of equal volume and equal volume is added in the bottle that is loaded with lyophilizing lipid, mixture is carried out to the hydration under room temperature, time is controlled at 20 minutes, carries out afterwards violent vibration under vortex agitator, and vibration duration is controlled at 3 minutes.After having vibrated, also to carry out to mixture the supersound process of 15 minutes.Utilize the method the envelop rate of mitoxantrone can be controlled on 90%.

The present invention is studied the sign of resulting liposome, first in appearance, by observing the particle diameter of visible liposome even under light microscopic, and without lumps thing, sees through electron microscopic observation and can see more clearly liposome particles of uniform size.

Secondly, for Liposomal formulation, the stability of particle diameter to product, pharmacokinetics behavior and drug effect impact are very large.In order to make the size of liposome can composite demand, can control with the polycarbonate membrane of small-bore, should select according to the liposome size that will obtain the specification of filter membrane.The present invention requires liposome can not leak in tissue through vascular space at the blood vessel place of normal structure, when treatment tumor, the vascular permeability of tumor tissues increases, make liposome to leak out to tumor tissues through tumor vessel, require to avoid again the generation of thrombosis phenomenon simultaneously, can bring into play better therapeutical effect.The present invention carries out particle size determination by employing ultramicroscope and particle size analyzer to resulting mitoxantrone Liposomal formulation, and the liposome particle size range of requirement should be below 150nm, and preferred scope is 100～120nm.

Again, can utilize several different methods to measure the envelop rate of liposome, for example, can adopt ultracentrifugal method, speed setting is arrived to 16000rpm, carry out the centrifugal of 30min～1h, supernatant is carried out to the concentration that Liquid Detection is measured mitoxantrone in supernatant, calculate the envelop rate of sample; Or by the method for dialysis, mitoxantrone liposome is placed among bag filter, the molecular cut off of dialyzer is probably at 8000-14000, bag filter is placed in the normal saline 16-20 hour that dialyses, after dialysis, utilize methanol to dissolve liposome, after sample dissolution, utilize HPLC to detect mitoxantrone in liposome or utilize spectrophotometer to detect, detection wavelength is 658nm.The computational methods of envelop rate are: dosage * 100% that is wrapped in mitoxantrone amount/mitoxantrone in liposome after the envelop rate of mitoxantrone liposome (%)=dialysis.In the present invention, require the envelop rate of mitoxantrone liposome should be controlled on 80%, further can bring up on 85%, further can bring up on 90%.Through study tour, find that the envelop rate of mitoxantrone is subject to the PH impact of buffer, through investigating, draw when pH value is within the scope of 7-10, can significantly measure mitoxantrone and have higher envelop rate, more preferred scope is that pH value is 8-10, most preferred scope is that pH value is 9, and mitoxantrone envelop rate now may reach more than 90%.

May there is multiple different variation in liposome, as phospholipid can be oxidized and be hydrolyzed, generate the phospholipid of short chain in put procedure, and may form and have deliquescent derivant; The physical changes such as liposome also can condense in addition, fusion, thus the seepage of encapsulate substances caused.Therefore Liposomal formulation must have good stability at duration of storage, brings into play in vivo its slow releasing function and should keep certain size and integrity before.In the present invention, investigated specially the stability of mitoxantrone novel form, its its envelop rate within 8 hours, in steady statue, has good stability.

In the present invention, detected especially the Zeta point position of liposome, because Zeta potential is the tolerance to mutual repulsion or captivation intensity between granule.Molecule or dispersed particle are less, and Zeta potential is higher, and system is more stable, dissolve or disperse to resist gathering.By the present invention, the research of the Zeta potential of novel lipid body preparation is found to the system of mitoxantrone novel lipide dosage form is more stable.

The mitoxantrone liposome arriving involved in the present invention, except introduce crude drug mitoxantrone and parcel adjuvant lipid in system, also relates to the excipient of medicine finished product.Excipient comprises various forms, can be liquid, semi-solid, solid-state; Can be filler, preparaton and liquid diluent.Selected excipient should be nontoxic.

Suitable excipient possesses following characteristics: the stability that can fully maintain material medicine mitoxantrone; Excipient can be water miscible also fat-soluble; The interpolation of excipient does not cause untoward reaction; The application of excipient does not affect the drug effect of medicine.The kind of excipient can be varied, for example, can be some lipoid material and Ester, Polyethylene Glycol etc., or the mixture of these materials all can be used as excipient and is utilized.The medicine of different dosage form can add excipient, comprises powder, water preparation, spray, cream, gel, tablet, electuary, capsule, pill, Emulsion, paste etc.

This research mitoxantrone liposome should preferably exist with above-mentioned dosage form, shared proportion should be at the 0.1%-50% of preparation toatl proportion, the scope that more has choosing is 0.1%-40%, and preferred scope should be positioned at 0.1%-30%, and most preferred scope should be positioned at 0.1%-25%.Can utilize conventional or generally acknowledged method that mitoxantrone is mixed with suitable excipient (single excipient or various ways admixed excipients), prepare the medicine of above dosage form.

Mitoxantrone liposome involved in the present invention can reduce the drug-resistant effect of cancerous cell to mitoxantrone.By the research of the overriding resistance mechanism to novel mitoxantrone Liposomal formulation, choose overriding resistance breast cancer tumour cell line, the mitoxantrone, the blank liposome that add wherein different dosage form, and introduce positive control, by fluorescence excitation method, investigate the picked-up situation of cell to mitoxantrone, found that and adopt Lipidosome can slow down the drug resistance tendency that mitoxantrone treatment tumor produces, the application of Lipidosome simultaneously also can be lowered the drug resistance tendency of tumor cell to other antitumor drug.Such as having lowered the drug resistance tendency of tumor cell to chemotherapeutics paclitaxel, amycin, daunorubicin etc.Therefore in tumor therapeutic procedure, can, by this mitoxantrone liposome with other chemotherapy drugs in combination application, also can in tumor therapeutic procedure, the mitoxantrone liposome of this research be applied respectively with other chemotherapeutics.

Route of administration of the present invention is varied, can select oral administration, intravenously administrable, topical, intraperitoneal administration etc., but first-selected parenteral route administration, more preferably administering mode is intravenously administrable.

In liposome, the amount of mitoxantrone is weighed with unit dose, and unit dose can be the dosage of every day, can be also dosage on the half, can be also dosage on the 1/3rd.Mitoxantrone in liposome can be one or more unit dose.For example, mitoxantrone can be 1,2,3,4 unit dose, can also be 1/4,1/3,1/2 unit dose.Unit dose is for weighing the dosage of single-dose mitoxantrone.

In the present invention, by the research of the novel preparation cells in vivo poison of mitoxantrone liposome, find that the mitoxantrone dosage of the leukemia tumor-bearing mice that weight average is 20-22g can be controlled in 10mg/kg-50mg/kg aspect dosage; More preferred dosage regimen is 15mg/kg-45mg/kg; More preferred dosage regimen can be controlled at 20mg/kg-40mg/kg by dosage; Highly preferred dosage regimen can be controlled at mitoxantrone dose in 30mg/kg-35mg/kg dosage range.For people, with the people's of 60kg body weight dosage, mitoxantrone dose can be controlled in the scope of 1.3mg/kg-6.5mg/kg; More preferred dosage can be controlled at mitoxantrone dose in the scope of 1.9mg/kg-5.8mg/kg; More preferred dosage can be controlled at mitoxantrone dose in the scope of 2.5mg/kg-5.2mg/kg; Highly preferred dosage can be controlled at mitoxantrone dose in the scope of 3.8mg/kg-4.5mg/kg.But can be according to specific circumstances, such as medication patient's disease severity, the factor of the aspects such as character of patient's body weight, the disease that takes a disease is adjusted dosage to adapt to the needs of concrete condition.The standard of dosage can be heightened under certain conditions, the standard of dosage can be turned down under certain conditions.Applicable dosage should be drug effect and the hypotoxic dosage having possessed.

The application of Lipidosome does not weaken mitoxantrone as the drug effect of antineoplastic agent, on the contrary, in the situation that mitoxantrone dosage is identical, the application of Lipidosome is compared and is possessed better drug effect with independent application mitoxantrone, and the inhibition that mitoxantrone liposome increases gross tumor volume is more remarkable.In addition utilize Lipidosome can weaken the drug toxicity that mitoxantrone causes, in the situation that mitoxantrone dosage is identical, Lipidosome can better reduce because drug toxicity causes laboratory animal mortality rate.

The mitoxantrone Liposomal formulation the present invention relates to can prolong drug action time.In research process, to having carried out pharmacokinetic in beasle dog body, by more known with the independent mitoxantrone medicine that dissociates that uses to using mitoxantrone Liposomal formulation, the Liposomal formulation of mitoxantrone can obviously increase the peak serum concentration of mitoxantrone.In addition, Lipidosome drug half-life and the peak area under drug-time curve in animal body all will be significantly higher than conventional mitoxantrone preparation.

Aspect medicine tissue distribution, in Mice Body, common mitoxantrone is compared accumulation in ， cardiac muscular tissue with lipid build mitoxantrone will be far away higher than the latter, and the former also will be significantly higher than the latter at the accumulation at lung, nephridial tissue position.And in the drug distribution of liver, spleen tissue, lipid build mitoxantrone will be higher than plain edition mitoxantrone.The main side reaction of mitoxantrone medicine is to cause experimenter's myocardial toxicity, and can reduce mitoxantrone in the accumulation of myocardial sites by the known lipid build of the drug metabolism accumulation distribution mitoxantrone in Mice Body, reduces its myocardial toxicity.

The Lipidosome relating in the present invention has improved the picked-up of mitoxantrone medicine greatly, has brought into play the curative effect of medicine, has obviously suppressed the growth of tumor, reduces the toxic and side effects of medicine, has extended the action time of medicine, has greatly improved oncotherapy effect.

The present invention is described in detail above, the present invention is described further now to utilize embodiment, and embodiment is only for explaining goal of the invention, rather than be used for limiting the present invention.

The specific embodiment

Embodiment 1

The present embodiment is a kind of preparation method of mitoxantrone liposome (LEM).

By lipid and cholesterol (distearoyl phosphatidylcholine/cholesterol=55/45 or dimyristoyl phosphatidyl choline/cholesterol=55/45, in formula, have a small amount of hydrocholesterol) be jointly dissolved in the middle of chloroform the dry adipose membrane that forms in the situation that passing into nitrogen.Add afterwards the 300mM citrate buffer solution of pH4.0 to carry out aquation.Resulting multilamellar vesicle liposome will, through 5 freeze thawing treatment, be extruded by the polycarbonate leaching film of 100nm afterwards.Under 632.8nm, carry out dynamic optical scanning scattering and detect liposome particle diameter.

Mitoxantrone is loaded and is encapsulated in liposome by the pH gradient of cross-film, first blank liposome is hatched to 10min at 65 ℃, then adding mitoxantrone to medicine fat mass ratio is 1: 10, get the medicine of 1.0ml and the mixture of liposome, add wherein 350ul, the N2HPO4 buffer of 0.5M is adjusted to pH=7.2 by pH=4.0.The mixture obtaining is hatched to 15min under 50 ℃ of conditions.

The liposome mean diameter that the method makes is 100-120nm, and envelop rate reaches medicine stability in 90%, 8h and reaches 94%.

Embodiment 2

The present embodiment is the another kind of preparation method of mitoxantrone liposome (LEM).

By mitoxantrone, cuorin, phosphatidylcholine, cholesterol according to 1: 1: 4.5: 3 mixed in molar ratio, in organic solvent, is evaporated organic solvent afterwards, obtains dry drug-to-lipid film.Utilize 6% aqueous trehalose to carry out aquation to film, by dialysis remove do not wrap into mitoxantrone, and envelop rate is measured, solution concentration is measured by HPLC.

The envelop rate of gained liposome of the present invention is 79.41 ± 1.26%.

Embodiment 3

The present embodiment is the another kind of preparation method of mitoxantrone liposome (LEM).

Cuorin, phosphatidylcholine, cholesterol be take to the ratio that mol ratio is 1: 6.8: 10 and be lyophilized into powder.Mitoxantrone be take concentration and is dissolved in normal saline as 1mg/ml, mixes with dried lipid.Medicine fat mol ratio is 1: 12.5.Mixture is aquation 2h under room temperature, ultrasonic 10min.Can obtain the liposome of parcel mitoxantrone.In liposome, the amount of mitoxantrone is measured in 658nm place by spectrophotometer.Entrapment efficiency calculates by following formula: envelop rate=(drug level before the drug level/dialysis after dialysis) * 100%.

The envelop rate of the preparation-obtained mitoxantrone liposome of said method is 94.62 ± 3.19%, and the particle diameter of gained liposome is 57.63 ± 1.24nm, and has good stability, and under room temperature, mitoxantrone liposome reaches more than 95% with interior stability for 8 hours.

Embodiment 4

The present embodiment is the another kind of preparation method of mitoxantrone liposome (LEM).

The formula of mitoxantrone liposome comprise the mitoxantrone of 3 μ M, the phosphatidylcholine of the Phosphatidylserine of 3 μ M, 12 μ M, the cholesterol of the cuorin of 2.5 μ M and 6.8 μ M.Specifically mitoxantrone is dissolved in to dichloromethane solution altogether with cuorin, phosphatidylcholine is dissolved in to hexane, cholesterol is dissolved in to chloroform, according to the amount in formula, phosphatidylcholine solution and cholesterol solution are added in mixed solution and are stirred, 37 ℃ or 37 ℃ of following vacuum evaporation organic solvents must be dried plasma membrane.Add 3ml physiological saline solution to carry out vortex mixed, aquation, supersound process obtains unilamellar liposome afterwards.

The envelop rate of the preparation-obtained mitoxantrone liposome of said method is 84.04 ± 0.53%, and the particle diameter of gained liposome is between 150-200nm, within 8 hours, with interior stability, reaches 90%.

Embodiment 5

The present embodiment is the another kind of preparation method of mitoxantrone liposome (LEM).

Mitoxantrone is dissolved in to the cuorin solution dissolving with chloroform, mitoxantrone and cuorin are 2.5 μ M.Evaporation, except organic solvent, obtains dry medicine-fat complexes.In complex, add the 15mol phosphatidylcholine that is dissolved in ethane and the 8mol cholesterol that is dissolved in chloroform.Stir the mixture, below 37 ℃ or 37 ℃, vacuum evaporation organic solvents obtain desciccator diaphragm.In desciccator diaphragm, add 3ml physiological saline solution, vortex vibration aquation obtains pastille multilamellar liposome.Multilamellar liposome is carried out to supersound process and obtain unilamellar liposome.

The envelop rate of the preparation-obtained mitoxantrone liposome of said method is 82.74 ± 2.52%, and the particle diameter of gained liposome is between 120-180nm, within 8 hours, with interior stability, reaches 92.5%.

Embodiment 6

The present embodiment is the another kind of preparation method of mitoxantrone liposome (LEM).

Cuorin, phosphatidylcholine, cholesterol be take after the ratio precise weighing that mol ratio is 1: 6.8: 10 and be dissolved in chloroform, under the effect of nitrogen current, form thin film, the vacuum of spending the night is except organic solvent.Utilize the ammonium sulfate (concentration is 0.12mol/L) of 300mM to carry out aquation 1h to adipose membrane, hydrating condition is 60 ℃.Probe type ultrasonic 10min, obtains blank liposome.

The foundation of ammonium sulphate gradient: blank liposome is loaded in bag filter, is placed in the conical flask that fills normal saline, and close plug in 37 ℃ of water-bath Turning motion instrument dialysed, and every 1h, with fresh normal saline, replaces medium outside bag filter.Medicine loads: after dialysis, in liposome, add mitoxantrone (mg) sodium chloride solution, the normal saline of take is adjusted in liposome solutions mitoxantrone concentration and put 37 ℃ of water bath heat preservation 15min and get final product as 1mgmL-1.

The liposome mean diameter that the method makes is 70-100nm, and envelop rate reaches more than 92%, and medicine stability is good, and under room temperature, mitoxantrone liposome reaches more than 95% with interior stability for 8 hours.

Embodiment 7

The present embodiment is the another kind of preparation method of mitoxantrone liposome (LEM).

This preparation method needs previously prepared go out mitoxantron solutions and blank liposome, afterwards both is mixed, and obtains mitoxantrone liposome.The composition of mitoxantrone liposome solutions comprises mitoxantrone hydrochloride, sodium acetate (0.004%), sodium chloride (0.75%), the acetic acid (0.043%) of 1.5mg/ml, and the pH of solution is 3.3-4.2.

First prepare blank liposome.Get the 11kg tert-butyl alcohol, add wherein 2.5g tocopherol acid succinate.The mixing of 10 minutes of mixture experience, dissolves tocopherol completely.In mixture, add 55g cuorin, mix 10 minutes.In mixture, add 120g cholesterol again, mix 10 minutes.In mixture, add 320g ovum Phosphorylcholine again, remix 10 minutes.The aqueous trehalose solution of 37 ℃ ± 2 ℃ (150g trehalose/1.5kg water) is added in mixture, until mixture becomes clarification.Utilize the filter in 0.22 μ m aperture to carry out aseptic filtration, will leach thing and be sub-packed in aseptic vial, every bottle of 10g, carries out lyophilizing.The Liposomal formulation of lyophilizing is white powder, and is easy to dissolve again.Keep the moisture of freeze-dried lipidosome preparation below 10%, lyophilized products is stored at 4 ℃.

Prepare after blank liposome, introduce mitoxantrone in preparation, concrete grammar is as follows: 15mg Novantrone (mitoxantrone hydrochloride) solution and 8ml physiological saline solution are together joined in freeze-dried lipidosome bottle.The vortex at a slow speed that bottle is carried out 30 minutes is processed, and realizes mitoxantrone with the hydration of liposome.Violent vortex is processed and within 3 minutes, is carried out the supersound process of 10 minutes again afterwards.Obtain final mitoxantrone liposome solutions, this liposome can be used for injection, should complete application in latter 8 hours in dissolving.

The envelop rate of the preparation-obtained mitoxantrone liposome of said method is 91.69 ± 3.59%, and gained liposome having good stability within 8 hours, can reach more than 95%.

Embodiment 8

The present embodiment has proved that from experiment in vitro angle the mitoxantrone of Lipidosome and the regular dosage form mitoxantrone of same concentrations compare and possess better therapeutic effect.

Get and without medicine, cultivate the HL60/ADM cell of 1 week, be made into the cell suspension of 5 * 104/ml, be inoculated in 96 orifice plates.Experiment is grouped into 3 groups: i.e. mitoxantrone liposome group, free mitoxantrone group, blank group (adding isopyknic normal saline).Concentration is grouped into: 0.025 μ g/ml, 0.025 μ g/ml, 0.25 μ g/ml, 2.5 μ g/ml, 25 μ g/ml.Each concentration group is established 3 multiple holes.Be placed in cultivation 24h in CO2 incubator, after 48h, suck culture fluid, add the culture fluid of serum-free and the MTT solution that concentration is 50mg/ml, in CO2 incubator, hatch 4h, centrifugal, abandon supernatant, add DMSO (dimethyl sulfoxine) 150 μ l/ holes, vibration 20min, spectrophotometric determination D (570) value.By following formula, ask cell survival rate, according to linear equation, ask IC50.

Cell survival rate={ processed group D (570) value/matched group D (570) value } * 100%

Cell inhibitory rate=1-cell survival rate.

Result is: mitoxantrone liposome and free mitoxantrone medicine to the IC50 of HL60/ADM cytosis 24h respectively: 12.42,27.31 μ g/ml, the IC50 of effect 48h respectively: 8.25,12.44 μ g/ml.Can find out that mitoxantrone liposome and free mitoxantrone medicine all have lethal effect to HL60/ADM cell, but kill rate exists larger difference, mitoxantrone liposome is strong to the lethal effect of the lethal effect specific ionization mitoxantrone of HL60/ADM cell.

Embodiment 9

The present embodiment proof lipid build mitoxantrone (LEM) is compared and is possessed lower toxicity with the regular dosage form mitoxantrone (MTO) of same concentrations.

The male CD2F1 Mus of choosing male 19-23g is experimental subject, carries out single-dose experiment, the toxicity in vivo of assessment medicine.Get altogether 64 of animals, make animal adapt to feeding environment 2 weeks, at random 64 animals are equally divided into 8 groups afterwards, every group 8, every Mus is accepted tail intravenously administrable, and experimental group gives respectively the lipid build mitoxantrone (LEM) of each treated animal 5.5,10.5,15.5mg/kg dosage (by mitoxantrone); Positive control treated animal gives 5.5,10.5, the conventional mitoxantrone (MTO) of 15.5mg/kg dosage; Tail vein injection blank liposome or normal saline are as blank, and the Lipid Concentration of blank liposome is consistent with the Lipid Concentration of 15.5mg/kg mitoxantrone liposome.

All will observe the state of Mus every day, all will carry out at least twice weigh to Mus weekly.Experiment ends at the 67th day, chooses when representational laboratory animal starts latter 3 days respectively at experiment and experiment finishes it is carried out to histopathological evaluation.

Single-dose toxicity test shows: the MTO of 5.5mg/kg or 10.5mg/kg and LEM to CD2F1 Mus all without lethal effect.The MTO of 15.5mg/kg dosage made the weight of animals obviously decline 30.7% in the time of the 9th day.In experiment all death of animal in the time of the 10th day.And the Mouse Weight of the LEM of 15.5mg/kg dosage group has certain minimizing for first 6 days in experiment, in the remaining time of experiment, it is normal that the body weight of animal is recovered gradually.The experimental result of histopathological evaluation shows: MTO administration group and LEM administration group laboratory animal are all found certain leucocytotoxicity.Test the 61st day to the 67th day, the leucocyte level of each LEM administration group is consistent with the leucocyte level of matched group, and every clinical chemistry index is all tending towards normal.Each medication group of MTO is all found the decay of bone marrow and spleen hematopoietic stem cell on the 3rd day in experiment, and finds the decay of spleen lymphocyte.Each administration group of LEM has also been found similar cytotoxicity, but the cytotoxicity all causing without MTO administration is serious, and to experiment the 67th day, the bone marrow of all surviving animals and spleen toxicity were all recovered.Each treatment group is not all found obvious liver, lung toxicity.

Embodiment 10

The present embodiment proof lipid build mitoxantrone (LEM) is compared and is possessed lower toxicity with the regular dosage form mitoxantrone (MTO) of same concentrations.

The CD2F1 Mus of choosing male 19-23g is experimental subject, carries out single-dose experiment, the toxicity in vivo of assessment medicine.Get altogether 40 of animals, make animal adapt to feeding environment 2 weeks, be equally divided into 4 groups afterwards with being about to 32 animals, 8 every group, every Mus is accepted tail intravenously administrable.LEM administration group gives respectively the dose (by mitoxantrone) of two groups of laboratory animal 25.5mg/kg and 35.5mg/kg; MTO administration group gives the dose of one group of laboratory animal 25.5mg/kg; With blank liposome (lipid content is with the LEM of 15.5mg/kg) with normal saline in contrast.

All will observe the state of Mus every day, all will carry out at least twice weigh to Mus weekly.Experiment ends at the 67th day equally, chooses when representational laboratory animal starts latter 3 days respectively at experiment and experiment finishes it is carried out to hematotoxicity and histopathological evaluation.

In experiment first 4 days, each treated animal had no untoward reaction and occurs, and at experiment 5-8 days, the animal of the MTO medication group of 25.5mg/kg is dead successively.Test the 5th day, have 2 animal deads, within the 6th day, have 2 animal deads, this group residue laboratory animal is in experiment all death in the 8th day.Except the 3rd group, the laboratory animal of other groups is without death, and do not find obvious clinical toxicity sign.The experimental result of hematotoxicity and histopathological evaluation shows: test the 3rd day, the animal that gives MTO25.5mg/kg and give LEM35.5mg/kg all finds that glutamate pyruvate transaminase (ALT) content in body has moderate rising.MTO administration group and LEM administration group laboratory animal are all found certain leucocytotoxicity, but the leucocytotoxicity of LEM group is starkly lower than MTO medication group, test the 61st day to the 67th day, the leucocyte level of each LEM administration group is consistent with the leucocyte level of matched group, and every clinical chemistry index is all tending towards normal.Each medication group of MTO is all found the decay of bone marrow and spleen hematopoietic stem cell on the 3rd day in experiment, and finds the decay of spleen lymphocyte.Each administration group of LEM has also been found similar cytotoxicity, but the cytotoxicity all not causing than MTO administration is more serious, and to experiment the 67th day, the bone marrow of all surviving animals and spleen toxicity were all recovered.Each treatment group is not all found obvious liver, lung toxicity.

By the observation to body weight, find, the 9th day the weight of animals of LEM single dose administration 25.0mg/kg group experiment has slight decline (having declined 4.8%), and the body weight of animal rises to again normally afterwards.And 35.5mg/kg LEM group and 25.5mg/kg MTO group when testing the 5th day, all see obvious reduction, reduced respectively 23% and 31%.MTO treated animal is all dead subsequently, and LEM treated animal body weight recovers normal gradually.In a word, there is obvious drug intoxication in the laboratory animal of 25.5mg/kg MTO group, although and there is certain toxic reaction in LEM treated animal, toxic reaction can recover later gradually.

Embodiment 11

The present embodiment proof lipid build mitoxantrone (LEM) is compared and is possessed lower toxicity with the regular dosage form mitoxantrone (MTO) of same concentrations.

The CD2F1 Mus of choosing male 19-23g is experimental subject, carries out multiple dosing experiment, the toxicity in vivo of assessment medicine.Get altogether 64 of animals, make animal adapt to feeding environment 2 weeks, be equally divided into 8 groups afterwards with being about to 64 animals, 8 every group, every Mus is accepted tail intravenously administrable.Multiple dosing experiment, is divided into administration in 5 days, and administration every day 1 time gives the lipid build mitoxantrone of laboratory animal 2.0,5.0,8.0mg/kg dosage (by mitoxantrone); Positive control treated animal gives 2,5.0, the conventional mitoxantrone of 8.0mg/kg dosage; Tail vein injection blank liposome or normal saline are as blank, and the Lipid Concentration of blank liposome is consistent with the Lipid Concentration of 8.0mg/kg mitoxantrone liposome.

All will observe the state of Mus every day, all will carry out at least twice weigh to Mus weekly.Experiment ends at the 64th day.Choose when representational laboratory animal starts latter 7 days respectively at experiment and experiment finishes animal is carried out to hematotoxicity and histopathological evaluation.

Multiple dosing toxicity test result shows: when the MTO of 2.0mg/kg dosage (every day 1 time, administration 5 days) causes experiment to finish, (64 days) have 3 CD2F1 Mus dead, 5 CD2F1 Mus survivals; The MTO of 5.0mg/kg dosage (every day 1 time, administration 5 days) declines rapidly the body weight of Mus and (within 7-9 days, loses weight 30.45 ± 6.0%, n=10), and all dead the 10th day Mus; The MTO of 8.0mg/kg dosage (every day 1 time, administration 5 days) declines rapidly the body weight of Mus, and (within the 7th day, lose weight 28.3%, n=10), and Mus is all dead subsequently.Mus death is not observed in the LEM administration of 2.0mg/kg dosage and 5.0mg/kg dosage (every day 1 time, administration 5 days).The LEM of 5.0mg/kg dosage causes the 8th day Mus body weight to reduce 6.7%, and body weight is gone up again subsequently; The LEM administration of 8.0mg/kg dosage (every day 1 time, administration 5 days) (64 days) when experiment finishes finds that there is 1 Mus death, all the other survivals.

Testing the 7th day, all there is the hemopoietic lymph regeneration function impairment of spleen and bone marrow in the MTO of each dosage and LEM.The MTO of multiple dosing 5.0mg/kg or 8.0mg/kg dosage, two treated animals all find that low grade is to moderate hepatocyte cavity sample degeneration.In addition, MTO administration also causes Mus small intestinal and large intestinal villus or crypts generation shrinkage.On the other hand, LEM administration does not produce intestinal toxicity.Each administration group is not all found lung, the heart, nephrotoxicity.

Embodiment 12

The present embodiment proof lipid build mitoxantrone (LEM) is compared and is possessed lower toxicity with the regular dosage form mitoxantrone (MTO) of same concentrations.

The CD2F1 Mus of choosing male 19-23g is experimental subject, carries out multiple dosing experiment, the toxicity in vivo of assessment medicine.Get altogether 56 of animals, make animal adapt to feeding environment 2 weeks, be equally divided into 7 groups afterwards with being about to 56 animals, 8 every group, every Mus is accepted tail intravenously administrable.Multiple dosing experiment, is divided into administration in 5 days, administration every day 1 time.Give the lipid build mitoxantrone of laboratory animal 5.5,8.0,11.0mg/kg dosage (by mitoxantrone); Positive control treated animal gives 2.0, the conventional mitoxantrone of 5.5mg/kg dosage; Tail vein injection blank liposome or normal saline are as blank, and the Lipid Concentration of blank liposome is consistent with the Lipid Concentration of 8.0mg/kg mitoxantrone liposome.

All will observe the state of Mus every day, all will carry out at least twice weigh to Mus weekly.Experiment ends at the 64th day.Choose when representational laboratory animal starts latter 7 days respectively at experiment and experiment finishes animal is carried out to hematotoxicity and histopathological evaluation.

Body weight change aspect, losing weight of the 3rd group (LEM11.0mg/kg/ days) and the 5th group of (MTO5.5mg/kg/ days) animal is comparatively obvious, the 3rd group of (LEM11.0mg/kg/ days) animal lost weight 33.7% on the 7th day at experiment 22.5%, the 5 treated animal (MTO5.5mg/kg/ days) of having lost weight for the 7th day in experiment.Experiment the 12nd day, the body weight of the 1st group (LEM5.5mg/kg/ days), the 2nd group (LEM8.0mg/kg/ days), the 4th group of (MTO2.0mg/kg/ days) animal has declined respectively 5.5%, 27%, 26.3%, but in experimentation subsequently, it is normal that the body weight of these several treated animals is recovered again.The body weight of each control animals is without obviously changing.

Drug toxicity lethal aspect, the 1st treated animal occurs without dead in whole experimentation, to there are two examples in the 2nd treated animal dead in whole experimentation, it is dead to there are 5 examples in the 4th treated animal, the 3rd group and the 5th treated animal all dead when experiment finishes.

Aspect hemocyte toxic and side effects, with the LEM administration of 11.0mg/kg/ days, all visible leukocyte, hematoblastic quantity declined in the MTO administration of 5.5mg/kg/ days.The neutrophilic granulocyte of visible two treated animals, lymphocyte quantity reduce and platelet levels reduces, and visible red cell counting level presents certain increase simultaneously.And this blood cell toxicity of each low dosage administration group not obvious.The MTO administration of 5.5mg/kg/ days all can be brought out hepatic injury to a certain degree with the LEM administration of 11.0mg/kg/ days, and this dying animal of two groups all shows the obvious rising of ALT, AST and the bilirubin level of two treated animals all apparently higher than control group mice.

In a word, clinical pathology data show, the MTO administration of 5.5mg/kg/ days presents similar drug toxicity with the LEM administration of 11.0mg/kg/ days.And dosage is that the MTO of 5.5mgmg/kg/ days is more high than the toxicity in vivo of the LEM of same dose and animal fatality rate.The application of this provable Lipidosome can obviously reduce the drug toxicity of mitoxantrone.

Embodiment 13

The blood drug level that the present embodiment proof lipid build mitoxantrone (LEM) is compared in animal blood with the regular dosage form mitoxantrone (MTO) of same concentrations is higher, and the LEM half-life in animal body will be longer than MTO, LEM clearance rate in animal body will be lower than MTO.

Using the CD2F1 mice of male 19-23g as experimental subject, and mice is divided into 2 groups, 36 every group.By the mode of tail vein injection, give the MTO of the 1st group of mice 5mg/ml, give the LEM (by mitoxantrone) of the 2nd group of mice 5mg/ml.After administration, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours modes by cardiac puncture are got blood to 4 mices on the same group not, mice are anaesthetized before getting blood.The blood taking out is placed in the test tube that is added with heparin, and adds wherein the 0.1M citrate buffer solution (pH3.0) of 20 μ l100mg/ml ascorbic acid.Blood sample under 4 ℃ of 2000rpm centrifugal 10 minutes with separated plasma.Get after blood the mice execution that dislocates.The different tissue (liver, spleen, heart, kidney, lung) of sharp separation carries out rinsing with low-temperature normal saline.Before analyzing for mitoxantrone, by blood and tissue samples in-20 ℃ of storages.

Utilize reverse hplc to analyze the mitoxantrone in blood and tissue samples.The ascorbic acid of the hexane sulfonic acid of 0.01mg/ml and 0.5mg/ml is joined in the plasma sample of 0.25ml, and add wherein the ametantrone of 0.25 μ g as internal reference thing.Vortex vibration 30 seconds, is adding the 0.1M borate buffer solution (pH9.5) of 0.5ml and the 1M cefpiramide hydride of 150 μ l afterwards wherein, and vortex vibration is 30 seconds again.Sample 10ml dichloromethane extraction, level is shaken 1 hour, the centrifugal 15min of 3000rpm.Isolate the organic layer of 9ml, evaporate passing under nitrogen.Sample is heavily dissolved in 100 μ l mobile phases, carries out afterwards HPLC and detects analysis.The 0.1M citrate buffer that tissue samples comprises 20% ascorbic acid with 1mlpH3.0 carries out homogenization processing, and extracts with said method.Utilize the separated mitoxantrone of reverse chromatograms (Waters-bondapak C18), the composition of mobile phase comprises the 0.16M ammonium formate of 33% acetonitrile, 67%pH2.7.Flow velocity is 1.0ml/min.Under 600nm, detect.The retention time of internal reference thing and mitoxantrone is respectively 3.7 and 4.3 minutes.

The pharmacokinetic parameters of blood plasma utilizes standard method to evaluate and test, and utilizes the linear regression analysis of plasma concentration-time graph to calculate clearance rate constant (K).Utilize external standard method to calculate area under curve (AUC).Half-life utilizes following formula to calculate: t1/2=0.693/K.

Drug metabolism aspect: the peak serum concentration after MTO administration is 0.41 ± 0.10 μ g/ml, correspondingly, after LEM administration, peak serum concentration is 20.95 μ g/ml, is 51.1 times of MTO.Peak area under the plasma concentration-time graph of LEM administration will be significantly higher than MTO administration (28.12vs0.14).The half-life of LEM administration will be significantly longer than MTO, and the LEM half-life is 67 minutes, and the MTO half-life is 7.3 minutes.The CLTB of LEM dosage form mitoxantrone will be significantly lower than MTO, and both are respectively 0.17 μ gh/ml and 22.3 μ gh/ml.

Aspect mitoxantrone medicine tissue distribution, give respectively after CD2F1 mice MTO (5mg/kg) or LEM (5mg/kg), within 5 minutes, at cardiac muscular tissue place, reach peak concentration.Giving the mitoxantrone accumulation of 1 gram of cardiac muscular tissue after MTO is 10.8 ± 0.2 μ g, and giving the mitoxantrone accumulation of 1 gram of cardiac muscular tissue after LEM is 5.6 ± 0.9 μ g.The mitoxantrone concentration of LEM administration cardiac muscular tissue is at least less than the twice of MTO administration; (P < 0.02) LEM administration is dirty at Hepar Mus will be significantly higher than MTO administration (heart: P < 0.001, spleen: P < 0.008) with accumulation spleen position; The accumulation of LEM administration Shu pulmonary and kidney portion will be significantly lower than MTO administration (lung: P < 0.001, kidney: P < 0.003).

Compare with MTO, the application of LEM makes mitoxantrone significantly reduce at the accumulation of CD2F1 rat heart, lung, kidney, at the accumulation of liver and spleen, is increased significantly.This proof lipid build mitoxantrone (LEM) can change the distribution in vivo of regular dosage form mitoxantrone.The toxicity of mitoxantrone is mainly reflected in myocardial toxicity aspect, utilize lipid build mitoxantrone (LEM) administration to compare with regular dosage form (MTO) administration and can significantly reduce mitoxantrone in the accumulation at cardiac muscular tissue place, particularly, after giving MTO, the myocardium accumulated concentrations of 5 minutes and 15 minutes mitoxantrones is 10.8-10.2 μ g/g, in experiment, within latter 48 hours, is down to 6.0 μ g/g; After giving LEM, the myocardium accumulated concentrations of 5 minutes and 15 minutes mitoxantrones is 5.6-4.7 μ g/g, in experiment, within latter 48 hours, is down to 1.5 μ g/g.The application of the provable LEM of above data can reduce the myocardial toxicity of mitoxantrone.

Embodiment 14

In dog body, pharmacokinetic parameters is measured, and 18 beasle dogs are divided into 3 groups by sex and Body Mass Index, are respectively mitoxantrone liposome group 0.2,0.4 and 1.0mg/kg group; 6 dogs of 0.2mg/kg group give free mitoxantrone 1.0mg/kg through the eluting after date of 3 weeks.Respectively at different time after administration (15,30,45min and 1,2,4,8,24,48,72,96,120h), from forelimb venous blood sampling 1ml, put anticoagulant heparin pipe.Adopt HPLC method to measure the drug level of mitoxantrone in blood plasma, chromatographic column is ZorbaxSB-C18 analytical column (150mm * 4.6mm, 5 μ m, Agilent company, SN:USCM014615), the preset post of C18 (4mm * 3.0mm, Phenonenex, Torrance, CA, USA); Detect wavelength 655nm; Mobile phase: acetonitrile and sodium heptanesulfonate buffer (sodium heptanesulfonate 2.01g, glacial acetic acid 4.5ml, water 500ml) 64: 36 (v/v); 30 ℃ of column temperatures; Flow velocity 1.0ml/min.Get dog plasma 200 μ l, add protein precipitant 400 μ l[acetonitrile-methanol (containing 0.5mol/LHCl)=10: 90, v/v], eddy current mixing 30s, room temperature is placed 30min, after centrifugal (11000r/min) 15min, get supernatant 20 μ l sample introductions, record chromatogram.Adopt DAS (ver2.1.1) pharmacokinetics software to calculate the blood drug level data of actual measurement, using SPSS (ver111.5) statistics software to carry out t check in groups, paired t-test and dependency check to pharmacokinetic parameters.

Pharmacokinetic parameters is measured with more known mitoxantrone liposome 0.2～1.0mg/kg in beasle dog body and is linear kinetics feature, compare with the free mitoxantrone of 1.0mg/kg, AUC and the t1/2 of isodose mitoxantrone liposome are higher, and clearance rate is lower, and distribution volume is less.

Embodiment 15

The present embodiment confirms lipid build mitoxantrone (LEM) preparation blood drug level after higher than conventional mitoxantrone (MTO) administration of the blood drug level in blood plasma after to beasle dog administration.The medicine removing speed of LEM is slow compared with MTO.

Body weight be the beasle dog of 10-14kg respectively through tail vein injection 0.25mg/kg, 0.5mg/kg, the mitoxantrone liposome of 1mg/kg, and the dosage free mitoxantrone that is 0.25mg/kg.And respectively at the 5min after drug injection, 15min, 30min, 1h, 2h, 4h, 8h, each time point of 24h and 48h is collected the blood of the beasle dog of each group.Blood is at 4 ℃, centrifugal 10min under 2000rpm condition, separated plasma.Utilize reverse high performance liquid chromatography to analyze the mitoxantrone in plasma sample.Mitoxantrone is separated by reverse-phase chromatography, and mobile phase is the ammonium formate buffer of acetonitrile (33%) and 0.16M, the compositions of mixtures of pH2.7 (67%), and flow velocity is 1.0ml/min.The detection wavelength of mitoxantrone is 600nm.This method is all good in therapeutic domain internal linear and the repeatability of mitoxantrone.Its sensitivity is 10ng/ml.

Result shows: free mitoxantrone and the half-life of mitoxantrone liposome when dosage is 0.25mg/kg are respectively 0.21h and 1.37h (p < 0.05); Free mitoxantrone and the clearance rate of mitoxantrone liposome when dosage is 0.25mg/kg are respectively 715.66ml/min/kg and 14.18ml/min/kg (p < 0.05); Free mitoxantrone and the AUC value of mitoxantrone liposome when dosage 0.25mg/kg are respectively 0.29mg.h/ml and 55.31mg.h/ml (p < 0.05).Concrete condition is in Table 1.

Pharmacokinetics comparison in table 1 different dosage form mitoxantrone beasle dog animal body

The present embodiment shows to give the beasle dog of lipid build mitoxantrone (LEM) and the conventional mitoxantrone (MTO) of same dose is compared, and the peak value of the mitoxantrone plasma concentration of its generation has improved 5-10 doubly.Liposomal formulation also can increase the AUC value of mitoxantrone and reduce its clearance rate, and along with the increasing of mitoxantrone introduction volume, AUC also can increase with dosage is linear.

Embodiment 16

The present embodiment has proved that mitoxantrone liposome (LEM) compared better active anticancer with free mitoxantrone (MTO).

The antitumaous effect of different dosage form mitoxantrone in CD2F1 mouse leukemia model: the 0th day, female CD1F1 mouse tail vein injection 1 * 10 ⁴individual L1210 leukaemia, after tumor inoculation 24h, mice is divided into four groups (n=10) at random, respectively by tail vein single dose administration LEM or MTO.The single-dose dosage of LEM is respectively 10.0mg/ml and 20.0mg/ml, and the single-dose dosage of MTO is 10.0mg/ml, and blank group is to the normal saline of respective volume.Observe mice state every day, experiment is carried out 60 days.

The antitumaous effect of different dosage form mitoxantrone in Balb/c human breast cancer in nude mice model: by the MCF-7 breast cancer cell (2 * 10 of exponential phase ⁶individual/0.2ml) subcutaneous vaccination is in the mammary gland region of female Balb/c nude mice.Treat that tumor growth is 50mm to average external volume ³time, start to treat.MCF-7 breast cancer model mice is divided into 5 groups (n=6) at random.Tail vein injection gives MTO0.5mg/kg respectively, MTO1.0mg/kg, and LEM2.5mg/kg, LEM5.0mg/kg treats, and blank is organized every day to isopyknic normal saline.Be administered once every other day, administration is 5 times altogether.The method for expressing of gross tumor volume: the gross tumor volume before treating (the 0th day time gross tumor volume) of take is 100%, surveys weekly volume twice after experiment starts, and does ratio respectively with the gross tumor volume of the 0th day, assesses the variation of gross tumor volume with the method.

In the present embodiment research process, the single-dose dosage of MTO is selected 10.0mg/kg, because the single-dose dosage of MTO while being greater than 10.0mg/kg, can show serious toxic reaction to CD2F1 mice; The single-dose dosage of LEM treatment nude mice is selected 20.0mg/kg, if because the single-dose dosage when MTO treatment nude mice is greater than 20.0mg/kg, nude mice will produce serious toxic reaction.In L1210 tumor model mice, normal saline mice is whole dead within 8 days after inoculation L1210 tumor cell, and death, all survival appearred in the mice of still accepting LEM (single-dose dosage is 20mg/kg) treatment in 60 days.This research has shown that mitoxantrone liposome has good therapeutic effect.The mice single mouse survival rate that mitoxantrone 10.0mg/kg treats of dissociating is 20%; Yet the mouse survival rate of accepting mitoxantrone liposome therapeutic is 40%, this phenomenon shows that mitoxantrone liposome compares and have higher active anticancer with free mitoxantrone.

The antitumaous effect experiment of different dosage form mitoxantrone in Balb/c nude mice model carried out 48 days, and the mouse tumor volume of the LEM treatment that the LEM that acceptable dose is 2.5mg/kg and dosage are 5mg/kg is obviously than the gross tumor volume of normal saline group little (p < 0.05); And the gross tumor volume of LEM group mice is compared also and is obviously reduced (p < 0.05) with the gross tumor volume of MTO group mice.The gross tumor volume of normal saline group is 1692.1 ± 198.5%; Dosage is that the gross tumor volume of the LEM group of 2.5mg/kg is 1046.5 ± 145.3%; Dosage is that the gross tumor volume of the LEM group of 5.0mg/kg is 815.7 ± 96.2%; The gross tumor volume of the free mitoxantrone treatment group of 0.5mg/kg is that the gross tumor volume of the free mitoxantrone treatment group of 1392.5 ± 245.3%, 1.0mg/kg is 1152.9 ± 22.7%.

Claims (10)

1. the liposome in the present invention is comprised of phospholipid, cholesterol, and the medicine of sealing is conventional chemotherapy medicine, and this liposome can be used for mammiferous oncotherapy, and wherein mammal can be people.

2. the mitoxantrone liposome in claim 1, is characterized in that in the phospholipid of composition, electronegative phospholipid is one or more in phosphatidylinositols, phosphatidic acid or Phosphatidylserine, Cor Bovis seu Bubali phospholipid; Wherein said cuorin can be selected from natural cuorin or synthetic cuorin; Neutral phospholipid is one or more in egg lecithin or hydrogenated soy phosphatidyl choline.

3. the mitoxantrone liposome in claim 1, the conventional chemotherapy medicine of sealing can be the Treated with Chemotherapeutic Drugs things such as mitoxantrone, amycin, paclitaxel epirubicin, cisplatin.

4. the mitoxantrone liposome described in claim 1, adds the protective agent that lyophilizing is relevant while it is characterized by freeze-drying process, and protective agent can be monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyhydric alcohol and other water-soluble high-molecular substances.

5. in claim 1, the common method of the preparation of liposome has a lot, for example: membrane process, ultrasonic method, ammonium sulphate gradient and lyophilization, removal of surfactant method, high pressure homogenization method etc., the most conventional is film dispersion method and ammonium sulphate gradient, and lyophilization can realize suitability for industrialized production scale.The present invention's preparation method used can be selected from above numerous methods.

6. in claim 1, the concentration range of the mitoxantrone of liposome is about 0.5-2mg/ml, and mitoxantrone should be 1 with the ratio of lipid: 10-1: in 20 scopes.

7. in claim 1, the envelop rate of the mitoxantrone of liposome is at least more than 90%, and particle size range should be within the scope of 10nm-0.5 μ m.

8. the mitoxantrone liposome route of administration in claim 1 can be oral administration, intravenously administrable, topical, intraperitoneal administration etc.Drug safety will be higher than conventional mitoxantrone, can comprise after medication the weight of animals alleviate low, mortality of animals is low, Leukocyte Killing is low in body, myocardial toxicity is low, bone marrow and spleen hemopoietic function suppress the aspect such as low.

9. in claim 1, mitoxantrone liposome can be used for the treatment of non-hodgkin's (family name) lymphoma, myeloma, advanced breast cancer, bladder cancer, ovarian cancer and hepatocarcinoma, acute non-lymphocyte is from disorders of blood, especially to acute lymphoblastic leukemia and advanced breast cancer and non-hodgkin's (family name) lymphoma by very high high tumor promotion, especially acute lymphoblastic leukemia and advanced breast cancer.

10. the liposome described in claim 1, if necessary can be together with being different from the second therapeutic agent of mitoxantrone use in conjunction, described the second therapeutic agent comprises antineoplastic agent, antifungal agent, antibiotic and other active medicine.