CN101926821B

CN101926821B - Pharmaceutical composition for targeted release of trace elements, and preparation method and application thereof

Info

Publication number: CN101926821B
Application number: CN2010102232823A
Authority: CN
Inventors: 周翔; 解慧琪; 康裕建
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2010-07-12
Filing date: 2010-07-12
Publication date: 2012-01-25
Anticipated expiration: 2030-07-12
Also published as: CN101926821A; WO2012006938A1

Abstract

The invention discloses a pharmaceutical composition with the function of ultrasonic targeted release of trace elements, which comprises the components with the concentration of more than 1 x 10⁶Each ml of hollow microspheres with the grain diameter of less than 10 mu m, at least one microelement with the physiological dose less than or equal to ten times of that of a human body, and a pharmaceutically acceptable carrier or auxiliary material; wherein the trace elements and the hollow microspheres exist in a combined or free form, and the hollow microspheres are prepared from a pharmaceutically acceptable film-forming material. The pharmaceutical composition of the invention is stable and safe, can directionally introduce trace element ions into the body under the irradiation effect of the ultrasound, thereby effectively leading the tissue vessel regeneration, the tissue regeneration or the anti-tumor biological effect of the part irradiated by the ultrasound, and has wide clinical prospect.

Description

A kind of pharmaceutical composition of targeted release of trace elements and method for preparing and application

Technical field

The invention discloses a kind of pharmaceutical composition of directed controlled release trace element, particularly can be by ultrasonic directed controlled release, but induced tissue regeneration, antitumor medicine composition.

Technical background

Trace element plays a part basic in keeping human health, and the major physiological function is in various enzyme systems, to play catalytic action, plays a role with the essential composition of hormone or vitamin or cofactor, forms metalloprotein with specific function etc.The meaning of trace element physiological action can be compared with vitamin, but body can synthesize some vitamin voluntarily and can't synthesize any element, sees that from this some essential trace element is even more important than vitamin to human body.The basic definition of essential trace element is meant that those have obvious Nutrition and physiological function, to keeping requisite elements such as body growth growth, vital movement and procreation.So-called " essential " promptly: 1. body must absorb this element from extraneous diet, and behind this element of removal from diet, the physiological deficiency state of this element will appear in body.2. after replenishing this element-specific, this deficiency state of body will be eased.3. this special element always has certain special biochemical function to body, and this effect can not be replaced by other any elements fully.When this element Deficiency of Intake can cause body biological function obstacle, and recover to alleviate or to prevent this dysfunction behind the physiological level of this element.In a single day body leaves this element, can not grow, and can not accomplish the life cycle that it should have again.In addition, essential trace element also has following characteristics: 1. this element is present in the tissue of different animals with similar concentration.No matter 2. the kind of animal how, remove and to occur similar physiology, biochemical unusual behind this element.Can alleviate or prevent above-mentioned unusual when 3. having this element to exist.4. this abnormal change also can be cured when shortage is controlled.

At present, the iodine in the trace element, selenium, zinc, ferrum, copper, manganese, chromium etc. are confirmed as " keeping the indispensable essential trace element of body normal activities " in the world.Along with the reach of science, the mankind more and more recognize the fundamental role of essential trace element in keeping health.Some element not only can infection, and relevant with many chronic, popular, endemicity even malignant change.The massive epidemiology investigation points out that body may make the crowd that the sensitivity of disease is increased if lack essential trace element, causes the generation and the development of sub-health state or disease.

Though it is trace element is few at the intravital content of people, closely bound up with health with people's existence.For many years, the importance of trace element in human body more and more obtains paying attention to.Trace element such as selenium, zinc, ferrum, copper be proved to be have multiple beneficial biological effect.According to scientific research, up to the present, being identified the essential trace element relevant with health and life has 18 kinds, and ferrum, copper, zinc, cobalt, manganese, chromium, selenium, iodine, nickel, fluorine, molybdenum, vanadium, stannum, silicon, strontium, boron, rubidium, arsenic etc. are promptly arranged.This every kind trace element all has its special physiological function.Although they are minimum at people's in-vivo content, they are very necessary to some conclusive metabolism of keeping in the human body.In case lacked these essential trace element, disease will appear in human body, even threat to life.As zinc deficiency can cause mouthful, eye, anus or external genital is rubescent, pimple, warm rash.And for example ferrum is one of main component that constitutes hemoglobin, and iron deficiency can cause iron deficiency anemia.Report was abroad once arranged: iron content, copper, zinc total amount reduce in the body, all can weaken body resistance against diseases, encourage bacterial infection, and metainfective mortality rate are also higher.Their excess intake, deficiency or shortage all can cause the unusual of Human Physiology to some extent or disease takes place.

Though the importance of trace element has obtained to generally acknowledge that the absorption of trace element and action effect are added and healthcare applications by the meals that mainly are confined to of extensive approval at present.This biological effect that mainly is limited by trace element has two kinds of contradiction property: 1. the contradiction of usefulness and dosage.Any trace element; For body; All must submit to the requirement of " trace ", though this metalloid element is most important as far as body, body content and intake all require extremely low; This just often causes body that the picked-up and the utilization of this dvielement are lacked efficient, and the easy minor metallic element that takes place is poisoned.2. the non-specific and dense contradiction of gathering demand of the specificity illness position that distributes in conventional picked-up and the body.Particularly when body is in the illness state, just more difficult to the utilization of trace element.Like heart infarction patient's cardiac muscular tissue the picked-up of copper ion is reduced in a large number, cause deficiency decorated albumino reaction, cause copper ion from heart, to lose.Early stage zoopery is found as rat heart being separated and at ECP, after perfusion stops 45 minutes, being poured into myocardial cell major injury, a large amount of release of copper ion in the heart more again.From the patient's of myocardial ischemia postmortem result also find myocardial ischemia and around the position, the concentration of copper ion obviously descends.And for example in pathological states such as hepatitis or liver fibers, generally lack selenium, zinc in hepatopath's body, and the state of an illness is serious more, the selenium in the blood, zinc level are low more.At this hepatopathy state, liver is difficult to make full use of selenium, the zinc of food, thereby causes the inefficiency of meal supplement: even the meal supplement of normal dose also is difficult to reach the effective demand of target organs, be prone to produce metal toxicity again like escalated dose.Situation like that, this predicament of body ubiquity: promptly damaged organize more the trace element that needs under pathological state, be difficult to more targeting, obtain efficiently.

Under the present circumstances, the picked-up of trace element must be directly or indirectly supplied with by soil, i.e. the oral health medicine picked-up of the various foods of the relevant content of administered through oral or similar kind pure and so on plyability.Improve trace element meal supplement efficient though createed distinct methods, such as the trace element that adopts Nano grade.Perhaps adopt the method for biological concentration: promptly utilize seaweeds to have the raw-food material of good bioconcentration, enrichment is the trace element of volume more, to improve the orally ingestible efficient of trace element.But these class methods still are limited by the functional status of certain organs, and are impaired like relevant organ dysfunction, still are difficult to reach its treatment concentration and biological physiology effect even trace element replenishes through meals in a large number.

Summary of the invention

In order to overcome the defective of prior art, the present invention provides a kind of pharmaceutical composition with ultrasonic targeted release of trace elements effect, and it comprises concentration greater than 1 * 10 ⁶Individual/ml, particle diameter is less than tiny balloon and at least a trace element that is less than or equal to ten times of physiological doses of human body of 10 μ m, and pharmaceutically acceptable carrier or adjuvant; Wherein trace element and tiny balloon are combining or free form exists, and said tiny balloon is prepared from pharmaceutically acceptable filmogen.

Further, said tiny balloon particle diameter is 1～5 μ m; Said tiny balloon includes the mist that gas is the combination in any of air, nitrogen, sulfur fluoride gas, fluoroalkane hydro carbons gas or other avirulence gases or above-mentioned one or more gas componants.

Said trace element is one or more the combination in any in ferrum, copper, zinc, cobalt, manganese, chromium, selenium, iodine, nickel, fluorine, molybdenum, vanadium, stannum, silicon, strontium, boron, rubidium or the arsenic ion, is preferably in copper, zinc, selenium, the ferrum one or more combination in any.

On the one hand; The trace element of said combining form and tiny balloon mutually combine through chemical action or physical action; Preferably; The trace element of combining form and tiny balloon are prepared by following method: 1) according to molar concentration rate be: 1: 0.05 to 1: 500 with the trace element ion and filmogen is water-soluble or organic solvent in, make the trace element ion combine formation trace element compound film material with the film material through physics or chemical action; 2) with conventional method with the compound film material that step 1) obtains, be prepared into the tiny balloon of particle diameter less than 10 μ m.

On the other hand; That the trace element of said free form is meant is not bonded with hollow micro capsule, can combine the compound formation complex of trace element to be present in carrier or the adjuvant with trace element ion and albumen, polypeptide, aminoacid, glucose or other; Preferably, said trace element and albumen, polypeptide, aminoacid, glucose, other can combine the molar concentration rate of the chemical compound of trace element to be: 1: 0.05 to 1: 500.

Further, filmogen according to the invention is human albumin, phospholipid or other high molecular polymers.

Described carrier or adjuvant are deionized water or normal saline or glucose solution or the solution that contains the trace element ion complex, and to be the trace element ion can combine the complex of the compound formation of trace element with albumen, polypeptide, aminoacid, glucose or other to described trace element ion complex.

Pharmaceutical composition of the present invention is preferably ejection preparation, is more preferably injectable powder.

The present invention also provides pharmaceutical composition of the present invention to promote the ultrasonic targeting of blood vessel or tissue regeneration and antineoplastic to discharge the purposes in the medicine in preparation

Described pharmaceutical composition utilizes ultrasound wave that therapentic part is carried out irradiation in human body is gone in intravenous injection, utilizes gassiness tiny balloon and hyperacoustic interaction to reach the ionic purpose of local release of trace elements.

Ultrasonic targeting of the present invention discharges the ultrasound wave that is meant certain energy to be made cavitation effect such as microbubble ruptures or vibration and impels and strengthens the associated metal ion and discharge and/or promote the respective metal ion component in the suspension to get into the irradiation tissue in that ultrasonic irradiation is local, reaches promotion blood vessel, tissue regeneration and/or the clinical purpose of antineoplastic.

But the trace element induced ultrasonic amplitude that ultrasonic targeting discharges produces the corresponding physiological function of trace element ion that carries according to the position.

The present invention also provides the method for preparation pharmaceutical composition of the present invention: at first preparation combines the ionic tiny balloon of trace element, and method for preparing comprises:

1) according to molar concentration rate be: 1: 0.05 to 1: 500 with the trace element ion and filmogen is water-soluble or organic solvent in, form by one or more trace element ions and combine to form the trace element compound film material through physics or chemical action;

2) compound film material that step 1) is obtained is prepared into the tiny balloon of particle diameter less than 10 μ m;

3) will prepare the ionic tiny balloon of accomplishing of combination trace element and mix formation suspendible pharmaceutical composition mutually with

claim

1,7,10 described carriers or adjuvant and complex;

4) directly stored refrigerated prepares injectable powder as suspendible drug injection or lyophilization.

Step 2) the method for preparing microsphere preparation commonly used of any pharmacy below capable of using: ultrasonic acoustic shake method, freeze-drying, spray drying, activity/controllable free-radical polymerisation, precipitation polymerization method, suspension polymerisation; Emulsion polymerisation; Seeding polymerization, heteropolymerization systems such as dispersin polymerization and precipitation polymerization, ionic cross-linking, emulsifying ionic gel method, ion precipitation-chemical crosslink technique, emulsifying-chemical crosslink technique, emulsion cross-linking method, heat cross-linking method, coacervation, emulsifying-solvent evaporated method.

Perhaps, pharmaceutical composition of the present invention can also be by following method preparation, and at first preparation does not combine the ionic tiny balloon of trace element, and method for preparing comprises:

1) by the step 2 of last method) described any pharmacy method for preparing microsphere commonly used, be prepared into the tiny balloon of particle diameter less than 10 μ m;

2) tiny balloon that step 1) is obtained can combine the complex solution of the compound formation of trace element to mix formation suspendible pharmaceutical composition with having combined ionic albumen of one or more trace element or polypeptide or aminoacid or glucose or other;

3) directly stored refrigerated prepares injectable powder as the composition of medicine injection or through lyophilization.

Because body has the contradiction property between the non-targeting property trace picked-up of trace element and the targeting property high concentration demand that medicinal organism is learned effector organ, we think that local orientation or the targeting of trace element discharges and have great clinical meaning.But still can't reach the purpose that administered through oral facilitates the orientation of trace element to discharge at present, because do not see that the oral drugs that can effectively implement the release of targeting trace element emerge at present as yet.Oral trace element multi-source absorbs through intestinal wall in food or with inorganic, organic state, after liver is handled, is with the non-specific characteristics that are distributed as of general.Therefore, reach the purpose of targeted release of trace elements, consider, being desirable approach through intravenous injection from clinical angle like need.

Yet the trace element ion salt of water-soluble state is even partial smearing also might produce serious IR.Therefore as carrying out the direct intravenous injection of metal ion salt solution, not only can't reach the purpose that orientation discharges and localized rich is gathered of target organs, the hidden danger that produces serious toxic and side effects is arranged on the contrary.In addition, the ionic salt ion state of this type trace element does not often possess BA, all need combine and transmit the competence exertion physiological action with the bio-compatible mode, such as with the combination of polypeptide, albumen, glucose etc.Therefore, reach the purpose of the trace element release of orientation or target organs, thinking of the present invention is: what the first step must be considered is trace element coalition or complexation body or the associated complex that generates effective protein proteins or polypeptide.Second step was considered method and the efficient that targeting discharges.Given this; What we at first considered is to utilize the most general metal ion of human body to carry albumen: albumin is as optimization protein; Because albumin is that topmost metal ion is conjugated protein in the human peripheral blood; It almost can effectively combine with all metal ions, and non-specificly is delivered to any organ.

Albumin is made up of 585 aminoacid, all links to each other with peptide chain between the aminoacid, and is twisted into Lumbricus shape or cellular, has countless netted spaces, carries medicine and has created favourable steric requirements for inlaying.What second step, we considered is how to accomplish that the method that targeting discharges or the directed tissue organ discharges selects.Targeting drug delivery system is one of emphasis of modern medicines research.With medicine and special carrier coupling, drug selectivity guiding diseased region, increase drug level, improve pharmacokinetic parameter, reduce the purpose of toxic and side effects to reach.So far, all these albuminoid microspheres product or experiments, the targeting that all is confined to antineoplastic chemotherapy medicine discharges or antibiotic controlled release.Applied research about albumin microsphere and albumin nano granular increases gradually in recent years; Mostly be to utilize the surface modification of microsphere and nanoparticle to obtain the better healing effect; Like glycyrrhizic acid finishing albumin nano granular, folacin coupled mitoxantrone albumin nano granular, magnetic adriamycin albumin nano granular etc., but still do not have the listing product; Only there is the paclitaxel albumin nano granular to get into clinical stage (U.S.), because they receive influence of various factors [1-14] in vivo.

In theory, have two kinds of targeting thinkings, first: the active targeting.The formation of this type targeting must at first be synthesized to a certain target organs or histiocytic albumin.The albumin microsphere of antibody-antigen mediation for example.Combine specific antibody or polypeptide at microsphere surface, this can make microsphere that certain cell is had special binding ability, thereby with this cell of drug targeting, realizes specific killing.Li Yuanchun etc. [5] are coupled to F (ab ') 2 fragments of human liver cancer-specific monoclonal antibody HAb18 on the doxorubicin albumin millimicro ball, process immune millimicro ball.The result shows that immune millimicro ball can combine and kill and wound this cell strain effectively, and its effect is dose dependent, and the millimicro ball of contrast then can not combine and kill and wound this cell strain significantly.Or the albumin microsphere of formation cell-specific receptors bind; Like [6] such as Cheng Yao with specific recognition and the picked-up of the asialoglycoprotein receptor on the hepatic parenchymal cells film to irreducibility galactose or IV-acetyl group galactose; The albumin microsphere that has prepared medicine carrying; In its surface parcel one deck galactose acylation chitosan derivant, finally obtain the 5-fluorouracil albumin microsphere that galactose acylation chitosan derivant coats, again in the hope of being attached to cell surface by specificity.

Second kind is so-called passive target.Promptly utilize physical action, make the local release of albumin microsphere.For example, the magnetic albumin microsphere is wrapped in formation magnetic albumin microsphere in the albumin jointly with medicine and magnetisable material.After magnetic and medicated microgranule intravascular is injected in the body; Utilize external magnetic field guide drugs microgranule to be stranded in a certain tissue or lesions position; Prolong drug release time, improve curative effect and the purpose that reduces toxic and side effects to reach, for drug targeting provides a new approach.Chatterjee etc. [4] are through the comparison of polystyrene magnetic microsphere and albumin magnetic microsphere; Find that the albumin magnetic microsphere has the proteinic ability of higher coupling, albumin magnetic microsphere that protein (biological agglutinin) is modified and the bonded ability of erythrocyte are considerably beyond the polystyrene magnetic microsphere.

All above-mentioned albumin microsphere products mainly focus on chemotherapy or anti-inflammatory treatment, but so far, do not see that as yet it is to application and exploitation in the release of the fixed point targeting of trace element.The effect purpose and this patent of the product of above-mentioned targeting modification or experiment are totally different first, all the localized rich of trace element are not gathered to discharge to produce the invention thinking.It two has combined the albumin behind the trace element as carrying out targeting modification once more, and its biological activity, stability and function are difficult to expection and hold.

Therefore thinking of the present invention is, desires to reach the dense effect of gathering of trace element that the second step targeting fixed point release of trace elements forms the local organization organ, and optimal state should be: reduce modification or modification to protein or polypeptide drugs molecule 1. as far as possible.The biomacromolecule that protein drug is made up of aminoacid with certain space conformation; Molecular weight is thousands of to hundreds of thousands often; Its activity depends on its correct structure; Comprise primary structure and space structure, and its structure receives the influence of various factors, such as various protease, heavy metal, organic solvent, temperature, pH value, inhibitor, mechanical force etc.Therefore, in its formulation preparation, storage and dispose procedure, be easy to receive external condition to influence and inactivation, so it is very important to keep proteinic stability.Therefore not having the modification of other drug, combine the albumin of trace element merely, possibly be at utmost to keep the bioactive form of melts combine albumin; 2. the albumin encapsulation that active targeting proteins that similar antigen-antibody is modified and magnetic material are modified; Because added accessory antigen or antibody protein or macromolecular substances and other magnetic metal elements; Its potential immunogenicity and pathogenic be to be difficult to prediction, and be unfavorable for the clinical practice approval of product and promote; 3. by 1,2, it is considered herein that passive target is that trace element albumin-binding or other biological film material are the directed optimization approach that discharges.

According to aforementioned thinking, the present invention has designed a cover biomembrane-trace element hollow micro capsule-ultrasonic drug-supplying system.Involved in the present inventionly be preferably albumin, preparation albumin-trace element hollow micro capsule to the biomembrane material.Its essence is and utilize albumin two specific characters: 1. the combination rate of high efficiency trace element; 2. the Action of Surfactant of albumin self.The innovation of this case is that albumin self promptly as the carrier that carries of trace element, is again the release vehicle of trace element.Albumin microcapsule of the present invention is actual to be the little air bag that includes gas componant that utilizes albumin surfactant characteristics to form.It both had been different from the albumin microballoons commonly used of the liquid phase core that encapsulates the pastille composition, also was different from the albumin microvesicle that is used for ultrasonic contrast imaging merely.Because its purpose had not both lain in conventional albumin microsphere capsule preparation--utilize the albumin encapsulation to carry the drug molecule in being coated on, also do not lie in ultransonic radiography contrast imaging.Its main purpose is to utilize ultrasonic destruction to microbubble that trace element-albumin film is discharged in partial fracture, and the fixed point that forms the trace element at ultrasonic irradiation position discharges and dense gathering, thereby reaches the physiological action of treatment and expectation.A large amount of physical study show, ultrasonic have a typical cavitation, and promptly the ultrasonic sound field part of higher-energy can produce high pressure and the low pressure of moment and replaces, and forms the partial cavitation of propagation, follows this process, can produce the HTHP of moment.When having microbubble in the ultrasonic sound field, this cavitation is more strengthened, and can make partial tissue or blood generation more intensive moment cavitation is HTHP.In the time of microbubbles rupture, this type of effect reaches peak value, the local transient energy of generation, even can make partial microvascular endothelial gap enlargement.This physical effect will make microvesicle and ultrasonication part form the effective blood turbulent flow and vascular space enlarges, and helps trace element in partial quick disperse and entering interstice.Therefore this cover system of the present invention in fact possesses the characteristics of typical passive target administration.Our experiment embodiment confirms: the albumin that has promptly combined trace element merely is its physiology combining form; The own physiological property that has kept trace element in theory to greatest extent; And passive ultrasonic irradiation targeting discharges the cavitation effect not only combined the directivity of ultrasonic acoustic beam but also to have combined ultrasonic sound field+microbubble, makes trace element more quick and direct in partial release and disperse.

Because the main delivery means that the present invention discharges as targeting the reinforcement of the cavitation of ultrasonic sound field with the microcapsule bubble; Therefore the present invention also comprises as selecting the dosage form scheme next time: with the trace element polypeptide complex as preparation host; Wrap up the microcapsule bubble as adjuvant with film; Form trace element-polypeptide complex+microbubble suspension, be injected in the body, breaking up with ultrasonic irradiation is the targeting releasing tool of fixing a point.Can reach the purpose that similar trace element fixed point discharges equally.But the inferior also microbubble due to albumin parcel microbubble or the polymer of scheme indication film parcel microcapsule bubble phospholipid parcel microbubble that selects.

Water miscible trace element salt generally maybe can't work or have the toxicity of height in vivo.Trace element ion (such as copper, zinc, selenium etc.) must be delivered with the mode of bio-compatible.This mode mainly is trace element and corresponding polypeptide or albumen is compound or the glucose associated complex forms polypeptide or albumin complex or glucose mixed liquor, and effectively pair cell produces the Physiology and biochemistry effect with tissue.Here institute's finger protein and polypeptide can refer to that albumin also can refer to other metal composite albumen or polypeptide.

Though the medicine carrying ultrasonic microbubble is to carry out being studied of macromolecular drug of targeting release to be used for thrombolytic, treatment of cancer, gene target conveying etc.This type pharmaceutical preparation is confined in the research and experiment of macromole synthetic drug release, and rests in theoretic discussion or the experimentation.This and the ionic application of basic trace element of arriving involved in the present invention are completely different.This is because metal ion is basic ion, and its toxicity and medical dosage are difficult to accurately to hold, and on the other hand except that the present invention, the targeting of not seeing ultrasonic microbubble prepares the pharmaceutical preparation appearance that thinking and method for preparing are applied to metal ion release.

Therefore important innovations of the present invention is invention and the thinking application to the pharmaceutical composition of the intravital targeted of trace element.The needed by human body trace element is an anthropoid requisite special preparation, and the physiological dose that the people can tolerate is little, and excessive free trace element ion is poisonous usually in vivo; It is not as the common disorder medicine; Need reach the purpose of removing focus, thereby overcome the defective of oral supplementation trace element, and then reach good curing and physiologic effect can't be expected fully like the direct administration of injection form how; The relevant report of also not having prior art; The inventor has created a kind of specific combined mode just, makes trace element with the ultrasonic directed mode that discharges in the body, has reached the satisfied treatment and the effect of adjusting physiological action.

As aforementioned, water miscible trace element salt generally maybe can't work or have the toxicity of height in vivo.Trace element ion (such as copper, zinc, selenium etc.) must be delivered with the mode of bio-compatible.This mode mainly is that trace element and corresponding polypeptide or albumen are compounded to form polypeptide or albumin complex, and effectively pair cell produces the Physiology and biochemistry effect with tissue.Here institute's finger protein and polypeptide can refer to that albumin also can refer to other metal composite albumen or polypeptide.

Specifically, the polypeptide trace element mixture smeared or sprayed generation to local organization therapeutical effect has obtained extensive recognition and acceptance, and has related agents to produce preparation.For example the copper polypeptide complex has been United States Patent (USP) U.S.Pat.Nos.4 for the treatment and the effect of skin and the shallow table wound of internal organs, 760,051; 4,665,054; , 877,770; 5,135,913and 5,348, and 943. grades are confirmed and announced.The copper polypeptide complex to the control of gastric ulcer and therapeutical effect also by United States Patent (USP) U.S.Pat.Nos.5,145,838; 4,767,753and 5,023,237 authorize and confirm.But all these patents all only relate to the body surface of trace element or smearing or the pad pasting therapeutical effect of hollow organ surface.All do not form the localized rich of the trace element of parenchymal viscera in the body is gathered and the therapeutical effect that causes.But these have all given the reasoning of an important logicality of the present invention; No matter the release of partial trace element be to superficial tissue or to the damaged tissues of intravital parenchymal viscera tissue; As long as can form dense gathering and the controlled release of fixing a point, just can produce effectively positive curative effect in the part.

As aforementioned, the related trace element polypeptide complex of this patent means following polypeptide complex (table 1 is that each amino acid whose English is called for short and the abbreviation corresponding tables).

Table 1: amino acid whose English is called for short and the abbreviation corresponding tables

The trace element polypeptide can be meant following complex (M refers to trace element):

glycyl-L-histidyl-L-lysine:M(″GHKM″)，L-alanyl-L-histidyl-L-lysine:M(″AHK-M″)，L-valyl-L-histidyl-L-lysine:M(″VHK-M″)，

L-leucyl-L-histidyl-L-lysine:M(″LHK-M″)，L-isoleucyl-L-histidyl-L-lysine:M(″IHK-M″)，L-pheny?lalanyl-L-histidyl-L-lysine:M(″FHK-M″)，

L-prolyl-L-histidyl-L-lysine:M(″PHK-M″)，L-seryl-Lhistidyl-L-lysine:M(″SHK-M″)，or?L-threonyl-Lhistidyl-L-lysine:M(″THK-M″)。

At this, the polypeptide trace element mixture is meant the coordination chemical compound generally, and it comprises a peptide molecule and trace element is non-covalent is compound on the aforementioned polypeptide.Peptide group is meant two or more aminoacid sequences or amino acid derivativges sequence covalent bond.In general, aminoacid that this patent is carried comprises an amino, a carboxyl, a hydrogen atom and an amino acid side chain conjugated group.The related polypeptide amino acid of this patent mainly is meant alpha, beta or gamma aminoacid, for example:

Alpha-aminoacid beta-aminoacid

Gamma-aminoacid

X represented amino acid side chain binding site in the said structure formula.The mentioned amino acid complex of this patent can be L type or D type or both mixing.The mentioned amino acid derivativges of this patent comprises the structure shown in the following chart.

The structure of the histidine derivative that the present invention comprised is following: n=1-20 in this structure (situation of getting rid of n=3).

The mentioned polypeptide trace element mixture of the present invention can be described according to following general formula: [R1-R2-R3]: M, M here is that the aminoacid or the amino acid derivativges of above-mentioned definition is connected in R through peptide bond at least.Here R single amino acids or amino acid derivativges, such polypeptide trace element mixture generally ranges tripeptides.The general formula of another polypeptide trace element mixture that this patent relates to describe and also can be [R ,-R ,-R ,]: M.But at this apoplexy due to endogenous wind R is that chemical group is connected in R through amido link.The chemical group here refers to comprise amino and can form amido link with any R that contains carboxyl terminal and is connected (such as the carboxyl terminal of histidine, arginic carboxyl terminal or related derivatives).Further set forth: R has 1-20 carbon atom-NH alkyl, and it links to each other through amido link with R, or the virtue that has 6-20 carbon atom is amino.The alkyl that reaches mentioned herein is not got rid of and is comprised amino, octylame, or propylamine.Similarly, fragrant amino is not got rid of and is comprised benzylamine or benzyl.Can produce the amino that amido link is connected with the carboxyl terminal of R for having, can comprise that polyamines is such as spermine and spermidine.

The present invention relates to the combination and the targeted of various trace elements, following embodiment is that the main target of implementing is carried effect with the fixed point of verifying the trace element that this patent is contained with copper.The copper ion of physiological dose possesses a lot of biological effectiveness, for example stimulates collagen and elastin laminin in wound or the damaged tissue to pile up (reference, Maquart et al., J.Clin.Invest.92:2368-2376 (1993); Maquart et al., FEBS Lett.238 (2): 343-346 (1988) and Wegrowski et al., Life Sci.51 (13): 1049-1056 (1992)).Regulate the activity (reference, Simeon et al., J.Invest.Dermatol.112 (6): 957-964 (1999)) of metalloproteases substrate.Improve angiogenic activity (reference, Ahmed et al., Biomaterials 20:201-209 (1999); Hu, G.F., J.Cell.Biochem.69:326-35 (1998); Lane et al., J.Cell.Biol.125 (4): 929-943 (1994); And Raju et al., JNCI69 (5): 1183-1188 (1982)).Improve speed and degree (reference, Counts et al, Federation of American Societies for Experimental Biology Journal 6 [5], the A1636 (1992) of repair in trauma; Downey et al., Surgical Forum 36:573-575 (1985); Fish et al., Wounds3:171-177 (1991); Mulder et al., Wound Repair and Regeneration 139 (1993); Swaim et al., Am.J.VetRes.57:394-399 (1996); And Swaim et al., J.Am.Anim.Hosp.Assoc.29:519-525 (1993)).

Yet as aforementioned, water miscible mantoquita ion is not have physiologically active even suitable toxicity and zest are arranged, and is under an embargo to directly act on wound or cicatrix zone.Copper ion must be delivered with the bio-compatible mode.

Thereby trace element ion of the present invention also can use the complex mode of glucose and trace element to carry, and its general structure is (X represents trace element) as follows:

Therefore, the invention discloses a kind of can be used for the directed release of trace elements ion of ultrasonic interaction so that promote the activation of the partial promotion cicatrix of ultrasonic irradiation, blood vessel, tissue regeneration, antineoplastic purpose combine the ionic pharmaceutical composition of trace element by respective metal ion modification or bonded film parcel gassiness microcapsule and with having combined trace element ionic film material solution or polypeptide.This has comprised that also simple film parcel microcapsule is with corresponding trace element solion or combined the ionic film material of corresponding trace element suspendible medicinal liquid.

This compound medicinal liquid can be under ultransonic effect of irradiation, and the directed trace element ion that imports gets in the body.Thereby the tissue blood vessel regeneration at the position that can effectively make ultrasound to, tissue regeneration or antineoplastic biological effect.Its clinical widely prospect comprises: the short angiogenesis that the revascularization of infarcted myocardium, the revascularization behind the coronary stenosis, various internal organs angiostenosis property or infarction sexually transmitted disease (STD) become (the short angiogenesis such as limb artery after the narrow or infarction).Potential application mainly is the certified physiology biological effect of corresponding trace element ion; The interior revascularization of cicatrix that for example comprises the internal organs of the body postoperative; Like cicatrix behind the various substantial visceras or promotion tissue regeneration; For example: liver postoperative, gastrointestinal postoperative, esophagus postoperative, uterus postoperative etc., the treatment of hepatic fibrosis also comprises such as to growth of tumor inhibition etc.

The mentioned regeneration of the present invention comprises many-sided implication: 1, the vascularization of scar tissue and elastic return; 2, the parenchyma regeneration at position, cicatrix place; 3, the blood vessel of location of necrosis and parenchyma regeneration; 4, the revascularization of narrow ishemic part; 5, tissue fibering reverse etc.

The drug combination preparation of ultrasonic targeted release of trace elements of the present invention has fabulous potential applicability in clinical practice, for blood vessel, tissue and the antineoplaston etc. of various pathological tissues provide a kind of new way of treating safely and effectively.

Description of drawings

Fig. 1 variable concentrations Cu ²⁺Facilitation to the human umbilical vein endothelial cell growth

Fig. 2 copper ion obviously is better than matched group to the increment facilitation of endotheliocyte

Fig. 3 agarose gel electrophoresis detects A group fluorescence quantitative RT-RCR product

Three groups of cell eNOS of Fig. 4 and Tie-1 differential expression

Fig. 5 A is a sham operated rats, and B is that matched group (does not add calcium alginate+CuSO ₄Pad pasting) local visible great amount of fat tissue of infraction and fibrosis tissue are piled up (arrow shows).C is that the revascularization of infarct area behind the pad pasting shows, the recovery of tangible cardiac muscle regeneration and cardiac structure has appearred in infarction tissue, and its form is more near the heart of sham-operation.It is thus clear that significantly capillary network forms.

Fig. 6 is above-mentioned heart infarction pathological section result's contrast.Histology's (HE dyeing, Masson three-color process) observes the more new vessels of cardiac muscle appearance that the Cu pad pasting is handled, and the recovery of a large amount of mononuclear cell and myocardial fibrosis is arranged.CuSO ₄Pad pasting makes sees significant little blood vessel and gliosis in the local heart infarction slough.Masson is a collagen staining.

The bonded fluorescent quenching of Fig. 7 human albumin and copper ion

Fig. 8 is chelating Cu not ²⁺The fluorescence peak of human albumin's microsphere

Fig. 9 adds 50 μ l CuSO ₄Back chelating Cu ²⁺The fluorescent absorption peak figure of microsphere

Figure 10 adds 100 μ l CuSO ₄Chelating Cu ²⁺The fluorescent absorption peak of microsphere

Figure 11 adds 200 μ l CuSO ₄Chelating Cu ²⁺The fluorescent absorption peak of microsphere

Figure 12 adds 300 μ l CuSO ₄Chelating Cu ²⁺The fluorescent absorption peak of microsphere

Figure 13 adds 400 μ l CuSO ₄Chelating Cu ²⁺Microsphere fluorescent absorption peak

Figure 14 adds 500 μ l CuSO ₄Chelating Cu ²⁺Microsphere fluorescent absorption peak

Figure 15 adds 600 μ l CuSO ₄Chelating Cu ²⁺The fluorescent absorption peak of microsphere

Figure 16 adds 700 μ l CuSO ₄Chelating Cu ²⁺Microsphere fluorescent absorption peak

Figure 17 adds 1000 μ l CuSO ₄Chelating Cu ²⁺Microsphere fluorescent absorption peak

The simple albumin microvesicle of Figure 18 increases with the amount of the copper ion that adds, and its fluorescent quenching increases gradually

Figure 19 chelated copper ion albumin film parcel microspherulite diameter distributes

Figure 20 description of drawings: after flow cytometer detect to show that microvesicle combines copper, leave standstill 2 months after, particle size distribution is stabilized in the normal state peak value of 1.5 μ m, mean diameter: 1.95 μ m; The microvesicle of particle diameter 0-3 μ m accounts for 90% of all microvesicle scopes, microbubble concentration 3 * 10 ⁸Individual/ml.There is not significant change with preparation preliminary phase ratio before February.Explain that the chelated copper microvesicle is reliable and stable.

Figure 21 adopts high-energy color Doppler mode continuous to break up albumin microsphere, forms the partial copper ion passive target of heart infarction is discharged.

Figure 22 A is a matched group, does not inject the general pathology picture of heart infarction rabbit model after 4 weeks of chelating albumin microvesicle.Heart front surface visible great amount of fat in a left side is piled up, and is the performance (arrow) of typical heart infarction cicatrix zone steatosis, athero.B is an experimental group, 4 weeks behind the injection chelating albumin microvesicle, and the obvious activation in heart infarction cicatrix district, the surface sees that a large amount of new lives' blood capillary generates (arrow)

Figure 23 A, B is a matched group, does not carry out ultrasonic irradiation targeted chelated copper ion.A: * 400Masson dyeing: degeneration of infarcted region significant quantities of fat and fat vacuole form, no blood vessel hypertrophy.B: * 100Masson dyeing: myocardial infarction sees still that with normal juncture area significant quantities of fat degeneration and fat vacuole form, and do not have obvious blood vessel hyperplasia.C, D are experimental group, and row ultrasonic irradiation targeted chelated copper ion produces obvious collagen and revascularization.C: * 400Masson dyeing: the visible significantly collagen fiber hypertrophy of infarcted region is accompanied little blood vessel hyperplasia.D: infarction and normal juncture area * 100Masson dyeing: see that tangible collagen fiber hypertrophy accompanies little blood vessel hyperplasia.

Figure 24 description of drawings: after leaving standstill February; The fluidic cell instrument show to combine behind the zinc ion microvesicle particle size distribution not have notable difference during with first the preparation with concentration change; Particle size distribution normal distribution peak value is at 1.5 μ m; The microvesicle that particle diameter 0-3 sympathizes accounts for 90.5% of all microvesicle scopes, mean diameter 2.5 μ m.Microbubble concentration 3.4 * 10 ⁸Individual/ml.Microvesicle in conjunction with zinc possesses reliability.

Figure 25 description of drawings: after leaving standstill February, after the demonstration microvesicle combined selenium, particle size distribution was stabilized in the normal state peak value of 1.5 μ m, mean diameter 2.1 μ m, and the microvesicle of particle diameter 0-3 μ m accounts for 91% of all microvesicle scopes, microbubble concentration 3 * 10 ⁸Individual/ml.With the bimester before the microvesicle particle size distribution compare with concentration and do not have significant change.Possesses reliable and stable characteristic in conjunction with selenic microvesicle.

Show that with the microvessel density contrast of only injecting in the phospholipid microsphere matched group heart infarction zone microvessel density that contains the heart infarction zone of the albuminous experimental group of chelated copper is significantly higher than matched group through ultrasonic irradiation phospholipid microsphere and the albuminous heart infarction of chelated copper zone in Figure 26 heart infarction zone.

In the heart infarction zone of ultrasonic irradiation phospholipid microsphere and chelated copper glucose, see tangible little revascularization in Figure 27 heart infarction zone.

Heart infarction zone through ultrasonic irradiation phospholipid microsphere and chelated copper glucose in Figure 28 heart infarction zone shows that with the microvessel density contrast of only injecting in the phospholipid microsphere matched group heart infarction zone the regional microvessel density of heart infarction that contains the experimental group of chelated copper glucose solution is significantly higher than matched group.

The specific embodiment

Below through the specific embodiment the present invention is done further explanation, but be not limitation of the present invention.Below among each embodiment; Our preferred copper ion in trace element carries out the confirming of isolated cells physiological effect, animal is tested local pad pasting controlled release at body and confirms that the generation of the physiological effect of isolated cells, chelated copper microvesicle produce isolated cells and showing consideration for the similar experiments such as physiological effect of film experiment cardiac muscle at body controlled release trace element, produce the science and the feasibility of topical therapeutic and corresponding physiological effect with the local controlled release trace element of proof microvesicle combining ultrasonic.

Embodiment 1 confirms irritation cell regeneration that copper ion is external and stem cell mobilization experiment and at the physiologic effect of the local pad pasting controlled release of body copper ion

(human umbilical vein endothelial cell HUVEC) can effectively strengthen propagation and differentiation to cell in vitro experiment confirm trace element-copper ion to human umbilical vein endothelial cell.The In vitro culture and the HUVEC that goes down to posterity.With HUVEC with 5 * 10 ³Individual/hole density is inoculated in 96 orifice plates, according to the difference that in orifice plate, adds solution concentration cell is divided into 3 groups at random, and A organizes 5 μ mol/L CuSO ₄, B organizes 25 μ mol/L CuSO ₄, the C group is blank, 4 every group multiple holes, and basal medium is MCDB131, adopts mtt assay detection cell proliferation and draws growth curve.Other gets HUVEC with 2 * 10 ⁵Individual/hole density is inoculated in 6 orifice plates, and it is the same to divide into groups, and fluorescence quantitative RT-RCR detects endothelial nitric oxide synthase (endothelial nitric oxide synthase, eNOS) gene and the Tie-1 gene expression of 3 groups of HUVEC.3d was the Exponential growth phase before growth curve showed as a result, and the 4th day begins to get into plateau.A group ability of cell proliferation is organized since the 3rd day apparently higher than B, C; The B group is higher than the C group in 2d, but significantly is lower than the C group since the 4th day, and comparing difference all has statistical significance (P＜0.05) to see Fig. 1; 2, copper ion obviously is better than matched group to the increment facilitation of endotheliocyte.After fluorescence quantitative RT-RCR detects and shows effect 48h; A, B, C group eNOS gene expression are respectively 7.294 soil 1.488,0.149 ± 0.044 and 1.000 ± 0.253, and the Tie-1 expression of gene is respectively 1.481 ± 0.137,1.131 ± 0.191 and 1.000 ± 0.177.A group and B, C organize relatively, and 2 expression of gene all have rise (P＜0.05); B, C organize relatively, eNOS down regulation of gene expression (P＜0.05), Tie-1 gene expression difference not statistically significant (P＞0.05) (Fig. 3,4, table 1).Conclusion: 5 μ mol/L Cu ²⁺Can effectively promote propagation and the differentiation of HUVEC.

The expression of three groups of cell eNOS of table 1 and Tie-1

^*The A group compares P＜0.05 with the C group; The #B group compares P＜0.05 with the C group

Embodiment 2 confirms irritation cell regeneration that copper ions are external and stem cell mobilization experiment and at the physiologic effect of the local pad pasting controlled release of body copper ion.

For confirming the activation and the partial result that stimulate cicatrix in revascularization of copper ion, local heart infarction animal model is carried out the copper-surfaced ionic gel, with the physiological role of checking local sustained release copper ion at the damage cicatrix.

1, preparation calcium alginate+CuSO ₄Pad pasting.With 10 milliliters of the sodium alginate solns of 2% mass fraction with contain 10mg CuSO ₄Solution fully mixes, Dropwise 5 ml CaCl ₂Film forming behind the solution.

2, preparation heart infarction model: under the narcotism, open the free and ligation Cor Leporis left anterior descending branch arteria coronaria of breast, preparation rabbit left side heart heart infarction model.Open under the breast state, preceding step institute's film forming is attached at left heart front surface.Close breast.After stablizing for 4 weeks, open breast once more and observe BIAO and BEN and pathological section difference substantially.

The result shows that the copper ion of local sustained release forms significant promotion revascularization and the activatory physiologic effect of cicatrix.On the myocardial infarction and ischemia model of rabbit; We are attached at the myocardial ischemia infarcted myocardium tissue after 4 weeks of performing the operation with preceding step institute's film forming; After treating for 2 weeks, the color ultra and heart muscle perfusion radiography of dynamic heart confirms can effectively improve myocardial ischemia and cardiac function after the copper ion part replenishes weekly; Pathologic finding finds to occur in the infarcted myocardium tissue a large amount of new vesselses (seeing Fig. 5,6).

Show among the figure: Fig. 5 A is a sham operated rats, and B is that matched group (does not add calcium alginate+CuSO ₄Pad pasting) local visible great amount of fat tissue of infraction and fibrosis tissue are piled up (arrow shows).C is that the revascularization of infarct area behind the pad pasting shows, the recovery of tangible cardiac muscle regeneration and cardiac structure has appearred in infarction tissue, and its form is more near the heart of sham-operation.It is thus clear that significantly capillary network forms.

Fig. 6 is above-mentioned heart infarction pathological section result's contrast.Histology's (HE dyeing, Masson three-color process) observes the more new vessels of cardiac muscle appearance that the Cu pad pasting is handled, and the recovery of a large amount of mononuclear cell and myocardial fibrosis is arranged.CuSO ₄Pad pasting makes sees in the local heart infarction slough that significant little blood vessel and gliosis Masson are collagen stainings.

Embodiment 3 copper ions and albumin bound experiment (preparation of copper ion chelating albumin microvesicle)

Used solution is prepared with deionized water in the experiment, uses 0.1molL ^-1NaCl keep ionic strength, as buffer, pH value is 7.4 with Tris-HCl, prepares HSA and copper-bath, concentration is followed successively by 8.76 * 10 ^-6MolL-1 and 6.48 * 10 ^-4MolL-1.Use the 1cm cuvette, measured (1) fixedly excitation wavelength be 280nm, copper sulfate carries out titration to HSA, the variation of the fluorescence emission peak of observation HSA.Can find out Cu among the figure ²⁺The excitation peak that obviously do not change of adding, the position at maximum fluorescence peak, but fluorescence intensity weakening in various degree with the increase of amount of copper sulfate.Because the volume of HSA solution is far longer than the volume of the metal ion solution that is dripped, therefore can ignore dilution effect.

Protein can send fluorescence, is that because these amino acid residue various structure, three's fluorescence intensity ratio is 100: 9: 0.5 usually because have three kinds of aromatic amino acid residue: Trp, Tyr and Phe residues in the protein.Whether contain tryptophan according to protein and can it be divided into two types: category-A albumen refers to not contain the protein that the Trp residue only contains Tyr and Phe residue; Category-B albumen: refer to not only contain the Trp residue but also contain Tyr and the protein of Phe residue.Serum albumin belongs to category-B albumen, and stronger fluorescence is arranged.Though all there is the characteristic fluorescence peak (the fluorescence peak position of Trp, Tyr and Phe lay respectively at 348,303 and 282nm) of oneself in three kinds of chromophores; But because in protein macromolecule; Very effective by Phe to the energy transfer of Tyr or rrrp; Therefore the absorption of whole molecule and emission neither these monomer component optical properties simply adds and subtracts, and often the fluorescence of Trp residue is in the ascendance.When in 270～290nm elicitor protein matter, can not ignore the contribution of Tyr residue, when excitation wavelength during, can think that fluorescence is all from the Trp residue greater than 290nm.Only contain one 214 Trp residue among the HSA, fluorescence emission peak is near 350nm.It is generally acknowledged that fluorescence mainly sends from 212 Trp residues, about 342nm.The maximum excitation peak of the two all is about 280nm.The Trp residue can behind materials such as adding metal ion, utilize the variation of the endogenous fluorescence of serum albumin as the probe that local conformation changes or aromatic amino acid residue microenvironment changes, can be to combining carry out qualitative and quantitative study.

This experiment embodiment shows that along with the adding of copper ion, more copper ion and human albumin's chelating make albuminous fluorescence generation cancellation.

Instance 4 copper ions and albumin microvesicle combine experiment, comprise fluorescence analysis result (preparation of copper ion chelating albumin microvesicle)

1. the preparation of albumin microvesicle: pipette 20ml 5% human albumin's solution to the 20ml disposable syringe that connects threeway; The Ultrasound Instrument probe is inserted 2cm place under the liquid level; With certain ultrasound intensity; The sound 20ml 1% human albumin's solution that shakes, the sound 10s that shakes fed a certain amount of C again in 5 seconds when sound shakes in advance ₃F ₈, continuation sound shakes, and stops sound after the temperature of breasting the tape and shakes, and gets C ₃F ₈Albumin microsphere suspension, suspension are transferred in the 50ml medicine bottle immediately, and medicine bottle upper end air is used C ₃F ₈Fully displacement, medicine bottle sternly seals with plug, under room temperature, leaves standstill 24h.Shake up the back and microsphere is carried out granularmetric analysis such as following table 2, microsphere average diameter 2.15 μ m.

Table 2: the particle size distribution of albumin microsphere

Table 2 shows that the microvesicle mean diameter is 2.15 μ m, and mean concentration is 3.7 * 10 ⁸Individual/ml.

Microvesicle film and copper ion combine experiment:

The accurate 5%HSA microvesicle solution 1ml that draws is diluted in the volumetric flask of 250ml, and pipet is respectively got in 10ml to 9 teat glass, drips Cu successively respectively ²⁺(the saturated copper solution of 1/50 dilution) solution is to be measured.Following table 3 is the bonded amount of each variable concentrations copper ion and HSA microvesicle.See Fig. 8-17, after albumin microvesicle adding different concentrations of copper sulphate, the fluorescent quenching reaction that takes place behind copper ion and the albumin microsphere vacuolar membrane chelating.All visible two absworption peaks of Fig. 8-17, the former is the characteristic peak (excitation wavelength is about 332nm) that adds behind the copper; The absworption peak (emission wavelength is 408nm) that the peak is the not ionic blank albumin microsphere of chelated copper itself in back; Method for reading data: there are two expression values at each peak, reads from top to bottom, and two data are used. and separate, the latter is the fluorescence spectrum absorption intensity.

This experiment proof copper ion combines with the albumin microsphere vacuolar membrane, causes albuminous fluorescence intensity gradually little.

Table 3: the variable concentrations copper sulfate of adding

The preparation of instance 5 copper ion albumin-binding microvesicles

Ultrasonic dispersing prepares the experiment of albumin copper microvesicle after copper ion and the albumin bound, comprises fluorescence analysis result and microvesicle metering result.

1. at first preparation combines the albumin solution of copper ion: by copper sulfate: albumin solution=1: 1mol carries out application of sample.The corresponding ratio of this instance is following: 1mg HSA adds 4.35 * 10 ^-6MolCu ²⁺

2. pipette 20ml 5% chelated copper-human albumin's solution to the 20ml disposable syringe that connects threeway; The Ultrasound Instrument probe is inserted 2cm place under the liquid level; With certain ultrasound intensity; The sound 20ml 5% human albumin's solution that shakes, the sound 10s that shakes fed a certain amount of C again in 5 seconds when sound shakes in advance ₃F ₈, continuation sound shakes, and stops sound after the temperature of breasting the tape and shakes, and gets C ₃F ₈Albumin microsphere suspension, suspension are transferred in the 50ml medicine bottle immediately, and medicine bottle upper end air is used C ₃F ₈Fully displacement, medicine bottle sternly seals with plug, under room temperature, leaves standstill 24h.Shake up the back and microsphere is carried out granularmetric analysis, particle size distribution such as Figure 19.The mean diameter of microvesicle is 2 μ m behind the demonstration chelated copper ion, and concentration is 3.1 * 10 ⁸Individual/ml

3. stability experiment: the microvesicle of aforementioned combination copper ion is injected in the 20ml vial, and sealing is preserved in the 5 degree refrigerators.To February, the measurement of resampling, microbubble concentration and particle size distribution and the microvesicle of just having prepared obviously do not change.Following Figure 20 is the detection of flow cytometer to the microvesicle change of size, detects explanation, and microbubble concentration and particle diameter are stable.

After Figure 20 flow cytometer detect to show that microvesicle combines copper, leave standstill 2 months after, particle size distribution is stabilized in the normal state peak value of 1.5 μ m, mean diameter: 1.95 μ m; The microvesicle of particle diameter 0-3 μ m accounts for 90% of all microvesicle scopes, microbubble concentration 3 * 10 ⁸Individual/ml.There is not significant change with preparation preliminary phase ratio before February.Explain that the chelated copper microvesicle is reliable and stable.

Experiment of instance 6 albumin copper ion microcapsular ultrasound irradiation myocardial infarction animal models and result

Prepare 15ml albumin C with instance 5 methods ₃F ₈, wherein be total to chelating 10mg copper ion.Under the narcotism, open the free and ligation Cor Leporis left anterior descending branch arteria coronaria of breast, preparation rabbit left side heart heart infarction model, stablized for 4 weeks after.The row auricular vein is injected chelated copper ion albumin microvesicle with the speed of 1ml/min continuously; With ultrasonic continuous irradiation left side pareordia; By doing back and forth sector scan continuously to the apex of the heart at the bottom of the heart, ultrasound wave output energy transfers to maximum and launches color Doppler pattern (Figure 21).Per 2 weeks repeat a shot and irradiation, and each dosage is identical.To 4 weeks, put to death experimental rabbit, the observation experiment rabbit is cardiac shape and heart infarction pathological section substantially, revascularization and activation situation in the heart infarction scar tissue of relative analysis matched group.The result shows that injection chelated copper ion albumin microsphere makes to produce in a large number and significantly revascularization and activation in the heart infarction local organization, sees Figure 22,23 under ultransonic irradiation.

Figure 21 breaks up albumin microsphere for adopting high-energy color Doppler mode continuous, forms the partial copper ion passive target of heart infarction is discharged.

Figure 22: A is a matched group, does not inject the general pathology picture of heart infarction rabbit model after 4 weeks of chelating albumin microvesicle.Heart front surface visible great amount of fat in a left side is piled up, and is the performance (arrow) of typical heart infarction cicatrix zone steatosis, athero.B is an experimental group, 4 weeks behind the injection chelating albumin microvesicle, and the obvious activation in heart infarction cicatrix district, the surface sees that a large amount of new lives' blood capillary generates (arrow).

Figure 23: A, B is a matched group, does not carry out ultrasonic irradiation targeted chelated copper ion.A: * 400Masson dyeing: degeneration of infarcted region significant quantities of fat and fat vacuole form, no blood vessel hypertrophy.B: * 100Masson dyeing: myocardial infarction sees still that with normal juncture area significant quantities of fat degeneration and fat vacuole form, and do not have obvious blood vessel hyperplasia.C, D are experimental group, and row ultrasonic irradiation targeted chelated copper ion produces obvious collagen and revascularization.C: * 400Masson dyeing: the visible significantly collagen fiber hypertrophy of infarcted region is accompanied little blood vessel hyperplasia.D: infarction and normal juncture area * 100Masson dyeing: see that tangible collagen fiber hypertrophy accompanies little blood vessel hyperplasia.

The preparation that combines experiment and chelated zinc albumin microsphere of instance 7 albumin and zinc ion (confirming the experiment of zinc ion chelating albumin microvesicle)

1. by zinc chloride: albumin=mol ratio was carried out in 1: 1.Accurate 5%HSA microvesicle solution 15ml, the liquor zinci chloridi of molar concentrations such as dropping drawn.Fluorescence analysis shows that after albumin solution added zinc chloride, the same fluorescent quenching reaction that takes place of zinc ion and albumin confirmed zinc ion and albumin generation chelating.According to mol ratio, prepare 15ml 5% chelated zinc human albumin altogether.

2. chelated zinc-human albumin's solution of producing of removing step 1 is to the 20ml disposable syringe that connects threeway; Ultrasound Instrument probe is inserted 2cm place under the liquid level, and with certain ultrasound intensity, human albumin's solution shakes; Sound shook 10 seconds in advance, when sound shakes, in 5 seconds, fed a certain amount of C again ₃F ₈, continuation sound shakes, and stops sound after the temperature of breasting the tape and shakes, and gets C ₃F ₈Albumin microsphere suspension, suspension are transferred in the 30ml medicine bottle immediately, and medicine bottle upper end air is used C ₃F ₈Fully displacement, medicine bottle sternly seals with plug, under room temperature, leaves standstill 24h.Shake up the back microsphere is carried out granularmetric analysis, particle size distribution is identical with instance 5, microsphere average diameter 2.5-3.0 μ m.

3. stability experiment: the microvesicle of aforementioned combination zinc ion is injected in the 20ml vial, and sealing is preserved in 4 ℃ of refrigerators.To February, the measurement of resampling, microbubble concentration and particle size distribution and the microvesicle of just having prepared obviously do not change.

Figure 24 is the granularmetric analysis that flow cytometer carries out microvesicle

After February is left standstill in Figure 24 demonstration; The fluidic cell instrument show to combine behind the zinc ion microvesicle particle size distribution not have notable difference during with first the preparation with concentration change; Particle size distribution normal distribution peak value is at 1.5 μ m, and the microvesicle of particle diameter 0-3 μ m accounts for 90.5% of all microvesicle scopes, mean diameter 2.5 μ m.Microbubble concentration 3.4 * 10 ⁸Individual/ml.Microvesicle in conjunction with zinc possesses reliability.

Instance 8 albumin and selenide combine experiment (confirming the experiment of plasma selenium chelating albumin microvesicle)

1. preparation 20ml combines selenic HSA: three selenium sulfide penicillaminees (4mM) are mixed in the phosphate buffer (pH7.4) of 0.01M with HSA (40mg/mL), and mixed liquor was hatched 10 minutes.Unreacted PenSePen removes through dialysis.Obtain combining selenic HSA.

2. selenium-human albumin's solution is to the 20ml disposable syringe that connects threeway, and the Ultrasound Instrument probe is inserted 2cm place under the liquid level, with certain ultrasound intensity, the sound human albumin's solution that shakes, the sound 10s that shakes in advance, again when sound shakes in 5 seconds a certain amount of C of feeding ₃F ₈, continuation sound shakes, and stops sound after the temperature of breasting the tape and shakes, and gets C ₃F ₈Albumin microsphere suspension, suspension are transferred in the 30ml medicine bottle immediately, and medicine bottle upper end air is used C ₃F ₈Fully displacement, medicine bottle sternly seals with plug, under room temperature, leaves standstill 24h.Shake up the back microsphere is carried out granularmetric analysis, particle size distribution is identical with instance 5, microsphere average diameter 2.5-3.0 μ m.

3. stability experiment: the microvesicle of aforementioned combination plasma selenium is injected in the 20ml vial, and sealing is preserved in the 5 degree refrigerators.To February, the measurement of resampling, microbubble concentration and particle size distribution and the microvesicle of just having prepared obviously do not change.Following Figure 25 is the granularmetric analysis that flow cytometer carries out.

After February was left standstill in Figure 25 demonstration, after the demonstration microvesicle combined selenium, particle size distribution was stabilized in the normal state peak value of 1.5 μ m, mean diameter 2.1 μ m, and the microvesicle of particle diameter 0-3 μ m accounts for 91% of all microvesicle scopes, microbubble concentration 3 * 10 ⁸Individual/ml.With the bimester before the microvesicle particle size distribution compare with concentration and do not have significant change.Possesses reliable and stable characteristic in conjunction with selenic microvesicle.

Instance 9 albumin bound copper+phospholipid microvesicle combined experiments

1. press the albumin of instance 3 preparation chelated coppers: the NaCl of 0.1M keeps ionic strength, and as buffer, pH value is 7.4 with Tris-HCl, preparation HSA and copper-bath, and concentration is followed successively by 8.76 * 10 ^-6M and 6.48 * 10 ^-4M.

2. inject 9.6ml SONOVUE phospholipid SF ₆Microvesicle.

3. 1 and 2 solution is mixed, form the suspension of albumin polypeptide chelated copper and phospholipid microvesicle.

The zoopery and the result of instance 10 albumin bound copper+phospholipid microvesicle heart infarction animal model

1.

press instance

2 and 6 preparation Cor Leporis stalk models.

2. the albumin polypeptide suspension of instance 9 made phospholipid microspheres and chelated copper is injected with the speed of 1ml/min through auricular vein continuously; Infusion is simultaneously with ultrasonic continuous irradiation left side pareordia; By doing back and forth sector scan continuously to the apex of the heart at the bottom of the heart, ultrasound wave output energy transfers to maximum and launches the color Doppler pattern.Per 2 weeks repeat a shot and irradiation, and each dosage is identical.To 4 weeks, put to death experimental rabbit, the observation experiment rabbit is cardiac shape and heart infarction pathological section substantially, revascularization and activation situation in the heart infarction scar tissue of relative analysis matched group.The result shows, the albumin polypeptide suspension of injection lipoid microsphere and chelated copper under ultransonic irradiation, makes and produces tangible revascularization and activation in the heart infarction local organization.Through the method for microvessel density counting, the heart infarction scar tissue is carried out pathology get sheet.Get 5 zones of every section stochastic sampling, 10 * 40 times of mirrors are counted down.Institute's blood vessel diameter of getting all in 20 μ m, is added up the microvessel quantity in the above-mentioned visual field.

Figure 26 shows: show that with the microvessel density contrast of only injecting in the phospholipid microsphere matched group heart infarction zone microvessel density that contains the heart infarction zone of the albuminous experimental group of chelated copper is significantly higher than matched group through ultrasonic irradiation phospholipid microsphere and the albuminous heart infarction of chelated copper zone in the heart infarction zone.

Instance 11 glucoses combine the zoopery and the result of copper+phospholipid microsphere heart infarction animal model

1.

press instance

2 and 6 preparation Cor Leporis stalk models.

2. the glucose suspension of instance 9 made phospholipid microspheres and chelated copper is injected with the speed of 1ml/min through auricular vein continuously; Infusion is simultaneously with ultrasonic continuous irradiation left side pareordia; By doing back and forth sector scan continuously to the apex of the heart at the bottom of the heart, ultrasound wave output energy transfers to maximum and launches the color Doppler pattern.Per 2 weeks repeat a shot and irradiation, and each dosage is identical.To 4 weeks, put to death experimental rabbit, the observation experiment rabbit is cardiac shape and heart infarction pathological section substantially, revascularization and activation situation in the heart infarction scar tissue of relative analysis matched group.The result shows, the glucose suspension of injection lipoid microsphere and chelated copper under ultransonic irradiation, makes and produces tangible revascularization and activation in the heart infarction local organization.Through the method for microvessel density counting, the heart infarction scar tissue is carried out pathology get sheet.Get 5 zones of every section stochastic sampling, 10 * 40 times of mirrors are counted down.Institute's blood vessel diameter of getting all in 20 μ m, is added up the microvessel quantity in the above-mentioned visual field.

Figure 27 shows: in the heart infarction zone of ultrasonic irradiation phospholipid microsphere and chelated copper glucose, see tangible little revascularization in the heart infarction zone.

Figure 28 demonstration: the heart infarction zone through ultrasonic irradiation phospholipid microsphere and chelated copper glucose in the heart infarction zone shows that with the microvessel density contrast of only injecting in the phospholipid microsphere matched group heart infarction zone the regional microvessel density of heart infarction that contains the experimental group of chelated copper glucose solution is significantly higher than matched group.

More than experiment proof; What the present invention prepared can the targeted release of trace elements preparation under ultrasonication, has reached at the local organization release of trace elements and produces the relevant biological effect of trace element therewith, ultrasonic targeted medicament composition determined curative effect of the present invention; Good stability; Safety is a kind of new pharmaceutical preparation, has fabulous clinical practice and industrial prospect.

List of references

[1]Coombes?A?G，Breeze?V，Lin?W，et?al.Lactic?acid-stabilised?albumin?for?microsphere?formulation?and?biomedical?coatings[J].Biomaterials，2001，22(1)：1-8.

[2]Almond?B?A，Hadba?A?R，Freeman?S?T，et?al.Efficacy?of?mitoxantrone-loaded?albumin?microspheres?for?intratumoral?chemotherapy?of?breast?cancer[J].J?Control?led?Release，2003，91(1-2)：147-155.

[3]Sahin?S，Selek?H，Ponchel?G，et?al.Preparation，characterization?and?in?vivo?distribution?of?terbutaline?sulfate?loaded?albumin?microspheres[J].J?Control?led?Release，2002，82：345-358.

[4]Chatterjee?J，Haik?Y，Chen?C?J.Modification?and?characterization?of?polystyrene-based?magnetic?microspheres?and?comparison?with?albumin-based?magnetic?microspheres[J].Magnetism?Magnetic?Materials，2001，225(1)：21-29.

[5] Li Yuanchun, Cai Meiying, Wang Zhongqiong kills and wounds cancerous cell effect [J] etc. the preparation of the anti-hepatocarcinoma amycin immunity of 2 sections guiding of. monoclonal antibody F (ab ') millimicro balls and external. Journal of West China University of Medical Sciences, 2002,33 (1): 8-11.

[6] Cheng Yao, Zhang Can, flat its ability, etc. the preparation [J] of the 5-fluorouracil albumin microsphere that galactose acylation chitosan derivant bag is multiple. China Medicine University's journal, 2004,35 (6): 517-520.

[7]Amedo?A，Espuelas?S，Irache?J?M.Albumin?nanoparticles?as?carriers?for?a?phosphodiester?oligonucleotide[J].Int?J?Pharm，2002，244(1-2)：59-67.

[8]Arnedo?A，Irache?J?M，Merodio?M，et?al.Albumin?nanoparticles?improved?the?stability，nuclear?accumulation?and?anticytomegaloviral?activity?of?aphosphodiester?oligonucleotide[J].J?Control?led?Release，2004，94(1)：217-227.

[9]Lin?W，Garnett?M?C，Schacht?E，et?al.Preparation?and?in?vitro?characterization?of?HAS-mPEG?nanoparticles[J].Int?J?Pharm，1999，189(2)：161-170.

[10] Gong Liansheng, Zhang Yangde, Liu Shu. the magnetic targeting property [J] of magnetic adriamycin albumin nano granular in the transplanted hepatoma model. Chinese Genneral Surgery magazine, 2003,9 (9): 543-546.

[11] Liu Xiaobo, Cai Meiying. the preparation of anti-human liver cancer immunity nanoparticle and the evaluation [J] of external immunological properties. Chinese Journal of Immunology, 2000,16:262-265.

[12] Zhang Liangke, Hou Shixiang, Mao Shengjun, etc. quinone albumin nano granular tumor cell targeting Journal of Sex Research [J] is feared in receptor-mediated rice holder. Sichuan University's journal (medicine), 2006,37 (1): 77-79.

[13] Zhang Yangde, Huang Lin, Zhang Lei, etc. glycosyl galactose magnetic adriamycin albumin nano granular is to the research [J] of rat implantation property liver cancer model bcl-2/bax protein expression. Chinese experimental surgery magazine, 2004,21 (11): 1298-1299.

[14] Yang Li, Hou Lianbing, Wang Xinya, etc. the preparation [J] of novel acoustic contrast agent perfluoropropane albumin microcapsule. Chinese Pharmaceutical Journal, 2005,49 (21): 1637-1640.

Claims

1. the pharmaceutical composition with ultrasonic targeted release of trace elements effect is characterized in that: comprise concentration greater than 1 * 10 ⁶Individual/ml, particle diameter is less than tiny balloon and at least a trace element that is less than or equal to ten times of physiological doses of human body of 10 μ m, and pharmaceutically acceptable carrier or adjuvant;

Wherein trace element and tiny balloon exist with bonded form, and said tiny balloon is prepared from pharmaceutically acceptable filmogen;

Said bonded form is a chelating;

Said trace element be in copper, zinc or the plasma selenium any one;

Said filmogen is the human albumin.

2. pharmaceutical composition according to claim 1 is characterized in that: said tiny balloon particle diameter is 1～5 μ m; Said tiny balloon includes the mist that gas is the combination in any of air, nitrogen, sulfur fluoride gas, fluoric ether gas or above-mentioned one or more gas componants.

3. pharmaceutical composition according to claim 1 is characterized in that: the trace element of said combining form and tiny balloon mutually combine through chemical action or physical action.

4. pharmaceutical composition according to claim 3; It is characterized in that: the trace element of combining form and tiny balloon are prepared by following method: 1) according to molar concentration rate be: 1: 0.05 to 1: 500 with filmogen and the trace element ion is water-soluble or organic solvent in, make the trace element ion through physics or chemical action and film material chelating formation trace element compound film material; 2) with conventional method with the compound film material that step 1) obtains, be prepared into the tiny balloon of particle diameter less than 10 μ m.

5. pharmaceutical composition according to claim 1 is characterized in that: described carrier or adjuvant are deionized water or normal saline or glucose solution or the solution that contains the trace element ion complex.

6. according to the arbitrary described pharmaceutical composition of claim 1～5, it is characterized in that: it is ejection preparation.

7. pharmaceutical composition according to claim 6 is characterized in that: said ejection preparation is injection or injectable powder.

8. any described pharmaceutical composition of claim 1～7 promotes the ultrasonic targeting of revascularization to discharge the purposes in the medicine in preparation; Wherein said trace element is a copper ion.

9. purposes according to claim 8; It is characterized in that: said pharmaceutical composition is in human body is gone in intravenous injection; Utilize ultrasound wave that therapentic part is carried out irradiation, utilize gassiness tiny balloon and hyperacoustic interaction to reach the ionic purpose of local release of trace elements.

10. purposes according to claim 8; It is characterized in that: said ultrasonic targeting discharges the ultrasound wave that is meant certain energy to be made cavitation effect such as microbubble ruptures or vibration and impels and strengthens the associated metal ion and discharge and/or promote the corresponding trace element ion component in the suspension to get into the irradiation tissue in that ultrasonic irradiation is local, reaches promotion blood vessel, tissue regeneration and/or the clinical purpose of antineoplastic.

11. purposes according to claim 10 is characterized in that: said ultrasonic targeting discharges drug-induced ultrasonic irradiation position and produces the corresponding physiological function of trace element ion that carries.

12. prepare the method for the described pharmaceutical composition of claim 1, it is characterized in that: at first preparation combines the ionic tiny balloon of trace element, and method for preparing comprises:

1) according to molar concentration rate be 1: 0.05 to 1: 500 with filmogen and the trace element ion is water-soluble or organic solvent in, make the trace element ion through physics or chemical action and film material chelating formation trace element compound film material;

3) will prepare the ionic tiny balloon of combination trace element accomplished and carrier or adjuvant and complex and mix formation suspendible pharmaceutical composition mutually.

13. according to the said method for preparing of claim 12; It is characterized in that: step 2) the hollow core microsphere utilize below the method for preparing microsphere preparation commonly used of any pharmacy: ultrasonic acoustic shake method, freeze-drying, spray drying, activity/controllable free-radical polymerisation, precipitation polymerization method, suspension polymerisation; Emulsion polymerisation; Seeding polymerization, dispersin polymerization and precipitation polymerization heteropolymerization system, ionic cross-linking, emulsifying ionic gel method, ion precipitation-chemical crosslink technique, emulsifying-chemical crosslink technique, emulsion cross-linking method, heat cross-linking method, coacervation, emulsifying-solvent evaporated method.