CN102302507B

CN102302507B - Pharmaceutical composition for directionally and controllably releasing trace elements and preparation method and application thereof

Info

Publication number: CN102302507B
Application number: CN201110277839.6A
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: 2014-02-05
Anticipated expiration: 2030-07-12
Also published as: CN102302507A

Abstract

The invention discloses a medicinal composition for directionally and controllably releasing trace elements, a preparation method and application thereof, which is characterized in that: containing more than 1 × 10 of⁶Each 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 microelements and the hollow microspheres exist in a 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

The pharmaceutical composition of directional controlled release of trace elements and preparation method and application

The application is the applying date: on July 12nd, 2010, and application number: 201010223282.3, denomination of invention: the dividing an application of a kind of pharmaceutical composition of targeted release of trace elements and preparation method and application's patent application.

Technical field

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

Technical background

Trace element plays a part basic in maintaining human health, and main Physiological Function is to play catalytic action in various enzyme systems, with the essential composition of hormone or vitamin or cofactor, plays a role, and forms the metalloprotein etc. with specific function.The meaning of trace element physiological action can be compared with vitamin, but body can synthesize some vitamin voluntarily and cannot synthesize any element, and from this point, essential trace element is even more important compared with vitamin to human body.The basic definition of essential trace element refers to that those have obvious Nutrition and physiological function, to maintaining the requisite elements such as body growth growth, vital movement and procreation.So-called " essential ": 1. body must absorb this element from extraneous diet, and when remove this element from diet after, body just there will be the physiological deficiency state of this element.2. supplement after this element-specific, this deficiency state of body will be eased.3. this special element always has certain species specific biochemical function to body, and this effect can not be replaced completely by other any elements.When this element Deficiency of Intake can cause body biological function obstacle, and recover can alleviate or prevent this dysfunction after the physiological level of this element.Once body leaves this element, can not grow, can not complete 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 there will be similar physiology, biochemical abnormal after this element.While 3. having this element to exist, can alleviate or prevent above-mentioned abnormal.4. this abnormal change also can be cured when shortage is controlled.

At present, the iodine in trace element, selenium, zinc, ferrum, copper, manganese, chromium etc. are confirmed as " maintaining 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 maintaining health.Some element not only can infection, and with many chronic, popular, endemicity, even malignant change is relevant.Massive epidemiology investigation is pointed out, if body lacks essential trace element, may make crowd increase the sensitivity of disease, causes generation and the development of sub-health state or disease.

Although the content of trace element in human body is few, closely bound up with people's existence and health.For many years, the importance of trace element in human body more and more paid attention to.The 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, has ferrum, copper, zinc, cobalt, manganese, chromium, selenium, iodine, nickel, fluorine, molybdenum, vanadium, stannum, silicon, strontium, boron, rubidium, arsenic etc.This every kind trace element has its special physiological function.Although they are minimum at people's in-vivo content, they are very necessary to some conclusive metabolism that maintain in human body.Once lacked these essential trace element, human body just there will be disease, 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 forming hemoglobin, and iron deficiency can cause iron deficiency anemia.Once there is report abroad: in body, iron content, copper, zinc total amount reduce, and all can weaken body resistance against diseases, and encourage antibacterial and infect, and metainfective mortality rate was also higher.Their excess intake, deficiency or shortage all can cause to some extent the abnormal of Human Physiology or disease occurs.

Although it is generally acknowledged that the importance of trace element has obtained, the meals that are mainly confined to that the absorption of trace element and action effect are extensively approved at present add and healthcare applications.This biological effect that is mainly limited by trace element has two kinds of paradoxes: 1. the contradiction of usefulness and dosage.Any trace element, for body, all must submit to the requirement of " trace ", although this metalloid element is most important for body, but body content and intake all require extremely low, this just often causes body to the picked-up of this dvielement and utilizes lacking efficiency, and it is poisoning that minor metallic element easily occurs.2. the contradiction of the dense poly-demand of specificity at the non-specific and illness position distributing in conventional picked-up and body.Particularly when body is during in illness state, just more difficult to the utilization of trace element.As heart infarction patient's cardiac muscular tissue reduces in a large number to the picked-up of copper ion, cause deficiency decorated albumino reaction, cause copper ion to lose from heart.Early animal experiment is found as rat heart is separated and perfusion in vitro, perfusion again again after perfusion stops 45 minutes, myocardial cell major injury, a large amount of release of copper ion in heart.From the patient's of myocardial ischemia postmortem result, also find that the concentration of copper ion obviously declines at myocardial ischemia and position around.And for example, in pathological states such as hepatitis or liver fibers, generally lack selenium, zinc, and the state of an illness is more serious in hepatopath's body, selenium, zinc level in blood are lower.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 if the meal supplement of normal dose is also difficult to reach the effective demand of target organs, as escalated dose easily produces again metal toxicity.Situation like that, this predicament of body ubiquity: damaged organize the trace element more needing under pathological state, be more difficult to targeting, obtain efficiently.

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

Summary of the invention

In order to overcome the defect of prior art, the invention provides a kind of pharmaceutical composition and preparation method and application of directional controlled release of trace elements, it comprises concentration and is greater than 1 * 10 ⁶individual/ml, particle diameter is less than the tiny balloon of 10 μ m, and at least one is less than or equal to the trace element of ten times of physiological doses of human body, and pharmaceutically acceptable carrier or adjuvant; Wherein trace element and tiny balloon exist with the form of dissociating, and described tiny balloon is prepared from by pharmaceutically acceptable filmogen.

Further, described tiny balloon particle diameter is 1～5 μ m; Described 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.

Described 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 arsenic ion, is preferably in copper, zinc, selenium, ferrum one or more combination in any.

On the other hand, the trace element of described free form refer to be not combined with hollow micro capsule, with trace element ion and albumen, polypeptide, aminoacid, glucose or other, can in conjunction with the compound formation complex of trace element, be present in carrier or adjuvant, preferably, described trace element and albumen, polypeptide, aminoacid, glucose, other can be in conjunction with the molar concentration rate of the compound of trace element: 1: 0.05 to 1: 500.

Further, filmogen of the present invention is human albumin, phospholipid or other high molecular polymers.

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

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 medicine in preparation

Described pharmaceutical composition, in intravenous injection enters human body, utilizes ultrasound wave to carry out irradiation to therapentic part, utilizes gassiness tiny balloon and hyperacoustic interaction to reach the object of local release of trace elements ion.

Ultrasonic targeting of the present invention discharges and refers to that the ultrasound wave of certain energy makes the cavitation effects such as microbubble ruptures or vibration and impels and strengthen associated metal ion in the local release of ultrasonic irradiation and/or promote the respective metal ion component in suspension to enter irradiation tissue, reaches and promotes blood vessel, tissue regeneration and/or the clinical object of antineoplastic.

The trace element that ultrasonic targeting discharges can induced ultrasonic amplitude produce the corresponding physiological function of trace element ion carrying according to position.

The present invention also provides the method for preparation pharmaceutical composition of the present invention:

First preparation is not in conjunction with the tiny balloon of trace element ion, and preparation method comprises:

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

2) by step 1) tiny balloon that obtains with combine the albumen of one or more trace element ions or polypeptide or aminoacid or glucose or other and can be mixed to form suspendible pharmaceutical composition in conjunction with the complex solution of the compound formation of trace element;

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

Step 1) hollow core microsphere can utilize the conventional method for preparing microsphere preparation of following any pharmacy: ultrasonic acoustic shakes that method, freeze-drying, spraying are dry, activity/controllable free-radical polymerisation, precipitation polymerization method, suspension polymerisation, emulsion polymerisation, seeding polymerization, the 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, emulsion solvent evaporation technique.

Because body has the paradox between the non-targeted property trace picked-up of trace element and the targeting high concentration demand of medicinal organism effector organ, we think that the local orientation of trace element or targeting release have great clinical meaning.But the method that there is no at present reaches the object of facilitating the targeted release of trace element by oral, because have not yet to see and can effectively implement the oral drugs that targeting trace element discharges and emerge.Oral trace element multi-source absorbs by intestinal wall in food or with inorganic, organic state, after liver is processed, is with the non-specific feature that is distributed as of general.Therefore, as need reach the object of targeted release of trace elements, from Point of View of Clinical, consider, take through intravenous injection is desirable approach.

Yet the trace element ion salt of water-soluble state, even partial smearing also likely produces serious irritant reaction.Therefore as carried out the direct intravenous injection of metal ion salt solution, not only cannot reach targeted release and the poly-object of localized rich of target organs, have on the contrary the hidden danger that produces serious toxic and side effects.In addition, the salt ion state of this class trace element ion does not often possess biologic activity, all need to be with the combination of bio-compatible mode and transmission competence exertion physiological action, such as the combination with polypeptide, albumen, glucose etc.Therefore, reach the object 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 albumen or polypeptide.Second step is considered method and the efficiency that targeting discharges.Given this, what first we considered is to utilize the most general metal ion of human body to carry albumen: albumin is as optimization protein, because albumin be in human peripheral blood topmost metal ion in conjunction with albumen, it almost can with the effective combination of all metal ions, and be non-specificly delivered to any organ.

Albumin is comprised of 585 aminoacid, all with peptide chain, is connected, and is twisted into Lumbricus shape or cellular between aminoacid, has countless netted spaces, for inlaying, carries medicine and has created favourable steric requirements.We consider second step is how to accomplish that the method that targeting discharges or directed tissue organ discharges selects.Targeting drug delivery system is one of emphasis of modern medicines research.By medicine and special carrier coupling, drug selectivity guiding diseased region, to reach, increase drug level, improve pharmacokinetic parameter, reduce the object of toxic and side effects.So far, all these albuminoid microspheres product or experiments, the targeting that is all 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 better healing effect, as glycyrrhizic acid finishing albumin nano granular, folacin coupled mitoxantrone albumin nano granular, magnetic adriamycin albumin nano granular etc., but there is no listing product, only there is paclitaxel albumin nano granular to enter clinical stage (U.S.), because they are subject to the impact [1-14] of many factors in vivo.

In theory, have two kinds of targeting thinkings, first: active targeting.First the formation of this class targeting, must synthesize for a certain target organs or histiocytic albumin.The albumin microsphere that for example antibody-antigen mediates.At microsphere surface, in conjunction with specific antibody or polypeptide, this can make microsphere have special binding ability to certain cell, thereby by this cell of drug targeting, realizes specific killing.Li Yuanchun etc. [5] are coupled to the F of human liver cancer-specific monoclonal antibody HAb18 (ab ') 2 fragments on doxorubicin albumin nanoparticles, make immune nanoparticles.Result shows, immune nanoparticles can in conjunction with and effectively kill and wound this cell strain, its effect is dose dependent, the nanoparticles of contrast can not in conjunction with kill and wound significantly this cell strain.Or the albumin microsphere of formation cell-specific receptors bind; as specific recognition and the picked-up to irreducibility galactose or IV-acetyl group galactose with the asialoglycoprotein receptor on hepatic parenchymal cells film such as Cheng Yao [6]; the albumin microsphere of having prepared medicine carrying; again at its surface parcel one deck N-Galactosylated chitosan derivative; finally obtain the coated 5-fluorouracil albumin microsphere of N-Galactosylated chitosan derivative, to can specific binding to cell surface.

The second is so-called passive target.Utilize physical action, make the local release of albumin microsphere.For example, magnetic albumin microspheres is jointly wrapped in medicine and magnetisable material and in albumin, forms magnetic albumin microspheres.After magnetic and medicated microgranule intravascular is injected in vivo, utilize external magnetic field guide drugs microgranule to be stranded in a certain tissue or lesions position, prolong drug release time, to reach the object that improves curative effect and reduce toxic and side effects, for drug targeting provides a new approach.Chatterjee etc. [4] are by the comparison of Magnetic Polystyrene Microsphere and albumin magnetic microsphere, find that albumin magnetic microsphere has the ability of higher coupling protein, the albumin magnetic microsphere that protein (biological agglutinin) is modified and the ability of erythrocyte binding are considerably beyond Magnetic Polystyrene Microsphere.

All above-mentioned albumin microsphere products mainly focus on chemotherapy or anti-inflammatory treatment, but so far, there is not yet its upper application and exploitation to the release of the fixed point targeting of trace element.Effect object and this patent of the product of above-mentioned targeting modification or experiment are totally different first, all poly-release of the localized rich of trace element are not produced to invention thinking.It two combines albumin after trace element as again carried out targeting modification, and its biological activity, stability and function are difficult to expection and hold.

Therefore thinking of the present invention is, wants to reach the dense poly-effect of trace element that second step targeting fixed point release of trace elements forms local organization organ, and optimal state should be: reduce modification or the modification to protein or polypeptide drugs molecule 1. as far as possible.The biomacromolecule with certain space conformation that protein drug is comprised of aminoacid, molecular weight is often thousands of to hundreds of thousands, its activity depends on its correct structure, comprise primary structure and space structure, and its structure is subject to the impact of various factors, such as various protease, heavy metal, organic solvent, temperature, pH value, inhibitor, mechanical force etc.Therefore, in its preparation preparation, storage and dispose procedure, be easy to be subject to external condition to affect and inactivation, so the stability of Protein requirement is very important.Therefore without the modification of other drug, in conjunction with the albumin of trace element, may be at utmost to maintain the bioactive form of melts combine albumin merely; 2. the albumin encapsulation that the active targeting proteins that similar antigen-antibody is modified and magnetic material are modified, because added attached antigen or antibody protein or macromolecular substances and other magnetic metal elements, the immunogenicity that it is potential 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 optimization approach of targeted release.

According to aforementioned thinking, the present invention has designed a set of biomembrane-trace element hollow micro capsule-ultrasonic drug-supplying system.Involved in the present inventionly to biomembrane material, be preferably albumin, prepare albumin-trace element hollow micro capsule.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, as the carrier that carries of trace element, is again the release vehicle of trace element.Albumin microcapsule of the present invention is actual is the micro-air bag that includes gas componant that utilizes albumin surfactant feature to form.It had both been different from the conventional albumin microballoons of the liquid phase core of coated pastille composition, was also different from the simple albumin microvesicle for ultrasonic contrast imaging.Because its object had not both lain in conventional albumin microsphere capsule preparation--utilize albumin encapsulation to carry the drug molecule in being coated on, also do not lie in ultrasonic radiography contrast imaging.Its main purpose is to utilize the 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 for the treatment of and expectation.A large amount of physical study show, ultrasonic have a typical cavitation, and the ultrasonic sound field part of higher-energy can produce the high pressure of moment and low pressure alternately, forms and propagates local cavitation, follows this process, can produce the High Temperature High Pressure of moment.When there is microbubble in ultrasonic sound field, this cavitation is more strengthened, and can make local tissue or blood produce more strong moment cavitation is High Temperature High Pressure.In the time of microbubbles rupture, this type of effect reaches peak value, and the local transient energy of generation even can make local microvascular endothelial gap enlargement.This physical effect will make microvesicle and the effective blood turbulent flow of the local formation of ultrasonication and vascular space expand, and contribute to trace element in the quick disperse of part and enter interstice.Therefore this cover system of the present invention in fact possesses the feature of typical passive target administration.Our experiment embodiment confirms: the albumin that combines merely trace element is its physiology combining form, the own physiological property that has kept to greatest extent in theory trace element, and passive ultrasonic irradiation targeting discharges the cavitation effect not only combine the directivity of ultrasonic acoustic beam but also to combine ultrasonic sound field+microbubble, make trace element more quick and direct in local release and disperse.

Because the main delivery means that microcapsule bubble discharges as targeting the strengthening of the cavitation of ultrasonic sound field are usingd in the present invention, therefore the present invention also comprises as selected dosage form scheme next time: using trace element polypeptide complex as preparation host, the film of usining parcel microcapsule bubble is as adjuvant, form trace element-polypeptide complex+microbubble suspension, be injected in body, with ultrasonic irradiation, break up as fixed point targeting releasing tool.Can reach equally the object that similar trace element fixed point discharges.But the inferior also microbubble due to albumin parcel microbubble or polymer of scheme indication film parcel microcapsule bubble phospholipid parcel microbubble that selects.

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

Although medicine carrying ultrasonic microbubble is to carry out targeting to discharge the studied for thrombolytic, treatment of cancer, gene target conveying etc. of macromolecular drug.This class pharmaceutical preparation is confined in the research and experiment of macromole synthetic drug release, and rests in theoretic discussion or experimentation.The application of this and the basic trace element ion arriving involved in the present invention is 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 the present invention, the targeting that has no ultrasonic microbubble is prepared the pharmaceutical preparation that thinking and preparation method be applied to Metal ion release and occurred.

Therefore important innovations of the present invention is the invention of the pharmaceutical composition of the targeted in trace element body and thinking application.Needed by human body trace element is an anthropoid requisite special preparation, the physiological dose that people can tolerate is little, excessive free trace element ion is conventionally poisonous in vivo, it is not as common disorder medicine, need to reach the object of removing focus, as thereby the direct administration of injection form how overcomes the defect of oral supplementation trace element, and then reach good treatment and physiologic effect and cannot expect completely, also without the relevant report of prior art, the inventor has created a kind of specific compound mode just, make trace element in the mode of ultrasonic targeted release in body, satisfied treatment and the effect of adjusting physiological action have been reached.

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

Specifically, polypeptide trace element mixture has obtained extensive recognition and acceptance to the therapeutical effect of local tissue blotting or spraying generation, and has related agents manufacture.For example copper polypeptide complex has been United States Patent (USP) U.S.Pat.Nos.4 for 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.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 pad pasting therapeutical effect of hollow organ surface.All do not form therapeutical effect poly-to the localized rich of the trace element of parenchymal viscera in body and that cause.But these have all given the reasoning of an important logicality of the present invention, no matter the release of local trace element is to superficial tissue or the damaged tissues to the parenchymal viscera tissue in body, as long as can form in part dense poly-and fixed point controlled release, just can produce effectively positive curative effect.

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 corresponding table of abbreviation).

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

Trace element polypeptide can refer to 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, polypeptide trace element mixture refers to coordination compound generally, and it comprises a peptide molecule and trace element is non-covalent is compound on aforementioned polypeptide.Peptide group refers to 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 refers to alpha, beta or gamma aminoacid, for example:

Upper figure X represented amino acid side chain binding site.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 following chart.

The structure of the histidine derivative that the present invention comprises is as follows: 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, is at least that an aminoacid defined above or amino acid derivativges are connected in R by peptide bond herein.R single amino acids or amino acid derivativges herein, 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 be that chemical group is connected in R by amido link at this apoplexy due to endogenous wind R.Chemical group herein refers to and comprises amino and can be connected (such as the carboxyl terminal of histidine, arginic carboxyl terminal or related derivatives) with any formation of the R containing carboxyl terminal amido link.Further set forth: R has 1-20 carbon atom-NH alkyl, and it is connected by amido link with R, or have the virtue amino of 6-20 carbon atom.Mentioned herein and alkyl do not get rid of and comprise amino, octylame, or propylamine.Similarly, fragrant amino is not got rid of and is comprised benzylamine or benzyl.For having, can produce the amino that amido link is connected with the carboxyl terminal of R, can comprise that polyamines is such as spermine and spermidine.

The present invention relates to combination and the targeted of various trace elements, following embodiment be take copper as main implementation goal is to verify the fixed point conveying effect of the trace element that this patent is contained.The copper ion of physiological dose possesses a lot of biological effectiveness, for example, stimulate collagen and elastin laminin in wound or 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)).The speed of raising repair in trauma and degree (reference, Counts et al, Federation of American Societies for Experimental Biology Journal 6[5], A1636 (1992); 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 to have suitable toxicity and zest, is prohibited to directly act on wound or cicatrix region.Copper ion must be delivered in bio-compatible mode.

Thereby trace element ion of the present invention also can be carried by the complex mode of glucose and trace element, its general structure following (X represents trace element):

Therefore, the invention discloses a kind of can for ultrasonic interaction targeted release trace element ion so that promote ultrasonic irradiation part the activation of promotion cicatrix, blood vessel, tissue regeneration, antineoplastic object by the film of respective metal ion modification or combination parcel gassiness microcapsule and with combine the film material solution of trace element ion or polypeptide and be combined the pharmaceutical composition of trace element ion.The film material suspendible medicinal liquid that this has also comprised simple film parcel microcapsule and corresponding trace element ion solution or has combined corresponding trace element ion.

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

The mentioned regeneration of the present invention comprises many-sided implication: 1, the vascularization of scar tissue and elastic return; 2, the parenchyma at position, cicatrix place regeneration; 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 for the treatment of safely and effectively.

Accompanying drawing explanation

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

The increment facilitation of Fig. 2 copper ion Human Umbilical Vein Endothelial Cells is obviously better than matched group

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

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

Fig. 5 A is sham operated rats, and B is that matched group (does not add calcium alginate+CuSO ₄pad pasting) the local visible a large amount of fatty tissue of infraction and fibrosis tissue are piled up (arrow shows).C is that the revascularization of infarct area after pad pasting shows, the recovery of obvious Myocardial Regeneration and cardiac structure has appearred in infarction tissue, and its form more approaches the heart of sham-operation.Visible obvious capillary network forms.

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

The fluorescent quenching that Fig. 7 human albumin is combined with copper ion

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

Fig. 9 adds 50 μ l CuSO ₄rear 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 adding, and its fluorescent quenching increases gradually

Figure 19 chelated copper ion albumin film parcel microspherulite diameter distributes

Figure 20 accompanying drawing explanation: flow cytometer detection display microvesicle in conjunction with copper after, after standing 2 months, 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.With before February, prepared preliminary phase ratio and there is no significant change.Illustrate that chelated copper microvesicle is reliable and stable.

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

Figure 22 A is matched group, the general pathology picture of the heart infarction rabbit model of not injecting chelating albumin microvesicle after 4 weeks.The visible a large amount of athero of left heart front surface, is the performance (arrow) of typical heart infarction cicatrix region steatosis, athero.B is experimental group, and after injection chelating albumin microvesicle 4 weeks, heart infarction cicatrix district obviously activated, and a large amount of new lives' angeogenesis (arrow) is seen on surface

Figure 23 A, B is matched group, does not carry out ultrasonic irradiation targeted chelated copper ion.A: * 400Masson dyeing: infarcted region significant quantities of fat degeneration and fat vacuole form, without blood vessel hyperplasia.B: * 100Masson dyeing: myocardial infarction is still shown in that with normal juncture area significant quantities of fat degeneration and fat vacuole form, and nothing is blood vessel hyperplasia obviously.C, D is experimental group, row ultrasonic irradiation targeted chelated copper ion produces obvious collagen and revascularization.C: * 400Masson dyeing: the visible significantly little blood vessel hyperplasia of collagen fiber hyperplasia accompanied in infarcted region.D: infarction and normal juncture area * 100Masson dyeing: see the little blood vessel hyperplasia of obvious collagen fiber hyperplasia accompanied.

Figure 24 accompanying drawing explanation: after standing February, fluidic cell instrument does not have notable difference while showing in conjunction with microvesicle particle size distribution and concentration change after zinc ion and just preparation, particle size distribution normal distribution peak value is at 1.5 μ m, 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.

Figure 25 accompanying drawing explanation: after standing February, after showing that microvesicle is in conjunction with selenium, particle size distribution is stabilized in the normal state peak value of 1.5 μ m, mean diameter 2.1 μ m, the microvesicle of particle diameter 0-3 μ m accounts for 91% of all microvesicle scopes, microbubble concentration 3 * 10 ⁸individual/ml.With bimester before microvesicle particle size distribution compare and there is no significant change with concentration.Microvesicle in conjunction with selenium possesses reliable and stable characteristic.

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

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

In Figure 28 heart infarction region, through the heart infarction region of ultrasonic irradiation phospholipid microsphere and chelated copper glucose, show with the microvessel density contrast of only injecting in phospholipid microsphere matched group heart infarction region, containing the microvessel density in the heart infarction region of the experimental group of chelated copper glucose solution, be significantly higher than matched group.

The specific embodiment

Below by the specific embodiment, the present invention is described further, but be not limitation of the present invention.Below in each embodiment, our preferred copper ion in trace element carry out isolated cells physiological effect really fixed and moving thing at body, test local pad pasting controlled release and confirm that the generation of the physiological effect of isolated cells, chelated copper microvesicle produce isolated cells and test the similar experiments such as physiological effect showing consideration for film cardiac muscle at body controlled release of trace elements, to prove that the local controlled release of trace elements of microvesicle combining ultrasonic produces science and the feasibility of topical therapeutic and corresponding physiological effect.

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

In vitro cell experiment confirms that trace element-copper ion can effectively strengthen propagation and differentiation to human umbilical vein endothelial cell (human umbilical vein endothelial cell, HUVEC).In vitro culture the HUVEC that goes down to posterity.By HUVEC with 5 * 10 ³individual/hole density is inoculated in 96 orifice plates, and according to adding the difference of solution concentration that cell is divided into 3 groups at random in orifice plate, A organizes 5 μ mol/L CuSO ₄, B organizes 25 μ mol/L CuSO ₄, C group is blank, 4 every group multiple holes, and basal medium is MCDB131, adopts mtt assay detect cell proliferation and draw growth curve.Separately get HUVEC with 2 * 10 ⁵individual/hole density is inoculated in 6 orifice plates, and grouping is the same, and fluorescence quantitative RT-RCR detects eNOS (endothelial nitric oxide synthase, eNOS) gene and the Tie-1 gene expression of 3 groups of HUVEC.Result growth curve shows that front 3d is Exponential growth stage, and the 4th day starts to enter plateau.A group ability of cell proliferation is since the 3rd day apparently higher than B, C group, B group in 2d higher than C group, but since the 4th day significantly lower than C group, comparing difference all has statistical significance (P < 0.05) to see Fig. 1,2, the increment facilitation of copper ion Human Umbilical Vein Endothelial Cells is obviously better than matched group.Fluorescence quantitative RT-RCR detects and shows after effect 48h, A, B, C group eNOS gene expression be respectively 7.294 ± 1.488,0.149 ± 0.044 and the expression of 1.000 ± 0.253, Tie-1 gene be respectively 1.481 ± 0.137,1.131 ± 0.191 and 1.000 ± 0.177.A group and B, C organize relatively, and the expression of 2 genes all has 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

* A group compares P < 0.05 with C group; #B group compares P < 0.05 with 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 determining copper ion in the activation of damage cicatrix and stimulating the partial result of revascularization in cicatrix, local heart infarction animal model is carried out to copper-surfaced ionic gel, to verify the physiological role of local sustained release copper ion.

1, prepare calcium alginate+CuSO ₄pad pasting.With 10 milliliters of the sodium alginate solns of 2% mass fraction and containing 10mg CuSO ₄solution fully mixes, and drips 5ml CaCl ₂film forming after solution.

2, prepare heart infarction model: under narcotism, open the free also ligation Cor Leporis left anterior descending branch arteria coronaria of breast, the left heart heart infarction of preparation rabbit model.Open under breast state, front step institute film forming is attached to left heart front surface.Close breast.Stablize after 4 weeks, again open breast and observe gross specimen and pathological section difference.

Result demonstration, the copper ion of local sustained release forms the physiologic effect of significant promotion revascularization and cicatrix activation.In the myocardial infarction and ischemia model of rabbit, we are attached at by front step institute film forming the infarcted myocardium tissue that myocardial ischemia was performed the operation after 4 weeks, treat after 2 weeks, dynamic heart color ultrasound and heart muscle perfusion radiography weekly, confirm can effectively improve myocardial ischemia and cardiac function after copper ion part supplements, pathologic finding is found appearance a large amount of new vesselses (seeing Fig. 5,6) in infarcted myocardium tissue.

In figure, show: Fig. 5 A is sham operated rats, B is that matched group (does not add calcium alginate+CuSO ₄pad pasting) the local visible a large amount of fatty tissue of infraction and fibrosis tissue are piled up (arrow shows).C is that the revascularization of infarct area after pad pasting shows, the recovery of obvious Myocardial Regeneration and cardiac structure has appearred in infarction tissue, and its form more approaches the heart of sham-operation.Visible obvious capillary network forms.

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

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

In experiment, solution used is prepared with deionized water, uses 0.1molL ^-1naCl keep ionic strength, with Tris-HCl, as buffer, pH value is 7.4, preparation HSA and copper-bath, concentration is followed successively by 8.76 * 10 ^-6mo lL-1 and 6.48 * 10 ^-4mo lL-1.Use 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.In figure, can find out Cu ²⁺add the excitation peak obviously changing, 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 dripped metal ion solution, so can ignore dilution effect.

Protein can send fluorescence, is because there is three kinds of aromatic amino acid residue: Trp, Tyr and Phe residue in protein, and due to the different structure of these amino acid residues, three's fluorescence intensity ratio is 100: 9: 0.5 conventionally.According to protein, whether contain tryptophan and can be divided into two classes: category-A albumen, refers to containing Trp residue, only not contain the protein of Tyr and Phe residue; Category-B albumen: refer to not only containing Trp residue but also containing the protein of Tyr and Phe residue.Serum albumin belongs to category-B albumen, has stronger fluorescence.Although there is the characteristic fluorescence peak (Fluorescent peal of Trp, Tyr and Phe lay respectively at 348,303 and 282nm) of oneself in San Zhong chromophore, but due in protein macromolecule, very effective to the energy transfer of Tyr or rrrp by Phe, therefore what the absorption of whole molecule and transmitting 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 is greater than 290nm, can think that fluorescence is all from Trp residue.In HSA, only contain the Trp residue of 214, fluorescence emission peak is near 350nm.It is generally acknowledged, fluorescence mainly sends from 212 Trp residues, in 342nm left and right.The maximum excitation peak of the two is all about 280nm.Trp residue can after adding the materials such as 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, and can carry out quantitative and qualitative analysis research to combination.

The demonstration of this experiment embodiment, along with adding of copper ion, more copper ion and human albumin's chelating, make albuminous fluorescence generation cancellation.

The combination experiment of example 4 copper ions and albumin microvesicle, comprises 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 of connecting tee, Ultrasound Instrument probe is inserted to 2cm place under liquid level, with certain ultrasound intensity, the sound 20ml 1% human albumin's solution that shakes, the pre-sound 10s that shakes, then when sound shakes, in 5 seconds, pass into a certain amount of C ₃f ₈, continuation sound shakes, and to stopping sound after outlet temperature, shakes, and obtains C ₃f ₈albumin microsphere suspension, suspension is transferred in 50ml medicine bottle immediately, medicine bottle upper end air C ₃f ₈fully displacement, medicine bottle sternly seals with plug, standing 24h under room temperature.Shake up and microsphere is carried out to granularmetric analysis as following table 2, microsphere average diameter 2.15 μ m afterwards.

Table 2: the particle size distribution of albumin microsphere

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

2. the combination of microvesicle film and copper ion 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 respectively successively Cu ²⁺(the saturated copper solution of 1/50 dilution) solution is to be measured.Following table 3 is the amount of the combination of each variable concentrations copper ion and HSA microvesicle.See Fig. 8-17, for albumin microvesicle adds after the copper sulfate of variable concentrations, copper ion reacts with the fluorescent quenching occurring after albumin microsphere vacuolar membrane chelating.Fig. 8-17 are visible two absworption peaks all, and the former is the characteristic peak (excitation wavelength is 332nm left and right) adding after copper; The absworption peak that a rear peak is the not blank albumin microsphere of chelated copper ion itself (emission wavelength is 408nm); 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 fluorescence spectrum absorption intensity.

This experimental results show that copper ion is combined with albumin microsphere vacuolar membrane, causes albuminous fluorescence intensity gradually little.

Table 3: the variable concentrations copper sulfate adding

The preparation of example 5 copper ion albumin-binding microvesicles

After copper ion and albumin bound, ultrasound wave disperses to prepare the experiment of albumin copper microvesicle, comprises fluorescence analysis result and microvesicle metering result.

1. first prepare the albumin solution in conjunction with copper ion: by copper sulfate: albumin solution=1: 1mol carries out application of sample.The corresponding ratio of this example is as follows: 1mg HSA adds 4.35 * 10 ^-6molCu ²⁺.

2. pipette 20ml 5% chelated copper-human albumin solution to the 20ml disposable syringe of connecting tee, Ultrasound Instrument probe is inserted to 2cm place under liquid level, with certain ultrasound intensity, the sound 20ml 5% human albumin's solution that shakes, the pre-sound 10s that shakes, then when sound shakes, in 5 seconds, pass into a certain amount of C ₃f ₈, continuation sound shakes, and to stopping sound after outlet temperature, shakes, and obtains C ₃f ₈albumin microsphere suspension, suspension is transferred in 50ml medicine bottle immediately, medicine bottle upper end air C ₃f ₈fully displacement, medicine bottle sternly seals with plug, standing 24h under room temperature.Shake up and rear microsphere is carried out to granularmetric analysis, particle size distribution is as Figure 19.After demonstration chelated copper ion, the mean diameter of microvesicle is 2 μ m, and concentration is 3.1 * 10 ⁸individual/ml

3. stability experiment: the microvesicle of aforementioned combination copper ion is injected in 20ml vial, and sealing, preserves in 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.Lower Figure 20 is the detection of flow cytometer to microvesicle change of size, detects explanation, and microbubble concentration and particle diameter are stable

Figure 20 flow cytometer detection display microvesicle in conjunction with copper after, after standing 2 months, 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.With before February, prepared preliminary phase ratio and there is no significant change.Illustrate that chelated copper microvesicle is reliable and stable.

Example 6 experiment of albumin copper ion microcapsular ultrasound irradiation myocardial infarction animal model and results

With example 5 methods, prepare 15ml albumin C ₃f ₈, be wherein total to chelating 10mg copper ion.Under narcotism, open the free also ligation Cor Leporis left anterior descending branch arteria coronaria of breast, the left heart heart infarction of preparation rabbit model, stablized after 4 weeks.Row auricular vein is injected chelated copper ion albumin microvesicle with the speed of 1ml/min continuously, with the left pareordia of ultrasonic continuous irradiation, by doing continuous sector scanning back and forth to the apex of the heart at the bottom of the heart, ultrasound wave output energy is adjusted to maximum and enables color Doppler pattern (Figure 21).Within every 2 weeks, repeat a shot and irradiation, each dosage is identical.After 4 weeks, put to death experimental rabbit, observation experiment rabbit is cardiac shape and heart infarction pathological section substantially, revascularization and Activation in the heart infarction scar tissue of relative analysis matched group.Result shows, injects chelated copper ion albumin microsphere under ultrasonic irradiation, makes to produce in a large number and significantly revascularization and activation in heart infarction local organization, sees Figure 22,23.

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

Figure 22: A is matched group, the general pathology picture of the heart infarction rabbit model of not injecting chelating albumin microvesicle after 4 weeks.The visible a large amount of athero of left heart front surface, is the performance (arrow) of typical heart infarction cicatrix region steatosis, athero.B is experimental group, and after injection chelating albumin microvesicle 4 weeks, heart infarction cicatrix district obviously activated, and a large amount of new lives' angeogenesis (arrow) is seen on surface.

Figure 23: A, B is matched group, does not carry out ultrasonic irradiation targeted chelated copper ion.A: * 400Masson dyeing: infarcted region significant quantities of fat degeneration and fat vacuole form, without blood vessel hyperplasia.B: * 100Masson dyeing: myocardial infarction is still shown in that with normal juncture area significant quantities of fat degeneration and fat vacuole form, and nothing is blood vessel hyperplasia obviously.C, D is experimental group, row ultrasonic irradiation targeted chelated copper ion produces obvious collagen and revascularization.C: * 400Masson dyeing: the visible significantly little blood vessel hyperplasia of collagen fiber hyperplasia accompanied in infarcted region.D: infarction and normal juncture area * 100Masson dyeing: see the little blood vessel hyperplasia of obvious collagen fiber hyperplasia accompanied.

The combination experiment of example 7 albumin and zinc ion and the preparation of chelated zinc albumin microsphere (confirming the experiment of Zinc Ions Chelated albumin microvesicle)

1. by zinc chloride: albumin=1: 1 mol ratio is carried out.Accurate 5%HSA microvesicle solution 15ml, the liquor zinci chloridi of the molar concentrations such as dropping drawn.Fluorescence analysis demonstration, albumin solution adds after zinc chloride, and zinc ion reacts with the same fluorescent quenching occurring of albumin, confirms zinc ion and albumin generation chelating.According to mol ratio, prepare altogether 15ml 5% chelated zinc human albumin.

2. chelated zinc-human albumin solution that removing step 1 is produced is to the 20ml disposable syringe of connecting tee, Ultrasound Instrument probe is inserted to 2cm place under liquid level, with certain ultrasound intensity, the sound human albumin's solution that shakes, pre-sound shakes 10 seconds, then in 5 seconds, passes into a certain amount of C when sound shakes ₃f ₈, continuation sound shakes, and to stopping sound after outlet temperature, shakes, and obtains C ₃f ₈albumin microsphere suspension, suspension is transferred in 30ml medicine bottle immediately, medicine bottle upper end air C ₃f ₈fully displacement, medicine bottle sternly seals with plug, standing 24h under room temperature.Shake up and rear microsphere is carried out to granularmetric analysis, particle size distribution is identical with example 5, microsphere average diameter 2.5-3.0 μ m.

3. stability experiment: the microvesicle of aforementioned combination zinc ion is injected in 20ml vial, and sealing, preserves 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

Figure 24 showed after standing February, fluidic cell instrument does not have notable difference while showing in conjunction with microvesicle particle size distribution and concentration change after zinc ion and just preparation, 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.

The combination experiment of example 8 albumin and selenide (confirming the experiment of plasma selenium chelating albumin microvesicle)

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

2. selenium-human albumin solution is to the 20ml disposable syringe of connecting tee, and Ultrasound Instrument probe is inserted to 2cm place under liquid level, with certain ultrasound intensity, and the sound human albumin's solution that shakes, the sound 10s that shakes in advance, then when sound shakes, in 5 seconds, pass into a certain amount of C ₃f ₈, continuation sound shakes, and to stopping sound after outlet temperature, shakes, and obtains C ₃f ₈albumin microsphere suspension, suspension is transferred in 30ml medicine bottle immediately, medicine bottle upper end air C ₃f ₈fully displacement, medicine bottle sternly seals with plug, standing 24h under room temperature.Shake up and rear microsphere is carried out to granularmetric analysis, particle size distribution is identical with example 5, microsphere average diameter 2.5-3.0 μ m.

3. stability experiment: the microvesicle of aforementioned combination plasma selenium is injected in 20ml vial, and sealing, preserves in 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.Lower Figure 25 is the granularmetric analysis that flow cytometer carries out.

Figure 25 shows after standing February, and after showing that microvesicle is in conjunction with selenium, particle size distribution is 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 bimester before microvesicle particle size distribution compare and there is no significant change with concentration.Microvesicle in conjunction with selenium possesses reliable and stable characteristic.

Example 9 albumin bound copper+phospholipid microvesicle combined experimentses

1. press the albumin of example 3 preparation chelated coppers: the NaCl of 0.1M keeps ionic strength, with Tris-HCl, as buffer, pH value is 7.4, 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.

Zoopery and the result of example 10 albumin bound copper+phospholipid microvesicle heart infarction animal models

1. by example 2 and 6, prepare Cor Leporis stalk model.

2. the albumin polypeptide suspension of the made phospholipid microsphere of example 9 and chelated copper is injected with the speed of 1ml/min continuously through auricular vein, infusion is simultaneously with the left pareordia of ultrasonic continuous irradiation, by doing continuous sector scanning back and forth to the apex of the heart at the bottom of the heart, ultrasound wave output energy is adjusted to maximum and enables color Doppler pattern.Within every 2 weeks, repeat a shot and irradiation, each dosage is identical.After 4 weeks, put to death experimental rabbit, observation experiment rabbit is cardiac shape and heart infarction pathological section substantially, revascularization and Activation in the heart infarction scar tissue of relative analysis matched group.Result shows, the albumin polypeptide suspension of injection lipoid microsphere and chelated copper, under ultrasonic irradiation, makes the obvious revascularization of generation and activation in heart infarction local organization.By the method for microvessel density, heart infarction scar tissue is carried out to pathology and get sheet.Get 5 regions of every section stochastic sampling, under 10 * 40 times of mirrors, count.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: in heart infarction region, through ultrasonic irradiation phospholipid microsphere and the albuminous heart infarction of chelated copper region, show with the microvessel density contrast of only injecting in phospholipid microsphere matched group heart infarction region, the microvessel density that contains the heart infarction region of the albuminous experimental group of chelated copper is significantly higher than matched group.

Example 11 glucoses are in conjunction with zoopery and the result of copper+phospholipid microsphere heart infarction animal model

1. by example 2 and 6, prepare Cor Leporis stalk model.

2. the glucose suspension of the made phospholipid microsphere of example 9 and chelated copper is injected with the speed of 1ml/min continuously through auricular vein, infusion is simultaneously with the left pareordia of ultrasonic continuous irradiation, by doing continuous sector scanning back and forth to the apex of the heart at the bottom of the heart, ultrasound wave output energy is adjusted to maximum and enables color Doppler pattern.Within every 2 weeks, repeat a shot and irradiation, each dosage is identical.After 4 weeks, put to death experimental rabbit, observation experiment rabbit is cardiac shape and heart infarction pathological section substantially, revascularization and Activation in the heart infarction scar tissue of relative analysis matched group.Result shows, the glucose suspension of injection lipoid microsphere and chelated copper, under ultrasonic irradiation, makes the obvious revascularization of generation and activation in heart infarction local organization.By the method for microvessel density, heart infarction scar tissue is carried out to pathology and get sheet.Get 5 regions of every section stochastic sampling, under 10 * 40 times of mirrors, count.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 heart infarction region, in the heart infarction region of ultrasonic irradiation phospholipid microsphere and chelated copper glucose, see obvious little revascularization.

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

More than experimental results show that, what the present invention prepared can targeted release of trace elements preparation under ultrasonication, reached at local organization release of trace elements and produced 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.

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Claims

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

Wherein trace element and tiny balloon exist with the form of dissociating, and described tiny balloon is prepared from by pharmaceutically acceptable filmogen;

Described trace element be in copper, zinc or selenium any one;

The trace element of described free form refer to be not combined with hollow micro capsule, with trace element ion and albumen, polypeptide, aminoacid, glucose or other, can in conjunction with the compound formation complex of trace element, be present in carrier or adjuvant;

Described filmogen is human albumin or phospholipid.

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

3. pharmaceutical composition according to claim 1, is characterized in that: described trace element and albumen, polypeptide, aminoacid, glucose, other can be in conjunction with the molar concentration rate of the compound of trace element: 1:0.05 is to 1:500.

4. pharmaceutical composition according to claim 1, it is characterized in that: described carrier or adjuvant are deionized water or normal saline or glucose solution, described trace element ion complex is that trace element ion and albumen, polypeptide, aminoacid, glucose or other can be in conjunction with the complex of the compound formation of trace element.

5. according to the pharmaceutical composition described in arbitrary one of claim 1 ~ 4, it is characterized in that: it is ejection preparation.

6. pharmaceutical composition according to claim 5, is characterized in that: described ejection preparation is injection or injectable powder.

7. the pharmaceutical composition described in claim 1～6 any one promotes the ultrasonic targeting of revascularization to discharge the purposes in medicine in preparation; Wherein said trace element is copper ion.

8. purposes according to claim 7, it is characterized in that: described pharmaceutical composition is in intravenous injection enters human body, utilize ultrasound wave to carry out irradiation to therapentic part, utilize gassiness tiny balloon and hyperacoustic interaction to reach the object of local release of trace elements ion.

9. purposes according to claim 8, it is characterized in that: described ultrasonic targeting discharges and refers to that the ultrasound wave of certain energy makes microvesicle produce cavitation effect and impels and strengthen associated metal ion in the local release of ultrasonic irradiation and/or promote the corresponding trace element ion component in suspension to enter irradiation tissue, reaches the clinical object that promotes revascularization.

10. purposes according to claim 9, is characterized in that: described ultrasonic targeting discharges drug-induced ultrasonic irradiation position and produces the corresponding physiological function of trace element ion carrying.

The method of 11. preparation pharmaceutical composition claimed in claim 1, is characterized in that:

1) filmogen is prepared into the tiny balloon that particle diameter is less than 10 μ m;

2) tiny balloon step 1) being obtained with combine the albumen of one or more trace element ions or polypeptide or aminoacid or glucose or other and can be mixed to form suspendible pharmaceutical composition in conjunction with the complex solution of the compound formation of trace element;

12. according to preparation method described in claim 11, it is characterized in that: step 1) hollow core microsphere can utilize the conventional method for preparing microsphere preparation of following any pharmacy: ultrasonic acoustic shakes that method, freeze-drying, spraying are dry, activity/controllable free-radical polymerisation, precipitation polymerization method, suspension polymerisation, emulsion polymerisation, seeding polymerization, 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, emulsion solvent evaporation technique.