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

Abstract

The invention discloses a pharmaceutical composition for directional and controlled release of trace elements, its preparation method and application, which is characterized in that it contains hollow microspheres with a concentration greater than 1×10 ⁶ /ml and a particle size less than 10 μm, and at least one Or trace elements equal to ten times the physiological dose of the human body, and pharmaceutically acceptable carriers or excipients; wherein the trace elements and hollow microspheres exist in a free form, and the hollow microspheres are prepared from pharmaceutically acceptable film-forming materials become. The pharmaceutical composition of the present invention is stable and safe, and can guide trace element ions into the body under the action of ultrasound irradiation, thereby effectively regenerating tissue blood vessels, tissue regeneration or anti-tumor biology at the site irradiated by ultrasound. effect, broad clinical prospects.

Description

Pharmaceutical composition for directional and controlled release of trace elements, preparation method and application

This application is a divisional application of a patent application with filing date: July 12, 2010, application number: 201010223282.3, and title of invention: a pharmaceutical composition for targeted release of trace elements, its preparation method and application.

technical field

The invention discloses a pharmaceutical composition for directional and controlled release of trace elements, in particular a pharmaceutical composition capable of directional and controlled release by ultrasound, capable of inducing tissue regeneration and anti-tumor.

technical background

Trace elements play a fundamental role in maintaining human health. The main physiological function is to play a catalytic role in various enzyme systems, play a role as an essential component or cofactor of hormones or vitamins, and form metalloproteins with special functions. The significance of the physiological effects of trace elements can be compared with that of vitamins, but the body can synthesize some vitamins by itself but cannot synthesize any elements. From this point of view, essential trace elements are more important to the human body than vitamins. The basic definition of essential trace elements refers to those elements that have obvious nutritional and physiological functions, and are essential for maintaining the growth and development, life activities and reproduction of the body. The so-called "essential" means: ① The body must take this element from the external diet. When this element is removed from the diet, the body will appear in a state of physiological deficiency of this element. ② After supplementing this specific element, the lack of the body will be alleviated. ③ This special element always has a specific biochemical function on the body, which cannot be completely replaced by any other element. Insufficient intake of this element will cause biological dysfunction of the body, and restoring the physiological level of this element can alleviate or prevent this dysfunction. Once the body leaves this element, it can neither grow nor complete the life cycle it should have. In addition, essential trace elements also have the following characteristics: ① This element exists in the tissues of different animals at similar concentrations. ②Regardless of the animal species, similar physiological and biochemical abnormalities will appear after removing this element. ③The presence of this element can alleviate or prevent the above-mentioned abnormalities. ④ This abnormal change can also be cured when the deficiency is under control.

At present, iodine, selenium, zinc, iron, copper, manganese, chromium, etc. among the trace elements have been recognized internationally as "indispensable essential trace elements to maintain normal life activities of the body". With the development of science, human beings are increasingly aware of the basic role of essential trace elements in maintaining health. Some elements are not only anti-infective, but also associated with many chronic, epidemic, endemic, and even malignant lesions. A large number of epidemiological investigations have pointed out that if the body lacks essential trace elements, it may increase the sensitivity of the population to diseases, leading to sub-health states or the occurrence and development of diseases.

Although the content of trace elements in the human body is not much, they are closely related to human survival and health. Over the years, the importance of trace elements in the human body has been increasingly recognized. Trace elements such as selenium, zinc, iron, and copper have been proven to have various beneficial biological effects. According to scientific research, so far, 18 kinds of essential trace elements have been confirmed to be related to human health and life, namely iron, copper, zinc, cobalt, manganese, chromium, selenium, iodine, nickel, fluorine, molybdenum, vanadium , tin, silicon, strontium, boron, rubidium, arsenic, etc. Each trace element has its special physiological function. Although they are present in very small amounts in the human body, they are necessary to maintain some crucial metabolisms in the human body. Once the lack of these essential trace elements, the human body will appear disease, even life-threatening. Such as zinc deficiency can cause mouth, eyes, anus or genital redness, pimples, warm rash. Another example is that iron is one of the main components of hemoglobin, and iron deficiency can cause iron deficiency anemia. It has been reported abroad that the reduction of the total amount of iron, copper, and zinc in the body can weaken the body's ability to resist disease, encourage bacterial infections, and the mortality rate after infection is also high. Excessive, insufficient or deficient intake of them will cause physiological abnormalities or diseases in different degrees.

Although the importance of trace elements has been recognized, the intake and effects of trace elements are currently widely recognized and mainly limited to dietary supplements and health care applications. This is mainly due to the fact that the biological effects of trace elements have two contradictions: 1. The contradiction between efficacy and dosage. Any trace element must be subject to the "trace" requirements for the body. Although this type of metal element is very important to the body, the body content and intake are extremely low, which often leads to The body lacks efficiency in the intake and utilization of such elements, and is prone to trace metal element poisoning. 2. The contradiction between the non-specificity of routine intake and distribution in the body and the specific concentration requirements of the disease site. Especially when the body is in a state of illness, it is even more difficult to utilize trace elements. For example, the uptake of copper ions in the myocardial tissue of patients with myocardial infarction is greatly reduced, causing ischemic modification of protein reactions, resulting in the loss of copper ions from the heart. Early animal experiments found that if the rat heart was isolated and perfused in vitro, when the perfusion was stopped for 45 minutes and then reperfused, the cardiomyocytes were severely damaged, and a large amount of copper ions in the heart were released. From the autopsy results of patients with myocardial ischemia, it was also found that the concentration of copper ions decreased significantly in myocardial ischemia and surrounding areas. Another example is that in pathological states such as hepatitis or liver fibrosis, patients with liver disease generally lack selenium and zinc in their bodies, and the more serious the disease, the lower the levels of selenium and zinc in the blood. In this state of liver disease, it is difficult for the liver to make full use of selenium and zinc in food, resulting in low efficiency of dietary supplementation: even with normal doses of dietary supplementation, it is difficult to meet the effective needs of target organs, and it is easy to cause metal toxicity if the dose is increased . In such situations, the body generally has this dilemma: that is, the more trace elements needed by damaged tissues, the more difficult it is to obtain them in a targeted and efficient manner under pathological conditions.

Under the current situation, the intake of trace elements must be directly or indirectly supplied by the soil, that is, through the oral intake of various foods with relevant content or compound oral health care drugs like Shanchun. Although different methods have been created to improve the efficiency of dietary supplementation of trace elements, such as the use of nano-scale trace elements. Or adopt the method of bioaccumulation: that is, use seaweed as a food raw material with excellent bioaccumulation effect to enrich more trace elements, so as to improve the oral intake efficiency of trace elements. However, such methods are still subject to the functional status of specific organs. For example, if the function of related organs has been damaged, it is still difficult to achieve the therapeutic concentration and biophysiological effects of trace elements even through a large amount of dietary supplementation.

Contents of the invention

In order to overcome the deficiencies of the prior art, the present invention provides a pharmaceutical composition for directional and controlled release of trace elements, a preparation method and application thereof, which comprises hollow microspheres with a concentration greater than 1× ¹⁰⁶ /ml and a particle size less than 10 μm, and At least one trace element less than or equal to ten times the physiological dose of the human body, and a pharmaceutically acceptable carrier or auxiliary material; wherein the trace element and hollow microspheres exist in a free form, and the hollow microspheres are composed of pharmaceutically acceptable ingredients Membrane material is prepared.

Further, the particle size of the hollow microspheres is 1-5 μm; the gas contained in the hollow microspheres is air, nitrogen, sulfur fluoride gas, fluoroalkane gas or other non-toxic gases or one or one of the above A mixed gas of any combination of the above gas components.

The trace elements are one or more of iron, copper, zinc, cobalt, manganese, chromium, selenium, iodine, nickel, fluorine, molybdenum, vanadium, tin, silicon, strontium, boron, rubidium or arsenic ions Any combination, preferably copper, zinc, selenium, iron or any combination of more than one.

On the other hand, the trace elements in the free form refer to those that are not combined with the hollow microcapsules, and exist in the carrier or auxiliary materials as complexes of trace element ions and proteins, polypeptides, amino acids, glucose or other compounds that can bind trace elements , Preferably, the molar concentration ratio of said trace elements to proteins, polypeptides, amino acids, glucose, and other compounds that can bind trace elements is: 1:0.05 to 1:500.

Further, the film-forming material of the present invention is human serum albumin, phospholipid or other polymers.

The carrier or auxiliary material is deionized water or physiological saline or glucose solution or a solution containing a trace element ion complex, and the trace element ion complex is a combination of trace element ions and proteins, polypeptides, amino acids, glucose or other Compounds formed by compounds of trace elements.

The pharmaceutical composition of the present invention is preferably an injection preparation, more preferably a powder injection.

The present invention also provides the use of the pharmaceutical composition described in the present invention in the preparation of ultrasonic targeted release drugs for promoting blood vessel or tissue regeneration and anti-tumor

The pharmaceutical composition is intravenously injected into the human body, the treatment site is irradiated by ultrasonic waves, and the purpose of locally releasing trace element ions is achieved by utilizing the interaction between air-containing hollow microspheres and ultrasonic waves.

Ultrasonic targeted release in the present invention refers to the cavitation effects such as microbubble rupture or oscillation caused by ultrasonic waves with a certain energy, which promotes and strengthens the local release of relevant metal ions in ultrasonic irradiation and/or promotes the corresponding metal ions in the suspension. The components enter the irradiated tissue to achieve the clinical purpose of promoting blood vessel, tissue regeneration and/or anti-tumor.

The trace elements released by ultrasound can induce the physiological effects corresponding to the trace element ions carried by the ultrasonic irradiation site.

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

First prepare hollow microspheres that are not bound to trace element ions, and the preparation method includes:

1) preparing the composite membrane material into hollow microspheres with a particle size of less than 10 μm;

2) Mix the hollow microspheres obtained in step 1) with a complex solution formed by a protein or polypeptide combined with one or more trace element ions, or an amino acid or glucose or other compounds that can bind trace elements to form a suspension drug combination thing;

3) Direct refrigerated storage as combined drug injection or freeze-dried to prepare powder injection.

Step 1) Hollow microspheres can be prepared by any one of the following commonly used pharmaceutical microsphere preparation methods: ultrasonic sonication, freeze drying, spray drying, active/controllable free radical polymerization, precipitation polymerization, suspension polymerization, emulsion Polymerization, seed polymerization, dispersion polymerization and precipitation polymerization and other heterogeneous polymerization systems, ion cross-linking method, emulsified ion gel method, ion precipitation-chemical cross-linking method, emulsification-chemical cross-linking method, double emulsion cross-linking method, thermal cross-linking method, coacervation method, emulsification-solvent evaporation method.

Due to the contradiction between the non-targeted trace intake of trace elements in the body and the targeted high concentration requirements of medicinal biological effect organs, we believe that the local directional or targeted release of trace elements has great clinical significance. However, the purpose of promoting the directional release of trace elements through oral administration cannot be achieved at present, because there is no oral drug that can effectively implement targeted release of trace elements. Oral trace elements are mostly derived from food or absorbed through the intestinal wall in the state of inorganic or organic matter, and are characterized by systemic non-specific distribution after being processed by the liver. Therefore, in order to achieve the purpose of targeted release of trace elements, from a clinical point of view, intravenous injection is the preferred way.

However, trace element ion salts in a water-soluble state may cause severe irritation even if they are applied topically. Therefore, such as direct intravenous injection of metal ion salt solution, not only the directional release and local concentration of targeted organs cannot be achieved, but there is a hidden danger of serious toxic and side effects. In addition, the salt ion state of such trace element ions is often not biologically active, and they all need to be combined and transported in a biocompatible manner to exert physiological effects, such as binding to peptides, proteins, glucose, etc. Therefore, in order to achieve the purpose of directional or targeted organ-targeted trace element release, the thinking of the present invention is: the first step must be considered to generate effective protein or polypeptide trace element conjugates or complexes or associations. The second step considers the method and efficiency of targeted release. In view of this, we first consider using the most common metal ion-carrying protein in the human body: albumin as the preferred protein, because albumin is the most important metal ion-binding protein in human peripheral blood, and it can effectively bind to almost all metal ions. And non-specific delivery to any organ.

Albumin is composed of 585 amino acids, all of which are connected by peptide chains, and twisted into an earthworm or honeycomb shape, with numerous mesh-like gaps, creating favorable space conditions for mosaic-carrying drugs. The second step we consider is how to achieve targeted release or targeted tissue and organ release method selection. Targeted drug delivery system is one of the focuses of modern drug research. The drug is coupled with a specific carrier, and the drug is selectively directed to the lesion to achieve the purpose of increasing drug concentration, improving pharmacokinetic parameters, and reducing toxic and side effects. So far, all such albumin microsphere products or experiments are limited to the targeted release of anti-tumor chemotherapy drugs or the controlled release of antibiotics. In recent years, research on the application of albumin microspheres and albumin nanoparticles has gradually increased, and most of them use the surface modification of microspheres and nanoparticles to obtain better therapeutic effects, such as glycyrrhizic acid surface modification of albumin nanoparticles, folic acid coupling Mitoxantrone albumin nanoparticles, magnetic doxorubicin albumin nanoparticles, etc., but there are no marketed products, and only paclitaxel albumin nanoparticles have entered the clinical stage (USA), because they are affected by various factors in the body[ 1-14].

In theory, there are two targeting ideas, first: active targeting. For the formation of this type of targeting, albumin targeting a certain target organ or tissue cell must first be synthesized. For example antibody-antigen mediated albumin microspheres. Binding specific antibodies or polypeptides on the surface of microspheres can make the microspheres have specific binding ability to certain cells, thereby directing the drug to the cells and achieving specific killing. Li Yuanchun et al[5] coupled the F(ab')2 fragment of the human liver cancer-specific monoclonal antibody HAb18 to doxorubicin albumin nanospheres to make immune nanospheres. The results showed that the immune nanospheres could bind and effectively kill the cell strain, and the effect was dose-dependent, while the control nanospheres could not bind and obviously kill the cell strain. Or form cell-specific receptor-bound albumin microspheres, such as Cheng Yao et al. Recognition and uptake, preparation of drug-loaded albumin microspheres, and then coating a layer of galactosylated chitosan derivatives on the surface, and finally obtaining 5-fluorouracil albumin coated with galactosylated chitosan derivatives Microspheres, in order to specifically bind to the cell surface.

The second is the so-called passive targeting. That is, the albumin microspheres are released locally by physical action. For example, magnetic albumin microspheres encapsulate drugs and magnetic substances in albumin to form magnetic albumin microspheres. After the magnetic drug particles are injected into the body through blood vessels, the magnetic field in vitro is used to guide the drug particles to stay in a certain tissue or lesion, prolonging the release time of the drug, so as to achieve the purpose of improving efficacy and reducing side effects, providing a new way for drug targeting . Chatterjee et al[4] compared polystyrene magnetic microspheres with albumin magnetic microspheres, and found that albumin magnetic microspheres have a higher ability to couple proteins, protein (biolectin) modified albumin magnetic microspheres and albumin magnetic microspheres The ability to bind red blood cells far exceeds that of polystyrene magnetic microspheres.

All the above-mentioned albumin microsphere products are mainly focused on chemotherapy or anti-inflammatory treatment, but so far, there has been no application and development for the site-specific and targeted release of trace elements. The purpose of the above-mentioned targeted modification products or experiments is quite different from that of this patent, and none of them has an inventive idea for the local concentration and release of trace elements. Second, if albumin combined with trace elements is subjected to targeted modification again, its biological activity, stability and function are difficult to predict and grasp.

Therefore, the idea of the present invention is to achieve the effect of the second step of targeted and fixed-point release of trace elements to form the concentration of trace elements in local tissues and organs. The most ideal state should be: 1. Minimize the modification or modification of protein or polypeptide drug molecules modified. Protein drugs are biological macromolecules composed of amino acids with a certain spatial conformation. The molecular weight is usually thousands to hundreds of thousands. The influence of factors, such as various proteases, heavy metals, organic solvents, temperature, pH value, inhibitors, mechanical forces, etc. Therefore, it is easy to be inactivated by external conditions during the preparation, storage and release of its preparation, so it is very important to maintain the stability of the protein. Therefore, without modification by other drugs, albumin that simply binds trace elements may be the form that maintains the biological activity of metal-binding albumin to the greatest extent; 2. Active targeting proteins similar to antigen-antibody modification and albumin encapsulation modified by magnetic materials, Because of the addition of attached antigens or antibody proteins or macromolecular substances and other magnetic metal elements, its potential immunogenicity and pathogenicity are difficult to predict, and it is not conducive to the clinical application approval and promotion of the product; 3. From 1, 2 , the present invention considers that passive targeting is the preferred way of directional release for trace elements combined with albumin or other biofilm materials.

According to the aforementioned idea, the present invention designs a biofilm-trace element hollow microcapsule-ultrasonic drug delivery system. The biofilm material involved in the present invention is preferably albumin, and albumin-trace element hollow microcapsules are prepared. Its essence is to utilize two characteristics of albumin: 1. Highly efficient binding rate of trace elements; 2. Surfactant effect of albumin itself. The innovation of this case is that the albumin itself is not only the carrying carrier of trace elements, but also the release carrier of trace elements. The albumin microcapsule of the present invention is actually a microair capsule containing gas components formed by utilizing the characteristics of the albumin surfactant. It is not only different from the commonly used albumin microsphere preparations coated with liquid phase cores containing drug ingredients, but also different from albumin microbubbles purely used for contrast-enhanced ultrasound imaging. Because its purpose is neither in the conventional albumin microcapsule preparations - the use of albumin encapsulation to deliver the coated drug molecules, nor in the contrast contrast imaging of ultrasound. Its main purpose is to make use of the destructive effect of ultrasound on microbubbles to partially rupture and release the trace element-albumin film, and form the fixed-point release and concentration of trace elements at the site of ultrasound irradiation, so as to achieve therapeutic and expected physiological effects. A large number of physical studies have shown that ultrasound has a typical cavitation effect, that is, the higher-energy ultrasonic sound field can locally generate instantaneous high-voltage and low-pressure alternating, forming a cavitation in the localized propagation, and with this process, instantaneous high temperature and high pressure will be generated. When there are microbubbles in the ultrasonic sound field, this cavitation effect is more intensified, which will cause more intense instantaneous cavitation, that is, high temperature and high pressure, in local tissues or blood. When the microbubbles burst, this type of action reaches its peak, and the local instantaneous energy generated can even expand the local microvascular endothelial gap. This physical effect will cause effective blood turbulence and expansion of blood vessel space locally by the action of microbubbles and ultrasound, which will help the local rapid diffusion of trace elements and enter the tissue space. Therefore, in essence, the system of the present invention possesses the characteristics of typical passive targeted drug delivery. Our experimental implementation has confirmed that: albumin that simply combines trace elements is its physiological binding form, which theoretically maintains the physiological characteristics of trace elements themselves to the greatest extent, and the targeted release of passive ultrasound irradiation not only combines ultrasound The directivity of the sound beam combined with the cavitation effect of the ultrasonic sound field + microbubbles makes the local release and dispersion of trace elements more rapid and direct.

Since the present invention uses the strengthening of the cavitation effect of microcapsules on the ultrasonic sound field as the main delivery method for targeted release, the present invention also includes the following sub-selection of dosage forms: use the trace element polypeptide complex as the main agent of the preparation, and use the membrane Encapsulated microcapsules are used as adjuvants to form trace element-polypeptide complex + microbubble suspension, injected into the body, and broken by ultrasonic irradiation as a targeted release tool. The purpose of similar fixed-point release of trace elements can also be achieved. The membrane-encapsulated microbubbles referred to in the second option can be phospholipid-encapsulated microbubbles, albumin-encapsulated microbubbles or microbubbles caused by polymers.

Since the present invention uses the strengthening of the cavitation effect of microcapsules on the ultrasonic sound field as the main delivery method for targeted release, the present invention also includes the following sub-selection of dosage forms: use the trace element polypeptide complex as the main agent of the preparation, and use the membrane Encapsulated microcapsules are used as adjuvants to form trace element-polypeptide complex + microbubble suspension, injected into the body, and broken by ultrasonic irradiation as a targeted release tool. The purpose of similar fixed-point release of trace elements can also be achieved. The membrane-encapsulated microbubbles referred to in the second option can be phospholipid-encapsulated microbubbles, albumin-encapsulated microbubbles or microbubbles caused by polymers.

Water-soluble trace element salts are generally either ineffective or highly toxic in the body. Trace element ions (such as copper, zinc, selenium, etc.) must be delivered in a biocompatible manner. This method is mainly to form a polypeptide or albumin complex or a glucose mixture by compounding trace elements and corresponding polypeptides or proteins or glucose associates, in order to effectively produce physiological and biochemical effects on cells and tissues. The protein and polypeptide referred to here can refer to albumin or other metal complex proteins or polypeptides.

Although drug-loaded ultrasonic microbubbles are used for targeted release of macromolecular drugs, they have been studied for thrombolysis, cancer treatment, gene targeted delivery, etc. This type of pharmaceutical preparation is limited to the research and experiment of macromolecule synthetic drug release, and stays in theoretical discussion or experimental research. This is completely different from the application of basic trace element ions involved in the present invention. This is because metal ions are basic ions, and its toxicity and medicinal dosage are difficult to accurately grasp. On the other hand, except for the present invention, there is no targeted preparation idea and preparation method of ultrasonic microbubbles applied to pharmaceutical preparations for metal ion release. Appear.

Therefore, the important innovation of the present invention lies in the invention and idea application of the pharmaceutical composition for the targeted delivery of trace elements in vivo. Trace elements required by the human body are a special kind of preparation that is essential for the human body. The physiological dose that can be tolerated by humans is small. Excessive free trace element ions are usually poisonous in the body. It is not the same as ordinary disease treatment drugs that need to achieve the removal of lesions The purpose of how to directly administer in the form of injection so as to overcome the defects of oral supplementation of trace elements, so as to achieve good therapeutic and physiological effects is completely unpredictable, and there is no relevant report in the prior art. The inventor just created a specific The combined method enables the trace elements to be released in the way of ultrasound in the body to achieve satisfactory treatment and physiological regulation effects.

As previously mentioned, water-soluble trace element salts are generally either ineffective or highly toxic in the body. Trace element ions (such as copper, zinc, selenium, etc.) must be delivered in a biocompatible manner. This method is mainly to compound trace elements and corresponding polypeptides or proteins to form polypeptide or albumin complexes, which can effectively produce physiological and biochemical effects on cells and tissues. The protein and polypeptide referred to here can refer to albumin or other metal complex proteins or polypeptides.

In particular, the therapeutic effect of the polypeptide trace element complex on local tissue smearing or spraying has been widely recognized and accepted, and related medicaments have been produced and prepared. For example, the treatment and effect of copper polypeptide complex on superficial wounds of skin and organs have been confirmed and published by U.S. Pat. Nos. 4,760,051; 4,665,054; , 877,770; The preventive and therapeutic effect of copper polypeptide complex on gastric ulcer has also been authorized and confirmed by U.S. Pat. Nos. 5,145,838; 4,767,753 and 5,023,237. But all these patents all only relate to the smearing of trace elements on the body surface or the cavity viscera surface or the therapeutic effect of sticking film. None of them formed the therapeutic effect caused by the local concentration of trace elements in the substantive organs in the body. But these all give the present invention an important logical reasoning, whether the release of local trace elements is to the superficial tissue or the damaged tissue of the substantive organ tissue in the body, as long as it can form a concentration and a fixed point locally Controlled release can produce effective positive curative effect.

As mentioned above, the trace element polypeptide complexes involved in this patent refer to the following polypeptide complexes (Table 1 is the corresponding table of English abbreviations and abbreviations of each amino acid).

Table 1: English abbreviations and abbreviations of amino acids

Trace element polypeptides may refer to the following complexes (M refers to trace elements):

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-phenyl 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").

Here, the polypeptide trace element complex generally refers to a type of coordination compound, which includes a polypeptide molecule and trace elements non-covalently complexed on the aforementioned polypeptide. A polypeptide group refers to the covalent bonding of two or more amino acid sequences or amino acid derivative sequences. In general, the amino acid mentioned in this patent contains an amino group, a carboxyl group, a hydrogen atom and an amino acid side chain binding group. The polypeptide amino acids involved in this patent mainly refer to alpha, beta or gamma amino acids, for example:

The X in the above figure represents the amino acid side chain binding site. The amino acid complexes mentioned in this patent can be L-type or D-type or a mixture of both. The amino acid derivatives mentioned in this patent include the structures shown in the following diagrams.

The structure of the histidine derivative included in the present invention is as follows: n=1-20 in this structure (excluding the case of n=3).

The polypeptide trace element complex mentioned in the present invention can be described according to the following general formula: [R1-R2-R3]: M, where M is at least one amino acid or amino acid derivative defined above linked to R through a peptide bond. Here R single amino acid or amino acid derivatives, such polypeptide trace element complexes are generally classified as tripeptides. The general formula description of another polypeptide trace element complex involved in this patent can also be [R, -R, -R,]:M. But in this class R is a chemical group attached to R through an amide bond. The chemical group herein refers to a group that contains an amino group and can form an amide bond with any carboxy-terminal R (such as the carboxy-terminus of histidine, carboxy-terminus of arginine or related derivatives). Further elaboration: R is an -NH alkyl group with 1-20 carbon atoms, which is connected to R through an amide bond, or an arylamino group with 6-20 carbon atoms. The alkyl mentioned here does not exclude the inclusion of amino, octylamine, or propylamine. Similarly, arylamino does not exclude benzylamine or benzyl. For amino groups having an amide linkage to the carboxy terminus of R, polyamines such as spermine and spermidine may be included.

The present invention involves the combination and targeted delivery of various trace elements. The following implementation examples take copper as the main implementation target to verify the effect of fixed-point delivery of trace elements covered by this patent. Physiological doses of copper ions have many biological effects, such as stimulating collagen and elastin accumulation in wounds or damaged tissues (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)). Modulates the activity of metalloprotease substrates (Reference, Simeon et al., J. Invest. Dermatol. 112(6):957-964 (1999)). Increase 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 the speed and extent of wound repair (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. 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)).

However, as mentioned above, water-soluble copper salt ions have no physiological activity and are even quite toxic and irritating, and are prohibited from directly acting on wounds or scars. Copper ions must be delivered in a biocompatible manner.

Thereby, the trace element ion of the present invention can also be transported by the complex mode of glucose and trace element, and its general structure formula is as follows (X represents trace element):

Therefore, the present invention discloses a membrane modified or bound by corresponding metal ions that can be used to interact with ultrasound to release trace element ions in a directional manner, thereby promoting local ultrasound irradiation to promote scar activation, blood vessel, tissue regeneration, and anti-tumor purposes. A pharmaceutical composition for encapsulating gas-containing microcapsules and membrane material solutions or polypeptides combining trace element ions. This also includes simple membrane-wrapped microcapsules and corresponding trace element ion solutions or membrane suspension medicinal solutions combined with corresponding trace element ions.

Under the action of ultrasonic irradiation, this composite medicinal solution can guide trace element ions into the body in a directional manner. Therefore, it can effectively regenerate blood vessels, tissue regeneration or anti-tumor biological effects in the tissue irradiated by ultrasound. Its broad clinical prospects include: angiogenesis in infarcted myocardium, angiogenesis after coronary stenosis, and angiogenesis in vascular stenosis or infarct lesions in various organs (such as angiogenesis in extremity arterial stenosis or infarction). Potential applications are mainly the proven physiological and biological effects of the corresponding trace element ions, for example, including angiogenesis in scars after internal organ surgery, such as scars after various solid organs or promotion of tissue regeneration, such as: liver surgery , gastrointestinal surgery, esophageal surgery, uterine surgery, etc., the treatment of liver fibrosis also includes such as tumor growth inhibition.

The regeneration mentioned in the present invention includes multiple meanings: 1. Vascularization and elastic recovery of scar tissue; 2. Regeneration of parenchymal cells at the site of the scar; 3. Regeneration of blood vessels and parenchymal cells at the site of necrosis; 4. Stenosis and ischemia 5. Reversal of tissue fibrosis, etc.

The pharmaceutical composition preparation for ultrasonic targeted release of trace elements of the present invention has excellent clinical application prospects, and provides a safe and effective new treatment approach for blood vessels, tissues and anti-tumor treatment of various diseased tissues.

Description of drawings

Figure 1 The promotion effect of different concentrations of Cu ²⁺ on the growth of human umbilical vein endothelial cells

Figure 2 The value-promoting effect of copper ions on endothelial cells was significantly stronger than that of the control group

Figure 3 Agarose gel electrophoresis detection of fluorescent quantitative RT-PCR products of group A

Figure 4 Differences in the expression of eNOS and Tie-1 among the three groups of cells

Figure 5A is the sham operation group, and B is the control group (no calcium alginate+CuSO ₄ patch). A large amount of adipose tissue and fibrotic tissue accumulation can be seen in the local infarction (arrows). C is the vascular regeneration of the infarcted area after patching. The infarcted tissue has obvious myocardial regeneration and recovery of cardiac structure, and its shape is closer to that of the sham-operated heart. Visible microvascular network formation.

Fig. 6 is a comparison of the results of the above-mentioned pathological slides of myocardial infarction. Histology (HE staining, Masson's trichrome method) observed that the myocardium treated with Cu film had more new blood vessels, a large number of mononuclear cells and the recovery of myocardial fibrosis. CuSO ₄ sticking film made obvious small blood vessels and glial hyperplasia in the necrotic tissue of local myocardial infarction. Masson is collagen staining.

Figure 7 Fluorescence quenching of human serum albumin bound to copper ions

Figure 8 Fluorescent peaks of unchelated Cu ²⁺ human albumin microspheres

Figure 9 Fluorescence absorption peaks of chelated Cu ²⁺ microspheres after adding 50 μl CuSO ₄

Fig.10 Fluorescence absorption peaks of Cu ²⁺ microspheres chelated with 100 μl CuSO ₄

Figure 11 Fluorescence absorption peaks of Cu ²⁺ microspheres chelated with 200 μl CuSO ₄

Fig. 12 Fluorescence absorption peaks of Cu ²⁺ microspheres chelated with 300 μl CuSO ₄

Figure 13 Adding 400μl CuSO ₄ to chelate Cu ²⁺ microspheres fluorescence absorption peak

Figure 14 Adding 500μl CuSO ₄ to chelate Cu ²⁺ microspheres fluorescence absorption peak

Fig. 15 Fluorescence absorption peaks of Cu ²⁺ microspheres chelated with 600 μl CuSO ₄

Figure 16 Adding 700μl CuSO ₄ to chelate Cu ²⁺ microsphere fluorescence absorption peak

Figure 17 Adding 1000μl CuSO ₄ to chelate Cu ²⁺ microsphere fluorescence absorption peak

Figure 18 The fluorescence quenching of pure albumin microbubbles gradually increases with the increase of the amount of copper ions added

Figure 19 particle size distribution of chelated copper ion albumin membrane-wrapped microspheres

Figure 20: The flow cytometer shows that after the microbubbles are bound to copper, after standing for 2 months, the particle size distribution is stable at the normal peak of 1.5 μm, and the average particle size is 1.95 μm; the particle size is 0-3 μm Microbubbles accounted for 90% of all microbubbles, and the concentration of microbubbles was 3×10 ⁸ /ml. Compared with the initial stage of preparation before February, there is no significant change. It shows that the chelated copper microbubbles are stable and reliable.

Fig. 21 Using high-energy color Doppler mode to continuously break down albumin microspheres to form passive targeted release of copper ions to the local myocardial infarction.

Fig. 22A is the gross pathological pictures of the myocardial infarction model rabbits not injected with chelated albumin microbubbles in the control group after 4 weeks. A large amount of fat accumulation can be seen on the front surface of the left heart, showing the typical appearance of fatty degeneration and fat accumulation in the scar area of myocardial infarction (arrow). B is the experimental group, 4 weeks after the injection of chelated albumin microbubbles, the myocardial infarction scar area was significantly activated, and a large number of new capillaries were formed on the surface (arrow)

Figure 23A, B is the control group, without ultrasonic irradiation targeted delivery of chelated copper ions. A: ×400Masson staining: massive fatty degeneration and fat vacuole formation in the infarct area, without vascular proliferation. B: ×100Masson staining: a large amount of fatty degeneration and fat vacuoles were still seen in the border area between myocardial infarction and normal, without obvious vascular hyperplasia. C and D are the experimental group, which underwent ultrasonic irradiation to deliver chelated copper ions to produce obvious collagen and angiogenesis. C: ×400Masson staining: obvious collagen fiber hyperplasia and small blood vessel hyperplasia can be seen in the infarct area. D: Masson staining at the border between the infarct and the normal area × 100: obvious collagen fiber hyperplasia accompanied by small blood vessel hyperplasia.

Figure 24: After standing for 2 months, the flow cytometry instrument showed that the microbubble particle size distribution and concentration changes after zinc ion binding were not significantly different from those at the initial preparation, and the normal distribution peak of the particle size distribution was at 1.5 μm. Microbubbles of 0-3 μm accounted for 90.5% of all microbubbles, with an average particle size of 2.5 μm. The microbubble concentration is 3.4×10 ⁸ /ml. Zinc-bound microbubbles are stable and reliable.

Description of Figure 25: After standing for 2 months, it shows that after the microbubbles are combined with selenium, the particle size distribution is stable at the normal peak of 1.5 μm, the average particle size is 2.1 μm, and the microbubbles with a particle size of 0-3 μm account for all the microbubbles. 91%, the microbubble concentration is 3×10 ⁸ /ml. Compared with two months ago, the size distribution and concentration of microbubbles did not change significantly. Selenium-incorporated microbubbles have a robust and reliable profile.

Figure 26 The comparison of the microvessel density in the myocardial infarction area of the myocardial infarction area irradiated by ultrasound with phospholipid microspheres and chelated copper albumin and only the injection of phospholipid microspheres in the control group shows that the experimental group containing chelated copper albumin The microvessel density in the myocardial infarction area was significantly higher than that in the control group.

Fig. 27 Obvious regeneration of small blood vessels was seen in the myocardial infarction area irradiated by ultrasound with phospholipid microspheres and chelated copper glucose.

Fig. 28 Comparison of the microvessel density in the myocardial infarction area of the myocardial infarction area irradiated by ultrasound with phospholipid microspheres and chelated copper glucose and the myocardial infarction area of the control group only injected with phospholipid microspheres shows that the heart of the experimental group containing chelated copper glucose solution The microvessel density in the stem area was significantly higher than that in the control group.

Detailed ways

The present invention will be further described below through specific embodiments, but the present invention is not limited thereto. In each of the following examples, we selected copper ions among the trace elements to determine the physiological effects of in vitro cells, in vivo experiments on animals to localize film-controlled release to confirm the occurrence of physiological effects of in vitro cells, and the use of chelated copper microbubbles in body control. The release of trace elements on myocardium produces similar physiological effects on isolated cells and in vivo membrane experiments, to prove the scientificity and feasibility of microbubbles combined with ultrasonic local controlled release of trace elements to produce local treatment and corresponding physiological effects.

Example 1 confirms that copper ions stimulate cell regeneration in vitro and stem cell mobilization experiments and the physiological effects of local film-controlled release of copper ions in the body

In vitro cell experiments have confirmed that trace element copper ions can effectively enhance the proliferation and differentiation of human umbilical vein endothelial cells (HUVEC). HUVECs were cultured and passaged in vitro. HUVEC were seeded in a 96-well plate at a density of 5×10 ³ cells/well, and the cells were randomly divided into 3 groups according to the concentration of the solution added to the well plate, group A 5 μmol/L CuSO ₄ , group B 25 μmol/L CuSO ₄ , Group C was the blank control, with 4 replicate wells in each group, the basal medium was MCDB131, and the MTT method was used to detect cell proliferation and draw the growth curve. Another HUVEC was inoculated on a 6-well plate at a density of 2×10 ⁵ cells/well, and the grouping was the same as before, and the endothelial nitric oxide synthase (eNOS) gene and Tie -1 gene expression. Results The growth curve showed that the first 3 days was an exponential growth period, and it entered a plateau period on the 4th day. The cell proliferation ability of group A was significantly higher than that of groups B and C from the 3rd day, and that of group B was higher than that of group C within 2 days, but was significantly lower than that of group C from the 4th day, and the differences were statistically significant (P< 0.05) see Figures 1 and 2, copper ions promote the proliferation of endothelial cells significantly stronger than the control group. Fluorescence quantitative RT-PCR detection showed that after 48 hours of treatment, the expression of eNOS gene in groups A, B, and C were 7.294±1.488, 0.149±0.044, and 1.000±0.253, and the expression of Tie-1 gene was 1.481±0.137, 1.131±0.191, and 1.000±0.177. Compared with groups B and C, the expressions of the two genes were up-regulated in group A (P<0.05); compared with groups B and C, the expression of eNOS gene was down-regulated (P<0.05), and there was no significant difference in the expression of Tie-1 gene ( P > 0.05) (Fig. 3, 4, Table 1). Conclusion: 5 μmol/L Cu ²⁺ can effectively promote the proliferation and differentiation of HUVEC.

Table 1 The expression of eNOS and Tie-1 in three groups of cells

＊Comparison between Group A and Group C, P<0.05; #Comparison between Group B and Group C, P<0.05

Example 2 confirms that copper ions stimulate cell regeneration and stem cell mobilization experiments in vitro and the physiological effects of local membrane-applied controlled release of copper ions in the body.

In order to determine the local effect of copper ions on the activation of scars and stimulate the regeneration of blood vessels in scars, the animal model of local myocardial infarction was pasted with copper ion gel to verify the physiological effect of local slow release of copper ions.

1. Prepare calcium alginate + CuSO ₄ film. Mix 10 ml of 2% sodium alginate solution with 10 mg of CuSO ₄ solution, add 5 ml of CaCl ₂ solution dropwise to form a film.

2. Preparation of the myocardial infarction model: under anesthesia, the left anterior descending coronary artery of the rabbit heart was opened and ligated to prepare the rabbit left myocardial infarction model. In the state of thoracotomy, attach the membrane formed in the previous step to the front surface of the left heart. Close chest. After 4 weeks of stabilization, the thoracotomy was performed again to observe the differences in gross specimens and pathological sections.

The results showed that the locally sustained release of copper ions formed a significant physiological effect of promoting angiogenesis and scar activation. In the myocardial ischemia model of rabbits, we used the membrane formed in the previous step to attach to the infarcted myocardial tissue 4 weeks after the myocardial ischemia operation. Supplementation can effectively improve myocardial ischemia and heart function, and pathological examination revealed a large number of new blood vessels in the infarcted myocardial tissue (see Figures 5 and 6).

The figure shows: Figure 5A is the sham operation group, and B is the control group (no calcium alginate+CuSO ₄ film was added). A large amount of adipose tissue and fibrotic tissue accumulation (arrows) can be seen in the local infarction. C is the vascular regeneration of the infarcted area after patching. The infarcted tissue has obvious myocardial regeneration and recovery of cardiac structure, and its shape is closer to that of the sham-operated heart. Visible microvascular network formation.

Fig. 6 is a comparison of the results of the above-mentioned pathological slides of myocardial infarction. Histology (HE staining, Masson's trichrome method) observed that the myocardium treated with Cu film had more new blood vessels, a large number of mononuclear cells and the recovery of myocardial fibrosis. CuSO ₄ sticking film made significant small blood vessels and glial hyperplasia in the necrotic tissue of local myocardial infarction. Masson's collagen staining.

Example 3 Copper ion and albumin binding experiment (preparation of copper ion chelated albumin microbubbles)

The solution used in the experiment was prepared with deionized water, 0.1mol L ^-1 NaCl was used to maintain the ionic strength, Tris-HCl was used as the buffer solution, the pH value was 7.4, HSA and copper sulfate solutions were prepared, and the concentrations were 8.76×10 ^{- 6} mol·L-1 and 6.48×10 ^-4 mol·L-1. Using a 1cm cuvette, measure (1) fix the excitation wavelength at 280nm, titrate HSA with copper sulfate, and observe the change of the fluorescence emission peak of HSA. It can be seen from the figure that the addition of Cu ²⁺ did not significantly change the position of the excitation peak and the maximum fluorescence peak, but the fluorescence intensity weakened to varying degrees with the increase of the amount of copper sulfate. Since the volume of the HSA solution is much larger than the volume of the metal ion solution added dropwise, the dilution effect can be ignored.

The protein can emit fluorescence because there are three aromatic amino acid residues in the protein: Trp, Tyr and Phe residues. Due to the different structures of these amino acid residues, the fluorescence intensity ratio of the three is usually 100:9:0.5. According to whether the protein contains tryptophan, it can be divided into two categories: type A protein refers to the protein containing no Trp residue but only Tyr and Phe residue; type B protein: refers to the protein containing both Trp residue and Tyr and Phe residues of the protein. Serum albumin is a class B protein with strong fluorescence. Although the three chromophores have their own characteristic fluorescence peaks (the fluorescence peaks of Trp, Tyr and Phe are located at 348, 303 and 282 nm, respectively), due to the energy transfer from Phe to Tyr or rrrp in protein macromolecules Very effectively, the absorption and emission of the whole molecule is therefore not a simple addition and subtraction of the optical properties of these monomeric components, often the fluorescence of Trp residues predominates. When the protein is excited at 270-290nm, the contribution of Tyr residues cannot be ignored. When the excitation wavelength is greater than 290nm, it can be considered that the fluorescence comes from Trp residues. There is only one Trp residue at position 214 in HSA, and the fluorescence emission peak is around 350nm. It is generally believed that the fluorescence is mainly emitted from the 212-position Trp residue at around 342nm. The maximum excitation peaks of both are about 280nm. Trp residues can be used as probes for local conformational changes or changes in the microenvironment of aromatic amino acid residues. When metal ions and other substances are added, the changes in the endogenous fluorescence of serum albumin can be used to conduct qualitative and quantitative studies on the binding.

This experimental example shows that with the addition of copper ions, more copper ions are chelated with human serum albumin, and the fluorescence of albumin is quenched.

The binding experiment of example 4 copper ions and albumin microbubbles, including fluorescence analysis result (preparation of copper ion chelated albumin microbubbles)

1. Preparation of albumin microbubbles: pipette 20ml of 5% human serum albumin solution into a 20ml disposable syringe connected to the tee, insert the probe of the ultrasonic instrument 2cm below the liquid surface, and vibrate 20ml with a certain ultrasonic intensity 1 % human serum albumin solution, pre-sound vibration for 10s, and then pass a certain amount of C ₃ F ₈ within 5 seconds while sonicating, continue the sonic vibration, stop the sonic vibration after reaching the end temperature, and obtain C ₃ F ₈ white Protein microsphere suspension, the suspension was immediately transferred to a 50ml vial, the air at the upper end of the vial was fully replaced with C ₃ F ₈ , the vial was tightly sealed with a rubber stopper, and stood at room temperature for 24 hours. After shaking well, the particle size analysis of the microspheres is shown in Table 2, and the average diameter of the microspheres is 2.15 μm.

Table 2: Particle size distribution of albumin microspheres

Table 2 shows that the average particle size of microbubbles is 2.15 μm, and the average concentration is 3.7×10 ⁸ /ml.

2. The binding experiment of microbubble membrane and copper ion:

Precisely draw 1ml of 5% HSA microbubble solution and dilute it into a 250ml volumetric flask, pipette 10ml each into 9 glass test tubes, and add Cu ²⁺ (1/50 diluted saturated copper solution) solution dropwise in turn for testing . Table 3 below shows the amount of copper ions with different concentrations bound to HSA microbubbles. See Figure 8-17, after adding different concentrations of copper sulfate to the albumin microbubbles, the fluorescence quenching reaction that occurs after the copper ions are chelated with the albumin microbubble membrane. Fig. 8-17 all can see two absorption peaks, and the former is the characteristic peak after adding copper (excitation wavelength is about 332nm); The latter peak is the absorption peak of the blank albumin microsphere itself of unchelated copper ion (emission wavelength is 408nm); data reading method: each peak has two representation values, which are read from bottom to top, and the two data are separated by ., which is the fluorescence spectrum absorption intensity.

This experiment proves that copper ions combine with the albumin microbubble membrane, resulting in the gradual decrease of the fluorescence intensity of albumin.

Table 3: Different concentrations of copper sulfate added

Preparation of Example 5 Copper Ions Binding Albumin Microbubbles

The experiment of preparing albumin copper microbubbles by ultrasonic dispersion after the combination of copper ions and albumin, including the results of fluorescence analysis and microbubble measurement results.

1. First prepare the albumin solution bound to copper ions: add the sample according to copper sulfate:albumin solution=1:1mol. The corresponding ratio in this example is as follows: 4.35×10 ^-6 mol Cu ²⁺ is added to 1 mg of HSA.

2. Pipette 20ml of 5% copper chelated-human albumin solution into a 20ml disposable syringe connected to the tee, insert the probe of the ultrasonic instrument 2cm below the liquid surface, and vibrate 20ml of 5% human blood with a certain ultrasonic intensity Albumin solution, pre-sonic vibration for 10s, then inject a certain amount of C ₃ F ₈ within 5 seconds while sonicating, continue sonication, stop sonication after reaching the end temperature, and obtain C ₃ F ₈ albumin microspheres Suspension, the suspension was immediately transferred to a 50ml medicine bottle, the air at the upper end of the medicine bottle was fully replaced with C ₃ F ₈ , the medicine bottle was tightly sealed with a rubber stopper, and stood at room temperature for 24 hours. After shaking well, the microspheres were subjected to particle size analysis, and the particle size distribution is shown in Figure 19. It shows that the average particle size of microbubbles after chelating copper ions is 2 μm, and the concentration is 3.1×10 ⁸ /ml

3. Stability test: inject the aforementioned microbubbles bound with copper ions into a 20ml glass bottle, seal it, and store it in a 5-degree refrigerator. After two months, re-sampled and measured, the microbubble concentration and particle size distribution did not change significantly from those just prepared. Figure 20 below is the detection of the change of microbubble particle size by flow cytometry. The test shows that the concentration and particle size of microbubbles are stable

Figure 20 Flow cytometry shows that after the microbubbles are bound to copper, after standing for 2 months, the particle size distribution is stable at the normal peak of 1.5 μm, with an average particle size of 1.95 μm; microbubbles with a particle size of 0-3 μm account for all 90% of the range of microbubbles, the concentration of microbubbles is 3×10 ⁸ /ml. Compared with the initial stage of preparation before February, there is no significant change. It shows that the chelated copper microbubbles are stable and reliable.

Example 6 Myocardial infarction animal model experiment and results of albumin copper ion microbubble ultrasonic irradiation

Prepare 15ml of albumin C ₃ F ₈ by the method of Example 5, in which 10mg of copper ions are co-chelated. Under anesthesia, the left anterior descending coronary artery of the rabbit heart was dissected by thoracotomy, and the left anterior descending coronary artery was ligated to prepare the rabbit model of left myocardial infarction, which was stabilized for 4 weeks. Continuous injection of chelated copper ion albumin microbubbles in the marginal ear vein at a rate of 1ml/min, continuous irradiation of the left precordial area with ultrasonic waves, and continuous fan-shaped scans from the bottom of the heart to the apex of the heart, with the output energy of the ultrasonic waves adjusted to the maximum and Enable color Doppler mode (Figure 21). Injection and irradiation were repeated every 2 weeks with the same dose each time. After 4 weeks, the experimental rabbits were killed, and the general heart shape and pathological sections of myocardial infarction were observed in the experimental rabbits, and the regeneration and activation of blood vessels in the myocardial infarction scar tissue of the control group were compared and analyzed. The results showed that the injection of chelated copper ion albumin microspheres, under the irradiation of ultrasound, produced a large number of obvious angiogenesis and activation in the local tissue of myocardial infarction, as shown in Figures 22 and 23.

Figure 21 shows the continuous breaking of albumin microspheres in high-energy color Doppler mode to form passive targeted release of copper ions to the local myocardial infarction.

Figure 22: A is the gross pathological pictures of the myocardial infarction model rabbits not injected with chelated albumin microbubbles after 4 weeks in the control group. A large amount of fat accumulation can be seen on the front surface of the left heart, showing the typical appearance of fatty degeneration and fat accumulation in the scar area of myocardial infarction (arrow). B is the experimental group, 4 weeks after the injection of chelated albumin microbubbles, the myocardial infarction scar area was significantly activated, and a large number of new capillaries were formed on the surface (arrow).

Figure 23: A and B are the control group, without ultrasonic irradiation for targeted delivery of chelated copper ions. A: ×400Masson staining: massive fatty degeneration and fat vacuole formation in the infarct area, without vascular proliferation. B: ×100Masson staining: a large amount of fatty degeneration and fat vacuoles were still seen in the border area between myocardial infarction and normal, without obvious vascular hyperplasia. C and D are the experimental group, which underwent ultrasonic irradiation to deliver chelated copper ions to produce obvious collagen and angiogenesis. C: ×400Masson staining: obvious collagen fiber hyperplasia and small blood vessel hyperplasia can be seen in the infarct area. D: Masson staining at the border between the infarct and the normal area × 100: obvious collagen fiber hyperplasia accompanied by small blood vessel hyperplasia.

Example 7 The binding experiment of albumin and zinc ions and the preparation of chelated zinc albumin microspheres (confirm the experiment of zinc ions chelated albumin microbubbles)

1. According to zinc chloride: albumin = 1: 1 molar ratio. Accurately draw 15ml of 5% HSA microbubble solution, and add dropwise zinc chloride solution of equimolar concentration. Fluorescence analysis showed that after zinc chloride was added to the albumin solution, the fluorescence quenching reaction of zinc ions and albumin also occurred, which confirmed the chelation of zinc ions and albumin. According to the molar ratio, a total of 15ml of 5% zinc chelated human serum albumin was prepared.

2. Pipette the chelated zinc-human serum albumin solution prepared in step 1 into a 20ml disposable syringe connected to the tee, insert the probe of the ultrasonic instrument 2cm below the liquid surface, and vibrate the human blood albumin with a certain ultrasonic intensity. For protein solution, pre-sonicate for 10 seconds, then inject a certain amount of C ₃ F ₈ within 5 seconds while sonicating, continue sonicating, stop sonicating after reaching the end temperature, and obtain C ₃ F ₈ albumin microspheres Suspension, the suspension was immediately transferred to a 30ml vial, the air at the upper end of the vial was fully replaced with C ₃ F ₈ , the vial was tightly sealed with a rubber stopper, and stood at room temperature for 24 hours. After shaking well, the microspheres were subjected to particle size analysis. The particle size distribution was the same as in Example 5, and the average diameter of the microspheres was 2.5-3.0 μm.

3. Stability test: the aforementioned microbubbles combined with zinc ions were injected into a 20ml glass bottle, sealed, and stored in a refrigerator at 4°C. After two months, re-sampled and measured, the microbubble concentration and particle size distribution did not change significantly from those just prepared.

Figure 24 is the particle size analysis of microbubbles by flow cytometry

Figure 24 shows that after standing for 2 months, the flow cytometer shows that the microbubble particle size distribution and concentration changes after zinc ion binding are not significantly different from those at the initial preparation, the normal distribution peak of the particle size distribution is at 1.5 μm, and the particle size is 0-3 μm The microbubbles accounted for 90.5% of all microbubbles, with an average particle size of 2.5 μm. The microbubble concentration is 3.4×10 ⁸ /ml. Zinc-bound microbubbles are stable and reliable.

The binding experiment of example 8 albumin and selenide (confirm the experiment that selenium ion chelates albumin microbubble)

1. Prepare 20ml of selenium-bound HSA: mix selenium penicillamine (4mM) with HSA (40mg/mL) in 0.01M phosphate buffer (pH7.4), and incubate the mixture for 10 minutes. Unreacted PenSePen was removed by dialysis. Selenium-bound HSA is obtained.

2. Put the selenium-human albumin solution into the 20ml disposable syringe connected to the tee, insert the probe of the ultrasonic instrument 2cm below the liquid surface, vibrate the human albumin solution with a certain ultrasonic intensity, pre-sonic vibration for 10s, and then Inject a certain amount of C ₃ F ₈ within 5 seconds while sonicating, continue sonicating, and stop sonicating after reaching the end point temperature to obtain a suspension of C ₃ F ₈ albumin microspheres, which is immediately transferred to In the 30ml medicine bottle, the air at the upper end of the medicine bottle was fully replaced with C ₃ F ₈ , the medicine bottle was tightly sealed with a rubber stopper, and stood at room temperature for 24 hours. After shaking well, the microspheres were subjected to particle size analysis. The particle size distribution was the same as in Example 5, and the average diameter of the microspheres was 2.5-3.0 μm.

3. Stability test: inject the aforementioned microbubbles combined with selenium ions into a 20ml glass bottle, seal it, and store it in a 5-degree refrigerator. After two months, re-sampled and measured, the microbubble concentration and particle size distribution did not change significantly from those just prepared. Figure 25 below is the particle size analysis performed by flow cytometry.

Figure 25 shows that after standing for 2 months, after microbubbles combined with selenium, the particle size distribution is stable at the normal peak of 1.5 μm, the average particle size is 2.1 μm, and the microbubbles with a particle size of 0-3 μm account for 91% of all microbubbles. , the microbubble concentration is 3×10 ⁸ /ml. Compared with two months ago, the size distribution and concentration of microbubbles did not change significantly. Selenium-incorporated microbubbles have a robust and reliable profile.

Example 9 albumin binding copper+phospholipid microbubble mixing experiment

1. Prepare copper-chelated albumin according to example 3: 0.1M NaCl maintains ionic strength, uses Tris-HCl as buffer solution, pH value is 7.4, prepares HSA and copper sulfate solution, the concentration is 8.76×10 ^-6 M successively and 6.48×10 ^-4 M.

2. Inject 9.6ml of SONOVUE phospholipid SF ₆ microbubbles.

3. Mix the solutions of 1 and 2 to form a suspension of albumin polypeptide chelated copper and phospholipid microbubbles.

Example 10 Animal experiments and results of albumin-binding copper+phospholipid microbubble myocardial infarction animal model

1. Prepare a rabbit myocardial infarction model according to Examples 2 and 6.

2. The phospholipid microspheres prepared in Example 9 and the albumin polypeptide suspension of chelated copper were injected continuously at a rate of 1 ml/min through the marginal ear vein, and the left precordial area was continuously irradiated with ultrasonic waves at the same time. Do a continuous sectoral scan back and forth to the apex of the heart, adjust the ultrasonic output energy to the maximum and enable the color Doppler mode. Injection and irradiation were repeated every 2 weeks with the same dose each time. After 4 weeks, the experimental rabbits were killed, and the general heart shape and pathological sections of myocardial infarction were observed in the experimental rabbits, and the regeneration and activation of blood vessels in the myocardial infarction scar tissue of the control group were compared and analyzed. The results showed that the injection of liposomes and copper chelated albumin polypeptide suspension, under the irradiation of ultrasound, caused obvious angiogenesis and activation in the local tissue of myocardial infarction. Pathological slices of myocardial infarction scar tissue were taken by microvessel density counting method. Randomly sample 5 areas per slice, and count under a 10×40 magnification lens. The diameters of the blood vessels were all within 20 μm, and the number of microvessels in the above field of view was counted.

Figure 26 shows: the comparison of the microvessel density in the myocardial infarction area of the myocardial infarction area irradiated by ultrasound with phospholipid microspheres and chelated copper albumin and only the injection of phospholipid microspheres in the control group shows that the experiment containing chelated copper albumin The microvessel density in the myocardial infarction area of the group was significantly higher than that of the control group.

Example 11 Animal experiments and results of glucose binding copper+phospholipid microspheres myocardial infarction animal model

1. Prepare a rabbit myocardial infarction model according to Examples 2 and 6.

2. The phospholipid microspheres prepared in Example 9 and the glucose suspension of chelated copper were injected continuously at a rate of 1ml/min through the marginal ear vein, and the left precordial area was continuously irradiated with ultrasound at the same time, from the bottom of the heart to the apex of the heart. Do a back and forth continuous sectoral scan, adjust the ultrasonic output energy to the maximum and enable the color Doppler mode. Injection and irradiation were repeated every 2 weeks with the same dose each time. After 4 weeks, the experimental rabbits were killed, the general heart shape and pathological sections of myocardial infarction were observed, and the regeneration and activation of blood vessels in the myocardial infarction scar tissue of the control group were compared and analyzed. The results showed that the injection of lipid microspheres and copper chelated glucose suspension, under the irradiation of ultrasound, caused obvious angiogenesis and activation in the local tissue of myocardial infarction. Pathological slices of myocardial infarction scar tissue were taken by microvessel density counting method. Randomly sample 5 areas per slice, and count under a 10×40 magnification lens. The diameters of the blood vessels were all within 20 μm, and the number of microvessels in the above field of view was counted.

Fig. 27 shows: in the myocardial infarction area, obvious small blood vessel regeneration can be seen in the myocardial infarction area irradiated by ultrasound with phospholipid microspheres and chelated copper glucose.

Figure 28 shows: the contrast of the microvessel density in the myocardial infarction area of the myocardial infarction area irradiated by ultrasonic phospholipid microspheres and chelated copper glucose with the myocardial infarction area of only injecting phospholipid microspheres control group in the myocardial infarction area shows that the experimental group containing chelated copper glucose solution The microvessel density in the myocardial infarction area was significantly higher than that in the control group.

The above experiments prove that the microelement preparation prepared by the present invention can release targeted trace elements under the action of ultrasound, and achieve the release of trace elements in local tissues and produce biological effects related to the trace elements. The ultrasonic targeted pharmaceutical composition of the present invention The curative effect is definite, the stability is good, and it is safe. It is a new pharmaceutical preparation and has excellent clinical application and industrialization prospects.

references

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Claims (12)

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:

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

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;

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

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.

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