CN1180304A - Method of magnetic resonance focused surgical and therapeutic ultrasound - Google Patents

Abstract

A novel method of magnetic resonance focused surgical ultrasound by administering to a patient a magnetic resonance imaging (MRI) contrast medium comprising gas filled vesicles, then scanning the patient with MRI techniques, and then applying ultrasound to effect surgery. These methods may also use an MRI contrast medium comprising gaseous precursor filled vesicles which undergo a phase transition from a liquid to gas in vivo after administration. Additionally, the MRI contrast medium may comprise a therapeutic compound.

Description

The method of the magnetic resonance focused ultrasonic art that is used for surgical operation and treatment

Applying for as a reference careful

The application is that what to submit March 9 nineteen ninety-five is 08/401 at careful application number; 974 continuation part; and this application is that what to submit on March 11st, 1994 is 08/212 at careful application number; 553 continuation part; these two applications all are incorporated herein by reference, and also claimed its priority application content.

What submit on June 11st, 1993 is 08/076 at careful application number, 250 application discloses the medicine delivery system of the microsphere that comprises the gas filling that contains a kind of therapeutic agent, this application lay special stress on utilize ultrasonic technique monitoring and be determined at the existence of described microsphere in patient's body, described microsphere is broken, so that discharge described therapeutic agent in intravital this zone of described microsphere that exists of patient.Described this application is that the U. S. application submitted on June 18th, 1991 number is 716,899 and 717,084 continuation part, and they be the U. S. application submitted August 20 nineteen ninety successively number are 569,828 continuation part, and the continuation part of the U. S. application that this application is in December, 1989 successively to be submitted on the 22nd numbers 455,707.

Is 08/076 with what proposed hereinbefore just now at careful application number, 250 application have identical the first to file source what submit on June 11st, 1993 is that 08/076,250 application discloses to be used for preparing and is applicable in the ultrasonography art and is used as contrast medium or is used as the method and apparatus of microsphere of the gas filling of drug release agent at careful application number.

What submitted in 16th in JIUYUE in 1994 is 08/307 at careful application number, 305 and what submit on November 30th, 1993 simultaneously is 08/159 at careful application number, 687 and 08/160, (it is respectively that the application number of submitting on June 11st, 1993 simultaneously is 08/076 successively in 232 application, 239 and application number be the continuation part of 08/076,250 application) disclose that preparation is used to diagnose and the gentle precursor-filled microsphere of gas for the treatment of and new the treatment delivery system and the method for multi-phase fluid and gas composition.

It is 08/307,305,08/159,687 that the application requires application number, 08/160,232,08/076,239 and 08/076,250 and its applying date in first to file be priority date, and they all are incorporated herein by reference.

The application number of submitting April 10 nineteen ninety 07/507,125 is also as the list of references of this paper, it disclose independent or with for example paramagnetic, super paramagnetic and the polymeric application of the blended bio-soluble of proton density contrast medium of one or more contrast medium.Multiple or a kind of polymer/contrast medium mixture can mix so that strengthen the laxity of the preparation that obtains with one or more bio-soluble gases non-imposedly.

Background of invention

Invention field

The present invention relates to the magnetic resonance imaging technical field, more particularly, relate to the purposes of the contrast medium of the supersonic operation that vesicle that the gas of stabilisation fills instructs as magnetic resonance imaging (MRI).

Existing various imaging techniques are used to diagnose people's disease.One of application imaging technique the earliest is the X-ray.For the X-ray, the image of the patient body that is produced reflects the different densities of body structure.In order to improve the diagnostic uses of this imaging technique, utilized contrast medium to improve the density of the relative surrounding tissue of required tissue, so that the required visibility of organizing is bigger under the X-ray.For example research can extensively utilize barium and iodinating contrast medium to observe esophagus, stomach, intestinal and rectum for X-ray gastrointestinal.Can utilize these contrast medium for the aspect X-radiography investigative technique (just computer assisted aspect X-radiography is CAT) of X-ray computerization in addition and be adjacent and organize for example contrast of blood vessel and lymph node so that improve the gastrointestinal visibility and for example improve gastrointestinal tract.Use described contrast medium can improve the density of esophagus, stomach, intestinal and rectum inside, and gastronintestinal system and surrounding structure can be distinguished.

The magnetic resonance imaging technology is newer imaging technique, and it is different with the X-ray, does not utilize Ionized radiation.Be similar to computer assisted aspect X-radiography (CAT), MRI prepares the X-section image of health, still, MRI have can make any plane of scanning motion (be axis, the crown, vow shape or orthogonal) other advantage of imaging.Unfortunately, owing to make MRI be restricted as the abundant application of the diagnostic mode of health to needs new or better contrast medium.Do not have suitable contrast medium, utilizing MRI to distinguish target tissue often is difficult with the tissue that links to each other.If obtain better contrast medium, will improve the comprehensive application of MRI, and improve the diagnosis accuracy of this mode widely as the imaging instrument.

MRI utilizes magnetic field, radio-frequency (RF) energy and magnetic field gradient so that make the health imaging.Different T1 (longitudinally) and the loose value of T2 (horizontal) and the proton densities (free water content effectively) that mainly reflect tissue of contrast and signal intensity between the tissue.When utilizing contrast medium to change patient's the signal intensity in a zone, can utilize several possible approach.For example, contrast medium can be designed so that change T1, T2 or proton density.

The summary of prior art

In the past, mainly be primarily focused on the paramagnetic contrast medium that is used for MRI.The paramagnetic contrast medium contains the unpaired electronics that has local little the action of a magnetic field in main magnetic field so that improve vertically (T1) and horizontal (T2) loose ratio.Most of paramagnetic contrast medium are deleterious metal ion as a rule.For reduced toxicity, utilize these metal ions of part complexation usually.The paramagnetic metal ion complex that obtains has the toxicity of reduction.To the metal-oxide as the contrast medium of MRI, the most significant is that the oxide of ferrum is tested.The oxide that for example is lower than the short grained ferrum of 20nm diameter may have the paramagnetic bulk characteristics, and its dominant effect is because big susceptibility.Therefore, magnetic-particle has mastery reaction to the T2 laxity.Nitroxide be another kind of also be paramagnetic MRI contrast medium.Their laxity is relatively low, and its effect as the MRI contrast medium is lower than paramagnetic ion usually.All these contrast medium have some toxic action when certain occasion is used, and it is ideal not having a kind of when pouring into contrast medium with regard to itself being used as.

Existing MRI contrast medium is subjected to many restrictions.For example, known positive contrast medium is because the motion that its inherent wriggling and breathing or cardiovascular activity produce increases visual noise.Positive contrast medium for example Gd-DTPA also has more complicated situation, and its signal intensity depends on the concentration of contrast medium and the continuity of used pulse.It is more complicated that gastrointestinal tract for example makes translating of image release to the absorption of contrast medium, particularly in the end portion of small intestinal, unless use paramagnet people such as (, Magn.Reson.Imaging, 6:124 (1988)) Kornmesser of enough high concentrations.Comparatively speaking, the variation of negative contrast medium paired pulses continuity is more insensitive, and more consistent comparative is provided.But when high concentration, for example the ferrite granule can produce false susceptibility, especially for example the intestinal juice absorption occurring and is making the spissated rectum of super paramagnetic meterial, and evidence is clearer and more definite.Common negative contrast medium shows the good comparative to fat, and still, for the image of T1 weighting, positive contrast medium is woven with good comparative with respect to normal group.Because most of pathological tissues have longer T1 and T2 than normal structure, so they show darkly on the image of T1 weighting, and show on the image of T2 weighting and become clear.This shows that ideal contrast medium should show on the image of T1 weighting bright, and show dark on the image of T2 weighting.The many MRI contrast medium that can buy now do not satisfy this double standards.

Toxicity is another problem of existing contrast medium.Any medicine all has some toxicity, and toxicity is relevant with dosage usually.For ferrite, after oral, often occur feeling sick and flatulence and serum levels of iron moment raising.Paramagnetic contrast medium Gd-DTPA is the organic metal complex of a kind of gadolinium and the penta acetic acid coupling of complexing agent diethylentriamine.Do not carry out coupling, free gadolinium ion is extremely deleterious.In addition, for example wherein stomachial secretion acid and the intestinal gastrointestinal uniqueness that discharges alkali produced decoupling and will dissociate gadolinium or other paramagnetic agent from the isolating probability of complex, thereby multiple pH value variation took place between the operating period at gastrointestinal tract.Certainly, making the dosage of paramagnetic agent be reduced to minimum is important for making potential toxicity be reduced to minimum.

It is essential using new and/or better contrast medium in magnetic resonance imaging and improved imaging technique.The present invention be more particularly directed to these important results.

In the research of the relevant MRI contrast medium that the application number of above submitting April 10 nineteen ninety 07/507,125 is described, how to disclose gas and polymer composition and paramagnetic or super paramagnetic agent combined and be used as the MRI contrast medium.Wherein verified by the stable gas of the said polymer how as the effect of the effective contrast medium of susceptibility so that be reduced in signal intensity on the T2 weighted image; It is effective especially that described system is used as gastrointestinal tract MRI contrast medium.

People such as Widder disclose the stable microbubble type ultrasonography agent that is produced by the biocompatible protein of thermal denaturation such as albumin, hemoglobin and collagen protein in disclosed European patent application EP-A-0324938.

The once report that to be people such as Moseley did on Californai is received the meeting of the medical magnetic resonance association that handkerchief holds in 1991 that also can mention, this report is summarized in a summary, and title is " microbubble: a kind of new magnetic resonance sensitivity contrast medium " (Microbubbles:ANovel MR Susceptibility Contrast Agent).The microbubble that is utilized comprises the air that is coated with the human albumin shell.But the document does not disclose the so stable vesicle that is filled with gas of the present invention.

But for using in the blood vessel, the inventor finds that making gas stable with the flexibility compound is favourable for obtaining optimum.Available protein for example albumin makes bubble stabilizes, but the bubble shell that obtains can be frangible with hard.Owing to several reasons, protein is unfavorable.At first, frangible cover layer has limited the ability of bubble expansion and decline.Because bubble runs into different pressure area (for example, moving to Arterial system via the circulation of heart from Venous system) in vivo, frangible shell fragmentation, gas will be run away.This has limited health obtains the time of useful comparative from the contrast medium of bubble effect duration.Described fragment frangible, fragmentation may also be deleterious.Frangible in addition cover layer for example makes the measuring body internal pressure very difficult from proteic rigid characteristic and the stiff bubble that obtains.

It is ideal for forming stable gas that the disclosed application of Quay WO93/05819 discloses the gas with high Q number, but its disclosed content limitation in stable gas, rather than its stabilisation and encapsulation, as described herein.In a preferred embodiment of the 31st page of description, utilize sorbic alcohol to improve viscosity, it is said that it can prolong the life-span of microbubble in solution successively.This is not a primary demand of the present invention, but related certain Q-value of gas tool or invasin.

People such as Lanza disclose sound reflecting lamella liposome in disclosed application WO93/20802, this liposome is at the multi-disc layer liposome of the aqueous space that has increase between the bilayer or contains the non-entad mode liposome that is nested between bilayer, and isolating bilayer in therefore containing.The document has also been narrated its application as ultrasonic contrast medium who strengthens ultrasonography and the medicine that the monitoring liposome discharges when giving patient's administration.

D ' Arrigo is at United States Patent (USP) 4,684, discloses gas-in-liquid emulsion and lipoid coating microbubble respectively in 479 and 5,215,680.

According to the present invention, have been found that the vesicle of the gas filling of stabilisation is very effective non-toxicity contrast medium for the ultrasonic art of magnetic resonance focused Noninvasive simultaneously.

Summary of the invention

The present invention relates to the method for the ultrasonic art that is used for surgical operation and treatment of magnetic resonance imaging focusing, this method comprises with the contrast medium that is used for magnetic resonance imaging gives the operating patient's administration of needs, this contrast medium contains the vesicle that gas is filled, utilize described contrast medium with the magnetic resonance imaging art to patient scan so that identify patient's the operating zone of needs, to carrying out the ultrasonic art of operating area applications.Using ultrasound is followed second scanning step later on, wherein with magnetic resonance imaging to patient scan.In applications of ultrasound, can use magnetic resonance imaging.Scanning and surgery ultrasonic step can repeat up to obtaining required effect.The vesicle that gas is filled contains the therapeutic agent of the specific region that is discharged into the patient when ultrasonic.

In addition, the ultrasonic art that the present invention includes the treatment that utilizes magnetic resonance imaging focusing is controlled the method that therapeutic agent is discharged into patient's a certain zone.This method comprises to contain vesicle that the gas for the treatment of chemical compound fills to patient's administration; Utilize magnetic resonance imaging monitoring vesicle so that be determined at the vesicle that this zone exists; And utilize ultrasonic art to make vesicle broken so that discharge therapeutic agent in this zone.

The invention still further relates to the method that is used for operating ultrasonic art of utilizing magnetic resonance imaging to focus on, comprise the contrast medium administration that is used for magnetic resonance imaging with the vesicle that contains gaseous precursors and fill to the patient of needs operations, make the mutually transformation of gaseous precursors generation from liquid to gas, with the magnetic resonance imaging art to patient scan so that identify the zone of patient's needs operation, and in this area applications ultrasonic surgical art.Phase conversion step and magnetic resonance imaging step can be carried out simultaneously.

Contrast medium comprises the vesicle that the gas of stabilisation is filled, wherein gas is bio-soluble gas, for example nitrogen or perfluoropropane, but it also can derive from gaseous precursors, for example, the perfluoro capryl bromine, and form by the chemical compound biological example solubility lipoid of Stabilization or polymer and stablize this vesicle.The present invention utilizes gaseous precursors to implement the gas of the vesicle of formation blanketing gas, often follows sizable advantage.These gaseous precursors can be by many factor activation, preferably by temperature-activated.Described gaseous precursors is a kind of chemical compound, and wherein when selected activation or phase transition temperature, the phase change from the liquid or solid to gas takes place this chemical compound.Therefore, by the temperature of chemical compound is brought up to and is higher than activation or phase transition temperature activates from being lower than activation or phase transition temperature.When forming vesicle with lipoid, lipoid can be monolayer or double-deck form, and can form a series of entad monolayers or bilayer with monolayer or bilayer lipid.Therefore, lipoid can be used for constituting unilamellar liposome (being made up of a monolayer or bilayer lipid), few layer liposome (being made up of two or three monolayers or bilayer lipid) or multilamellar liposome (by forming more than trilaminar monolayer or bilayer lipid).Preferably, the biocompatibility lipoid comprises phospholipid.Non-imposed ground, contrast medium comprises paramagnetic and/or super paramagnetic contrast medium, preferably by vesicle bag quilt.Non-imposed ground, contrast medium also further comprises liquid-fluorination carbon compound, for example a kind of perfluocarbon is so that further make this vesicle stable.Preferably, liquid-fluorination carbon has vesicle bag quilt.

These and other aspect of the present invention will become more apparent according to following detailed description.

Detailed Description Of The Invention

The present invention relates to the ultrasonic art method that is used for surgical operation and treatment that magnetic resonance imaging focuses on, this method comprises with the contrast medium that is used for magnetic resonance imaging the operating patient's administration of needs, this contrast medium contains the vesicle that gas is filled, utilize described contrast medium with the magnetic resonance imaging art to patient scan so that identify patient's the operating zone of needs, and in the ultrasonic art of area applications of implementing operation.In ultrasonic step, can use magnetic resonance imaging.Using ultrasound is followed second scanning step later on, wherein with magnetic resonance imaging to patient scan.The vesicle that gas is filled contains the therapeutic agent of the specific region that is discharged into the patient when ultrasonic.

Scanning and surgery ultrasonic step can repeat up to obtaining required effect.According to the present invention, be meant simultaneously with ultrasonic and magnetic resonance imaging can exist simultaneously or use synchronously; Use successively or continuously; So that can be observed ultrasonic fragmentation to vesicle and tissue.Therefore, ultrasonic and magnetic resonance can be carried out simultaneously, or one is followed another and carries out.The magnetic resonance imaging technology is used the accuracy of the imaging therapy that can improve present acquisition with ultrasonic art.The accuracy that the magnetic resonance imaging technology is used with ultrasonic art confirms the position of vesicle, because available magnetic resonance imaging scans whole health, provides visible big zone, and in case the location, can make the vesicle fragmentation with ultrasonic in the given area of health.

The invention still further relates to the method for the ultrasonic surgical art of utilizing magnetic resonance imaging focusing, comprise the contrast medium administration that is used for magnetic resonance imaging with the vesicle that contains gaseous precursors and fill to the patient of needs operations, make the mutually transformation of gaseous precursors generation from liquid to gas, with magnetic resonance imaging art and described contrast medium to patient scan so that identify the zone of patient's needs operation, and in this area applications ultrasonic surgical art.

In addition, the present invention includes the method that the ultrasonic art control therapeutic agent that utilizes magnetic resonance focused treatment is discharged into patient's a certain zone.This method comprises with the contrast medium that contains the vesicle that the gas for the treatment of chemical compound fills patient's administration; Utilize magnetic resonance imaging monitoring vesicle so that be determined at the vesicle that this zone exists; And utilize the broken vesicle of ultrasonic art so that discharge therapeutic agent in this zone.

As disclosed in above-mentioned and whole description, except as otherwise noted, otherwise following term will have following meanings.

" magnetic resonance imaging art " (MRI) uses static main magnetic field; As seen the radio-frequency (RF) energy of pulse and the magnetic gradient of pulse promptly, make vesicle to prepare image.Can utilize radio frequency and electrical gradient to cause local energy to store, and the activation vesicle, be preferred energy still in order to activate vesicle ultrasonic.When implementing magnetic resonance imaging method of the present invention, contrast medium can be used in combination separately or with other diagnostic agent, therapeutic agent or other medicament.Described other medicament comprises excipient, for example increases flavor and hyperchromic material.The ultrasonic art of applied magnetic resonance is conventional and is described in for example D.M.Kean and M.A.Smith " magnetic resonance imaging art: principle and application " (Magnetic Resonance Imaging:Principles and Applications) (William and Wilkins, Baltimore 1986).Described MRI technology includes but not limited to, nuclear magnetic resonance, NMR (NMR) and electronic spin resonance (ESR) and magnetic resonance angioblast art (MRA).Preferred imaging mode is NMR.Certainly, except that MRI, also can utilize the magnetic Imaging to detect the interior vesicle of the scope of the invention.The magnetic Imaging utilizes magnetic field, does not need to utilize pulsed gradient or radio-frequency (RF) energy.The magnetic Imaging can be used for detecting the magnetic vesicle, such as but not limited to ferromagnetic vesicle.Utilize gaussmeter superconduction quantum to finish the magnetic Imaging at downside measuring device (SQUID).SQUID can screen all systemic magnetic granules apace; Then with localization by ultrasonic in those zones.For the application of this respect, the magnetic resonance imaging art comprises the magnetic Imaging, but should to understand the magnetic Imaging be the image of magnetic vesicle and do not comprise its nuclear resounce.

" ultrasonography art " is to carry out at required tissue, and just utilizes ultransonic energy activated or broken vesicle in case arrive its predetermined organizational goal ground.That focus on or relate to ultrasonicly be meant the specific region that ultrasonic energy is applied to health, thereby ultrasonic energy is focused in selected zone or the target circle.In addition, focus on and be meant magnetic resonance, it is ultrasonic by as seen vesicle and target circle are guided; Thereby observe simultaneously and broken described tissue with ultrasonic.Noninvasive is meant and does not cut skin and make the broken or imbalance of in-vivo tissue.Ding Yi the ultrasonic surgical operation that causes tissue necrosis that is meant in the present invention, i.e. tissue broken and destroying; Structural hole, cave, rent or the reparation (for example hernia) of scratching, whole or the alleviating of part diseased tissue (for example tumor); By vesicle activation or the fragmentation of ultrasonic energy with subjacent tissue.Ultrasonic art is and the nuclear medicine diagnostic imaging technology different with the X-ray, because it does not contact the patient with the toxic action that the ionizing radiation produces.In addition, different with the magnetic resonance imaging technology, ultrasonic art is relatively cheap, can be used as the detection of being convenient to move.When utilizing ultrasonic technique, sound is sent to patient or animal by means of a pick off.When sonic propagation was crossed health, they ran into the interface of tissue and body fluid.Based on the acoustic characteristic of in-vivo tissue and body fluid, ultrasound wave partly or entirely is reflected or absorbs.When sound wave detects and be processed to form image by the receptor in the pick off during by boundary reflection.The acoustic characteristic of intravital tissue and body fluid has been determined comparative to have occurred in the image that obtains.Another kind of optionally method is utilized ultrasonicly to make vesicle as seen, and the magnetic resonance imaging technology can be used for activating vesicle.In addition, the strong intensity of ultrasonic energy can cause the broken and activation of vesicle.The broken successively subjacent tissue of the activation of vesicle is so that cause this tissue necrosis.

The diagnostic ultrasound image device of any kind can be used for implementing the present invention, and the particular type of this device or model are not the keys of the inventive method.It also is suitable being designed for the Hyperthermic device of ultrasound detection, and described unit describe is in United States Patent (USP) 4,620,546,4,658,828 and 4,586,512, and its all disclosed contents are as the list of references of this paper.Preferably, this device has resonance (RF) spectroanalysis instrument frequently.The pick off survey meter can outsidely use or be transplanted.Usually start ultrasonic art than low-intensity the time, and preferably continue when the peak resonant frequency, improve intensity then, time and/or resonant frequency are up to the microsphere fragmentation.

" vesicle " is meant and is characterized as the spherical integral body that has internal voids.Preferred vesicle is to comprise that with lipoid the various lipoids of describing are formulated herein.For given vesicle, lipoid can be monolayer or double-deck form, and can form one or more monolayers or bilayer with monolayer or bilayer lipid.For more than one monolayer or bilayer, monolayer or double-deck normally entad.The vesicle of Miao Shuing comprises the entity of so-called vesicle for liposome, micella, bubble, microbubble, aeroge, cage type constraint or the like herein.Therefore, lipoid can be used for constituting monolayer vesicle (being made up of a monolayer or bilayer lipid), few layer vesicle (being made up of two or three monolayers or bilayer lipid) or multilamelar vesicles (by forming more than trilaminar monolayer or bilayer lipid).As required, the internal voids of vesicle can be used liquid, comprises for example liquid, aqueous, gas, gaseous precursors and/or solid or solute substance, comprises that for example target hits part and/or bio-activator is filled.

" liposome " typically refers to the ball bundle or the aggregation of amphiphilic compound, comprises usually the lipids of one or more entad layer form.The liposome that most preferred gas is filled is built into the single monolayer of simple lipoid layer (being monolayer) or lipoid.Can make liposome with various lipoids, comprise phospholipid and non-ionic surface active agent (for example niosomes).Most preferredly comprise that the lipoid of the liposome that gas is filled is to be gel state under physiological temp.Can crosslinked or polymerization with liposome, and carry for example Polyethylene Glycol of polymer on its surface.To hit the surface that part is attached to the liposome of gas filling at the target of epidermis cell.It is a kind of material that is attached to vesicle that target hits part, and it leads specific cell type such as but not limited to epidermal tissue and/or cell with vesicle.Target hits part can be attached to vesicle by covalent bond or non-covalent bond.Liposome is also referred to as the lipoid vesicle herein.Most preferred liposome portion within it comes down to water-free.

" micella " is meant the colloid that the concentration when the lipids laurilsulfate is formed by lipids when critical temperature is above.Also have surfactant properties (promptly can reduce surface tension and like water and the hydrophilic and lipophilic zone of fat), also available these material stabilise bubbles owing to form the chemical compound lot of micella.Usually, these micella materials like adopting monolayer or hexagon H2 phase configuration, yet adopt double-deck configuration.When forming the vesicle that gas fills with the micella material, this chemical compound adopts and contains towards aliphatic (fat is liked) composition of vesicle and the radially configuration of the hydrophilic area on vesicle surface dorsad usually.For target hits epidermis cell, target is hit part be attached on the micella chemical compound or be attached on the amphiphilic species with the micella compound.Another kind of optionally method, it is adsorbable to making the stable micella material surface of vesicle that target hits part.

" hydrogel " is meant the structure that is similar to microsphere, and the internal structure of different is common hydrogel is made up of a plurality of little spaces rather than a space.In addition, preferred hydrogel is formed (for example from toasting the foams of resorcinol and prepared formaldehyde) by synthetic, but for example poly-polysaccharide or proteinic natural materials also can be used to prepare hydrogel.Target hits the adsorbable surface to hydrogel of part.

" cage type body " typically refers to solid matter, and it combines with vesicle as the host, rather than covers the surface of vesicle.Solid, half porous or porous cage type body can be used as the medicament of stablizing vesicle, but cage type body itself can not cover the whole surface of vesicle.But the cage type body forms the fixedly spatial structure of vesicle that has that is known as " cage ", the adsorbable one or more vesicles of cage type body.Similar with microsphere, one or more surfactants can mix the cage type body and these surfactants will help to stablize vesicle.Usually surfactant covers vesicle and help and keeps combining of vesicle and cage type body.The useful cage type body material of stablizing vesicle comprises porous apatite for example the precipitation for example alginic acid and the calcium salt of calcium hydroxy apetite and polymer and metal ion.Hitting part at the target of epidermis cell can be incorporated into cage type body itself or be incorporated into and the bonded surfactant materials of cage type body.

Be not subjected to the constraint of any specific theory of operation, it is believed that the present invention partly depends on the following fact at least: gas, liquid and solid have different susceptibility mutually.At the interface of gas and water, for example magnetic area is changed, after this causes the phase shift of for example spin of proton.Can see that when imaging the signal intensity that shows as with gas/water interface adjacency reduces.This effect is more obvious in the image of T2 weighting, and the main gradient echo pulse continuity that shows.The pulse continuity of utilizing the extension of narrow band width to read can improve this effect.ET to gradient echo pulse continuity is long more, acts on big more (degree and the size that are the loss of signal are big more).

It is believed that the vesicle of the gas filling of the stabilisation that is used for the present invention depends on other characteristic different and that describe in this article of this phase susceptibility, and the effective fragmentation that the mri contrast medium of high efficiency levels is provided and makes the vesicle of contrast medium.Form from the substrate of the chemical compound of stabilisation and promptly to prepare vesicle, described chemical compound is set up the vesicle that gas fills and is used in the magnetic resonance imaging technology afterwards and keeps its size and shape in the required time.This chemical compound also allows at the broken vesicle of certain energy level, and preferred described energy is a ultrasonic energy.Most typical these stabilisation chemical compounds are those chemical compounds with hydrophobic/hydrophilic character, so that they form monolayer or bilayer etc. in the presence of water, and vesicle.Therefore, often be called the water of diluent usually at this paper, saline or other aqueous medium are the vesicle contrast medium that stabilisation gas of the present invention is filled.

In fact, the stabilisation chemical compound can be a mixture of authorizing the chemical compound of the various required characteristics of the vesicle of stabilisation.For example, have been found that the dissolving of the chemical compound that helps basic stabilisation and dispersive chemical compound have advantage.Other element of the vesicle of stabilisation is a gas, and it is gas in preparation during vesicle, maybe can be gaseous precursors, it to activation factor for example temperature easily play induction, and change gas phase mutually into from liquid or solid.Use description to the various aspects of the contrast medium that the gas of stabilisation of the present invention fills now.Using method

According to the present invention, provide the magnetic resonance focused ultrasonic art method of synchronous Noninvasive.Contrast medium by will being used for magnetic resonance imaging is to the operating patient's administration of needs, this contrast medium contains the vesicle that gas is filled, give patient scan so that identify patient's the operating zone of needs with the magnetic resonance imaging art, and, thereby implement image formation method of the present invention at ultrasonic art of this area applications and mr techniques.Patient's zone is meant whole patient or patient's specific region or part.

After to patient's administration, adopt MRI to observe visible vesicle in magnetic resonance imaging.When the position of determining vesicle according to MRI is a desired zone the patient, then with energy preferably ultrasonic energy be applied to this zone.This energy makes vesicle activation, heating and directly and apace make surrounding tissue coagulation necrosis (the ultrasonic art of surgery).Simultaneously, if desired, also can observe this zone by the magnetic resonance imaging technology.Preferably, being used for the activatory energy of vesicle is high-octane successive ultrasound wave, preferably is higher than 50 milliwatts/cm ², 100 milliwatts/cm more preferably ²Depend on required therapeutic effect, energy can be higher, is up to 10 watts/cm ²The magnetic resonance compatible sonac that most preferably utilizes palm to hold stores energy in this tissue.Sonac is by non-ferrous ion and the preparation of nonferromagnetic material.Be supplied to the cable of sonac to have the Faraday constant guard shield energy and pass the possibility that cable supply pick off causes the breast picture so that reduce by electric energy.

The ultransonic energy that is used for the treatment of and the persistent period of pulse will be depended on therapeutic purposes.Preferably ultrasonic energy is concentrated and selected focus circle so that target hits predetermined vesicle zone.

The supersonic operation that focuses on is with about 2 watts/cm ²Energy carry out.Focus supersonic operation energy can be at least 2 watts/cm ²-Yue 10 watts/cm ²Cause organizing directly and coagulation necrosis fast.Simultaneously to being used to observe the vesicle enforcement MRI in target circle or zone.Strengthen the effect of this operation then at this target circle vesicle with ultrasonic technique.

From about 500 milliwatts/cm ²-Yue 10 watts/cm ², preferably about 1 watt energy range is effective to the disorganization of cavitation.Vesicle has reduced the marginal value of cavitation, so that cavitation occurs with the low-energy marginal value that causes disorganization in the scope of target tissue.

From about 50 milliwatts/cm ²-500 milliwatts/cm ²Energy range in the broken or activation of vesicle takes place.Can broken vesicle by non-cavitation.When vesicle by ultrasonic energy enough fast and consumingly during pulse, the vesicle film is degenerated.When energy and pulse are used with specified energy range, improve appearance with the temperature of vesicle film micro area of broken relevant moment, and this method is not damaged surrounding tissue.This effect of broken vesicle helps being used for the treatment of the location release of agent.Thus, utilize this technology can see that therapeutic agent is discharged into certain zone of health.Moreover, can be used for making shock wave from the energy of vesicle fragmentation, so that also the therapeutic agent that stores is discharged into subjacent tissue.This is specially adapted to gene therapy, wherein can utilize shock wave to open the hole in adjacency cell membrane moment, helps cell to absorb hereditary material.

As nuclear, can utilize about 500 milliwatts/cm with vesicle ²-5 watts/cm ²The energy of scope improves high-energy sound in the transformation that is localized tissue, thereby will organize heating and induce hyperpyrexia.

For the situation of gaseous precursors, when ultrasonic energy concentrated on this precursor, it can cause that precursor changes gaseous state into.The gas cavities that enlarges has been made the zone that susceptibility improves, and is monitored apace in magnetic resonance imaging.For example perflexane is at 56 ℃ to change the temperature of gas into by the liquid of selecting suitable qualification, and selected precursor can be strengthened monitoring effect especially.Therefore the present invention also is used in monitoring Noninvasive temperature in the MRI process.When from gaseous precursors formation vesicle, the material around the gaseous precursors is broken.In addition, therapeutic agent can be discharged into subjacent tissue in the part, and therapeutic agent is by broken in vesicle altogether.The energy that is absorbed by the vesicle interface when vesicle forms increases.This can be used for increasing Hyperthermic heating and broken vesicle.

Contrast medium is specially adapted to provide the imaging of cardiovascular and gastrointestinal regional and allows implements supersonic operation and/or drug release at cardiovascular and gastrointestinal regional, but also can more be widely used for the imaging of vascular system for example or use with the conspicuous alternate manner of those skilled in that art.The term cardiovascular zone of Shi Yonging is meant by heart and guiding heart with from the patient's of the vascular system of heart zone in this article.Term gastrointestinal regional of Shi Yonging or gastrointestinal tract comprise the patient's who is limited by esophagus, stomach, small intestinal and large intestine and rectum zone in this article.The term vascular system of Shi Yonging is meant the blood vessel (tremulous pulse, vein or the like) of organ health or health or part in this article.The patient can be the mammal of any kind, but people most preferably.

Will find to be used as in the synchronous ultrasonic art of magnetic resonance focused Noninvasive the vesicle that the gas of the new stabilisation of contrast medium fills is suitable for using the zone of MRI to use at all.

Will recognize that as those skilled in that art the administration that is used for the vesicle that the present invention's stabilisation gas fills can be in various mode, for example blood vessel is interior, oral, internal rectum or the like mode, with various dosage form administrations.When zone to be scanned was the cardiovascular zone, contrast medium preferably of the present invention was with intravascular administration.When zone to be scanned was gastrointestinal regional, contrast medium preferably of the present invention was with oral or internal rectum form administration.Effectively the ad hoc fashion of dosage and administration depends on patient's age, weight, specific mammal and zone to be scanned and the specific contrast medium of the present invention that will use.Usually, supply with the dosage of reduced levels when initial, bring up to then and obtain required contrast reinforcement.Can utilize the vesicle that the gas of various bonded stabilisations fills so that modify the laxity behavior of medium or the characteristic of Change Example such as viscosity, permeability or palatability (for the situation of the material of oral administration).When implementing synchronous magnetic resonance focused Noninvasive ultrasonic method of the present invention, contrast medium can be used separately or combine administration with other diagnosis, treatment or other medicament.Described other medicament comprises excipient for example flavoring agent or toner.Employed magnetic resonance imaging technology is a routine techniques, can be referring to for example D.M.Kean and M.A.Smith " magnetic resonance imaging technology: principle and application " (William and Wilkins, Baltimore 1986).Described MRI technology includes, but is not limited to nuclear magnetic resonance, NMR (NMR) and electric rotating resonance (ESR).Preferred imaging mode is NMR.

As mentioned above, the application region and the administering mode of the vesicle of gas filling are not limited only to blood volume space, i.e. vascular system.If take in vesicle,, utilize the vesicle of the gas filling of using in the present invention can finish the Noninvasive ultrasonic technique that synchronous magnetic resonance imaging technology focuses on so so that make gastrointestinal tract imaging and fragmentation vesicle wherein by mouth.Another kind of optionally method is, the rectally of the vesicle of filling with the gas of these stabilisations can cause making more rudimentary gastrointestinal tract to comprise the imaging admirably of rectum, descending colon, transverse colon and ascending colon and vermiform appendix, and broken wherein vesicle.Also can obtain the imaging and the ileum imaging imaginably of jejunum by means of the intestinal approach; And broken these regional vesicles.Directly the intraperitoneal administration can be observed peritoneum and fragmentation vesicle wherein.The gas vesicle that also relates to stabilisation directly is administered in the auditive catheter, if so that people can see pipeline and pharyngotympanic tube and have perforation, can see internal ear.The activation and the fragmentation of in ear vesicle also can take place in addition.Also relate to the gas vesicle intranasal administration of stabilisation so that help to observe nasal septum film and nasal sinuses, and broken wherein vesicle.Also can interstice's administration.

Therefore can increase the other administration route of vesicle contrast medium of the present invention and the tissue regions of imaging and broken vesicle, include, but is not limited to 1) intranasal administration, so that make nasal passage and nasal sinuses comprise nose region and hole and the imaging of hole shape gland; 2) intranasal and mouthful interior administration are so that make the remainder of respiratory tract comprise trachea, bronchus, bronchioles and lung imaging; 3) administration in the membranous cochlea, so that make auditory channel and pharyngotympanic tube, tympanum film and outer and inner ear and syrinx imaging; 4) eye drops, so as with the imaging of visual correlation zone; 5) as seen intraperitoneal administration so that make peritoneum; And 6) intravesical is promptly by the bladder administration, so that make the genitourinary tract zone include but not limited to the vascular system of urethra, bladder, ureter, kidney and kidney and farther position imaging by means of this zone, for example, implement the existence of cystography or the anti-stream of confirmation ureter.In addition, also similarly method make brain, spinal column, lung zone and for example and not comprise soft tissue, muscle and the organ imaging of obesity tissue and can utilize ultrasonic technique to finish these regional surgical operations.

Utilize program of the present invention can implement the surgical operation of ultrasonic mediation.The surgical operation of ultrasonic mediation is meant the surgical operation that causes tissue necrosis effectively, and is promptly broken, destroy or the reparation (for example hernia) of for example little damage in the tissue film of the reparation of tissue (hole, rent or scratch); Whole or the alleviating of part diseased tissue (for example tumor); By vesicle activation or the fragmentation of ultrasonic energy with subjacent tissue.Gas and gaseous precursors

Vesicle parcel gas of the present invention and/or gaseous precursors.Term used herein " gas and/or gaseous precursors are filled " is meant that vesicle of the present invention has inner chamber, this inner chamber contains 10% gas and gaseous precursors at least, preferably contain have an appointment 25% gas and gaseous precursors at least, even more preferably contain at least about 50% gas and gaseous precursors, more preferably contain 75% gas and gaseous precursors, most preferably contain gas and gaseous precursors at least about 90%.The existence of gas in use is important, is preferably the vesicle inner chamber and contains 10% gas of having an appointment at least, preferably contains at least and has an appointment 25%, 50%, 75% and most preferably contain 90% gas at least.

Any bio-soluble gas and gaseous precursors may be used to the gentle precursor-filled vesicle of gas of the present invention.Described gas comprises (high degree of polarization) xenon of for example air, nitrogen, carbon dioxide, oxygen, argon, fluorine gas, xenon, neon, helium, rubidium reinforcement, the argon that rubidium is strengthened, the helium that rubidium is strengthened, the neon of rubidium reinforcement or their combination.For example, NMR with ¹⁹F uses together than using liquid or solid can improve more sensitive visibility.In addition, for example various perfluocarbons of various fluorizated chemical compounds, hydrofluorocarbons and sulfur hexafluoride gas can be used to prepare the vesicle that gas is filled.Also can use gas in the disclosed application of Quay WO93/05819, comprise " Q " factor gas (document is incorporated herein by reference) of describing herein.In addition, can use paramagnetic gas and for example ¹⁷The gas of O.Oxygen should be stable, because oxygen dissolves in blood.Adopt the preferably polymeric or crosslinked liposome or the impermeability shell of cyanoacrylate microsphere; Or with perfluocarbon for example perflenapent or perfluorinated butane use and can reach stabilisation.In the used gas, perfluocarbon and sulfur hexafluoride are preferred.Suitable pfc gas for example comprises perfluorinated butane, Freon C318, perfluoromethane, hexafluoroethane, perfluoropropane, perflenapent, perflexane, most preferably is perfluoropropane.The mixture of dissimilar gas also is preferred, for example the gas of pfc gas and other type oxygen or the like for example.Really, the combination that it is believed that gas is particularly suitable for the accumulative Noninvasive ultrasonic technique of synchronous magnetic resonance.

Gaseous precursors also can be a solid form.Based on the activation of solid precursor form, crystals of sodium carbonate produces carbon dioxide.Solid and liquefied gas precursor are specially adapted to Hyperthermic supersound process, can be gaseous state with precursor activation.

No matter be requirement by the vesicle of prepared inflation of stable compound and gaseous precursors, the preferably same gas that uses with suitable high stability.High stability gas is meant that those are selected from have low (limited) dissolubility and diffusible gas in water-bearing media.Gas such as perfluocarbon have lower diffusibility with relative insoluble, therefore the easier bubble form of stablizing in water-bearing media.

The using gases precursor is a kind of optional scheme of the present invention.Found that particularly perfluocarbon is suitable as gaseous precursors.As what the professional recognized, when vesicle used among the present invention was at first formed, known perfluocarbon can be used as gaseous precursors, is liquid state, or can directly be used as gas, was gaseous state, to constitute inflation and gaseous precursors vesicle.Certainly, a kind of like this perfluocarbon is that gas or liquid or solid depend on its liquid/gas phase or solid/gas phase alternating temperature degree, or boiling point.For example, more preferably one of perfluocarbon is a perflenapent, and its liquid/gas phase transition temperature or boiling point are 27 ℃, this means that this chemical compound is a liquid form under common room temperature, but will become gas under the environment of human body, this is because human body temperature is higher than its liquid/gas phase transition temperature or boiling point.Therefore, under normal circumstances, perflenapent is a gaseous precursors.As for additional embodiments, perfluorinated butane and perflexane are arranged, they are the hithermost homologue of perflenapent.The liquid/gas phase transition temperature of perfluorinated butane is that 4 ℃ and perflexane are 57 ℃, make the former become the effective gas precursor, but may be more useful as gas, yet the latter but has to as gaseous precursors usually, only under unique situation, because its high boiling cause.

Another aspect of the present invention is to use fluorizated chemical compound, and special perfluocarbon helps or strengthen the stability of described inflation and gaseous precursors vesicle, and described perfluocarbon is liquid under the serviceability temperature of vesicle of the present invention.Described fluorinated compound comprises various liquid-fluorination chemical compounds, and for example (Wilmington, DE) fluorinated surfactant of Zhi Zaoing is ZONYL by E.I.Du Pont Company ^TM, and Liquid perfluorocarbon.Thisly comprise perfluoro capryl bromine (PFOB), perfluorodecalin, perfluorodo naphthalane, perfluoro octyl iodide, perfluamine, perfluorotributylamine as the useful perfluocarbon of additional stability agent.Usually, the perfluocarbon that length surpasses six carbon atom will not be a gas under normal human's temperature, i.e. on-gaseous, but liquid is liquid state.Yet, use in stable/inflation that these chemical compounds can use in preparation the present invention again and the gaseous precursors vesicle.Best this perfluocarbon is full n-octyl bromide or perflexane, and it at room temperature is liquid.The gas that exists can be as perfluoropropane or nitrogen, or can be derived by gaseous precursors and obtain, and this gaseous precursors can be a perfluocarbon also, as perflenapent.If at that rate, vesicle of the present invention will be by the preparation of perfluocarbon mixture, and the perfluocarbon example that can provide has perfluoropropane (gas) and perflenapent (gaseous precursors) and fluorine n-octyl bromide (liquid).Although do not plan to quote any theory, it is theorized that the liquid-fluorination compound is positioned on the interface between gas and vesicle film surface.Thereby be to form the perfluocarbon stabilized zone on the inner surface of bio-soluble lipoid at the stabilisation chemical compound that is used to form vesicle, this perfluorinate carbon-coating can be used for also preventing that gas from diffusing through the vesicle film.Gaseous precursors within the scope of the invention is a liquid under the temperature of preparation and/or storage, but at least in use or become gas between the operating period.

For example, found the liquid-fluorination chemical compound for example perfluocarbon when combining, can give stability than independent using gases or the unavailable additional degree of gaseous precursors with gas that is generally used for preparing vesicle of the present invention or gaseous precursors.Therefore, within the scope of the present invention can with or gaseous precursors such as pfc gas precursor, as still keeping the perfluocarbon of liquid together to use after perflenapent and the patient's administration, promptly the perfluocarbon that is higher than patient temperature with its liquid to the phase transition temperature of gas together uses, as the perfluoro capryl bromine.

Any biological dissolubility gas or gaseous precursors all can be used for forming stable inflation and gaseous precursors vesicle." bio-soluble " is meant that gas or gaseous precursors importing patient's tissue will can not produce the unacceptable toxicity of any degree when interior, comprise that allergen is replied and morbid state, and be preferably inert.A kind of gas like this or gaseous precursors also should be suitable for preparing the vesicle that can be used as described inflation and gaseous precursors herein.

Use stable chemical compound herein, the gentle precursor-filled vesicle of gas becomes more stable, and the size of adjusting vesicle then is to adapt to the application of specific MRI.For example, the magnetic resonance imaging Technology Need of vascular system is not more than the vesicle of 30 μ diameters, preferably less vesicle, for example be not more than the vesicle of the diameter of about 12 μ, if desired, the big I of the gentle precursor-filled vesicle of gas is regulated by a series of measures, and these measures comprise microemulsified, vortex, extruding, filtration, supersound process, homogenize, repeat to solidify and thaw cycle, extrude by the micropore pressurization of giving sizing, and similar method.

For using in the blood vessel, the average diameter of vesicle is lower than 30 μ usually, and preferably average diameter is lower than 12 μ.Use in the blood vessel that hits for target, for example, be attached to certain tissue such as tumor, the diameter of vesicle can be lower than 1um, in addition be lower than 100nm can observable scope.For enteral is gastrointestinal administration, can use the vesicle of some greatly, for example be up to 1 millimeter size, but average diameter is preferred between 20 μ and 100 μ.

As mentioned above, with respect to their preparation, formation and purposes, this embodiment also can comprise by the activated gaseous precursors of temperature.List in the following Table 1 a series of liquid state to the gaseous state phase transition temperature near political party's body temperature (37 ℃) or lower gaseous precursors with form the size of 10 microns required emulsion droplets of maximum sized microbubble.

Table 1

The physical property of gaseous precursors and formation

The diameter of the emulsion droplet of 10 μ m vesicles ^*

Chemical compound	Molecular weight	Boiling point (℃)	Density	Form the diameter (μ m) of the emulsion droplet of 10 microns microspheres
					Perflenapent	??288.04	???28.5	???1.7326	????2.9
1-fluorine butane	??76.11	???32.5	???6.7789	????1.2
					2-methybutane (isopentane)	??72.15	???27.8	???0.6201	????2.6
2-methyl-1-butene alkene	??70.13	???31.2	???0.6504	????2.5
					The 2-methyl-2-butene	??70.13	???38.6	???0.6623	????2.5
Valylene	??66.10	???34.0	???0.6801	????2.4
					3-methyl isophthalic acid-alkynes	??68.12	???29.5	???0.6660	????2.5
Perfluorocyclobutane	??200.04	???-5.8	???1.48	????2.8
					Ten fluorine butane	??238.04	???-2	???1.517	????3.0
Perfluoroethane	??138.01	???-78.1	???1.607	????2.7

^*Draw certainly: Chemical Rubber Company Handbook ot Chemistry andPhysics, Robert C.Weast and David R.Lide, eds., CRC Press, Inc.BocaRaton, Florida (1989-1990).

Hereinafter listed possible gaseous precursors catalogue, these gaseous precursors can be used for forming the vesicle of prescribed level.Yet this catalogue is also non-limiting, because might use other gaseous precursors to realize this purpose.Phase transfer to gas phase in fact, is directed to various different application, in fact can uses any liquid to prepare gaseous precursors, as long as they can take place so that provide gas constantly in some that use when being in proper temperature.The suitable gas precursor of Shi Yonging is in the present invention: Hexafluoro acetone, isopropyl-acetylene, allene, the tetrafluoro allene, boron trifluoride, iso-butane, 1, the 2-butadiene, 2, the 3-butadiene, 1, the 3-butadiene, 1,2,3-three chloro-2-fluoro-1, the 3-butadiene, the 2-methyl isophthalic acid, the 3-butadiene, hexafluoro-1, the 3-butadiene, diacetylene, 1-fluoro-butane, the 2-methybutane, ten butyl fluorides, 1-butylene, 2-butylene, 2-methyl-1-butene alkene, 3-methyl-1-butene, perfluoro-1-butylene, perfluoro-2-butylene, 4-phenyl-3-butene-2-ketone, valylene, the nitric acid butyl ester, ethyl acetylene, 2-butyne, 2-chloro-1,1,1,4,4,4-hexafluoro butine, 3-methyl isophthalic acid-butine, perfluoro-2-butyne, the 2-bromobutyraldehyde, carbonyl sulfide, butene nitrile, Tetramethylene., methyl cyclobutane, Perfluorocyclobutane, the perfluor cyclobutane, 3-chlorine cyclopentenes, the octafluoro cyclopentenes, cyclopropane, 1, the 2-dimethylcyclopropane, 1, the 1-dimethylcyclopropane, 1, the 2-dimethylcyclopropane, the ethyl cyclopropane, methyl cyclopropane, diacetylene, 3-ethyl-3-methyl two ethylene imines, 1,1,1-trifluoro aziethane, dimethylamine, the hexafluoro dimethylamine, dimethylethyl amine, two (dimethylphosphine) amine, perflexane, 2,3-dimethyl norbornane, the perfluoro dimethyl amine, dimethyl oxygen father-in-law chloride, 1,3-dioxolanes-2-ketone, the 4-methyl isophthalic acid, 1,1, the 2-tetrafluoro is for ethane, 1,1, the 1-HFC-143a, 1,1,2, the 2-tetrafluoroethane, 1,1,2-three chloro-1,2, the 2-HFC-143a, 1, the 1-dichloroethanes, 1,1-two chloro-1,2,2, the 2-tetrafluoroethane, 1, the 2-Difluoroethane, 1-chloro-1,1,2,2, the 2-pentafluoroethane, 2-chloro-1, the 1-Difluoroethane, 1,1-two chloro-2-fluoroethanes, 1-chloro-1,1,2, the 2-sym-tetrachloroethane, 2-chloro-1, the 1-Difluoroethane, ethyl chloride, the chloro pentafluoroethane, dichlorotrifluoroethane, fluoroethane, perfluoroethane, nitro-pentafluoroethane, the nitroso-group pentafluoroethane, perfluoro ethamine, ethyl vinyl ether, 1, the 1-dichloroethanes, 1,1-two chloro-1, the 2-Difluoroethane, 1, the 2-Difluoroethane, methane, three fluoridize mesyl chloride, three fluoridize Fumette, bromination bifluoride nitroso-group methane, Bromofluoromethane, Bromochlorofluoromethane, bromotrifluoromethane, the nitro difluorochloromethane, dinitro-chlorine methane, fluorochloromethane, trifluorochloromethane, difluorochloromethane, difluorodibromomethane, the dioxy dichloromethane, dichlorodifluoromethane, difluoromethane, the iodine difluoromethane, disilane alcohol methane, fluoromethane, iodomethane, CF3I, the trifluoro Nitrocarbol., trifluoronitrosomethane, tetrafluoromethane, fluoro trichloromethane, fluoroform, the 2-methybutane, methyl ether, methyl isopropyl ether, methyl lactate, methyl nitrite, methyl sulfide, methyl vinyl ether, neon, neopentane, nitrogen (N ₂), nitrous oxide, 1,2,3-nonadecane tricarboxylic acids-2-hydroxyl trimethyl ester, 1-nonene-3-alkynes, oxygen (O ₂), 1, the 4-pentadiene, pentane, perflenapent, 4-amino-4-methyl-2 pentanone, the 1-amylene, 2-amylene (suitable), 2-amylene (instead), 3-bromo-1-amylene, perfluorinate-1-amylene, tetrachloro is for phthalic acid, 2,3, the 6-trimethyl-piperidine, propane, 1,1,1,2,2, the 3-hexafluoorpropane, 1, the 2-expoxy propane, 2, the 2-difluoropropane, 2-aminopropane, 2 cbloropropane isopropyl chloride, 1-nitro heptafluoro-propane, 1-nitroso-group seven fluoro-propanes, perfluoropropane, propylene, HFC-236fa, 2,3-two chloro-1,1,1,2,3, the 3-HFC-236fa, the 1-chloropropene, chloropropene (instead), the 2-chloro-propane, the 3-fluoro-propane, propine, 3,3,3-three fluoro propine, 3-fluorobenzene ethylene, sulfur hexafluoride, ten fluoridize two sulfur (S ₂F ₁₀), 2,4 di amino toluene, trifluoro acetonitrile, trifluoromethyl peroxide, trifluoromethyl sulfide, tungsten hexafluoride, vinylacetylene, vinyl ethers, argon.

As pointed out already the front, perfluocarbon was the preferred component as gaseous precursors and additional stability component.This class perfluocarbon composition comprises saturated perfluocarbon, unsaturated perfluocarbon, and ring-type perfluocarbon.Usually preferred saturated perfluocarbon, it has the CnF2n+2 molecular formula, and wherein n is 1 to 12, and is preferred 2 to 10, more preferably 4 to 8, and most preferably 5.The example of suitable saturated perfluocarbon comprises: tetrafluoromethane, perfluoroethane, octafluoropropane, ten fluorine butane, R-4112, perflexane and Fluorinert PF 5070.The ring-type perfluocarbon that also preferably has the CnF2n molecular formula, wherein n is 3 to 8, and is preferred 3 to 6, and comprises as hexafluoro cyclopropane, Perfluorocyclobutane and ten fluorine Pentamethylene..

Using limited dissolved gas to make effect the best of vesicle also is a part of the present invention.Term " limited dissolubility " be meant gas around rely on its dissolubility from vesicle, to diffuse out in the water-bearing media ability.Higher dissolubility in water-bearing media has been given barometric gradient to the gas in the vesicle, makes gas have the trend that diffuses out like this from described vesicle.On the other hand, lower dissolubility can reduce or cut down the gradient between vesicle and the interface in water-bearing media, and making gas diffuse out from vesicle like this will be obstructed.The gas that preferably is wrapped in the vesicle has the dissolubility lower than oxygen, and promptly 1 part of gas is dissolved in 32 parts of water.Referring to Matheson Gas Data Book, 1996Matheson Company Inc..The gas that more preferably is wrapped in the vesicle has than dissolubility in the low water of air; Even the gas in the vesicle comprises the gas that has than dissolubility in the low water of nitrogen in more preferably wrapping up.Stable compound

Use one or more stable compounds to constitute vesicle, and guarantee the follow-up gas or the parcel of gaseous precursors.Even for insoluble relatively, indiffusion gas, as perfluoropropane or sulfur hexafluoride, when be filled with in formation that gas is stopped and the process of the vesicle of gaseous precursors in one or more stable compounds of use, the vesicle goods can be improved.With respect to size, shape and/or other characteristic of vesicle, these stable compounds keep its stability and integrity.

Term used herein " is stablized " or " stable " means that vesicle is degradation-resistant basically in the useful time cycle, the gas of promptly anti-microsphere structure or parcel or gaseous precursors loss.General vesicle of the present invention has the good shelf life, under the home condition during at least about two or three weeks in, usually can keep volume at least about 90% original structure.But preferred is during this period at least one month, more preferably at least two months, even more preferably at least 6 months, further preferred 18 months, and most preferably 3 years.Therefore, the vesicle that is filled with gas and gaseous precursors generally has the gratifying shelf life, sometimes even under unfavorable conditions, as is higher or lower than that those are also like this under resultant empirical temperature and pressure under the home condition.

The stability of the vesicle of Shi Yonging to small part is attributable to the material of the described vesicle of preparation in the present invention, and usually need not use the additional stability additive, although this use is so use that choose wantonly and usually preferred, this class additional stability agent and characteristic thereof hereinafter will give more detailed description.The material that constitutes vesicle of the present invention is preferably bio-soluble lipoid or polymeric material, wherein eugenic especially thing solubility lipoid.In addition, owing to prepared, promptly produce the simplicity of microsphere or foams ability before administration, these vesicles can prepare easily then and there.

Used lipoid and polymer is that biology mixes in preparation vesicle of the present invention." biology mixes " is that they will can not produce the unacceptable toxicity of any degree, comprise that allergen is replied and morbid state when referring to that lipoid or polymer import in the tissue of patient.Preferred above-mentioned lipoid or compound are inert.The biocompatibility lipoid

For the biocompatibility lipid materials, preferred this class usually is known as " amphipathic " lipid materials (being the polarity lipoid) at occurring in nature, they are meant that having lipotropy on the one hand is hydrophobicity, and have arbitrary component that lipophobia is hydrophilic material simultaneously on the other hand.

Hydrophilic group can be that live part or other have the group of affinity to glassware for drinking water.The example that preparation is used for the useful lipoid of stable vesicle of the present invention comprises natural and synthetic phospholipid.They comprise charged phosphate " head " group, and this group is hydrophilic, are connected hydrophobic long carbochain tail end.This structure is allowed phospholipid realization single double-deck (monolithic layer) arrangement, and wherein all water-insoluble hydrocarbon ends are in contact with one another, and reserve multi-charge phosphate header area freedom and polarity aqueous environment and interact.Recognize that easily a series of entad bilayers are possible, promptly few layer and multilamellar, and this arranging also constituted one aspect of the present invention.Form ability that this bilayer arranges and be a feature of lipid materials useful among the present invention.

Lipoid also can be form of single sheet on the other hand, and this monolayer lipoid can be used for forming single monolayer (monolayer) arrangement.On the other hand, the monolayer lipoid also can be used for forming a series of entad monolayers, promptly few layer or multilamellar, and this arrangement also is considered as included within the scope of the invention.

We also find to obtain the importance of stable vesicle of the present invention, and these vesicles can prepare to the liquid crystal state phase transition temperature at the gel state that is lower than as the lipoid of stable compound.This phase transition temperature is meant that the class lipid bilayer is transformed into mesomorphic temperature from gel state.For example referring to (J.Biol.Chem.) 1974 the 249th phase 2512-2521 pages or leaves of people such as Chapman " journal of biological chemistry ".

It is generally acknowledged that gel state is high more to mesomorphic phase transition temperature, good more in the gas impermeability of giving next inflation of fixed temperature and gaseous precursors vesicle.Referring to Derek Marsh, CRC Handbook ofLipid Bilayers (FL 1990 for CRC Press, Boca Raton), the 139th page, the main chain fusing point that has provided saturated diacyl-Sn-glyceryl-3-phosphocholine changes.The gel state of various lipoids to mesomorphic phase transition temperature obviously is conspicuous for those skilled in the art, for example, sees the Liposome Technology that Gregoriadis edits, and Vol.I is listed among the 1-18 (CRC Press, 1984).Listed some representative lipoid and its phase transition temperature in the following table 2:

Table 2

Saturated diacyl-Sn-glyceryl (3)

Phosphocholine main chain phase transition temperature ^*

Carbon atom in the acyl chain	Main phase transition temperature ℃
		?????1，2-(12：0) ?????1，2-(13：0) ?????1，2-(14：0) ?????1，2-(15：0) ?????1，2-(16：0) ?????1，2-(17：0) ?????1，2-(18：0) ?????1，2-(19：0) ?????1，2-(20：0) ?????1，2-(21：0) ?????1，2-(22：0) ?????1，2-(23：0) ?????1，2-(24：0)	???????-1.0 ???????13.7 ???????23.5 ???????34.5 ???????41.4 ???????48.2 ???????55.1 ???????61.8 ???????64.5 ???????71.1 ???????74.0 ???????79.5 ???????80.1

^*Derek?Marsh“CRC?Handbook?of?Lipid?Bilayers”，CRC?Press，Boca?Raton，Florida(1990)，P.139.

Found might improve the stability of the used vesicle of the present invention by mix a small amount of at least electronegative lipoid in the lipoid that is used to form inflation and gaseous precursors vesicle, " a small amount of at least " is meant about 1 to 10 molar percentage of total lipoid amount.Suitable negative electricity lipoid comprises as Phosphatidylserine, phosphatidic acid and fatty acid.This class negative electricity lipoid merges and disruptive trend together by hindering vesicle, and additional stability can be provided, and promptly the negative electricity lipoid helps to constitute an even negative charge layer on the vesicle outer surface, and it will be repelled by similar charged skin on other vesicle.Like this, can prevent vesicle close contact each other, this contact usually causes the film of relevant vesicle or cortex to break and cause touching vesicle being merged into single bigger vesicle.The lasting of certain this merging process will cause vesicle significantly to be degraded.

Be used to constitute the lipid materials of vesicle or other stable compound and also be preferably flexiblely, the flexibility that is filled with vesicle complete before gas and the gas in this application is meant that its shape of structural change is with by the ability of size less than the hole of vesicle.

When selecting suitable preparation the present invention to stablize the lipoid of vesicle, find that various lipoids all are suitable for the formation of vesicle.The material of any suitable liposome preparation known to those skilled in the art or its combination are all particularly useful.Used lipoid can be natural, synthetic or semisynthetic.

The lipoid that can be used for preparing used inflation of the present invention and gaseous precursors vesicle includes but not limited to: lipoid such as fatty acid; the haemolysis lipoidis; phosphatidylcholine with saturated and unsaturated lipoid; it comprises the dioleoyl phospholipid phatidylcholine; dimyristoyl phosphatidyl choline; two (pentadecanoyl) phosphatidylcholine; two lauroyl phospholipid phosphatidylcholines; dipalmitoyl phosphatidyl choline (DPPC); distearoyl phosphatidylcholine (DSPC); PHOSPHATIDYL ETHANOLAMINE such as DOPE and two palmityl PHOSPHATIDYL ETHANOLAMINE (DPPE); Phosphatidylserine; phosphatidyl glycerol; phosphatidylinositols; sphingolipid such as sphingomyelin; glycosyl lipoid such as ganglioside CM1 and CM2; glycolipid; sulfatide; TANGSHEN is through sphingolipid; phosphatidic acid is as two palmityl phosphatidic acid; Palmic acid; stearic acid; arachidonic acid; oleic acid; be loaded with the lipoid of polymer; described polymer such as Polyethylene Glycol are the PEGYization lipoid; chitin; hyaluronic acid or polyvinylpyrrolidone; be loaded with sulfonated list-; two-; few-or the lipoid of polysaccharide; cholesterol; cholesterol sulfuric ester and CH; vitamin E hemisuccinic acid ester; contain lipoid by ether and the banded fatty acid of ester; the polymerization lipoid is a various lipoid known in those skilled in the art; the diacetyl phosphate ester; two cetyl phosphate esters; octadecane amine; cuorin; contain the phospholipid that chain length is the short-chain fatty acid of 6-8 carbon atom; have asymmetric acyl chain synthetic phospholipid (as one be 6 carbon atom acyl chains and another is the synthetic phospholipid of twelve carbon atom acyl chain); ceramide; the nonionic lipoid comprises niosomes such as polyoxyethylene fatty acid ester; the polyoxyethylene aliphatic alcohol; polyoxyethylene aliphatic alcohol ether; polyoxyethylene anhydro sorbitol fat acid esters; poly-hydroxy stearic acid glycol glycerin ester; poly-castor oil acid glycol glycerin ester; the ethoxyquin soyasterol; the ethoxyquin Oleum Ricini; polyoxyethylene; polyoxypropylene polymer and polyoxyethylene fatty acid stearate; sterol aliphatic ester comprises the sulphuric acid cholesterol ester; the butanoic acid cholesterol ester; the isopropylformic acid. cholesterol ester; the cholesteryl palmitate ester; cholesteryl stearate; acetic acid lanosterol ester; the Palmic acid ergosterol ester; and n-butyric acie plant sterol ester; the furamide of sterol comprises the glucosiduronic acid cholesterol ester; glucosiduronic acid lanosterol ester; glucosiduronic acid 7-dehydrocholesterol ester; the glucosiduronic acid ergosterol ester; the gluconic acid cholesterol ester; gluconic acid lanosterol ester and gluconic acid ergosterol ester; the ester that saccharic acid and alcohol form comprises the glucosiduronic acid lauryl; the glucosiduronic acid stearyl; glucosiduronic acid tetradecane ester; the gluconic acid lauryl; gluconic acid tetradecane ester; and gluconic acid octadecane ester; sugar comprises Surfhope SE Cosme C 1216 with the ester that fatty acid forms; the fructose laurate; sucrose palmitate; sucrose stearate; glucuronic acid; gluconic acid; accharic acid; and polyuronic acid; saponarin comprises sarsasapogenin; smilagenin; hederagenin; caryophyllin and digitoxigenin; Dilaurin; trilaurin; dipalmitin; glycerol and glyceride comprise tripalmitin; glycerol distearate; glycerol tristearate; glycerol two myristinates; myristin; long-chain alcohol comprises Decanol; lauryl alcohol; myristyl alcohol; spermol; and octadecanol; 6-(5-cholestene-3 beta-yl oxygen base)-1-sulfo--β-D-galactopyranoside; digalactosyl two glyceride; 6-(5-cholestene-3 beta-yl oxygen base)-6-amino-6-deoxidation-1-sulfo--β-D-galactopyranose; 6-(5-cholestene-3 beta-yl oxygen base) hexyl-6-amino-6-deoxidation-1-sulfo--α-D-mannopyranose glycosides; 12-(((7 '-diethyl amino coumarin-3-yl) carbonyl) methylamino) octadecanoid acid; N-[12-(((7 '-diethyl amino coumarin-3-yl) carbonyl) methylamino) octadecanoyl]-the amino Palmic acid of 2-; 4 '-trimethylammonio butanoic acid cholestene ester; N-succinyl DOPE; 1; 2-two oleoyls-Sn-glycerol; 1; 2-two palmityls-Sn-3-succinyl glycerol; 1,3-two palmityls-2-succinyl glycerol; 1-cetyl-2-palmityl glycerophosphorylethanolamine and palmityl homocysteine and/or their combination.

If desired; can use various cation lipoids such as DOTMA, N-[1-(2; 3-two oily acyloxy) propyl group]-N; N; N-trimethyl ammonium chloride, DOTAP, 1; 2-two oily acyloxy-3-(trimethylammonio) propane and DOTB, 1,2-dioleoyl-3-(4 '-the trimethyl ammonium) bytyry-Sn-glycerol.Generally speaking, the mol ratio of cation lipoid and non-cationic lipoid can be as 1: 1000 in the liposome, 1: 100, preferably between 2: 1 to 1: 10, more preferably between 1: 1 to 1: 2.5, and most preferably be 1: 1 (ratio of the mole of cation lipoid and non-cationic lipoid mole, for example DPPC).When cation lipoid was used to constitute vesicle, various lipoids can be made up of the non-cationic lipoid.This non-cationic lipoid is preferably dipalmitoyl phosphatidyl choline, two palmityl PHOSPHATIDYL ETHANOLAMINE or DOPEs.Replace above-mentioned cation lipoid, have the lipoid of cationic polymer such as polylysin or poly arginine, and alkyl phosphonates, inferior this acid alkyl ester of phosphorus and alkyl phosphite also can be used to constitute vesicle.

Most preferred lipoid is a phospholipid, preferred DPPC, DPPE, DPPA and DSPC, and DPPC most preferably.

In addition, saturated and example unsaturated fatty acid that can be used for making the stable vesicle that uses among the present invention (for being filled with the mixing microvesicle form of gas and gaseous precursors) can comprise the molecule of the straight or branched form that preferably contains 12 to 22 carbon atoms.Also can use by the isoprenoid unit and form and/or prenyl alkyl.Suitable satisfied fatty acid example includes, but is not limited to lauric acid, myristic acid, Palmic acid and stearic acid, the example of spendable unsaturated fatty acid includes, but is not limited to lauroleic acid, physeteric acid, myristoleic acid, palmitoleic acid, petroselinic acid and oleic acid, and the example of available branched chain fatty acid includes, but is not limited to different lauric acid, different myristic acid, different Palmic acid and isostearic acid.Except that saturated and unsaturated group, the mixing vesicle that is filled with gas and gaseous precursors also can be made of the group of different diene of 5 carbon classes and isoprene containing.In addition, partially fluorinated phospholipid can be used as the stable compound of parcel vesicle.The biocompatibility polymer

As the biocompatibility polymer of the stable compound of preparation inflation of the present invention and gaseous precursors vesicle can be natural, semisynthetic (modified natural goods) or synthetic.Term " polymer " herein " expression is by two or more repeated monomers unit, the chemical compound that preferred 10 or more a plurality of repeated monomers unit are formed.Term " semi synthetic polymer (or natural polymer of modification) " is meant in some aspects by the natural polymer of chemical modification herein.The typical natural polymer that is suitable for the present invention's use comprises naturally occurring polysaccharide.This class polysaccharide comprises as araban, levan, fucan, galactan, polygalacturonic acid, glucosan, mannan, xylose (as glucose), Gum levan, fucoidan, carrageenin, galatocarolose, pectic acid, pectase, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, pustulan, chitin, agarose, Keratin, chrondroitin, dermatan, hyaluronic acid, alginic acid, the mountain xanthan, starch and various natural homopolymer or heteropolymer such as those contain one or more following aldoses, ketose, the polymer of acid or amine: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, Erythrulose, ribulose, xylulose, psicose, fructose, sorbose, talose, mannitol, Sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, agedoite, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucamine, aminogalactose, with neuraminic acid and their naturally occurring derivants.Typical semi synthetic polymer comprises carboxymethyl cellulose, hydroxy methocel, hydroxypropyl emthylcellulose, methylcellulose and methoxyl group cellulose.The typical synthetic polymer that is fit to use in the present invention comprise polyethylene kind (as Polyethylene Glycol, polyoxyethylene, and polyethylene terephthalate), polypropylene type (as polypropylene glycol), polyurethane (as polyvinyl alcohol (PVA), polrvinyl chloride and polyvinylpyrrolidone), the polyamide that comprises nylon, polystyrene, polylactic acid, fluorohydrocarbon, perfluorocarbon (as politef) and polymethyl methacrylate, and their derivant.In a single day those skilled in the art have grasped teachings herein, in conjunction with the application and the known data of this specialty, as see the United States Patent (USP) 5,205 of Unger, content described in 290 (this literature content can all be incorporated this paper into for referencial use), the method for preparing this polymer-matrix vesicle just becomes apparent.

Preferably, when being intended for use gastrointestinal tract, employed polymer is the polymer with strong relatively water binding ability.In use, for example at gastrointestinal regional, the polymer with strong water binding ability makes polymer carry a large amount of liquid and passes gastrointestinal tract in conjunction with a large amount of free waters, expands thereby be full of gastrointestinal tract and make it.Being filled with expansible gastrointestinal tract makes this zone have image more clearly.In addition, when the imaging of needs gastrointestinal region, preferably employed polymer is a kind ofly not to be degraded basically and non-absorbent polymer at gastrointestinal region.It is preferred making interior metabolism of gastrointestinal region and absorption be reduced to minimum, so that avoid contrast medium to remove and avoid to produce gas owing to its degraded in gastrointestinal tract from gastrointestinal region.In addition, the dosage that uses for gastrointestinal tract particularly, preferred polymer are that they can displaced air, and make the formation of a large amount of bubbles in this polymer composition be reduced to minimum.

Particularly preferred embodiment of the present invention comprises vesicle, the stabilisation chemical compound that wherein forms the gentle precursor-filled vesicle of stable gas comprises three kinds of compositions: (1) is neutral (for example, nonionic and amphion) lipoid, (2) electronegative lipoid and (3) lipoid that carries hydrophilic polymer.Preferably, the amount of described electronegative lipoid is greater than 1 mole of % of the total lipoid that accounts for existence, and the amount of lipoid of carrying hydrophilic polymer is greater than 1 mole of % of the total lipoid that accounts for existence.Described electronegative lipoid is that a kind of phosphatidic acid also is preferred.Gratifying is that the lipoid that carries hydrophilic polymer is a kind of lipoid that is covalently bound on the described polymer, and described polymer preferably has from 400 to 100,000 mean molecule quantity.Described hydrophilic polymer preferably is selected from following group: Polyethylene Glycol, polypropylene glycol, polyvinyl alcohol and polyvinylpyrrolidone and their copolymer.PEG and other polymer can combine with DPPE or other lipoid by covalent bond, for example amide, carbaminate or amine bonding.Another kind of optionally method can be with ester, ether, and thioesters, sulphamide or disulfide bond (thioesters) use with PEG or other polymer so that this polymer scale is incorporated into for example cholesterol or other phospholipid.If hydrophilic polymer is a Polyethylene Glycol, the lipoid that carries a kind of like this polymer is considered to " PEGization ", it is abbreviated as " PEG " for the existing document of Polyethylene Glycol.The described lipoid that carries hydrophilic polymer is two palmityl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol 5000 preferably, promptly have mutually to carry the two palmityl PHOSPHATIDYL ETHANOLAMINE lipoids (DPPE-PEG5000) that mean molecule quantity is 5000 Polyethylene Glycol on it; Or DSPE-Polyethylene Glycol 5000.

The example of preferred vesicle of the present invention comprises the two palmityl phosphatidic acid (DPPA) that for example have 12.5 moles of % and has the dipalmitoyl phosphatidyl choline (DPPC) of 77.5 moles of % of two palmityl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol 5000 (DPPE/PEG5000) of 10 moles of %.It also is preferred that molar percentage is respectively these compositionss of 82/10/8.Effectively the DPPC component is neutral, because phosphatidyl partly is electronegative, and choline partly is positively charged.Therefore, electronegative DPPA composition is added with enhanced stability, further relate to electronegative lipoid as additive according to the principle of describing.The third composition DPPE, PEG provide the material that is attached to the PEGization on lipid membrane or the vesicle skin by the DPPE composition, wherein free PEG composition is around the film or the skin of vesicle, thereby and to have formed the enzyme and the function that stop various healths be other physical barriers of endogenous dose of degraded exogenous material.The theoretical material of also thinking PEGization can be defeated the effect of the macrophage of human immune system because similar with water-bound, otherwise described macrophage around with the removal allogenic material.The result improves the time that the stabilisation vesicle plays a role during agent as a comparison.Other and auxiliary stable compound

The material composition of use except that biocompatibility lipoid recited above and polymer prepares stable inflation and the gaseous precursors vesicle also is a part of the present invention, and its condition is that the vesicle of so preparation satisfies stability as herein described and other requirement.These compositions can be bases and basic, promptly form the main base material that produces or prepare stable inflation and gaseous precursors vesicle.On the other hand, they can be complementary, and promptly as auxiliary or enriching substance, they can strengthen the effect of basicly stable chemical compound, perhaps can provide some required character except that the characteristic that basicly stable chemical compound provided in addition.

Yet, be difficult to determine given chemical compound be basic chemical compound or adjuvant, this is because the effect of described chemical compound is by stablizing the result that vesicle produces and test definite according to generating.As the example how these fundamental sum auxiliary compounds work, observed and shaken biocompatibility lipoid and water or normal saline and carry out simple in conjunction with usually obtaining turbid solution, autoclaving subsequently.This turbid solution can play contrast medium, but aestheticly is tedious and has and be unstability insoluble or indiffusion lipoid particle form.Therefore, can add propylene glycol and remove muddiness by promoting particulate dissolving of lipoid or diffusion.Propylene glycol also plays a part thickening agent, by increasing the surface tension on vesicle film or top layer, can improve the formation and the Stabilization of vesicle.Might further play a part extra play by propylene glycol, therefore the film or the top layer of this layer parcel vesicle provide additional stability.The example of the basic or auxiliary stable compound of relevant other this class can use conventional surfactants; United States Patent (USP) 4,684,479 and 5,215,680 referring to D ' Arrigo.

Auxiliary and basicly stable chemical compound in addition comprises this class material such as Oleum Arachidis hypogaeae semen, Canola oil, olive oil, safflower oil, Semen Maydis oil or any other is known usually and according to requirement listed in this description and explanation, suitable edible oil as stable compound.

In addition, the chemical compound that is used to prepare the mixed micelle system also is suitable for use as basic or auxiliary stable compound, and these include, but is not limited to: lauryl trimethylammonium bromide (dodecyl-), cetyl trimethylammonium bromide (cetyl-), myristyl trimethylammonium bromide (myristyl-), zephiran (alkyl=C ₁₂, C ₁₄, C ₁₆), benzyl dimethyl dodecyl bromide/ammonium chloride, benzyl dimethyl cetyl bromine/ammonium chloride benzyl dimethyl myristyl bromine/ammonium chloride, cetyl dimethyl ethyl bromine/ammonium chloride or cetyl bromide/pyridinium chloride.

Found can control size, dissolubility and the heat stability of inflation used among the present invention and gaseous precursors vesicle by selecting the additional or auxiliary stabilizer described in different the application.These stabilizing agents are not only by them and the physical action of lipoid coating, and are filled with the viscosity of vesicle of gas and gaseous precursors and these parameters that capillary ability influences vesicle by their improvement.Therefore, the inflation that the present invention is used and the vesicle of gaseous precursors can suitably be improved and further stablized by adding one or more following various compositions: (a) viscosity modifier includes, but is not limited to carbohydrate and phosphorylation thereof and sulfonated derivant; And polyethers, the polyethers of preferred molecular weight between 400 and 100,000, two-and three-hydroxyl alkane and polymer thereof, preferably have molecular weight between 200 and 50,000; (b) emulsifying agent and/or solubilizer also can be used in combination with lipoid, this obtains ideal improvement and advances one Stabilization, this class material includes, but is not limited to arabic gum, cholesterol, diethanolamine, glyceryl monostearate, lanolin alcohol, lecithin, mono-and diglycerides, monoethanolamine, oleic acid, oleyl alcohol, Poloxamer (as Poloxamer188, Poloxamer 184 and Poloxamer 181), stearic acid polyoxyethylene 50, poly-oxyl 35 Oleum Ricini, poly-oxyl 10 oily ethers, poly-oxyl 20 whale stearyl ethers, poly-hydrocarbon oxygen 40 stearates, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propylene-glycol diacetate, propylene glycol monostearate, sodium lauryl sulphate, sodium stearate, anhydro sorbitol list lauryl, Arlacel-80, Arlacel-40, Arlacel-60, stearic acid, triethanolamine and emulsifing wax; (c) can include, but is not limited to arabic gum with suspending agent and/or the viscosity increasing agent that lipoid together uses, agar, alginic acid, aluminum monostearate, bentonite, magma, carbopol preparation 934P, carboxymethyl cellulose, calcium and sodium and sodium 12, carrageenin, cellulose, dextrin, gelatin, guar gum, locust bean gum, aluminium-magnesium silicate, hydroxyethyl-cellulose, hydroxypropyl emthylcellulose, Magnesiumaluminumsilicate, methylcellulose, pectin, poly(ethylene oxide), polyvinyl pyrrolidone, propylene glycol alginate, silicon dioxide, sodium alginate, Tragacanth and xanthan gum, α-d-Fructus Vitis viniferae acid lactone, glycerol, mannitol, (d) the synthetic suspending agent that also can be used is as Polyethylene Glycol (PEG), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polypropylene glycol and Spheron MD 30/70; And (e) also can comprise the tension force improving agent, these reagent comprise (but being not limited to) Sorbitol, propylene glycol and glycerol.Aqueous diluent

As above already as described in, when vesicle was natural quasi-ester, the specific composition of stablizing vesicle was some kind aqueous environment, this environmental induction lipoid (because basic hydrophobic/hydrophilic character) forms vesicle (in this environment available structural stability).The diluent that can be used for producing this aqueous environment includes, but is not limited to water, can be to take off ionic or contain any amount of dissolving salt etc., they will be not can the interference stability microsphere generation and maintenance or influence their application, and conventional saline and normal saline as the MRI contrast medium.Paramagnetic and super paramagnetic contrast medium

In another embodiment of the invention, the contrast medium of vesicle of filling based on stabilisation gas of the present invention further contains other contrast medium, Chang Gui contrast medium for example, they play a part to improve the effectiveness of the contrast medium that is used for the ultrasonic art of the accumulative Noninvasive of synchronous magnetic resonance.Many such contrast medium are that those skilled in that art are known, and comprise paramagnetic and super paramagnetic contrast medium.

The group that the example that is applicable to paramagnetism contrast medium of the present invention comprises stable free (for example, stable nitro oxide), and comprise transition, group of the lanthanides and actinides, if desired, they can be salt form or with complexing agent (comprising its lipophilic derivant), or covalently or non-covalently combine with protein macromolecule.

Preferably transition, group of the lanthanides and actinides comprise Gd (III), Mn (II), Cu (II), Cr (III), Fe (II), Fe (III), Co (II), Er (II), Ni (II), Eu (III) and Dy (III).Preferred described element comprises Gd (III), Mn (II), Cu (II), Fe (II), Fe (III), Eu (III) and Dy (III), especially Mn (H) and Gd (III).

These elements can be salt forms if desired, for example for example manganese gluconate and hydroxyapatite manganese salt of manganese salt such as manganese chloride, manganese carbonate, manganese acetate and manganese acylate; And iron salt for example, for example iron sulfide and ferrous salt ferrous chloride for example.

If desired, these elements can and complexing agent (comprising its lipophilic derivant), or covalently or non-covalently combine with protein macromolecule.Preferred complexing agent comprises for example diethylenetriamine-valeric acid (DTPA), edathamil (EDTA), 1,4,7,10-tetraazacyclododecanand-N, N ', N ', N -tetraacethyl (DOTA), 1,4,7,10-tetraazacyclododecanand N, N ', N "-triacetic acid (DO3A); 3; 6,9-three azepines-12-oxa--3,6; 9-three carboxymethyls alkene-10-carboxyl-13-phenyl-three capric acid glyceric acid (B-19036); hydroxybenzyl vinyl-ethylenediamine-N,N'-diacetic acid (EDDA) (HBED); N, N '-two (pyridoxyl-5-phosphoric acid) ethylene diamine, N, N '-diamidogen (DPDP), 1,4,7-7-triazacyclononane-N, N ', N "-triacetic acid (NOTA); 1; 4; 8,11-tetraazacyclododecane four decane N, N '; N ', N -tetraacethyl (TETA); kryptands (being the macro ring mixture), with desferrioxamine.More preferably, complexing agent is EDTA, DTPA, DOTA, DO3A and kryptands, most preferably DTPA.Preferred lipophilic complex comprises the alkyl derivative of complexing agent EDTA, DOTA etc., for example, and EDTA-DDP, i.e. N, N '-two-(carboxyl-decyl amide methyl-N-2,3-dihydroxypropyl)-1-N, N ' diacetin; EDTA-ODP, i.e. N, N '-two-(carboxyl-octadecyl amide methyl-N-2,3-dihydroxypropyl)-1-N, N ' diacetin; EDTA-LDP, i.e. N, N '-two-(carboxyl-dodecane amide methyl-N-2,3-dihydroxypropyl)-1-N, N ' diacetin or the like; As the U. S. application submitted on May 22nd, 1992 number is those disclosed complexing agent in 887,290, and the document is incorporated herein by reference document.Preferred protein macromolecule comprises albumin, collagen, pR60, many lysine, polyhistidine, gamma globulin and betaglobulin.More preferably, protein macromolecule comprises albumin, pR60, many lysine, polyhistidine.

Suitable complex comprises Mn (II)-DTPA, Mn (II)-EDTA, Mn (II)-DOTA, Mn (II)-DO3A, Mn (II)-kryptand, Gd (III)-DTPA, Gd (III)-DOTA, Gd (III)-DO3A, Gd (III)-kryptands, Cr (III)-EDTA, Cu (II)-EDTA, or ferrum-desferrioxamine, especially Mn (II)-DTPA or Gd-(III)-DTPA.

The paramagnetism chelating agen comprises for example at United States Patent (USP) 5,312, the alkylation chelating agen of the disclosed paramagnetic ion of 617 (being incorporated herein by reference), as at United States Patent (USP) 5,385, the disclosed paramagnetism combined polymerization chelating agen that is used to be attached to the liposome that gas fills and is attached to the surface of the polymer liposome that gas fills in 719, the stabilisation free radical (NSFRs) that is used to be attached to the phospholipid of the liposome that gas fills and is attached to the nitro oxide that is used to make up the liposome that gas fills, with the chelating agen composition that approaches one or more NSFRs most that contains one or more paramagnetic ions (as at United States Patent (USP) 5,407, generalized in 657) the heterozygosis complexing agent, described chelating agen can be used for making up the liposome that paramagnetism gas is filled.These heterozygosis complexing agents have improved lusitropic widely, have therefore improved the sensitivity to vesicle in magnetic resonance widely.

Nitro oxide is the paramagnetism contrast medium, and it improves laxity ratio between T1 and the T2 by means of the unpaired electronics of of nitro molecule.Paramagnetism effect as the given chemical compound of MRI contrast medium is relevant with the quantity of the not sharing electron of paramagnetism nuclear or molecule to small part, particularly sharing electron quantity square relevant not.For example, cadmium has seven unpaired electronics, and the nitro oxide molecule only has a not sharing electron; Therefore cadmium is the MRI contrast medium stronger than nitro oxide usually, still, effectively correlation time, is used to estimate another parameter that contrast medium is renderd a service, and the laxity of nitro oxide is improved.Be in close proximity to proton Larmour frequency when effective correlation time, the laxity ratio can be increased sharply.When the decline ratio is slowed down, for example since the paramagnetism contrast medium be attached on the macrostructure, make descend slack-off, thereby more effectively energy is shifted, promote proton loose of water.But for cadmium, the electronics rotation is fast the laxity time, and will be restricted to the degree that rotational time at a slow speed can improve laxity.But for nitro oxide, the electronics spin correlation time is then better, and is slack-off by the spin correlation time that makes these molecules, can make laxity trend towards improving.The vesicle that gas of the present invention is filled is ideal for arriving slack-off spin correlation time with the purpose that obtains the raising of laxity.Though do not plan to combine with specific theory of operation, for example by its alkyl derivative of preparation, nitro oxide can wrap the periphery of the vesicle of being filled by gas, so that make the best correlation time that obtains.In addition, can see that obtaining contrast medium of the present invention is the magnetic ball, be the geometric configuration of laxity maximum.

If desired, can be prepared into derivant with the nitro oxide alkylation or with it, for example nitro oxide 2,2,5,5-tetramethyl-1-pyrrolidinyl oxygen, free radical and 2,2,6,6-tetramethyl-1-piperidines oxygen, free radical (TMPO).

The example that is applicable to super paramagnetic contrast medium of the present invention comprises the metal-oxide metal sulfide that lives through field regions, ferromagnetic chemical compound, for example pure iron, Magnet oxide (magnetic iron ore), γ-Fe ₂O ₃, Fe ₃O ₄, iron sulfide, magnetic ferrous acid manganese, cobalt ferrite and nickel ferrite based magnetic loaded and the ferritin or for example ferromagnetic and super paramagnetic meterial of other magnetic active substance of filling with magnetic iron ore.

Above for example paramagnetic of Miao Shuing and super paramagnetic contrast medium can be used as the composition in the vesicle or are used to contain the contrast medium of vesicle.They are used as the inner composition of vesicle,, or are incorporated in the chemical compound of the stabilisation that forms blister wall with the vesicle administration with the solution form.

To surpass paramagnetic agent and be used as clathrate, to be adsorbed onto vesicle and to make vesicle stable.For example, the emulsion of various perfluocarbons, for example perflexane or with the blended full chloro-fluorocarbon of erose iron oxides.The millimicro droplet of the hydrophobic crack electric liquid gaseous precursors in the iron oxides is adsorbed onto the surface of solid matter.

For example paramagnetic or super paramagnetic contrast medium can be incorporated into stable compound with the form of alkylation or other derivant if desired, especially the lipoid wall of vesicle.Specifically, by means of many different keys acetoxyl group for example, nitro oxide 2,2,5,5-tetramethyl-1-pyrrolidinyl oxygen, free radical and 2,2,6,6-tetramethyl-1-piperidines oxygen, free radical can form adduct with the position that long-chain fatty acid is not captured by methyl on ring.Described adduct is very suitable for being incorporated in the stable compound, and the characteristic of lipoid especially can form the wall of vesicle of the present invention.

The mixture of same one or more paramagnetic agents and/or super paramagnetic agent can be used for contrast medium.

If desired, above-mentioned paramagnetic agent and super paramagnetic agent also can be respectively applied for common administration.

Be used for for example iron oxides of effective carrier that vesicle that the present invention's gas fills not only can be used as super paramagnetic agent, and demonstration has the effect that can increase contrast medium susceptibility.Super paramagnetic contrast medium comprises metal-oxide, iron oxides but comprise manganese oxide particularly, and as iron oxides, contain various quantity experience the manganese of field regions, cobalt and nickel.These medicaments are millimicro or microparticle, have very high susceptibility and horizontal laxity ratio.Bigger granule is the 100nm diameter for example, has the R2 laxity more much higher than R1 laxity, but smaller particles 10-15nm diameter for example has lower R2 laxity, but has more equilibrated R1 and R2 value.Minimum granule for example diameter is that the iron oxide particle of the monocrystalline of 3-5nm has lower R2 laxity, but may have best balanced R1 and R2 laxity ratio.Also ferritin may be prepared so that wrap by the nuclear of the super paramagnetic iron of a very high laxity ratio.Have been found that in the present invention vesicle that the gas that uses is filled can improve effectiveness and the safety based on the MRI contrast medium of these conventional iron oxides.

Iron oxides is incorporated into simply the stable compound that is used for preparing vesicle.Particularly, iron oxides is incorporated into wall based on the vesicle of lipoid, for example is adsorbed onto the surface of vesicle, or is bound by the inside of vesicle, as what describe in the United States Patent (USP) 5,088,499 of authorizing on February 18th, 1992.

Though do not plan way of act of the present invention is limited to specific theory, it is believed that vesicle improves the effectiveness of super paramagnetic contrast medium by several principles.At first, it is believed that the function of vesicle improves the apparent magnetic concentration of iron oxide particle, the second, it is believed that vesicle improves the apparent spin correlation time of the MRI contrast medium that comprises paramagnetic and super paramagnetic agent.At last, vesicle seems with the new mechanism performance function of the magnetic area that improves apparent contrast medium, and the mode hereinafter and then to describe that it is believed that plays a role.

Vesicle is considered for the elastic ball zone with different suspension media susceptibility, described suspension media is aqueous contrast medium suspension, with the situation for gastrointestinal administration be gastro-intestinal Fluid, for intravascular injection with to be expelled to another body space be blood or other body fluid.When considering ferrite or iron oxide particle, should notice that the effect of these contrast medium comparative depends on granular size, promptly it depends on the particle diameter of iron oxide particle.This phenomenon is very general, and often is called " secular " laxity of hydrone.Describe in the mode of physicsization more, laxity mechanism is the effect size that depends on this molecular complex, the paramagnetic atom of having resided in this complex, or a paramagnetic molecule, or several paramagnetic molecule.A physical interpretation has been described in the SolomonBloembergen equation below, wherein with the relation of the gyromagnet ratio g that perplexed by paramagnetic ion definition paramagnetism to the T1 that rotates 1/2 nuclear and T2 correlation time:

1/T ₁M＝(2/15)S(S+1)γ ²g ²β ²/r ₆[3T _c/(1+ω _l ²T _c ²)+

7T _c/ (1+ ω _s ²T _c ²)]+(2/3) S (S+1) A ²/ h ²[T _e/ (1+ ω _s2T _e ²)] and

1/T ₂M＝(1/15)S(S+1)γ ²g ²β ²/r ⁶[4T _c+3T _c/(1+ω _l ²T _c ²)+

13T _c/ (1+w _s ²T _c ²)]+(1/3) S (S+1) A ²/ h ²[T _e/ (1+ ω _s2T _e ²)] wherein

S=electronics rotational quantum number;

The g-factor of g=electronics;

β=Bohr magneton;

ω _lAnd ω _s(=657w _lThe Larmor angle operation frequency of rotation of)=nuclear and electronics rotation;

The distance of γ=ion-nuclear;

The atomic little coupling constant of A=;

_TcWith _Te=be respectively two utmost points and interactional correlation time scalariform; With

The h=Planck's constant.

Referring to for example Solomon, I.Phys.Rev.99,559 (1955) and Bloembergen, N.J.Chem.Phys.27,572,595 (1957), be incorporated herein by reference.

Usually some bulky grains have bigger effect than more substantial much smaller granule, and this is mainly due to bigger correlation time.If the granule of iron oxides is made bigger, yet they will be deleterious, stop up lung and activate complicated train.In addition, it doesn't matter for particulate total size, and still particularly the particulate diameter of its edge or outer surface is crucial.Magnetized area and susceptibility effect are with respect to being index decreased with particulate surface.In general, for the situation of two utmost points (by the surface) laxity mechanism, be index decreased and show and r ⁶Dependency.According to document, leave the influence that the hydrone of 4 dusts in paramagnetic surface is subjected to and hang down 64 times than the influence that the hydrone that leaves 2 dusts in paramagnetic surface is subjected to.For the comparative effect being realized maximum ideal case is iron oxide particle hollow, flexible, and big as much as possible.Up to now, also possibility is not accomplished this point; In addition, may have realized that these interests up to now.By with the contrast medium bag by the inside of vesicle or outer surface, even one contrast medium for example iron oxides nanoparticles or paramagnetic ion be relatively little structure, the effect of contrast medium is strengthened widely.Wherein, contrast medium works as rendeing a service much bigger ball, determines magnetized effective coverage by the diameter of vesicle in this ball, and is maximum in the surface magnetization zone of vesicle.These contrast medium have elasticity, i.e. compliance.And hard vesicle is loaded into lung or other organ, and causes the electronics toxic reaction, the easier capillary tube that slips over of these elastic vesicles.Preparation method

Being used for the gentle precursor-filled vesicle of stabilisation gas of the present invention can prepare with multiple appropriate method.These are inflated for vesicle, are that the situation that gaseous precursors is filled is described below respectively, are parts of the present invention although have the vesicle of gas and gaseous precursors.Use gas

Preferred scheme be included in be lower than lipoid gel under the temperature of the phase transition temperature of liquid crystal, in the presence of gas, stir and contain stable compound, the aqueous solution of preferred lipoid is to form the gentle precursor-filled vesicle of gas.Term used herein stirs and its flexible form, means to shake aqueous solution and make gas be imported into any motion the aqueous solution from surrounding.Shaking must have enough dynamics to cause vesicle, the particularly formation of the vesicle of stabilisation.Shaking can be vortex, and a side is to opposite side, or moves up and down.Multi-form motion can in conjunction with.And shaking can be by shaking the container of containing the lipoid aqueous solution, or does not shake container itself and carry out by shaking the aqueous solution in the container.

And shaking can be manual or motor-driven.Available mechanical shaker comprises, for example, shaker table such as VWR Scientific (Cerrtos, CA) shaker table, or be found can produce excellent results from Crescent Dental Mfg.Ltd., the Wig-L-Bug that Lyons, Ill. obtain ^Shaking machine.A preferred version of the present invention is the stable vesicle that shaking of some type or vortex are used to be manufactured on the preferred size scope.It is preferred shaking, and preferably this shakes and uses Wig-L-Bug ^Mechanical shaker.According to preferable methods, it is preferred that oscillating motion is used to produce the gentle precursor-filled vesicle of gas.More preferably move and swing with arc.More preferably move with the swing of the arc between about 2 ° to 20 °, further preferably radian is between about 5 ° to about 8 °.Most preferably move and between about 6 ° to about 7 °, swing the most about 6.5 °.What can expect is, rate of oscillation and radian thereof are crucial for amount and the size of determining the gentle precursor-filled vesicle of the gas that forms.A preferred version of the present invention is a swing numerical value, i.e. the numerical value of complete alternation vibration is in the scope of about per minute 1000 and about 20000.The numerical value of preferred swing or vibration will be between 2500 to 8000.Above-mentioned Wig-L-Bug ^Being meant provided pound in per 10 seconds 2000, i.e. 6000 vibrations of per minute.Certainly, vibration number depends on the content of being stirred, and quality is big more, and vibration number is more little.Other means that generation is shaken are included at a high speed or the effect of the gas that distributes under the high pressure.

Should be understood that also preferably, the aqueous solution volume is big more, the also corresponding increase of the total amount of strength.Acutely shake and be restricted to per minute, and be preferred at least about 60 swing movements.The vortex that changes of 60-300 is more preferably at least for per minute.The vortex of per minute 300-1800 is most preferred.Formation based on the gentle precursor-filled vesicle of the gas that shakes can be looked survey.The concentration of lipoid that need be used to form the vesicle level of required stabilisation will change with the type of used lipoid.And can easily measure by normal experiment.For example; in preferred scheme; the method according to this invention be used to form stabilisation vesicle 1; the concentration of 2-two palmityl phosphatidylcholines (DPPC) is the saline solution of about 0.1mg/ml to about 30mg/ml; preferred about 0.5mg/ml is to about 20mg/ml saline solution, and most preferred is that about 1mg/ml is to about 10mg/ml saline solution.The concentration that is used for the DSPC (DSPC) of preferred version is the common salt aqueous solution of about 0.1mg/ml to about 30mg/ml; be more preferably the saline solution of about 0.5mg/ml, the most preferred saline solution that is about 1mg/ml to about 10mg/ml to about 20mg/ml.

Except above-mentioned simply shaking method, more complicated but since this former thereby not too preferable methods also can be employed, for example, liquid crystal shakes the gas instillation, with the vacuum dried gas instillation, described in the U. S. application submitted on June 11st, 1993 number 076250, the document is drawn the reference of making this paper in full.When these class methods are used, will can before gas injects, be equipped with technology by the vesicle of the gentle precursor-filled stabilisation of gas and prepare with the conspicuous any conventional liposome of those skilled in the art.These technology comprise freezes-melts, and such as sonication, chelating dialysis, homogenize, solvent diffuse, microemulsified, spontaneous formation, solvent evaporationization, French pressure born of the same parents technology, detergent dialysis and other method, every kind comprises in every way preparation vesicle in the solution that contains required active ingredient so that treatment, beauty treatment or other medicament are wrapped, net is caught, or lies in the vesicle of polarity lipoid base of generation.Referring to, for example, people such as Madden, Chemistry and Physics of Lipids, 1990,53,37-46, the document is drawn the reference of making this paper in full.

The scope of the vesicle of filling according to the gas of method for preparing is being lower than 1 μ to the size that is higher than 100 μ.In addition, should be noted that, behind extruding and sterilization process, stir or shake the vesicle that step produces the gas filling, seldom or do not have residual anhydrous lipoid phase (Bangham, A.D., Standish, a M.M ， ﹠amp; Watkins, J.C. (1965) J.Mol.Biol.13 238-252) is present in the residue of solution.The vesicle that the gas that produces is filled was stablized when room temperature preservation 1 year or even longer.

If desired, the big I of the vesicle of gas filling is regulated by hole and similar method that microemulsified, vortex, extruding, filtration, soundization, homogenize, repeated freezing and fusion circulation, pressurization push through fixed size.Yet, it would be desirable that generally the state with formation as described in back more uses microsphere of the present invention and foam, and do not do any attempt to revise its size.

The vesicle that gas is filled can be determined size with the straightforward procedure that pushes through filter; The size distribution of the gentle precursor-filled vesicle of the gas that the control of the pore size of filter produces.By placed in-line with two or more, i.e. the filter of superposition, for example 10 μ then are 8 μ, the vesicle that gas is filled has the extremely narrow size distribution that concentrates on about 7-9 μ m.After the filtration, it is stable above 24 hours that the vesicle that the gas of these stabilisations is filled keeps.

By before using when sterile tube is removed suspension, utilize filter membrane can finish the screening and the filtration of size, preferably during use, filter membrane is incorporated in the syringe.The method of the vesicle of screening certain size comprises that utilization contains a socket, the syringe of at least one filter membrane and syringe needle, and adopt the step of squeezing out to finish, the described step of squeezing out comprises from described socket pushes described vesicle, make it socket by being installed in syringe and the filter membrane between the syringe needle, thereby be applied to according to the present invention before, select the vesicle of certain size with the patient who treats of vesicle as the MRI contrast medium.The step of squeezing out comprises also described vesicle is squeezed into described syringe that can wherein filter membrane works in an identical manner, based on entering into the vesicle that syringe screens a certain size.Another kind of optionally method is, fill described syringe with vesicle, described vesicle has otherwise been selected size, in this case the function of filter membrane be guarantee by after the syringe extruding only the vesicle in the vesicle in the required size scope or the required full-size scope be used for administration.

The typical devices that is used to implement the screening of size and filtration step is to be shown in the syringe of accompanying drawing 2 of Application No. 08/401,974 (application on March 9 nineteen ninety-five) and the combination (document is incorporated herein by reference) of filter membrane.

In preferred scheme, stable compound solution or suspension are pushed through filter, and said solution or suspension are heated sterilization before shaking.The vesicle that gas is filled is in case form, and they will be filtered with sizeization as described above.Form gentle precursor-filled vesicle these steps before of gas and can bring advantage, for example, reduce the amount of the stable compound of non-hydrated, thereby the vesicle of the gas filling of obvious more high yield is provided, and the vesicle that is directly used in the aseptic gas filling of patient's administration is provided.For example, the mixer of bottle or syringe and so on can be used stable compound filling, especially the lipoid suspension after filtering, and suspension for example, is sterilized by autoclaving in mixer then.Gas can be injected into the vesicle that forms the gas filling in the lipoid suspension by shaking sterile chamber.Preferably, sterile chamber is equipped with filter so that the vesicle that gas is filled passes through filter before the contact patient.

The first step of this method for optimizing pushes through filter with the especially class lipoprotein solution of stabilisation, by the exsiccant chemical compound of fragmentation and expose the amount that bigger surface area hydration reduces the non-hydrated chemical compound.Preferably, filters has hole size about 0.1 to about 5 μ m, and more preferably, about 0.1 to about 4 μ m, and more more preferably, about 0.1 to about 2 μ m, most preferably, and about 1 μ m.The chemical compound of non-hydrated, especially lipoid show as the amorphous crumb of non-unified size, and are unfavorable.

Second step, sterilization provide can be directly to the compositions of patient's administration of needing the MRI imaging.Preferably sterilize and finish, preferably by at least about 100 ℃ of autoclavings, more preferably by heat sterilization, about 100 ℃ to about 130 ℃ of autoclavings, also more preferably about 110 ℃ to about 130 ℃, more preferably about again 120 ℃ to about 130 ℃, most preferably be about 130 ℃.Preferred heating at least 1 minute approximately more preferably about 1 to about 30 minutes, more preferably about 10 to about 20 minutes again, most preferably is about 15 minutes.

If desired, above generalized first and second the step can reverse the right order, perhaps have only one in two steps to be employed.

When sterilization with not being when the heat-killed method of the temperature that causes the gentle precursor-filled microsphere explosion of gas is carried out, sterilization and then the gas vesicle of filling formation and carry out, and be preferred.For example, gamma-rays can and/or be used before the vesicle formation that gas is filled afterwards.Use gaseous precursors

Except that aforementioned schemes, the also available interior gaseous precursors of vesicle that is contained in the lipoid base, it can be by being activated by the temperature of the patient tissue of administration, light or pH or other character, liquid or solid in the vesicle that is captured in the lipoid base becomes gaseous state mutually, expand to produce the vesicle that is used for stable inflation of the present invention.This technology is described in detail in the number of patent application 08/160232 and 08/159687 that proposed on November 30th, 1993, and they are all drawn the reference of making this paper in full.The technology of the vesicle that the preparation gaseous precursors is filled is similar to the technology that being used to of describing prepares the vesicle that gas fills usually herein, and different is to use the gaseous precursors place of gas.

The method for optimizing of activated gas precursor is a temperature.Activation or transition temperature and similar terms are meant the boiling point of gaseous precursors, and under this temperature, gaseous precursors takes place from the phase transformation of liquid to gas phase.Useful gaseous precursors is that those have at about-100 ℃ of gases to the boiling point of 70 ℃ of scopes.Activation temperature is specific for each gaseous precursors.About 37 ℃, or the activation temperature of human body temperature is preferred for gaseous precursors of the present invention.Like this, the liquefied gas precursor is activated at 37 ℃ and becomes gas.Yet, gaseous precursors can liquid or gas phase be used for method of the present invention.Preparation is used for the method for MRI contrast medium of the present invention can be carried out being lower than the gaseous precursors boiling point, so that liquid is impregnated in the vesicle.In addition, said method can be carried out so that gas is impregnated in the vesicle at the boiling point of gaseous precursors.For the gaseous precursors with low boiling temperature, Liquid precursor can be with being chilled to the emulsifying of cryogenic miniflow attitude agent device.Solvent in the also available liquid medium of boiling point reduces so that form the application precursor with liquid.This method also can temperature be increased and carry out in whole process, and technology is begun with the gaseous precursors of gas, finishes with gas.

Gaseous precursors can be selected to form gas on the spot in destination organization or fluid, promptly enters patient or animal body when it, before the use, between the storage life and during making.The method of the gentle precursor-filled vesicle of the gas of production temperature-activated can be carried out under the temperature that is lower than the gaseous precursors boiling point.In this scheme, gaseous precursors become trapped in phase transformation is not taken place at production period.In fact, the gentle precursor-filled vesicle of gas is with the liquid phase production of gaseous precursors.The activation of phase transformation can be allowed to surpass whenever the carrying out of temperature of the boiling point of precursor in temperature.And in the microdroplet of known liquefied gas precursor during the amount of liquid, it is determined to reach after the gas phase the big I of vesicle.

In addition, gaseous precursors can be used to be created in preceding preformed stable inflated bladder.In this scheme, gaseous precursors is added under the temperature that is lower than the liquid of each gaseous precursors-gas phase alternating temperature degree and is equipped with in container suspension and/or stable.If temperature exceeds, emulsion forms between gaseous precursors and liquid solution, gaseous precursors from liquid to the gaseous state phase transformation.As the result that the gentle body of heating forms, the air in the gas displacement liquid suspension upper space to be forming the gas of captured gas precursor, ambient gas (for example air), or capture the inflation lipoid ball of the turnover air of gas phase gaseous precursors jointly.This phase transformation mixes and stabilisation applicable to the best of MRI contrast medium.For example, gaseous precursors, perfluorinated butane can be trapped in biological codissolved lipoid or other stable compound, surpasses 4 ℃ (boiling point of perfluorinated butane) along with temperature raises, and the perfluorinated butane gas that stable compound captures produces.As additional example, the gaseous precursors perfluorinated butane can be suspended in the water slurry that contains emulsifying agent and stabilization agent such as glycerol or propylene glycol, and with commercial scroll machine vortex.Vortex is that the temperature of liquid begins in enough low gaseous precursors that makes, and continues, and the temperature of sample rises through the gasiform phase transition temperature of liquid omnidirectional of associating.In this generic operation, in the microemulsified process, precursor conversion is a gaseous state.In the presence of suitable stabilization agent, unexpectedly, produce stable inflated bladder.

Correspondingly, gaseous precursors can be selected forming the vesicle of inflation in vivo or to be designed in process of production, or when storing, or certain time before using produce inflated bladder on the spot.

As another program of the present invention, enter in the aqueous emulsion and keep known dimensions by the liquid phase that is pre-formed gaseous precursors, can determine with perfect gas law in case realize the maximum volume that becomes the gaseous state microvesicle mutually.For the vesicle from gaseous precursors manufacturing inflation, gas phase is supposed moment formation, and does not have the gas of the new vesicle that forms to consume owing to being diffused in the liquid (normally natural aqueous).Thereby the volume of known liquid can be predicted the maxsize of inflated bladder from emulsion.

According to the present invention, the emulsion of stable compound such as lipoid, gaseous precursors, the microdroplet that contains fixed size liquid can be prepared, and to reach specific temperature, the boiling point of gaseous precursors, microdroplet will expand into the inflated bladder of determining size.Determine that size represents the upper limit to actual size because be diffused in the solution such as gas, lose gas in atmosphere and the factor of the effect of increase pressure and so on be the indeterminable factor of perfect gas law.

Perfect gas law and when being used to calculate from liquid to the gaseous state phase transformation increase equation of bubble volume as follows:

PV=nRT wherein

P=is the pressure of unit with the atmospheric pressure

V=is with the volume of the unit of being upgraded to

The n=number of moles of gas

T=is the temperature of unit with ° K

R=ideal gas constant=22.4L atmospheric pressure degree ^-1Mole ^-1

By volume, density and the temperature etc. of liquid in the liquid emulsion, the amount of Liquid precursor (for example molal quantity) can be calculated with the volume of collective's one precursor.When being converted into gas, the vesicle of known volume will be expanded to.The volume that calculates will reflect the upper limit of the size of inflated bladder, suppose that instantaneous expansion is vesicle of inflating and the diffusion that can ignore gas in the expansible time.

Like this, for the stabilisation that is in liquid precursor in emulsion, wherein the precursor microdroplet is spheric, and the volume of precursor microdroplet is measured by following equation:

Volume (ball)=4/3 π r ³Wherein

The radius of r=ball

Like this, in case volume is scheduled, and know liquid in temperature required density, the amount of liquid in the microdroplet (gaseous precursors) can be determined.For illustrating further, can adopt following formula to calculate:

V gas=4/3 π (r _Gas) ³

By perfect gas law

PV＝nRT

Substitution

V _Gas=nRT/P _Gas

Or

(A) n=4/3[π r _Gas ³] P/RT

Quantity n=4/3[π r _Gas ³P/RT] ^*MWn

Become again and be liquid volume

(B) V _Liquid=[4/3[π r _Gas ³] P/RT] ^*MWn/D]

The density of D=precursor wherein

Separate the diameter of liquid droplet

(C) diameter/2=[3/4 π [4/3 ^*[π r _Gas ³] P/RT] MWn/D] ^1/3

Be reduced to

Diameter=2[[r _Gas ³] P/RT[MWn/D]] ^1/3

As the other means of preparation as the vesicle of the required size of MRI contrast medium of the present invention, with little volume of stable compound/precursor liquids and half specific warp, can be the spheroid of suitable radius with the size of determining the gaseous precursors microdroplet with the filter of suitable size.

Representational gaseous precursors can be used for forming for example vesicle of 10 μ diameters of given size.Form vesicle in the present embodiment in people's blood flow, therefore typical temperature is 37 ℃ or 310 ° of K.Under 1 atmospheric pressure and utilize equation in (A), the volume of filling the vesicle of 10 μ diameters needs 7.54 * 10 ^-17The gaseous precursors of mole.

The gaseous precursors of the amount that utilization calculates above and have 76.11 molecular weight, 32.5 ℃ boiling point and 0.7789g/ml in the time of 20 ℃ ^-1The 1-fluorine butane of density, further calculating prediction needs 5.74 * 10 for the vesicle of 10 μ ^-15This precursor of gram.Further extrapolation, and by density with equation (B) further prediction to form maximum be that the vesicle of 10 μ need 8.47 * 10 ^-16The Liquid precursor of milliliter.

At last, utilize equation (C), the preparation maximum is that the vesicle that the gaseous precursors of 10 μ is filled need form the drop emulsion that radius is the diameter of 0.0272 μ or corresponding 0.0544 μ.

The emulsion of specific size can easily realize by the filter with suitable size.In addition, as can be seen, must be used to form the size of filter of the gaseous precursors microdroplet of prescribed level, the filter of this specification also is enough to remove any possible germ contamination, thereby, also can be used as sterilising filtration.

This scheme that is used to prepare the Noninvasive ultrasonic contrast medium's who focuses on as magnetic resonance imaging synchronously inflated bladder in the method for the invention can be used to all gas precursor by temperature-activated.In fact, the gaseous precursors of carrying out liquid-gas phase change in the temperature that is lower than 0 ℃ is used in the freezing point permission that reduces solvent system.Solvent system can be selected to be provided for the medium of levitation gas precursor.For example, can be miscible the freezing point that is lower than independent water that shows of 20% propylene glycol in buffered saline.Amount by increasing propylene glycol or add such as muriatic material, freezing point can further reduce.

The selection of appropriate solvent system can be determined by physical method etc.When material, solid or liquid, this paper is called solute, is dissolved in solvent, such as water base buffer, and freezing point is lowered, and its amount depends on solution composition.Like this, as the Wall definition, can express the depression of the freezing point of solvent by establishing an equation down:

Inx _a=In (1-x _b)=Δ H _Molten/ R (1/T ₀-1/T)

Wherein:

x _aThe molar fraction of=solvent

x _bThe molar fraction of=solute

Δ H _MoltenThe melting heat of=solvent

T ₀The normal freezing point of=solvent

Produce the normal freezing point of solvent by solving an equation.If x _bRelatively less than x _a, above-mentioned equation can be write:

x ^b=Δ H _Molten/ R[T-To/ToT] ≈ Δ H _MoltenΔ T/RT ₀ ²

The change Delta T of top assumed temperature is less than T ₂, this equation can further be simplified, and supposes that the concentration (with the molal quantity in every kilogram of solute) of solute can be represented by weight-molality m, then x _b=m/[m+1000/m _a] ≈ mMa/1000 wherein:

The molecular weight of Ma=solvent and

M=is the weight-molality of the solute of unit with per 1000 gram moles.

Substitution mark x _b:

Δ T=[MaRTo ²/ 1000 Δ H _Molten] m

Or Δ T=K _fM, wherein

K _f=MaRTo ²/ 1000 Δ H _Molten

K _fRefer to the mole freezing point and under 1 atmospheric pressure, equal per unit molar concentration 1.86 degree for water.Above-mentioned equation can be used to accurately measure the mole freezing point of the vesicle that is used for gaseous precursors filling of the present invention.

Therefore, above-mentioned equation can be used to estimate depression of the freezing point and measure the debita spissitudo that the solidification temperature of solvent is dropped to the required liquid or solid solute of appropriate value.

The method of the gentle precursor-filled vesicle of the activatory gas of preparation temperature comprises:

(a) vortex is used for the water slurry of the vesicle of gaseous precursors filling of the present invention; The variable factor of the method comprise shake before autoclaving at random, the water slurry of heated air precursor and lipoid optionally, optionally give the vessel port of dress suspension, optionally shake or make spontaneous formation of gaseous precursors vesicle and the precursor-filled vesicle suspension of cold gas, optionally the water slurry of gaseous precursors and lipoid is pushed through the filter of about 0.22 μ, in addition, filtration can be carried out during the vivo medicine-feeding of the vesicle that produces, and uses the filter of about 0.22 μ;

(b) carry out microemulsified, the aqueous suspension of the gentle precursor-filled vesicle of gas wherein of the present invention by before giving patient's administration, stir and heating emulsified to form vesicle; With

(c), and/or stir to form the suspension of gaseous precursors in lipoid by heating, the gentle precursor-filled vesicle of wherein low close spirit body by expand and the conversion container in other vesicle float to the top of solution, and with vessel port to discharge air; With

(d) in arbitrary said method, contain the water slurry of gaseous precursors and stable compound such as the codissolved lipoid of biology with airtight container, said suspension is retained under the phase transition temperature of gaseous precursors, then high temperature sterilize moves on to temperature and is higher than phase transition temperature, optionally shake, or make the spontaneous formation of gaseous precursors vesicle, wherein the pressure in the said container of gaseous precursors expansion increase in airtight container, the vesicle suspension of cooling inflation also can shake then.

Before shaking the gas injection method, lyophilizing can be used for removing from stable compound anhydrates and Organic substance.Drying-gas injection method can be used to remove from vesicle and anhydrate.By pre-captured gas precursor in exsiccant vesicle (promptly dry preceding), warm after, gaseous precursors will expand and fill vesicle.Gaseous precursors also can be used to fill exsiccant vesicle after it carries out the tool sky.Be maintained at as exsiccant vesicle and be lower than its gel state to the temperature of liquid crystal temperature; exsiccant cavity can be filled lentamente with being in gasiform gaseous precursors; for example; perfluorinated butane can be used to fill the drying be made up of two palmityl phosphatidylcholines (DPPC) at 4 ℃ (boiling points of perfluorinated butane) be lower than 4 ℃, between the phase transition temperature of biological codissolved lipoid.In the case, it will be most preferred filling vesicle at about 4 ℃ to about 5 ℃ temperature.

The method for optimizing that is used for the vesicle that the activatory gaseous precursors of preparation temperature fills is included in the gel state that the is lower than lipoid temperature to the liquid crystal state phase transformation, shakes to have such as the mix aqueous solution of stabilisation chemical compound of lipoid of biology in the presence of gaseous precursors.The present invention also expects to prepare the application of the method for the vesicle that gaseous precursors fills, and is included in gaseous precursors and exists down and shake the aqueous solution that comprises stable compound such as biocompatibility lipoid, and isolate the vesicle that the resultant gaseous precursors that is used for the MRI imaging is filled.Vesicle by method for preparing is attributed to the vesicle that shakes the gaseous precursors filling of gaseous precursors injection method preparation by gel state here.

Common ground, the water-filling liposome of the prior art temperature on the phase transition temperature of the lipoid that is used to make them routinely form, because they are more flexible, thereby are liquid crystal state to be used for living things system.Referring to, for example, Szoka and Papahadjopoulos, Proc.Natl.Acad.Sci.1978,75,4194-4198.Relatively, be that gaseous precursors is filled according to the vesicle of preferred version manufacturing described herein, it has bigger elasticity, than aqueous solution compressibility and tortuosity is arranged more because gaseous precursors forms the back at gas.Therefore, the vesicle that gaseous precursors is filled can be used to living things system when the temperature of the phase transition temperature that is lower than lipoid forms, although gel phase more has rigidity.

The gaseous precursors that is provided at temperature-activated by the method for the present invention expectation exists to stir down and contains stable compound, as the aqueous solution of biocompatibility lipoid.Used herein shaking is defined as agitate water solution and makes gaseous precursors be imported into motion the aqueous solution from surrounding.The agitate water solution of any kind and the motion that causes importing gaseous precursors all can be used to shake.Shaking must be the enough strong vesicle that forms suitable quantity with after date the time.Preferably, it is enough strong shaking, and makes vesicle in short period, as 30 minutes, in preferred 20 minutes, more preferably forms in 10 minutes.Shake and can pass through microemulsified, by Micro Fluid, for example, swirl, as vortex, a side is to opposite side, or moves up and down.Be in adding under the situation of liquid gaseous precursors, except that the shaking that proposes above, sonication can be employed.And dissimilar motions can be made up, and shaking can be by shaking the container of dress lipoid aqueous solution, or does not shake container itself and carry out by shaking the aqueous solution in the container.Further, shaking can be manual or motor-driven.The mechanical shaker that can be employed comprises that for example, shaker table is as VWR Scientific (Crritos, CA) shaker table, microfluidization device, Wig-L-Bug ^TM(Crescent Dental Manufacturing, Inc., Lyons, IL), the latter has been found and can have produced good especially result and mechanical paint mixer, and other known machine.Other means that generation is shaken are included at a high speed or the effect of the gaseous precursors of distributing under the high pressure.Also should understand, preferably, for the aqueous solution of larger volume, total strength correspondingly increase.Acutely shake and be restricted to per minute, and be preferred at least about 60 swing movements.An example that acutely shakes, it is preferred changeing vortex with per minute at least 1000.It is most preferred changeing vortex with per minute 1800.

The formation of the vesicle of filling by the gaseous precursors of shaking can detect by the foamy existence at the aqueous solution top.Follow reducing of the aqueous solution volume that produces owing to foamy formation like this.The final volume of preferred foams be lipoid aqueous solution initial volume at least about twice; More preferably foamy final volume be the aqueous solution initial volume at least about three times; More preferably foamy again final volume be the aqueous solution initial volume at least about four times; Most preferably all lipoid aqueous solutions all are converted into foam.

Required shake time bar and can measure by detecting foamy formation.For example 10ml class lipoprotein solution by the about 15-20 of vortex minute, becomes enough thick until the viscosity of the gentle precursor-filled vesicle of gas and no longer is bonded on the sidewall when it is being swirled in the 50ml centrifuge tube.At this moment, foam will cause that the solution that contains the gentle precursor-filled vesicle of gas rises to 30 to 35ml level.

The type of the lipoid that the concentration of stable compound, the especially lipoid of required formation preferred foam level will mix with used stable compound such as biology changes, and in case prepare by content disclosed herein, will be easy to be measured by the professional and technical personnel.For example, in preferred scheme, be used to form according to by 1 of the gentle precursor-filled vesicle of the gas of the method for the present invention's expectation, the concentration of 2-two palmityl phosphatidylcholines (DPPC) is about 20mg/ml about 30mg/ml saline solution extremely.The concentration that is used for the DSPC (DSPC) of preferred version is that about 5mg/ml is to about 10mg/ml saline solution.

Particularly, DPPC is with concentration 20mg/ml to 30mg/ml, and by shaking, it is big four times to produce the independent suspension vol of the gaseous precursors volume ratio of total suspension and capture.DSPC is with concentration 10mg/ml, by shaking, produces fully without any the liquid suspension volume and contains whole foamy cumulative volumes.

Those skilled in the art should understand, as long as prepared by content disclosed herein, as lipoid and other stable compound of raw material, or the vesicle end product, can operate before or after the method for the present invention carrying out.For example, the codissolved lipoid of stable compound such as biology can be undertaken freezing and the fusion circulation by hydration lyophilizing then, or simple hydration.In preferred scheme, before forming the gentle precursor-filled vesicle of gas, lipoid is by hydration, lyophilizing then.

The method of expectation according to the present invention, the existence such as, but not limited to the gas of air also can be provided by surrounding atmosphere.Surrounding atmosphere can be the atmosphere in the hermetic container, or in non-hermetic container, can be external environment condition.In addition, for example, gas can be injected into or alternate manner or adding have in the container of lipoid aqueous solution or in the lipoid aqueous solution self so that the gas of non-air to be provided.Not heavier-than-air gas is added in the airtight container, and heavier-than-air gas is added in the airtight or unsealed container.Therefore, the present invention includes air and/or other common capture with gaseous precursors.

Described in relevant with stable compound in the above paragraph, the method for optimizing of being expected by the present invention carries out to the temperature of liquid crystal state phase transition temperature at the gel state that is lower than used lipoid.To " gel state is to the liquid crystal state phase transition temperature ", the meaning is meant that the class lipid bilayer will be from the temperature of gel state to the liquid crystal state conversion.Referring to, for example, people such as Chapman, J.Biol.Chem.1974,249,2512-2512.

Therefore, the The pro-vesicle of aforementioned stableization can the mode identical with being used for other stabilisation vesicle of the present invention be used, in case activate by being applied to patient tissue, wherein can be used to cause the generation of gas such as factors such as temperature or pH.Preferably this programme wherein gaseous precursors carrying out phase transformation from liquid to gaseous state near said patient's normal body temperature, and by the temperature-activated of said patient tissue to become gas phase mutually.More preferably, patient tissue is the tissue with about 37 ℃ of normal temperatures in this method, and wherein gaseous precursors is in the tissue near 37 ℃ of normal temperatures, and wherein gaseous precursors is being carried out from liquid to gasiform phase transformation near 37 ℃.

All comprise that preparation is used for the such scheme of the gentle precursor-filled vesicle of stabilisation gas of the present invention, can be by autoclaving or sterilising filtration, if these processes transform before the gas implantation step or prior to the gas of temperature sensitive gaseous precursors in the suspension of temperature mediation.In addition, one or more antibacterial and/or antiseptic can be included in the foamy preparation of stabilisation, as sodium benzoate, all quaternary ammonium salts, Hydrazoic acid,sodium salt, methyl paraben, propyl paraben, sorbic acid, ascorbyl palmitate, butylated BHA, butylated hydroxy-methylbenzene, methaform, dehydroactic acid, ethylenediamine, monothioglycerol, Potassium Benzoate, inclined to one side Potassium acid sulfite, potassium sorbate, sodium sulfite, sulfur dioxide and organic mercury salt.The sterilization of this class also can be realized by other conventional means such as radiation, for the stabilisation microsphere being used under the invasive situation as being used in the blood vessel or intraperitoneal will be necessary.The suitable sterile means will be conspicuous for the gentle precursor-filled vesicle of stabilized gas and the professional involved in the present invention of its application.Usually contrast medium is stored with the aqueous suspensions form, but under the situation of exsiccant vesicle or exsiccant lipoid ball, contrast medium can store with the preceding dry powder of getting reorganization in advance ready.

Further confirmed among the example 1-11 shown in the present invention below.But the scope that the embodiment that is provided does not limit the present invention in any way.

Embodiment

Embodiment 1

Transferrin is attached on the glucosan, and it is joined in the iron salt solutions.Survey (Heat System in the hot system that is equipped with pressure chamber, Farmingdale, N.Y.) being dissolved into water and pH by the mixture with ferrous ion and ferric ion in the cabin of the anaerobic environment of sonicated machine is in 1.0 the hydrochloric acid, and the solution of the super paramagnetic iron oxide of preparation.Utilize standard-sized horn under medium/high-power situation, to activate the sonicated machine, when oxygen passes the solution bubble, 0pH is brought up to pH=12 suddenly.The result obtains by magnetic iron ore, Fe ₃O ₄The nanoparticles of the iron oxides of forming.Nanoparticles is washed in normal saline, and utilize the centrifugal results diameter of difference level to be 20nm and littler nanoparticles.The concentration of these nanoparticles with the nanoparticles of 10 mg/ml is suspended in the normal hexane of the dipalmitoyl phosphatidyl choline that contains 10 mg/ml.With the normal hexane evaporation and the nanoparticles lyophilizing of iron oxides of phospholipid of will wrap quilt.Therefore, preparation iron oxides nanoparticles, the described granule glucosan bag quilt that carries transferrin.Be that the perflenapent of super paramagnetic iron oxide and 2 mg/ml of 10 mg/ml and the Pluronic F-68 of 20 mg/ml that contains the dioleoyl phospholipid phatidylcholine of 10 mg/ml mix in the sterilized water of the mannitol that contains 5.5% weight with concentration.With its microfluidization, and caused producing bag by the soliquid of perflenapent of transferrin, described transferrin with magnetite ore particles as representative.With the under a cloud extrauterine female patient of its intravenously administrable (dosage=5 milliliter) to 25 years old age.Because transferrin is attached to placenta tissue, the vesicle of magnetic mark is positioned ectopic pregnancy, is observed by MRI.The uterus placenta tissue is used the high energy continuous wave ultrasound, 2MHz, 2.5 watts/cm, because the acoustic wave energy absorbtivity that vesicle causes improves, ultrasonic energy destroys the placenta tissue in uterus then.This has been avoided use incision program, and for example laparotomy or more invasive operational example be as the mirror of cutting open the belly, because the common percutaneous of ultrasonic therapeutic method under magnetic resonance is instructed is finished, there is no need to carry out surgical operation and enters.

Embodiment 2

The colloidal suspension of preparation perflexane (0.2 mg/ml) and perflenapent (0.2 mg/ml) in the phospholipid of 10 mg/ml of the mannitol that contains 20 mg/ml Pluronic F-68 and 5.5% weight (82 moles of %DPPC, 7 moles of %DPPE-PEG 5000 and 10 moles of %DPPA and 1 mole of %DPPE-PEG 5000 antiplasmin protein antibodies).To the iron oxides nanoparticles that wherein adds 5 mg/ml and as described in the embodiment of front with this material microfluidization.Patient's intravenously administrable of this material being suffered from vascular thrombosis to suspection.With superconduction quantum INFEROMETRY device (SQUID) magnetometer, a kind of magnetic imaging type scans patient body, determines the frontal bone vein of the obvious zone of improving of susceptibility the patient.Confirm the existence of grumeleuse by ultrasonography.Then with 500 milliwatts/cm ²The high-energy ultrasound wave be used for grumeleuse zone in conjunction with vesicle.Under the guidance of SQUID or magnetometer, finish supersound process.SQUID is installed, as the part of pick off on sonac.When supersound process occurs, can detect the variation of susceptibility with magnetometer.The microvesicle capsule causes the absorption of acoustic energy to improve; In ultrasonography or magnetic imaging, see the mutually transformation of perflexane from liquid to gas in emulsion easily, because vesicle expansion during thermal enlargement causes the local cracking of grumeleuse, and alleviates the Noninvasive surgical operation that uses the treatment thrombosis.

Embodiment 3

To on the microvesicle cyst membrane, soak into microvesicle injection capsule that the gas of paramagnetic iron oxide particle the fills nest before the elbow.Vesicle is at first admitted by lymphatic vessel, is accredited as the profile clearly that is loaded with the tumor lymphatic knot in the magnetic resonance of carrying out with superconduction quantum INFEROMETRY device (SQUID).According to evaluation to lymph node, use the sonac that is loaded with the focusing of tumor at evaluation then, under instructing, uses SQUID successive ultrasound wave ordering.Preparation microvesicle capsule is so that resonance and discharge the energy of heat energy form, thereby heating destroys tumor subsequently.Utilize SQUID, carry out magnetic resonance imaging once more, the imaging that is loaded with tumor in lymphsystem no longer can be identified, illustrates that tumor is destroyed, thereby has improved the Noninvasive surgery operating technology.

Embodiment 4

At United States Patent (USP) 5,312, disclosed in 617 by N, N '-two-(carboxyl-decyl amide methyl-N-2, the 3-dihydroxypropyl)-and 1-N, N ' oxalic acid manganese (MN-EDTA-DDP) and Mn-EDTA-ODP, a kind of paramagnetic complex has prepared the microvesicle capsule (document is incorporated herein by reference) that gas is filled, and this vesicle is expelled among the patient who suffers from malignant tumor.The accurate dimension and the site that utilize MRI to identify the tumor in the lymph chain, and carry out the ultrasonic technique processing simultaneously with magnetic resonance imaging with pick off that magnetic resonance imaging mixes, operation is at 1.5 watts/cm ²Carry out with the frequency of 0.75MHz.The existence of bubble causes sedimentary energy to improve, this tissue heating and local hole.Employing is carried out synchronous magnetic resonance imaging and the degree of Noninvasive ground monitoring tissue necrosis and the temperature of tissue on the machine of having equipped echo plane imaging gradient, therefore realized the Noninvasive surgical operation.

Embodiment 5

For the patient who suffers from arteriovenous malformation of brain (AVM), preparation skull baffle plate, surgical exposure cerebral dura mater.The patient is placed MRT-0.5 tesla, interfere the magnetic resonance imaging technological system, (GE Medical System, Milwaukee, WI).Utilize this magnetic resonance system during the surgical operation that synchronous magnetic resonance imaging art is arranged, to enter the patient.Aerosomes with 0.2 ml/kg ^TMBe expelled to the patient, described Aerosomes ^TMLipoid by 2 mg/ml is formed, described lipoid contains 75 moles of %DPPC, the Alabaster Alabama that the mixture of 8 moles of %DPPE-PEG 5000 and 8 moles of %DPPA and 9 moles of % platelet activating factors (PAF), Avanti Polar Lipids, air and perfluorinated butane gas is caught.The purpose of using PAF is from Aerosomes ^TMDischarge PAF afterwards with platelet activation, so that stimulate the thrombosis of AVM.In the magnetic resonance imaging process, equipped the sonac that the high-octane magnetic resonance of imaging and treatment function mixes and be positioned AVM.At intravenous injection Aerosomes ^TMAfterwards, they pass big conduit, and carry out the microcirculation of AVM.Utilize magnetic resonance imaging technology and ultrasonic art to see microvesicle easily.When magnetic resonance imaging, microvesicle is described as the signal blank in the bright blood imaging of the gradient echo that obtains during shifting by AVM, and bubble shows as speculum reflection like the snowstorm in ultrasonic art.Sonac is concentrated on AVM, so that the focus zone of ultrasonic demonstration is equivalent to the focus of blood vessel.(for example up to several watts) implement synchronous magnetic resonance imaging technology and ultrasonic art when the motivation level of ultrasonic art improves.Hindered the observation of many skeletons in ultrasonic art from the radiation in hole, magnetic resonance is the detailed surrounding tissue of display abnormality still.The unusual surgeon who carries out the ultrasonic art of high-energy can control target better and hit, the energy level of shooting and high-energy pick off and avoid damaging crucial cerebral blood vessel structure on every side.This step causes AVM, agglomerative necrotic zone, and the ablation of blood vessel focus thrombosis.When finishing, this step in ultrasonic art, is used for agglomerative necrosis in the tissue, the set of bubble, and the imaging of most skeleton is hindered, but magnetic resonance imaging has shown that whole confined surgical areas and surgical operation are to influence that handle and tissue on every side.

Embodiment 6

There is hemorrhage injured victim to scan with MRI to suspection.Scanning shows that spleen is hemorrhage.With the Aerosomes intravenous injection of using in the foregoing description, different is the thrombin that Aerosomes also catches 1 mg/ml, carries out synchronous magnetic resonance imaging technology and ultrasonography technology simultaneously.When Aerosomes passes the tremulous pulse of spleen, the surgeon is increased to about 1.0 watts/cm with the ultrasonic power of the sonac of magnetic resonance compatible ², and Aerosomes pops and discharges PAF and thrombin.Obtain thrombosis and hemorrhage being stopped.Synchronous magnetic resonance angiography confirms the formation of splenic artery thrombosis.This invasive degree is hanged down to minimum step and is more cheap and cuts less causing of surgical operation than routine and fall ill.

Embodiment 7

Perflenapent vesicle to 100 milliliters of patient's intravenous injections of suffering from breast carcinoma, above-mentioned vesicle is with phospholipid, 82 moles of %DPPC, 8 moles of %DPPE-PEG 2000 and 10 moles of %DPPA (average diameter=about 1 micron) and the 10 moles of alkylating complex (Mn-EDTA-ODP) of the antibody of manganese, the monoclonal antibody of anti-breast cancer (people) CA-15-3, IgG1 (SIGNET LABS of anti-human breast carcinoma of carrying of %, Dedham, MA) bag quilt.Vesicle also contains the alkyl derivative that is attached to the amycin on the vesicle film of 10 moles of %.Give patient scan with MRI after four hours.Identify the lymph node of reinforcement in the oxter, show metastatic disease.The 1MHz ultrasonic probe that magnetic resonance mixes is positioned lymph node, with 200 milliwatts/cm ²High-octane continuous ultrasound ripple be added to this lymph node.With commodity Magnet for example 1.5 teslas (GE SigmaMilwaukee WI), utilizes pulse train fast, Spoiled GRASS for example, TR=30 millisecond and TE=5 millisecond carry out the magnetic resonance imaging art of real number time synchronized with 30 ° racing angle.

When vesicle is controlled between the period of heating, can see on the magnetic resonance imaging art that vesicle is the increase zone of low signal intensity, it is corresponding to the susceptibility zone that is caused by vesicle.When vesicle pops, can see the moment zone of the inferior intensity that more can illustrate.Vesicle disappears after vesicle pops and removes.The amycin prodrug is released and is activated in neoplastic lymph node because vesicle pops.

Embodiment 8

Survey (Heat System in the hot system that is equipped with pressure chamber, Farmingdale, N.Y.) in the cabin of the anaerobic environment of sonicated machine, be dissolved into the hydrochloric acid of water and pH1.0, prepare super paramagnetic iron oxide by mixture with ferrous ion and ferric ion salt.Utilize standard-sized horn under medium/high-power situation, to activate the sonicated machine, when oxygen passes the solution bubble, pH is brought up to pH=12 suddenly.This result obtains by magnetic iron ore, Fe ₃O ₄The nanoparticles of the iron oxides of forming.Nanoparticles is washed in normal saline, and utilize the centrifugal results diameter of difference level to be 20nm and littler nanoparticles.The concentration of these nanoparticles with the nanoparticles of 10 mg/ml is suspended in the normal hexane of the dipalmitoyl phosphatidyl choline that contains 10 mg/ml.With the normal hexane evaporation and the nanoparticles lyophilizing of iron oxides of phospholipid of will wrap quilt.Then with the bag of 10% weight by the milliwatt granule of iron oxides of phospholipid join in the phospholipid of 1 mg/ml, described phospholipid is by 82 moles of % dipalmitoyl phosphatidyl choline (DPPC), 8 moles of % two palmityl PHOSPHATIDYL ETHANOLAMINE-PEG 5000 (DPPE-PEG 5000) and 8 moles of % two palmityl phosphatidic acid (DPPA) and 2 moles of % palmityl cisplatin derivants.The mixture of the iron oxides of phospholipid, phospholipid bag quilt and palmitoylation prodrug is suspended in the phospholipid total concentration of 1 mg/ml in the common saline in the sterile chamber of sealing, in this container all spaces perfluorinated butane gas is arranged.With this material at Wig-L-Bug ^TMIn with 4200rpm jolting 2 hours, cause vesicle to carry the prodrug of phospholipid bag quilt, at its incrustation ferrum liquefied gas nanoparticles.Vesicle (average diameter=about 2 microns, bubble concentration=1 * 10 with 20 milliliters of dosage ⁹/ milliliter) intravenous injection utilizes the magnetic resonance imaging technology to obtain GRASS sequence fast to the patient.The vesicle of seeing the iron oxides labelling in the magnetic resonance imaging technology easily is the magnetization agent that causes time intensity area.Proof relates to accumulation in lymph node and other tissue based on the contrast medium of prodrug vesicle in the zone of the tumor that forms blood vessel.By under magnetic resonance is instructed, using the 1MHz ultrasound wave that vesicle is popped as mentioned above and obtaining the release of topical remedy.

Embodiment 9

With antimyosin antibody, Chicken Muscle, Catalogue No.476123, (CALBIOCHEM, Jolla CA) are attached to two palmityl PHOSPHATIDYL ETHANOLAMINE.The concentration that its 0.1 mg/ml is joined in the mannitol of 5.5% weight in the sterilized water is the phospholipid (DPPC of 90 moles of % and 10 moles of %DPPA) of 0.1 mg/ml perflenapent and 5 mg/ml.Make this mixture produce bubble with oxygen and oxygen-17, (Microfluidics, Newton MA) make its microemulsified 20 rounds with 16000psi, cause carrying the colloid of the perflenapent of emulsifying antibody to utilize the microfluidization meter then.These materials of 10 milliliters are expelled to the patient of incredulity muscle infarction and carry out the magnetic resonance imaging art.Is the zone that susceptibility improves in the proof of GRASS magnetic resonance imaging technology fast in the myocardium zone of infraction.At the successive ultrasonic sensor of myocardium location magnetic resonance compatible, with 0.100 watt/cm ²Ultrasonic energy is handled myocardium.This causes bubble to pop and the oxygen part is discharged into ischemic tissue.

Embodiment 10

According to embodiment 9 described preparation vesicles, different is to use cationic phospholipid DOTMA, N-[1 (2,3-two oleoyl oxygen) propyl group] N, N '-chlorination trimethyl ammonium substitutes DPPA and substitutes DPPC with DPPE.Prepare the colloidal solid of the perflenapent that carries antibody then as mentioned above, in perflenapent, loaded oxygen-17 gas.Then the DNA of the gene of the coding vascular epidermis somatomedin (VEGF) of 10 mg/ml is added and at 4 ℃, low power set were with this suspension rotation 2 minutes.Patient to the incredulity muscle infarction carries out the magnetic resonance imaging technology.About 30 minutes zones after the contrast medium administration in myocardium evaluation low signal intensity.The sonac of magnetic resonance compatible is concentrated on the myocardium zone of ischemic/infraction, utilize the dull and stereotyped imaging technique of echo of gradient echo equipping the resonance gradient, (Advanced NMR, Woburn, MA), 1.5 teslas (GE Sigma System, the Milwaukee that is retrofited, WI)) magnetic resonance system carries out the synchronous magnetic resonance imaging technology of real number time, and the perflenapent microvesicle takes place by " popping ".The gene of this VEGF that causes encoding infraction/ischemic myocardium in local the integration and local gene expression.New blood vessel is replied VEGF and is bred, and the growth of neovascularity improves.This causes more healthy regional myocardium.

Embodiment 11

Shake 82 moles of %DPPC of lipoid by using, the mixture of 7 moles of %DPPE-PEG 5000 and 5 moles of %DPPA and 6 moles of %DOTMA and prepare the cation vesicle from the front space of the gas of the mixture of perfluorinated butane and oxygen-16 preparation.After shaking, as using Wig-L-Bug in front ^TMEmbodiment in disclosed, add the DNA of gene of the coding VEGF of 100 mg/ml, with the slowly rotation 1 minute of this mixture.This causes DNA to absorb the surface of microvesicle capsule.For observing under magnetic resonance, the vesicle of comparatively high amts is more necessary especially than vesicle magnetic labelling, but still detects vesicle the spin-echo of T2 weighting or fast rotational echo or gradient echo pulse sequence.For the diabetics of suffering from lower end points of relating to of peripheral blood vessel, utilize the Flight pulse train of 2D Time to carry out magnetic resonance angiography (MRA), one type magnetic resonance imaging technology.Zai popliteal tremulous pulse shows significant stenosis.To carry the microvesicle capsule intravenously administrable of DNA and, under the guidance of the magnetic resonance imaging technology of skin surface, use high-octane ultrasonic Treatment so that with the stenosis zone of energy focusing Yu popliteal tremulous pulse based on vesicle signal from MRI and ultrasonic art.This causes catching with gene at the partial vesicle in tremulous pulse stenosis site and discharges.The local expression of VEGF has promoted the formation of collateral branch and new conduit, thereby has improved blood flowing to remote leg.

All patents of putting down in writing in this article and describing, its whole references introduced as this paper of patent application and publication.

Except describing herein, according to the description of front the present invention being done various modifications is conspicuous to those skilled in that art.Described modification is all in the scope of appended claim.

Claims (31)

1. magnetic resonance focused method that is used for the ultrasonic surgical art comprises:

Give the operating patient's administration of needs with the contrast medium that is used for the magnetic resonance imaging technology, described contrast medium contains the vesicle that gas is filled,

Use the magnetic resonance imaging technology, utilize described contrast medium to scan described patient, with evaluation patient's the operating zone of needs, and

Give the ultrasonic art of described area applications so that implement described surgical operation.

2. method according to claim 1, the application of wherein said ultrasonic technique is carried out synchronously with second scanning step, thereby with the described patient of magnetic resonance imaging technology to analyze.

3. method according to claim 1 is followed second scanning step after the application of wherein said ultrasonic technique, thereby with the described patient of magnetic resonance imaging technology to analyze.

4. method according to claim 1, wherein said surgical operation are that one of infra column region is finished: blood vessel, cardiovascular, gastrointestinal tract, intranasal pipeline, audition pipeline, ophthalmic zone, intraperitoneal zone, kidney, urethra, Genito-urinary pipeline, brain, spinal column, lung zone and soft tissue.

5. method according to claim 1, wherein said vesicle comprise target and hit medicament.

6. method according to claim 1, the vesicle that wherein said gas is filled further comprises therapeutic agent, described therapeutic agent is released when ultrasonic technique is used.

7. method according to claim 6, wherein said therapeutic agent are selected from following group: oligonucleotide sequence, antisense sequences, antibody and chemotherapeutic agents.

8. method according to claim 1, wherein said ultrasonic art is repaired the hole in described patient's described zone.

9. method according to claim 8, wherein said hole are the vascular systems described patient.

10. method according to claim 1, the vesicle that wherein said gas is filled is an intravenous administration.

11. method according to claim 1, wherein said gas are to be selected from following one group of gas: air, nitrogen, carbon dioxide, oxygen, fluorine, helium, argon, xenon and neon.

12. method according to claim 1, wherein said gas are fluorizated gas.

13. method according to claim 12, wherein said fluorinated gas are to be selected from following one group of gas: the hexaflarate of perfluocarbon and sulfur.

14. method according to claim 13, wherein said pfc gas are selected from following one group of gas: perfluoropropane, perfluorinated butane, Freon C318, perfluoromethane, hexafluoroethane, perflexane and perflenapent.

15. method according to claim 1, wherein said gas is ¹⁷O.

16. method according to claim 1, wherein said contrast medium further comprise paramagnetic agent or super paramagnetic agent.

17. method according to claim 16, wherein said contrast medium is a paramagnetic agent.

18. comprising, method according to claim 17, wherein said paramagnetic agent be selected from following one group paramagnetic ion: transition elements, group of the lanthanides and actinides.

19. method according to claim 18, wherein said paramagnetic ion are to be selected from a following group element: Gd (III), Mn (II), Cu (II), Cr (III), Fe (II), Fe (III), Co (II), Er (II), Ni (II), Eu (III) and Dy (III).

20. method according to claim 19, wherein said paramagnetic ion are Mn (II).

21. method according to claim 17, wherein said paramagnetic agent comprises nitroxide.

22. method according to claim 16, wherein said contrast medium are super paramagnetic agents.

23. method according to claim 22, wherein said super paramagnetic agent comprises metal-oxide or metal sulfide.

24. method according to claim 23, wherein said super paramagnetic agent comprises metal-oxide, and wherein metal is a ferrum.

25. method according to claim 22, wherein said super paramagnetic agent are ferritin, ferrum, Magnet oxide, γ-Fe ₂O ₃, ferrous acid manganese, cobalt ferrite and nickel ferrite based magnetic loaded.

26. a magnetic resonance focused method that is used for operating ultrasonic art comprises:

Give the operating patient's administration of needs with the contrast medium that is used for the magnetic resonance imaging technology, described contrast medium contains the vesicle that gaseous precursors is filled,

Make the mutually transformation of gaseous precursors generation from liquid to gas,

Use the magnetic resonance imaging technology, utilize described contrast medium to scan described patient, with evaluation patient's the operating zone of needs, and

Give the ultrasonic art of described area applications so that implement described surgical operation.

27. method according to claim 26, wherein said gaseous precursors changing mutually and using magnetic resonance imaging from liquid to gas is to take place synchronously.

28. utilize magnetic resonance focused treatment ultrasonic technique to make the method for treatment chemical compound at patient's specific region sustained release, this method comprises:

Give the operating patient's administration of needs with the contrast medium that is used for the magnetic resonance imaging technology, described contrast medium contains vesicle and the treatment chemical compound that gas is filled;

Use the magnetic resonance imaging technology, utilize described contrast medium to scan described patient, determining in the existing of this district's vesicle, and

Give the ultrasonic art of described area applications so that thereby described vesicle is broken at this zone release treatment chemical compound.

29. method according to claim 1, the vesicle that wherein said gas is filled are to use ¹⁹F fills, and described magnetic resonance imaging technology is a nuclear magnetic resonance technique.

30. method according to claim 1, it confirms but the vesicle that gas is filled is interstice's administration.

31. method according to claim 1, wherein said gas are selected from following one group of gas: the xenon that rubidium is strengthened, the argon that rubidium is strengthened, the neon that helium that rubidium is strengthened and rubidium are strengthened.