CN1180304A - Ultrasound methods of magnetic resonance focusing for surgery and therapy - Google Patents

Abstract

A novel method of magnetic resonance focused surgical ultrasound involves administering a magnetic resonance imaging (MRI) contrast medium to a patient, followed by scanning the patient with MRI and then performing surgery using ultrasound. The contrast medium comprises gas-filled vesicles. These methods can also use MRI contrast media containing gaseous precursor-filled vesicles that undergo a phase transition from liquid to gas in vivo after administration. Additionally, the MRI contrast medium may contain a therapeutic compound.

Description

Ultrasound methods of magnetic resonance focusing for surgery and therapy

Pending applications as references

This application is a continuation-in-part of co-pending application serial number 08/401,974, filed March 9, 1995, which in turn is a continuation-in-part of co-pending application serial number 08/212,553, filed March 11, 1994, both Each application is incorporated herein by reference in its entirety, and the priority application content is also claimed.

Copending Application Serial No. 08/076,250, filed June 11, 1993, discloses a therapeutic drug delivery system comprising gas-filled microspheres containing a therapeutic agent, with particular emphasis on the use of ultrasound techniques to monitor and measure The presence of the microspheres in the patient then ruptures the microspheres to release the therapeutic agent in the area of the patient in which the microspheres are present. Said application is a continuation-in-part of U.S. Application Nos. 716,899 and 717,084, filed June 18, 1991, which in turn are continuations of U.S. Application No. 569,828, filed August 20, 1990, which In turn, is the continuation-in-part of US Application No. 455,707, filed December 22, 1989.

Copending Application No. 08/076,250, filed June 11, 1993, having the same prior application source as the pending application No. 08/076,250 just filed above, discloses a method for preparing applicable Method and apparatus for gas-filled microspheres for use as contrast agents or as drug-releasing agents in ultrasound imaging.

Pending application No. 08/307,305 filed September 16, 1994 and concurrently pending application Nos. 08/159,687 and 08/160,232 filed on November 30, 1993 (which in turn were concurrently filed in 1993 Application Serial Nos. 08/076,239 and 08/076,250, filed June 11, 1999) disclose the preparation of gas and gas precursor filled microspheres and multiphase liquid and gas applications for diagnostic and therapeutic applications. Novel therapeutic delivery systems and methods of compositions.

This application claims the filing dates of Application Nos. 08/307,305, 08/159,687, 08/160,232, 08/076,239 and 08/076,250 and their earlier applications as priority dates, and are incorporated herein by reference in their entirety.

Application No. 07/507,125, filed April 10, 1990, also incorporated herein by reference, discloses biological Application of soluble polymers. The polymer/contrast mixture or mixtures may optionally be mixed with one or more biosoluble gases to enhance the bulk of the resulting formulation.

Background of the Invention

field of invention

The present invention relates to the field of magnetic resonance imaging, and more particularly to the use of stabilized gas-filled vesicles as a contrast medium for magnetic resonance imaging (MRI) guided ultrasound procedures.

A variety of imaging techniques have been used to diagnose disease in humans. One of the earliest imaging techniques used was X-rays. With x-rays, the resulting image of the patient's body reflects the varying densities of body structures. To enhance the diagnostic utility of this imaging technique, contrast agents are used to increase the density of the desired tissue relative to surrounding tissue so that the desired tissue is more visible under x-rays. For example for X-ray gastrointestinal studies barium and iodinated contrast media are widely used to visualize the esophagus, stomach, bowel and rectum. Also for X-ray computerized tomography research techniques (i.e. computer-aided tomography or CAT) these contrast agents can be used in order to improve the visibility of the gastrointestinal tract and, for example, to enhance Contrast of adjacent tissues such as blood vessels and lymph nodes. The use of such contrast agents can increase the density of the interior of the esophagus, stomach, intestine, and rectum, and can differentiate the gastrointestinal system from surrounding structures.

Magnetic resonance imaging is a relatively new imaging technique that, unlike X-rays, does not use ionizing radiation. Similar to computer-aided tomography (CAT), MRI produces cross-sectional images of the body, however, MRI has the ability to make any scan plane (ie, axial, vertex, sagittal, or orthogonal) Other advantages of the image. Unfortunately, the full application of MRI as a diagnostic modality of the body has been limited by the need for new or better contrast agents. Without a suitable contrast agent, it is often difficult to distinguish target tissue from adjacent tissue using MRI. The availability of better contrast agents would improve the overall utility of MRI as an imaging tool and greatly improve the diagnostic accuracy of this modality.

MRI uses magnetic fields, radiofrequency energy, and magnetic field gradients in order to image the body. Differences in contrast and signal intensity between tissues primarily reflect the tissue's T1 (longitudinal) and T2 (transverse) bulk values and proton density (effectively free water content). When changing the signal intensity of an area of a patient by using a contrast medium, several possible approaches are available. For example, contrast media can be designed so as to vary T1, T2 or proton density.

Brief Description of the Prior Art

In the past, the main focus has been on paramagnetic contrast media for MRI. Paramagnetic contrast agents contain unpaired electrons with localized small magnetic fields within the main magnetic field to increase the longitudinal (T1) and transverse (T2) loose ratios. Most paramagnetic contrast agents are toxic metal ions in most cases. To reduce toxicity, ligands are often used to complex these metal ions. The resulting paramagnetic metal ion complex has reduced toxicity. Metal oxides, most notably iron oxides, have been tested as contrast agents for MRI. Iron oxides of small particles, eg below 20 nm in diameter, may have paramagnetic loose properties, with the dominant effect being due to the large magnetic susceptibility. Therefore, magnetic particles have a dominant effect on T2 looseness. Nitroxides are another class of MRI contrast agents that are also paramagnetic. They are relatively less bulky and are generally less effective as MRI contrast agents than paramagnetic ions. All of these contrast agents have some toxic effects when used in certain settings, and none is ideal in itself as a perfusion contrast agent.

Existing MRI contrast agents suffer from a number of limitations. For example, positive contrast agents are known to increase image noise due to their inherent peristalsis and motion generated by respiratory or cardiovascular activity. There is a more complex situation with positive contrast agents such as Gd-DTPA, where the signal intensity depends on the concentration of the contrast agent and the coherence of the pulses used. Absorption of contrast agents from the gastrointestinal tract, for example, complicates the interpretation of images, especially in the distal parts of the small intestine, unless sufficiently high concentrations of paramagnetic substances are used (Kornmesser et al., Magn. Reson. Imaging, 6:124( 1988)). In contrast, negative contrast agents are less sensitive to changes in pulse coherence and provide more consistent contrast. However, at high concentrations eg ferrite particles can produce spurious magnetic susceptibility, especially eg in the rectum where absorption of intestinal fluid occurs and concentrates superparamagnetic substances, the evidence is more definitive. Usually negative contrast agents show good contrast to fat, however, positive contrast agents give good contrast to normal tissue on T1-weighted images. Since most pathological tissues have longer T1 and T2 than normal tissues, they appear dark on T1-weighted images and bright on T2-weighted images. This suggests that an ideal contrast agent should appear bright on T1-weighted images and dark on T2-weighted images. Many MRI contrast media that are available for purchase today do not meet this double standard.

Toxicity is another problem with existing contrast agents. Any drug has some toxicity and usually toxicity is dose related. With ferrite, nausea and flatulence and a transient increase in serum iron are often present after oral administration. The paramagnetic contrast agent Gd-DTPA is an organometallic compound in which gadolinium is coupled with diethylenetriaminepentaacetic acid. Without coupling, free gadolinium ions are extremely toxic. In addition, the uniqueness of the GI tract, for example, where the stomach secretes acid and the gut releases base, creates the possibility of decoupling and separating free gadolinium or other paramagnetic agents from the complex, resulting in multiple occurrences during GI tract use. pH changes. Of course, minimizing the dose of paramagnetic agent is important to minimize potential toxicity.

The use of new and/or better contrast agents in magnetic resonance imaging as well as improved imaging techniques is necessary. The present invention is particularly concerned with these important results.

In the studies on MRI contrast agents described above in Application No. 07/507,125, filed April 10, 1990, it has been disclosed how to use gases in combination with polymer compositions and paramagnetic or superparamagnetic agents as MRI contrast agents. agent. It has been demonstrated therein how the gas stabilized by the polymer acts as an effective susceptibility contrast agent to reduce the signal intensity on T2-weighted images; the system is particularly effective as a gastrointestinal MRI contrast medium.

Widder et al. in Published European Patent Application EP-A-0324938 disclose stable microbubble-type ultrasonic imaging agents produced from heat-denatured biocompatible proteins such as albumin, hemoglobin, and collagen.

Mention may also be made of a report given by Moseley et al. at a meeting of the Society for Medical Magnetic Resonance in Napa, California, in 1991, summarized in an abstract entitled "Microbubbles: A New Magnetic Resonance Susceptibility Contrast Agents" (Microbubbles: ANovel MR Susceptibility Contrast Agent). The microbubbles utilized consisted of air coated with a shell of human albumin. However, this document does not disclose such stable gas-filled vesicles according to the present invention.

For intravascular use, however, the inventors have found that gas stabilization with a flexible compound is advantageous for best results. Bubbles can be stabilized with proteins such as albumin, but the resulting bubble shells can be brittle and hard. Protein is not ideal for several reasons. First, the fragile covering limits the ability of the bubbles to expand and decay. As the gas bubbles encounter different pressure zones in the body (for example, moving from the venous system to the arterial system via the circulation of the heart), the fragile shell breaks and the gas will escape. This limits the shelf life of the time the body obtains useful contrast from the bubbled contrast agent. The fragile, broken fragments may also be toxic. Additionally fragile coatings such as air bubbles resulting from the firm nature of protein and stiffness make measuring in vivo pressure very difficult.

Quay Published Application WO93/05819 discloses that gases with high Q numbers are ideal for forming stable gases, but its disclosure is limited to stable gases rather than their stabilization and encapsulation as described in the present invention . In a preferred embodiment described on page 31, sorbitol is used to increase the viscosity, which in turn is said to prolong the lifetime of the microbubbles in solution. This is not an essential requirement of the present invention, the gases involved have a certain Q value or diffusibility factor.

Lanza et al. in published application WO 93/20802 disclose acoustically reflective lamellar liposomes which are multilamellar liposomes with an enlarged aqueous space between bilayers or containing nested Liposomes have a non-centripetal pattern between bilayers, and thus contain inner separated bilayers. The document also describes its use as an ultrasound contrast agent to enhance ultrasound imaging and as a drug to monitor liposomal release when administered to a patient.

D'Arrigo discloses gas-in-liquid emulsions and lipid-coated microbubbles, respectively, in U.S. Patents 4,684,479 and 5,215,680.

According to the present invention, it has been found that stabilized gas-filled vesicles are very effective non-toxic contrast agents for simultaneous magnetic resonance focused non-invasive ultrasound.

Summary of Invention

The present invention relates to a method of magnetic resonance imaging focused ultrasound for surgery and therapy comprising administering to a patient in need of surgery a contrast medium for magnetic resonance imaging comprising a gas-filled Blisters, the patient is scanned with magnetic resonance imaging using the contrast medium to identify areas of the patient requiring surgery, and ultrasound is applied to the area where surgery is to be performed. The application of ultrasound is followed by a second scanning step in which the patient is scanned with magnetic resonance imaging. Magnetic resonance imaging can be applied concurrently with the application of ultrasound. Scanning and surgical ultrasound steps can be repeated until the desired effect is achieved. The gas-filled vesicles contain therapeutic agents that are released to specific areas of the patient upon ultrasound.

In addition, the present invention includes a method of controlling the release of a therapeutic agent to a region of a patient using magnetic resonance imaging-focused therapeutic ultrasound. The method includes administering to a patient gas-filled vesicles containing a therapeutic compound; monitoring the vesicles using magnetic resonance imaging to determine the presence of vesicles in the area; and disrupting the vesicles using ultrasound to release the therapeutic agent in the area. agent.

The invention also relates to a method of ultrasound for surgery using magnetic resonance imaging focusing, comprising administering to a patient in need of surgery a contrast medium for magnetic resonance imaging comprising a gas precursor-filled vesicle such that The gaseous precursor undergoes a phase transition from liquid to gas, the patient is scanned with magnetic resonance imaging to identify the area of the patient requiring surgery, and surgical ultrasound is applied to the area. The phase transition step and the magnetic resonance scanning step can be performed simultaneously.

Contrast media consist of stabilized gas-filled vesicles, where the gas is a biosoluble gas such as nitrogen or perfluoropropane, but it can also be derived from gas precursors such as perfluorooctyl bromide, as well as by stabilizing compounds For example, biosoluble lipids or polymers form to stabilize the vesicles. The present invention utilizes gas precursors to implement gas formation of gas-filled vesicles, often with considerable advantages. These gas precursors can be activated by many factors, preferably temperature. The gas precursor is a compound that undergoes a phase change from a liquid or solid to a gas at a selected activation or phase transition temperature. Thus, activation occurs by increasing the temperature of the compound from below the activation or phase transition temperature to above the activation or phase transition temperature. When lipids are used to form vesicles, the lipids can be in the form of unilayers or bilayers, and a series of centripetal unilayers or bilayers can be formed with unilayers or bilayers of lipids. Thus, lipids can be used to construct unilamellar liposomes (consisting of a single or bilayer of lipids), oligolamellar liposomes (consisting of two or three monolayers or bilayers of lipids) or multilamellar lipids Body (consisting of more than three layers of lipid monolayer or bilayer). Preferably, the biocompatible lipids include phospholipids. Optionally, the contrast medium comprises paramagnetic and/or superparamagnetic contrast agents, preferably coated by vesicles. Optionally, the contrast medium also further comprises a liquid fluorocarbon, such as a perfluorocarbon, to further stabilize the vesicles. Preferably, the liquid fluorocarbon is vesicle coated.

These and other aspects of the invention will become more apparent from the following detailed description.

                    Invention Details

The present invention relates to magnetic resonance imaging-focused ultrasound methods for surgery and therapy comprising administering to a patient in need of surgery a contrast medium for magnetic resonance imaging comprising a gas-filled bleb The patient is scanned with magnetic resonance imaging using the contrast medium to identify areas of the patient requiring surgery, and ultrasound is applied to the area where surgery is to be performed. Magnetic resonance imaging may be applied concurrently with the ultrasound procedure. The application of ultrasound is followed by a second scanning step in which the patient is scanned with magnetic resonance imaging. The gas-filled vesicles contain therapeutic agents that are released to specific areas of the patient upon ultrasound.

Scanning and surgical ultrasound steps can be repeated until the desired effect is achieved. According to the present invention, simultaneously means that ultrasound and magnetic resonance scanning can exist simultaneously or be used simultaneously; sequentially or continuously; so that the ultrasound can observe the fragmentation of vesicles and tissues. Thus, ultrasound and magnetic resonance can be performed simultaneously, or one can follow the other. The use of magnetic resonance imaging in conjunction with ultrasound can improve the precision of imaging therapies now available. The precision with which magnetic resonance imaging is used in conjunction with ultrasound confirms the location of the vesicles, as magnetic resonance imaging can be used to scan the entire body, providing a large area of view, and once localized, ultrasound can be used in a given area of the body break up the vesicles.

The present invention also relates to a method of focused surgical ultrasound using magnetic resonance imaging comprising administering to a patient in need of surgery a contrast medium for magnetic resonance imaging comprising a gas precursor-filled vesicle such that the gas precursor A phase transition from liquid to gas occurs, the patient is scanned with magnetic resonance imaging and the contrast medium to identify the area of the patient requiring surgery, and surgical ultrasound is applied to that area.

In addition, the present invention includes a method of controlling the release of a therapeutic agent to a region of a patient using magnetic resonance focused therapeutic ultrasound. The method comprises administering to a patient a contrast medium of gas-filled vesicles containing a therapeutic compound; monitoring the vesicles using magnetic resonance imaging to determine the presence of vesicles in the area; Release the therapeutic agent.

As disclosed above and throughout the specification, the following terms shall have the following meanings unless otherwise stated.

"Magnetic Resonance Imaging" (MRI) uses a static main magnetic field; pulsed radiofrequency energy and pulsed magnetic gradients to prepare images, ie, to visualize vesicles. Radio frequency and electric gradients can be used to cause local energy storage and activate vesicles, but ultrasound is the preferred energy for vesicle activation. In practicing the magnetic resonance imaging method of the present invention, the contrast medium may be used alone or in combination with other diagnostic, therapeutic or other agents. Such other agents include excipients such as flavor and color enhancing substances. Magnetic resonance ultrasonography as applied is conventional and described, for example, in D.M. Kean and M.A. Smith "Magnetic Resonance Imaging: Principles and Applications" (William and Wilkins, Baltimore 1986). The MRI techniques include, but are not limited to, nuclear magnetic resonance (NMR) and electron spin resonance (ESR), and magnetic resonance angioblastography (MRA). The preferred imaging modality is NMR. Of course, in addition to MRI, magnetic imaging can also be used to detect vesicles within the scope of the present invention. Magnetic imaging uses magnetic fields and does not require the use of pulsed gradients or radiofrequency energy. Magnetic imaging can be used to detect magnetic vesicles, such as but not limited to ferromagnetic vesicles. Magnetic imaging is performed using the magnetometer Superconducting Quantum Underlying Measurement Device (SQUID). SQUID quickly screens all body tissue for magnetic particles; ultrasound is then localized to those areas. For this application, magnetic resonance imaging includes magnetic imaging, but it should be understood that magnetic imaging is the image of magnetic vesicles and does not include nuclear resonance thereof.

"Sonography" is performed on the desired tissue and utilizes the energy of ultrasound to activate or disrupt the vesicle once it reaches its intended tissue destination. Focused or referred ultrasound refers to the application of ultrasound energy to specific areas of the body so that the ultrasound energy is concentrated within a selected area or target circle. Additionally, focusing refers to magnetic resonance, which guides ultrasound by making the vesicles and target circle visible; thereby simultaneously viewing and disrupting the tissue with ultrasound. Non-invasive means breaking or disabling internal tissue without cutting the skin. Ultrasound as defined in the present invention refers to surgical operations leading to tissue necrosis, i.e. fragmentation and destruction of tissue; repair of holes, cavities, ruptures or cuts in tissue (such as hernias), diseased tissue in whole or in part (such as tumors) ) relief; vesicle activation or disruption of adjacent tissue by ultrasonic energy. Ultrasound is a diagnostic imaging technique that differs from nuclear medicine and X-rays because it does not expose the patient to the toxic effects of ionizing radiation. Furthermore, unlike magnetic resonance imaging techniques, ultrasound is relatively inexpensive and can be used as a mobile-friendly test. With ultrasound technology, sound is transmitted to the patient or animal by means of a transducer. As sound waves travel through the body, they encounter the interface of tissues and body fluids. Based on the acoustic properties of tissues and fluids in the body, ultrasonic waves are partially or fully reflected or absorbed. When the sound wave is reflected by the interface, it is detected by the receiver in the sensor and processed to form an image. The acoustic properties of tissues and fluids in the body determine the appearance of contrast in the resulting images. As an alternative to the use of ultrasound to visualize the vesicles, magnetic resonance imaging can be used to activate the vesicles. Additionally, the intense intensity of the ultrasonic energy can lead to fragmentation and activation of the vesicles. Activation of the vesicles in turn fragments adjacent tissue to cause necrosis of that tissue.

Any type of diagnostic ultrasound imaging device may be used in the practice of the present invention, and the particular type or model of device is not critical to the methods of the present invention. Also suitable are devices designed for ultrasonic detection of hyperthermia as described in US Patent Nos. 4,620,546, 4,658,828 and 4,586,512, the entire disclosures of which are incorporated herein by reference. Preferably, the device has a resonance frequency (RF) spectrum analyzer. Sensor probes can be used externally or be implanted. Sonication is usually initiated at a lower intensity, and preferably continued at the peak resonance frequency, and then increased in intensity, time and/or resonance frequency until the microspheres are broken.

"Vesicle" refers to a spherical entity characterized by the presence of an internal void. Preferred vesicles are formulated with lipids, including various lipids described herein. For a given vesicle, the lipid can be in the form of a unilayer or a bilayer, and one or more unilayers or bilayers can be formed from unilayer or bilayer lipids. For more than one single or double layer, the single or double layer is usually centripetal. Vesicles described herein include entities commonly referred to as liposomes, micelles, bubbles, microbubbles, aerogels, caged vesicles, and the like. Thus, lipids can be used to construct unilamellar vesicles (consisting of a single or bilayer lipid), oligolamellar vesicles (consisting of two or three monolayer or bilayer of lipids with more than three layers). As desired, the internal void of the vesicle can be filled with a liquid, including, for example, an aqueous liquid, a gas, a gas precursor, and/or a solid or solute substance, including, for example, a targeting ligand and/or a bioactivator.

"Liposome" generally refers to bundles or assemblies of amphiphilic compounds, including lipid compounds, usually in the form of one or more centripetal layers. Most preferred gas-filled liposomes are structured as a simple lipid layer (ie, monolayer) or as a single monolayer of lipid. Liposomes can be prepared from a variety of lipids, including phospholipids and nonionic surfactants (eg, niosomes). Most preferred lipids, including gas-filled liposomes, are in the gel state at physiological temperature. Liposomes can be cross-linked or polymerized and carry polymers such as polyethylene glycol on their surface. A targeting ligand for epidermal cells was bound to the surface of gas-filled liposomes. A targeting ligand is a substance that binds to a vesicle and directs the vesicle to a particular cell type such as but not limited to epidermal tissue and/or cells. Targeting ligands can be bound to vesicles either covalently or non-covalently. Liposomes are also referred to herein as lipid vesicles. Most preferred liposomes are substantially free of water in their interior.

"Microcell" refers to the colloid formed by a lipid compound when the concentration of the lipid compound lauryl sulfate is above the critical temperature. Since many of the compounds that form micelles also have surfactant properties (ie, the ability to lower surface tension and have hydrophilic and lipophilic regions that prefer water and fat), these substances can also be used to stabilize the bubbles. Typically, these cellular materials prefer to adopt a monolayer or hexagonal H2 phase configuration, and also adopt a bilayer configuration. When forming gas-filled vesicles with cellular material, generally the compound adopts a radial configuration with an aliphatic (fat-loving) component facing the vesicle and a hydrophilic region facing away from the vesicle surface. To target epidermal cells, the targeting ligand is bound to the microcellular compound or to an amphiphilic substance mixed with the microcellular compound. Alternatively, targeting ligands can be adsorbed to the surface of the vesicle-stabilizing micelle material.

"Hydrogel" refers to a structure similar to a microsphere, except that usually the internal structure of a hydrogel consists of multiple small voids instead of a single void. In addition, preferred hydrogels are composed of synthetic substances (such as foams prepared from baked resorcinol and formaldehyde), but natural substances such as polysaccharides or proteins can also be used to prepare hydrogels. Targeting ligands can be adsorbed to the surface of the hydrogel.

"Cathrate" generally refers to a solid material that is associated with a host vesicle rather than covering the surface of the vesicle. Solid, semiporous, or porous clathrates can be used as agents to stabilize vesicles, but clathrates themselves cannot cover the entire surface of a vesicle. Rather, the clathrates form structures known as "cages" with spaces to hold the vesicles to which the clathrates can adsorb one or more vesicles. Similar to microspheres, one or more surfactants can be incorporated into clathrates and these surfactants will help stabilize the vesicles. Typically the surfactant coats the vesicles and helps maintain the association of the vesicles with the clathrates. Useful clathrate materials for stabilizing vesicles include porous apatites such as calcium hydroxyapatite and precipitates of aggregates and metal ions such as alginic acid and calcium salts. Targeting ligands for epidermal cells can be incorporated into the clathrate itself or into a surfactant substance bound to the clathrate.

Without being bound by any particular theory of operation, it is believed that the present invention relies, at least in part, on the fact that gaseous, liquid and solid phases have different magnetic susceptibilities. At the interface of gas and water, eg the magnetic domain is altered, which results in a phase shift of eg the spins of the hydrogen nuclei. When imaged it can be seen as a decrease in signal intensity adjacent to the gas/water interface. This effect is more pronounced on T2-weighted images and is most evident in gradient echo pulse coherence. This effect can be enhanced by the pulse coherence of the extended readout with a narrow bandwidth. The longer the echo time on the continuity of the gradient echo pulse, the greater the effect (ie, the greater the degree and magnitude of the signal loss).

It is believed that the stabilized gas-filled vesicles used in the present invention rely on the difference in magnetic susceptibility of the phases, as well as other properties described herein, and provide efficient levels of magnetic resonance imaging contrast media as well as making the vesicles of the contrast media Effective fragmentation of capsules. Vesicles are formed from a matrix of stabilized compounds that establish gas-filled vesicles and then retain their size and shape for the desired time of use in magnetic resonance imaging techniques. The compound also allows disruption of vesicles at a certain energy level, preferably said energy is ultrasonic energy. Most typical of these stabilizing compounds are those having hydrophobic/hydrophilic properties such that they form monolayers or bilayers etc., as well as vesicles in the presence of water. Thus, water, saline or other water-based medium, often referred to herein as a diluent, is the stabilized gas-filled vesicle contrast medium of the present invention.

In fact, the stabilizing compound may be a mixture of compounds that impart various desired properties to the stabilized vesicle. For example, it has been found to be advantageous to have compounds that aid in the dissolution and dispersion of the basic stabilizing compounds. Other elements of stabilized vesicles are gases, which are gases when the vesicles are prepared, or which may be gas precursors, which are responsive to activating factors such as temperature and which transition from a liquid or solid phase to a gaseous phase. Various aspects of the stabilized gas-filled contrast media used in the present invention will now be described. Instructions

According to the present invention, a magnetic resonance focused synchronized non-invasive sonographic method is provided. by administering to a patient in need of surgery a contrast medium for magnetic resonance imaging, the contrast medium comprising a gas-filled vesicle, scanning the patient with magnetic resonance imaging to identify areas of the patient requiring surgery, and Ultrasound and magnetic resonance techniques are used in this area to implement the imaging method of the present invention. A region of a patient refers to an entire patient or a specific region or portion of a patient.

After administration to the patient, MRI was used to observe the vesicles visible on the magnetic resonance imaging. When the location of the vesicle is determined to be in the desired area of the patient from the MRI, then energy, preferably ultrasound energy, is applied to that area. This energy activates the vesicles, heats and directly and rapidly coagulates and necroses the surrounding tissue (surgical ultrasound). At the same time, this area can also be visualized by magnetic resonance imaging, if desired. Preferably, the energy used for vesicle activation is high energy continuous ultrasound, preferably higher than 50 mW/cm ² , more preferably 100 mW/cm ² . Depending on the desired therapeutic effect, the energy can be higher, up to 10 W/cm ² . Most preferably, energy is stored in the tissue using a magnetic resonance compatible ultrasound transducer in the palm of the hand. Ultrasonic sensors are prepared from non-ferrous ions and non-ferromagnetic substances. The cable supplying energy to the ultrasonic sensor has a Faraday constant shield to reduce the possibility of artifacts caused by power passing through the cable supplying the sensor.

The energy and duration of the pulses of ultrasound used for therapy will depend on the purpose of the therapy. It is preferred to focus the ultrasound energy and select a focal circle in order to target a predetermined vesicle area.

Focused ultrasound procedures are performed at an energy of about 2 watts/ ^cm2 . The focused ultrasound procedure energy can be at least 2 watts/cm ² to about 10 watts/cm ² . Causes immediate and rapid agglutination and necrosis of tissue. Simultaneously MRI is performed on the vesicles for viewing the target circle or area. The effect of the procedure is then enhanced at the target circle vesicle together with ultrasound technology.

An energy range of from about 500 milliwatts/ ^cm2 to about 10 watts/ ^cm2 , preferably about 1 watt, is effective for destruction of cavitated tissue. The vesicles lower the cavitation threshold so that cavitation occurs within the confines of the target tissue at a low energy threshold leading to tissue destruction.

Disruption or activation of vesicles occurs at energies ranging from about 50 mW/ ^cm2-500 mW/ ^cm2 . Vesicles can be disrupted by noncavitation. When a vesicle is pulsed sufficiently quickly and intensely with ultrasonic energy, the vesicle membrane degrades. When energies and pulses were applied in the specified energy ranges, transient microregional temperature increases associated with vesicle fragmentation occurred without damage to surrounding tissue by the method. This effect of disrupting the vesicles is advantageous for targeted release of therapeutic agents. Thus, release of a therapeutic agent to a certain area of the body can be seen using this technique. Again, the energy from vesicle disruption can be used to create a shock wave to release stored therapeutic agent to adjacent tissue as well. This is particularly useful in gene therapy, where shock waves can be used to instantaneously open pores in adjacent cell membranes, helping cells to take up genetic material.

With the vesicle as the nucleus, energy in the range of about 500 mW/cm ² -5 W/cm ² can be used to enhance the transformation of high-energy sound in the localized tissue, thereby heating the tissue and inducing hyperthermia.

In the case of a gaseous precursor, when ultrasonic energy is focused on the precursor it can cause the precursor to transform into a gaseous state. Enlarged gas cavities create a region of increased magnetic susceptibility and are rapidly monitored in magnetic resonance imaging. The monitoring effect can be particularly enhanced by selecting the precursor by choosing a suitably defined liquid-to-gas transition temperature such as perfluorohexane at 56°C. The present invention can therefore also be used to monitor non-invasive temperature during MRI procedures. When a vesicle is formed from a gas precursor, the material surrounding the gas precursor is fragmented. Additionally, the therapeutic agent can be released locally to adjacent tissues while the therapeutic agent is co-disrupted within the vesicle. The energy absorbed by the vesicle interface increases as vesicles form. This can be used to increase the heating of hyperthermia as well as to break up the vesicles.

Contrast media are particularly useful for providing imaging of and allowing ultrasound procedures and/or drug delivery in the cardiovascular and gastrointestinal areas, but can also be used more generally, for example, for imaging of the vascular system or in the field of art. Other ways obvious to those skilled in the art can be used. The term cardiovascular region as used herein refers to the region of the patient consisting of the heart and the vascular system leading to and from the heart. The term gastrointestinal region or gastrointestinal tract as used herein includes the region of a patient bounded by the esophagus, stomach, small and large intestines and rectum. The term vasculature as used herein refers to the blood vessels (arteries, veins, etc.) of the body or an organ or part of the body. The patient can be any type of mammal, but is most preferably a human.

It will be found that the new stabilized gas-filled vesicles for use as a contrast medium in synchronized magnetic resonance focused non-invasive ultrasound are suitable for use in all areas where MRI is used.

As will be appreciated by those skilled in the art, administration of the stabilized gas-filled vesicles for use in the present invention may be in a variety of different ways, such as intravascularly, orally, intrarectally, etc., in various Dosage Form Administration. When the area to be scanned is a cardiovascular area, it is preferred that the contrast medium of the invention is administered intravascularly. When the area to be scanned is the gastrointestinal area, it is preferred that the contrast medium of the invention is administered orally or intrarectally. The effective dose administered and the particular mode of administration will depend on the age, weight of the patient, the particular mammal and area to be scanned, and the particular contrast medium of the invention to be used. Typically, lower levels of dosage are administered initially and then increased to achieve the desired contrast enhancement. Various combinations of stabilized gas-filled vesicles can be utilized in order to modify the bulk behavior of the medium or to alter properties such as viscosity, permeability or palatability (in the case of orally administered materials). The contrast medium may be administered alone or in combination with other diagnostic, therapeutic or other agents in practicing the synchronized magnetic resonance focused non-invasive ultrasound method of the present invention. Such other agents include excipients such as flavoring or coloring agents. The magnetic resonance imaging technique used is conventional, see eg D.M. Kean and M.A. Smith "Magnetic Resonance Imaging: Principles and Applications" (William and Wilkins, Baltimore 1986). Such MRI techniques include, but are not limited to, nuclear magnetic resonance (NMR) and electrospin resonance (ESR). A preferred imaging modality is NMR.

As mentioned above, the area of application and mode of administration of gas-filled vesicles is not limited to the blood volume space, ie the vascular system. If the vesicles are ingested by mouth in order to image the gastrointestinal tract and disrupt the vesicles therein, then simultaneous magnetic resonance imaging focused non-invasive ultrasound can be accomplished using the gas-filled vesicles used in the present invention technology. Alternatively, rectal administration of these stabilized gas-filled vesicles can result in excellent formation of the lower gastrointestinal tract, including the rectum, descending colon, transverse colon, and ascending colon, as well as the appendix. Like, and break the vesicles in it. Imaging of the jejunum and conceivably the ileum can also be obtained by means of the enteral route; and the vesicles of these regions are disrupted. Direct intraperitoneal administration allows visualization and disruption of vesicles in the peritoneum. It also involves administering stabilized gas vesicles directly into the ear canal so that one can visualize the duct as well as the Eustachian tube and, if there is a perforation, the inner ear. In addition, activation and fragmentation of intraauricular vesicles can also occur. It also involves the intranasal administration of stabilized gas vesicles to aid visualization of the nasal septum and sinuses, and to disrupt the vesicles therein. Interstitial administration can also be used.

Additional routes of administration and imaging of the vesicular contrast agents of the present invention and disrupted tissue areas of the vesicles may thus be added, including (but not limited to) 1) intranasal administration to allow nasal passages and sinuses including nasal areas and sinuses 2) Intranasal and oral administration to image the rest of the respiratory tract including the trachea, bronchi, bronchioles, and lungs; 3) Intracochlear administration to image the auditory passage and Eustachian tube, tympanic membrane and outer and inner ear and ear canal imaging; 4) intraocular for imaging of areas associated with vision; 5) intraperitoneal for visualization of the peritoneum; and 6) intravesical for administration via the bladder, In order to image the genitourinary tract region including, but not limited to, the urethra, bladder, ureters, kidneys and renal vasculature, and beyond by means of this region, for example, to perform cystography or to confirm the presence of ureteral reflux. In addition, the brain, spine, lung regions, and soft tissues, muscles, and organs such as and excluding obese tissue can also be imaged in a similar manner and surgery in these regions can be accomplished using ultrasound techniques.

Ultrasound-mediated surgery may be performed using the procedures of the present invention. Ultrasound-mediated surgery refers to surgical procedures that effectively cause tissue necrosis, that is, fragmentation, destruction, or repair of tissue such as repair of small lesions (holes, ruptures, or lacerations) in tissue membranes (eg, hernias); whole or in part Relief of diseased tissue (eg tumors); activation or disruption of vesicles of adjacent tissue by ultrasound energy. Gases and Gas Precursors

The vesicles of the invention encapsulate gases and/or gas precursors. The term "gas and/or gas precursor filled" as used herein means that the vesicles of the present invention have a lumen containing at least 10% gas and gas precursor, preferably at least about 25% Gases and gas precursors, more preferably at least about 50% gases and gas precursors, more preferably 75% gases and gas precursors, most preferably at least about 90% gases and gas precursors. The presence of gas is important in use, preferably the vesicle lumen contains at least about 10% gas, preferably at least about 25%, 50%, 75%, and most preferably at least 90% gas .

Any biosoluble gas and gas precursor can be used in the gas and gas precursor-filled vesicles of the invention. Such gases include, for example, air, nitrogen, carbon dioxide, oxygen, argon, fluorine, xenon, neon, helium, rubidium-enhanced (highly polarized) xenon, rubidium-enhanced argon, rubidium-enhanced helium, rubidium Intensified neon gas or a combination thereof. For example, the use of NMR ^with19F provides more sensitive visibility than liquids or solids. Additionally, various fluorinated compounds such as various perfluorocarbons, hydrofluorocarbons, and sulfur hexafluoride gases can be used to prepare gas-filled vesicles. Gases in Quay published application WO 93/05819 may also be used, including the "Q" factor gases described herein (which document is incorporated herein by reference). In addition, paramagnetic gases and gases such as ¹⁷ O can be used. Oxygen should be stable because oxygen is soluble in blood. Stabilization can be achieved using the impermeable shell of preferably polymerized or cross-linked liposomes or cyanoacrylate microspheres; or with perfluorocarbons such as perfluoropentane or perfluorobutane. Of the gases used, perfluorocarbons and sulfur hexafluoride are preferred. Suitable perfluorocarbon gases include, for example, perfluorobutane, perfluorocyclobutane, perfluoromethane, perfluoroethane, perfluoropropane, perfluoropentane, perfluorohexane, most preferably perfluoropropane . Mixtures of different types of gases are also preferred, such as perfluorocarbon gases and other types of gases such as oxygen and the like. Indeed, it is believed that the combination of gases is particularly suitable for use in non-invasive ultrasound techniques with simultaneous magnetic resonance focus.

The gaseous precursor can also be in solid form. Based on the activation of the solid precursor form, the sodium carbonate crystals generate carbon dioxide gas. Solid and liquid gaseous precursors are particularly suitable for hyperthermic sonication, which activates the precursors into a gaseous state.

Irrespective of the requirements for gas-filled and gas-precursor vesicles prepared from stabilizing compounds, it is also advantageous to use gases with a relatively high stability. High stability gases are those selected from the group of gases having low (limited) solubility and diffusivity in aqueous media. Gases such as perfluorocarbons are less diffusible and relatively insoluble, and thus more stable in the form of bubbles in aqueous media.

The use of gaseous precursors is an optional aspect of the present invention. In particular perfluorocarbons have been found to be suitable as gas precursors. As recognized by those skilled in the art, when the vesicles used in the present invention are first formed, known perfluorocarbons can be used as gas precursors, i.e. in liquid form, or can be used directly as gases, i.e. Gaseous state to form gas-filled and gas precursor vesicles. Of course, whether such a perfluorocarbon is a gas or a liquid or a solid depends on its liquid/gas or solid/gas phase transition temperature, or boiling point. For example, one of the more preferred perfluorocarbons is perfluoropentane, which has a liquid/vapor phase transition temperature or boiling point of 27°C, which means that the compound is in liquid form at ordinary room into a gas due to the temperature of the human body above its liquid/vapor phase transition temperature or boiling point. Therefore, under normal conditions, perfluoropentane is a gaseous precursor. As further examples, there are perfluorobutane and perfluorohexane, which are the closest homologues of perfluoropentane. The liquid/vapor phase transition temperature of perfluorobutane is 4°C and that of perfluorohexane is 57°C, making the former an effective gas precursor, but perhaps more useful as a gas, whereas the latter usually has to be used as a gas precursor, Except in unique circumstances, because of its high boiling point.

Another aspect of the present invention is the use of fluorinated compounds, in particular perfluorocarbons, to aid or enhance the stability of the gas-filled and gas precursor vesicles, at the use temperature of the vesicles of the present invention in the range of liquid. The fluorinated compounds include various liquid fluorinated compounds such as ZONYL( ^TM ), a fluorinated surfactant manufactured by DuPont (Wilmington, DE), and liquid perfluorocarbons. Such perfluorocarbons useful as additional stabilizers include perfluorooctyl bromide (PFOB), perfluorodecalin, perfluorododecalin, perfluorooctyl iodide, perfluorotripropylamine, perfluorotributylamine. In general, perfluorocarbons longer than six carbon atoms will not be gases at normal body temperature, i.e. not in the gaseous state, but liquids, i.e. in the liquid state. However, these compounds can in turn be used in the preparation of the stabilized/gas-filled and gas precursor vesicles used in the present invention. Preferably the perfluorocarbon is peroctyl bromide or perfluorohexane which is liquid at room temperature. The gas present may be, for example, perfluoropropane or nitrogen, or may be derived from a gas precursor which may also be a perfluorocarbon, such as perfluoropentane. In that case, the vesicles of the present invention will be prepared from a mixture of perfluorocarbons, examples of which may be given are perfluoropropane (gas) and perfluoropentane (gas precursor) and fluorooctyl bromide (liquid) . While not intending to be bound by any theory, it is theorized that the liquid fluorine compound is located at the interface between the gas and the vesicle membrane surface. A perfluorocarbon stabilizing layer is thus formed on the inner surface of the stabilizing compound used to form the vesicle, the biosoluble lipid, which also serves to prevent gas diffusion through the vesicle membrane. Gas precursors within the scope of the present invention are liquid at the temperature of manufacture and/or storage, but become gaseous at least on or during use.

For example, it has been found that liquid fluorinated compounds, such as perfluorocarbons, when combined with the gases or gas precursors commonly used in the preparation of the vesicles of the present invention can impart an additional degree of stabilization not obtained with the gases or gas precursors alone. sex. Thus, it is within the scope of the present invention to use either a gas precursor such as a perfluorocarbon gas precursor, such as perfluoropentane, with a perfluorocarbon that remains liquid after administration to the patient, i.e. its liquid to gas phase Use with perfluorocarbons, such as perfluorooctyl bromide, that have temperature changes above the body temperature of the patient.

Any biosoluble gas or gas precursor can be used to form stable gas-filled and gas-precursor vesicles. By "biosoluble" is meant that the gas or gas precursor will not produce any degree of unacceptable toxicity, including allergenic responses and disease states, when introduced into the tissues of a patient, and is preferably inert. Such a gas or gas precursor should also be suitable for preparing vesicles that can be used as gas-filled and gas precursors as described herein.

Using the stabilized compounds herein, gas and gas precursor-filled vesicles are made more stable, and the vesicles are then sized to suit specific MRI applications. For example, magnetic resonance imaging of the vasculature requires vesicles no greater than 30 μ in diameter, preferably smaller vesicles, such as vesicles no greater than about 12 μ in diameter, gas and gas precursor filled vesicles if desired Capsule size can be adjusted by a range of measures including microemulsification, vortexing, extrusion, filtration, sonication, homogenization, repeated cycles of solidification and thawing, pressurized extrusion through micropores of a given size, and similar method.

For intravascular use, typically the vesicles will have a mean diameter of less than 30[mu], preferably a mean diameter of less than 12[mu]. For targeted intravascular use, for example, binding to certain tissues such as tumors, the diameter of the vesicles may be below 1 um, even in the visible range below 100 nm. For enteral, ie gastrointestinal, larger vesicles may be used, for example up to 1 mm in size, but average diameters between 20[mu] and 100[mu] are preferred.

As noted above, embodiments of the present invention may also include gaseous precursors that are activated by temperature with respect to their preparation, formation, and use. Listed in Table 1 below is a list of gaseous precursors with liquid to gas phase transition temperatures near body temperature (37°C) or lower and the emulsified droplet sizes required to form microbubbles with a maximum size of 10 microns.

Table 1

Physical properties and formation of gaseous precursors

Diameter of emulsified droplets of 10 μm vesicles ^* compound molecular weight Boiling point (°C) density Diameter of emulsified droplets forming 10 micron microspheres (μm) Perfluoropentane 288.04 28.5 1.7326 2.9 1-fluorobutane 76.11 32.5 6.7789 1.2 2-Methylbutane (Isopentane) 72.15 27.8 0.6201 2.6 2-Methyl-1-butene 70.13 31.2 0.6504 2.5 2-Methyl-2-butene 70.13 38.6 0.6623 2.5 2-Methyl-1-buten-3-yne 66.10 34.0 0.6801 2.4 3-methyl-1-yne 68.12 29.5 0.6660 2.5 Octafluorocyclobutane 200.04 -5.8 1.48 2.8 Decafluorobutane 238.04 -2 1.517 3.0 Hexafluoroethane 138.01 -78.1 1.607 2.7

^* Quoted from: Chemical Rubber Company Handbook ot Chemistry and Physics, Robert C. Weast and David R. Lide, eds., CRC Press, Inc. Boca Raton, Florida (1989-1990).

Below is a list of possible gas precursors that can be used to form vesicles of a defined size. However, this list is not limiting as it is possible to use other gas precursors for this purpose. In fact, for various applications, virtually any liquid can be used to prepare the gas precursors as long as they are capable of phase transition to the gas phase when at the appropriate temperature to provide the gas at some point of use. Suitable gas precursors for use in the present invention are: hexafluoroacetone, isopropylacetylene, propadiene, tetrafluoropropadiene, boron trifluoride, isobutane, 1,2-butadiene, 2, 3-butadiene, 1,3-butadiene, 1,2,3-trichloro-2-fluoro-1,3-butadiene, 2-methyl-1,3-butadiene, hexafluoro Substituent-1,3-butadiene, butadiyne, 1-fluoro-butane, 2-methylbutane, decafluorobutane, 1-butene, 2-butene, 2-methyl-1 -butene, 3-methyl-1-butene, perfluoro-1-butene, perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl -1-butene-3-yne, butyl nitrate, 1-butyne, 2-butyne, 2-chloro-1,1,1,4,4,4-hexafluorobutyne, 3-methyl- 1-butyne, perfluoro-2-butyne, 2-bromobutyraldehyde, carbonyl sulfide, crotononitrile, cyclobutane, methylcyclobutane, octafluorocyclobutane, perfluorocyclobutene, 3-Chlorocyclopentene, Octafluorocyclopentene, Cyclopropane, 1,2-Dimethylcyclopropane, 1,1-Dimethylcyclopropane, 1,2-Dimethylcyclopropane, Ethylcyclopropane , methylcyclopropane, diacetylene, 3-ethyl-3-methyldiaziridine, 1,1,1-trifluorodiazoethane, dimethylamine, hexafluorodimethylamine, dimethyl Ethylamine, bis(dimethylphosphine)amine, perfluorohexane, 2,3-dimethylnorbornane, perfluorodimethylamine, dimethyloxonium chloride, 1,3-dioxo Pentancycline-2-one, 4-methyl-1,1,1,2-tetrafluoroethane, 1,1,1-trifluoroethane, 1,1,2,2-tetrafluoroethane, 1,1,2-Trichloro-1,2,2-trifluoroethane, 1,1-dichloroethane, 1,1-dichloro-1,2,2,2-tetrafluoroethane, 1 , 2-difluoroethane, 1-chloro-1,1,2,2,2-pentafluoroethane, 2-chloro-1,1-difluoroethane, 1,1-dichloro-2-fluoro Ethane, 1-chloro-1,1,2,2-tetrachloroethane, 2-chloro-1,1-difluoroethane, chloroethane, chloropentafluoroethane, dichlorotrifluoroethane , fluoroethane, hexafluoroethane, nitro-pentafluoroethane, nitrosopentafluoroethane, perfluoroethylamine, ethyl vinyl ether, 1,1-dichloroethane, 1,1 -Dichloro-1,2-difluoroethane, 1,2-difluoroethane, methane, trifluoromethanesulfonyl chloride, trifluoromethanesulfonyl fluoride, brominated difluoronitrosomethane, bromine Fluoromethane, bromochlorofluoromethane, trifluorobromomethane, nitrodifluorochloromethane, dinitrochloromethane, fluorochloromethane, trifluorochloromethane, difluorochloromethane, difluorodibromomethane, dioxydichloromethane, Fluorodichloromethane, difluoromethane, iododifluoromethane, disilanolmethane, fluoromethane, iodomethane, trifluoroiodomethane, trifluoronitromethane, trifluoronitrosomethane, tetrafluoromethane, fluorotrichloromethane Methane, trifluoromethane, 2-methylbutane, methyl ether, methyl isopropyl ether, methyl lactate, methyl nitrite, methyl sulfide, methyl vinyl ether, neon, neopentane, nitrogen (N ₂ ), nitrous oxide, 1,2,3-nonadecanetricarboxylic acid-2-hydroxytrimethyl ester, 1-nonen-3-yne, oxygen (O ₂ ), 1,4 -pentadiene, n-pentane, perfluoropentane, 4-amino-4-methyl-2-pentanone, 1-pentene, 2-pentene (cis), 2-pentene (trans), 3 -Bromo-1-pentene, perfluorinated-1-pentene, tetrachlorophthalic acid, 2,3,6-trimethylpiperidine, propane, 1,1,1,2,2, 3-hexafluoropropane, 1,2-epoxypropane, 2,2-difluoropropane, 2-aminopropane, 2-chloropropane, 1-nitroheptafluoropropane, 1-nitrosoheptafluoropropane, all Fluoropropane, propene, hexafluoropropane, 2,3-dichloro-1,1,1,2,3,3-hexafluoropropane, 1-chloropropene, chloropropene (trans), 2-chloropropane, 3-fluoropropane, propyne, 3,3,3-trifluoropropyne, 3-fluorostyrene, sulfur hexafluoride, disulfur decafluoride (S ₂ F ₁₀ ), 2,4-diamino Toluene, trifluoroacetonitrile, trifluoromethyl peroxide, trifluoromethyl sulfide, tungsten hexafluoride, vinyl acetylene, vinyl ether, argon.

As already indicated above, perfluorocarbons are preferred components for use as gas precursors and additional stabilizing components. Such perfluorocarbon constituents include saturated perfluorocarbons, unsaturated perfluorocarbons, and cyclic perfluorocarbons. Saturated perfluorocarbons, having the formula CnF2n+2, where n is 1 to 12, preferably 2 to 10, more preferably 4 to 8, and most preferably 5, are generally preferred. Examples of suitable saturated perfluorocarbons include: tetrafluoromethane, hexafluoroethane, octafluoropropane, decafluorobutane, dodecafluoropentane, perfluorohexane, and perfluoroheptane. Also preferred are cyclic perfluorocarbons having the formula CnF2n, where n is 3 to 8, preferably 3 to 6, and include, for example, hexafluorocyclopropane, octafluorocyclobutane and decafluorocyclopentane.

Optimizing the efficacy of the vesicles using a gas of limited solubility is also part of the invention. The term "limited solubility" refers to the ability of a gas to diffuse out of a vesicle by virtue of its solubility in the surrounding aqueous medium. Higher solubility in aqueous media imparts a pressure gradient to the gas in the vesicles, such that the gas has a tendency to diffuse out of the vesicles. On the other hand, lower solubility in aqueous media reduces or cuts the gradient between the vesicle and the interface, such that gas diffusion out of the vesicle will be hindered. Preferably the gas encapsulated in the vesicles has a lower solubility than oxygen, ie 1 part gas dissolves in 32 parts water. See Matheson Gas Data Book, 1996 Matheson Company Inc. More preferably the gas enclosed in the vesicles has a lower water solubility than air; even more preferably the gas in the encapsulated vesicles comprises a gas with a lower water solubility than nitrogen. stable compound

One or more stabilizing compounds are used to form the vesicle and ensure subsequent encapsulation of the gas or gas precursor. Even for relatively insoluble, nondiffusible gases, such as perfluoropropane or sulfur hexafluoride, improved vesicle formation can be obtained when one or more stabilizing compounds are used during the formation of vesicles filled with gas and gas precursors. Capsule products. These stabilizing compounds maintain the stability and integrity of the vesicle with respect to its size, shape and/or other properties.

As used herein, the terms "stable" or "stable" mean that the vesicles are substantially resistant to degradation, ie, loss of the microsphere structure or encapsulated gas or gas precursor, for a useful period of time. Vesicles of the present invention generally have a good shelf life, often retaining at least about 90% of their original structural volume for a period of at least about two or three weeks under normal environmental conditions. Preferably, however, the period is at least one month, more preferably at least two months, even more preferably at least 6 months, still more preferably 18 months, and most preferably three years. Thus, vesicles filled with gases and gas precursors generally have a satisfactory shelf life, sometimes even under unfavorable conditions, such as temperatures and pressures above or below those experienced under normal ambient conditions.

The stability of the vesicles used in the present invention is at least in part attributable to the material from which the vesicles are made, and it is often not necessary to use additional stabilizing additives, although such use is optional and often preferred, such additional stabilizing Agents and their properties are described in more detail below. The material constituting the vesicle of the present invention is preferably biosoluble lipid or polymer material, among which biosoluble lipid is particularly preferred. Furthermore, due to the simplicity of formulation prior to administration, ie the ability to generate microspheres or foams, these vesicles can be conveniently prepared on site.

The lipids and polymers used in the preparation of the vesicles of the invention are biocompatible. "Biocompatible"means that the lipid or polymer will not produce any degree of unacceptable toxicity, including allergenic responses and disease states, when introduced into patient tissue. Preferably the above-mentioned lipid or compound is inert. biocompatible lipids

For biocompatible lipid materials, it is preferred that this class is often referred to in nature as "amphiphilic" lipid materials (i.e. polar lipids), which refer to lipophilicity on the one hand, i.e. hydrophobicity, and on the other hand Any component of a material that is lipophobic or hydrophilic at the same time.

A hydrophilic group can be a charged moiety or other group that has an affinity for water. Examples of lipids useful in preparing stable vesicles for use in the present invention include natural and synthetic phospholipids. They contain a charged phosphate "head" group, which is hydrophilic, attached to the tail of a long hydrophobic carbon chain. This structure allows the phospholipids to achieve a single-bilayer (monolayer) arrangement in which all water-insoluble hydrocarbon ends are in contact with each other, leaving the multiply charged phosphate head region free to interact with the polar aqueous environment. It will readily be appreciated that a range of centripetal bilayers is possible, ie, oligolayers and multilayers, and such arrangements also form an aspect of the invention. The ability to form this bilayer arrangement is a characteristic of the lipid materials useful in the present invention.

Lipids on the other hand can also be in the form of a single layer, and such a single layer of lipids can be used to form a single monolayer (monolayer) arrangement. On the other hand, unilamellar lipids can also be used to form a series of centripetal monolayers, ie, oligolamellar or multilamellar, and such arrangements are also considered to be within the scope of the present invention.

We have also found the importance of obtaining stable vesicles of the present invention which can be prepared below the gel-to-liquid crystal phase transition temperature of the lipid used as the stabilizing compound. This phase transition temperature refers to the temperature at which the lipid bilayer transitions from a gel state to a liquid crystal state. See, eg, Chapman et al., J. Biol. Chem. 1974, No. 249, pp. 2512-2521.

It is generally believed that the higher the gel to liquid crystalline phase transition temperature, the better the gas impermeability of the gas-filled and gas precursor vesicles at a given temperature. See Derek Marsh, CRC Handbook of Lipid Bilayers (CRC Press, Boca Raton, FL 1990), p. 139, giving the backbone melting point transitions for saturated diacyl-Sn-glycero-3-phosphocholine. The phase transition temperatures from the gel state to the liquid crystalline state of various lipids are clearly apparent to those skilled in the art, see, for example, those listed in Liposome Technology, Vol. 1, 1-18 (CRC Press, 1984), edited by Gregoriadis . Some representative lipids and their phase transition temperatures are listed in Table 2 below:

Table 2

  Saturated Diacyl-Sn-Glyceryl (3)

Choline Phosphate Main Chain Phase Transition Temperature ^* carbon atoms 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) -1.013.723.534.541.448.255.161.864.571.174.079.580.1

^* Derek Marsh, "CRC Handbook of Lipid Bilayers," CRC Press, Boca Raton, Florida (1990), p. 139.

It has been found that it is possible to increase the stability of the vesicles used in the present invention by incorporating at least a small amount of negatively charged lipids into the lipids used to form gas-filled and gas precursor vesicles, "at least a small amount" referring to the total lipid The amount is about 1 to 10 mole percent. Suitable negatively charged lipids include, for example, phosphatidylserine, phosphatidic acid and fatty acids. Such negatively charged lipids can provide additional stability by hindering the tendency of the vesicles to fuse together and rupture, i.e., the negatively charged lipids help form a uniform negative charge layer on the outer surface of the vesicle, which will be absorbed by similar vesicles on other vesicles. repelled by the charged outer layer. In this way, close contact of the vesicles with each other is prevented, which often leads to rupture of the membrane or skin of the involved vesicles and to coalescence of contact vesicles into a single larger vesicle. Of course the continuation of this incorporation process will lead to significant degradation of the vesicles.

The lipid material or other stabilizing compound used to make up the vesicles is also preferably flexible, in this application the flexibility of a vesicle filled with gas and gas prefilled refers to the ability of the structure to change its shape to pass through pores that are smaller in size than the vesicle ability.

In selecting lipids suitable for preparing stable vesicles according to the invention, it was found that a variety of lipids were suitable for vesicle formation. Any material or combination of materials known to those skilled in the art to be suitable for liposome preparation is particularly useful. The lipids used may be natural, synthetic or semi-synthetic.

Lipids that can be used to prepare gas-filled and gas precursor vesicles for use in the present invention include, but are not limited to: lipids such as fatty acids, lysolipids, phosphatidylcholines with saturated and unsaturated lipids, which include dioleoyl Phosphatidylcholine, Dimyristoylphosphatidylcholine, Dipentadecanoylphosphatidylcholine, Dilauroylphosphatidylcholine, Dipalmitoylphosphatidylcholine (DPPC), Distearoyl Phosphatidylcholine (DSPC), phosphatidylethanolamines such as dioleoylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine (DPPE), phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, sphingolipids such as sphingomyelin , glycosyl lipids such as gangliosides CM1 and CM2, glycolipids, sulfatides, glycosphingolipids, phosphatidic acids such as dipalmitoylphosphatidic acid, palmitic acid, stearic acid, arachidonic acid, oleic acid, Lipids loaded with polymers such as polyethylene glycol or PEGYlated lipids, chitin, hyaluronic acid or polyvinylpyrrolidone, loaded with sulfonated mono-, di-, oligo- or poly Lipids of sugars, cholesterol, cholesterol sulfate and cholesterol hemisuccinate, vitamin E hemisuccinate, lipids containing fatty acids linked by ethers and esters, polymeric lipids are known to those skilled in the art Synthesis of various lipids, diacetyl phosphate, dicetyl phosphate, stearylamine, cardiolipin, phospholipids containing short-chain fatty acids with a chain length of 6-8 carbon atoms, with asymmetric acyl chains Phospholipids (such as synthetic phospholipids with one acyl chain of 6 carbon atoms and the other with acyl chain of 12 carbon atoms), ceramides, nonionic lipids including niosomes such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohols, polyoxyethylene Fatty Alcohol Ether, Polyoxyethylated Sorbitan Fatty Acid Ester, Polyglycol Glyceryl Hydroxystearate, Polyethylene Glycol Glyceride Ricinoleate, Ethoxylated Soy Sterol, Ethoxylated Castor Oil, Polyoxyethylene, Polyoxypropylene polymer and polyoxyethylene fatty acid stearate, sterol fatty acid ester including cholesteryl sulfate, cholesteryl butyrate, cholesteryl isobutyrate, cholesteryl palmitate, cholesteryl stearate , lanosteryl acetate, ergosteryl palmitate, and phytosteryl n-butyrate, sugar esters of sterols include cholesterol glucuronide, lanosteryl glucuronide, 7-dehydrocholesteryl glucuronide , ergosteryl glucuronate, cholesteryl glucuronate, lanosteryl gluconate and ergosterol gluconate, esters of sugar acids and alcohols including lauryl glucuronate, stearyl glucuronate , myristyl glucuronate, lauryl gluconate, myristyl gluconate, and octadecyl gluconate, esters of sugar and fatty acids include sucrose laurate, fructose laurate, sucrose Palmitate, sucrose stearate, glucuronic acid, gluconic acid, accharic acid, and polyuronic acid, saponins including sarsasapogenin, smilagenin, hecuragene, caryophyllin and Digoxigenin, Glyceryl Dilaurate, Glyceryl Trilaurate, Glyceryl Dipalmitate, Glyceryl and Glyceryl Esters including Glyceryl Tripalmitate, Glyceryl Distearate, Glyceryl Tristearate, Glyceryl dimyristate, glyceryl trimyristate, long chain alcohols including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, and stearyl alcohol, 6-(5-cholestene-3β- yloxy)-1-thio-β-D-galactopyranoside, digalactose diglyceride, 6-(5-cholesten-3β-yloxy)-6-amino-6-deoxy -1-thio-β-D-galactopyranose, 6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-α-D-pyridine Mannoside, 12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic acid, N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic acid, N-[12-(((7′-diethylaminocoumarin Facilin-3-yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmitic acid, cholestenyl 4'-trimethylammoniobutyrate, N-succinyldioleoylphosphatidylethanolamine, 1,2 - Dioleoyl-Sn-glycerol, 1,2-Dipalmitoyl-Sn-3-succinylglycerol, 1,3-Dipalmitoyl-2-succinylglycerol, 1-hexadecyl-2-palmitoyl Glycerophosphoethanolamine and palmitoylhomocysteine and/or combinations thereof.

Various cationic lipids such as DOTMA, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, DOTAP, 1, 2-dioleoyloxy-3-(trimethylammonio)propane and DOTB, 1,2-dioleoyl-3-(4'-trimethylammonio)butyryl-Sn-glycerol. In general, the molar ratio of cationic lipids to non-cationic lipids in liposomes can be, for example, 1:1000, 1:100, preferably between 2:1 and 1:10, more preferably between 1:1 and 1 : between 2.5, and most preferably 1:1 (the ratio of the molar weight of cationic lipid to the molar weight of non-cationic lipid, such as DPPC). When cationic lipids are used to form vesicles, the various lipids can be composed of non-cationic lipids. Such non-cationic lipid is preferably dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine or dioleoylphosphatidylethanolamine. Instead of the cationic lipids mentioned above, lipids with cationic polymers such as polylysine or polyarginine, as well as alkyl phosphonates, alkyl phosphinates and alkyl phosphites can also be used to form vesicle.

The most preferred lipids are phospholipids, preferably DPPC, DPPE, DPPA and DSPC, and most preferably DPPC.

In addition, examples of saturated and unsaturated fatty acids that can be used in the manufacture of stable vesicles (in the form of mixed microbubbles filled with gas and gas precursors) used in the present invention may include straight-chain fatty acids preferably containing 12 to 22 carbon atoms. or branched chain forms of molecules. It is also possible to use hydrocarbyl groups consisting of isoprenoid units and/or containing isoprenyl groups. Examples of suitable saturated fatty acids include, but are not limited to, lauric acid, myristic acid, palmitic acid, and stearic acid, and examples of unsaturated fatty acids that may be used include, but are not limited to, lauric acid, spermidic acid, myristicole Acids, palmitoleic acid, 6-octadecenoic acid, and oleic acid, examples of useful branched chain fatty acids include, but are not limited to, isolauric acid, isomyristic acid, isopalmitic acid, and isostearic acid. In addition to saturated and unsaturated groups, mixed vesicles filled with gases and gas precursors can also be constructed from 5-carbon isodiene- and isoprene-containing groups. In addition, partially fluorinated phospholipids can be used as stabilizing compounds for encapsulating vesicles. biocompatible polymer

Biocompatible polymers used as stabilizing compounds in the preparation of the gas-filled and gas precursor vesicles of the present invention may be natural, semi-synthetic (modified naturals) or synthetic. The term "polymer" here means a compound consisting of two or more repeating monomer units, preferably 10 or more repeating monomer units. Herein the term "semi-synthetic polymer (or modified natural polymer)" refers to a natural polymer that has been chemically modified in some way. Typical natural polymers suitable for use in the present invention include naturally occurring polysaccharides. Such polysaccharides include, for example, arabinan, fructan, deoxygalactan, galactan, polygalacturonic acid, dextran, mannan, xylose (such as glucose), levan, Fucoidan, carrageenan, galatocarolose, pectic acid, pectinase, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, ascocin, chitin, agar Sugars, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, bengal gum, starch and various natural homopolymers or heteropolymers such as those containing one or more of the following aldoses, ketones Polymers of sugars, acids, or amines: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, semi Lactose, talose, erythrulose, ribulose, xylulose, allulose, fructose, sorbose, talose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, fiber Disaccharides, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid , galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid and their naturally occurring derivatives. Typical semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose and methoxycellulose. Typical synthetic polymers suitable for use in the present invention include polyethylenes (such as polyethylene glycol, polyoxyethylene, and polyethylene terephthalate), polypropylenes (such as polypropylene glycol), polyurethanes (such as polyvinyl alcohol (PVA), polyvinyl chloride, and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acid, fluorocarbons, fluorinated carbons (such as polytetrafluoroethylene), and polymethacrylic acid Methyl esters, and their derivatives. Once the skilled artisan has grasped the teachings of the present application, combining the present application and the material known in the art, as described in Unger, U.S. Patent 5,205,290 (the content of which is incorporated herein by reference in its entirety), such polymers can be prepared. The approach to vesicle-based vesicles becomes apparent.

Preferably, when intended for use in the gastrointestinal tract, the polymer used is a polymer with a relatively strong water binding capacity. In use, for example in the gastrointestinal region, a polymer with strong water binding capacity binds a large amount of free water, causing the polymer to carry a large amount of fluid through the gastrointestinal tract, thereby filling and distending the gastrointestinal tract. A full and distended GI tract allows for a clearer picture of the area. Additionally, when imaging of the GI tract region is desired, it is preferred that the polymer used is one that is substantially non-degradable and non-absorbable in the GI tract region. Minimizing metabolism and absorption in the gastrointestinal region is preferred in order to avoid removal of the contrast agent from the gastrointestinal region and to avoid gas generation in the gastrointestinal tract due to its degradation. In addition, particularly for dosages for gastrointestinal use, polymers are preferred such that they are capable of displacing air and minimizing the formation of bulk air bubbles in the polymer composition.

Particularly preferred embodiments of the invention include vesicles wherein the stabilizing compound that forms the stable gas and gas precursor filled vesicles comprises three components: (1) a neutral (e.g., nonionic and zwitterionic) lipid, (2) negatively charged lipids, and (3) lipids carrying hydrophilic polymers. Preferably, the negatively charged lipid is present in an amount greater than 1 mole % of the total lipid present and the hydrophilic polymer-bearing lipid is present in an amount greater than 1 mole % of the total lipid present. It is also preferred that the negatively charged lipid is a phosphatidic acid. Desirably, the lipid carrying the hydrophilic polymer is one covalently bound to said polymer, and said polymer preferably has an average molecular weight of from 400 to 100,000. The hydrophilic polymer is preferably selected from the group consisting of polyethylene glycol, polypropylene glycol, polyvinyl alcohol, and polyvinylpyrrolidone and their copolymers. PEG and other polymers can be covalently bonded to DPPE or other lipids, such as amide, carbamate or amine bonds. Alternatively, esters, ethers, thioesters, sulfamides or disulfide bonds (thioesters) can be used with PEG or other polymers to bind the polymer to, for example, cholesterol or other phospholipids. Lipids carrying such a polymer are said to be "PEGylated" if the hydrophilic polymer is polyethylene glycol, for which the literature has abbreviated "PEG". The lipid carrying the hydrophilic polymer is preferably dipalmitoylphosphatidylethanolamine-polyethylene glycol 5000, that is, a dipalmitoylphosphatidylethanolamine lipid having polyethylene glycol with an average molecular weight of 5000 on it. (DPPE-PEG5000); or distearoylphosphatidylethanolamine-polyethylene glycol 5000.

Examples of preferred vesicles according to the present invention include, for example, vesicles with 12.5 mole % of dipalmitoylphosphatidic acid (DPPA) and with 10 mole % of dipalmitoylphosphatidylethanolamine-polyethylene glycol 5000 (DPPE/PEG5000). 77.5 mole % dipalmitoylphosphatidylcholine (DPPC). These compositions having mole percentages of 82/10/8 respectively are also preferred. Effectively the DPPC component is neutral since the phosphatidyl moiety is negatively charged and the choline moiety is positively charged. Therefore, a negatively charged DPPA component was added to enhance stability according to the described principles, which further involved negatively charged lipids as additives. The third component, DPPE, PEG, provides a PEGylated material bound by the DPPE component to the lipid membrane or outer layer of the vesicle, wherein the free PEG component surrounds the membrane or outer layer of the vesicle and thus forms a barrier to each This body's enzymes and functions act as a physical barrier to other endogenous agents that degrade exogenous materials. It is also theorized that PEGylated materials, due to their structural resemblance to water, are able to defeat the action of macrophages of the human immune system, which otherwise surround and remove foreign substances. The result is an increased time for the stabilized vesicles to function as a contrast agent. Other and auxiliary stabilizing compounds

It is also part of the invention to prepare stable gas-filled and gas precursor vesicles using ingredients other than the biocompatible lipids and polymers described above, provided that the vesicles so prepared meet the stability requirements described herein. and other requirements. These ingredients can be basic and fundamental, ie form the main base material that creates or prepares stable gas-filled and gas-precursor vesicles. On the other hand, they may be adjunctive, that is, as auxiliary or supplementary agents, which enhance the action of the basic stabilizing compound, or otherwise provide some desired property in addition to that provided by the basic stabilizing compound.

However, it is difficult to determine whether a given compound is an essential compound or an adjuvant since the effect of the compound is determined experimentally as a result of the formation of stable vesicles. As an example of how these primary and auxiliary compounds work, it has been observed that a simple combination of shaking a biocompatible lipid and water or saline often results in a cloudy solution, followed by autoclaving. This cloudy solution can function as a contrast agent, but is aesthetically objectionable and has instability in the form of insoluble or non-diffusing lipid particles. Therefore, propylene glycol can be added to remove turbidity by promoting the dissolution or diffusion of lipid particles. Propylene glycol also acts as a thickener, improving vesicle formation and stabilization by increasing the surface tension of the vesicle membrane or surface layer. It is possible that the propylene glycol further acts as an additional layer that wraps around the membrane or skin of the vesicle, thus providing additional stability. For examples of other such primary or secondary stabilizing compounds, conventional surfactants can be used; see US Patents 4,684,479 and 5,215,680 to D'Arrigo.

Additional auxiliary and primary stabilizing compounds include such substances as peanut oil, canola oil, olive oil, safflower oil, corn oil or any other generally known and suitable for stabilizing Compound edible oil.

In addition, compounds used to prepare mixed micellar systems are also suitable as primary or secondary stabilizing compounds, these include (but are not limited to): lauryltrimethylammonium bromide (lauryl-), cetyltrimethylammonium bromide Methylammonium bromide (hexadecyl-), myristyltrimethylammonium bromide (tetradecyl-), alkyl dimethyl benzyl ammonium chloride (alkyl=C ₁₂ , C ₁₄ , C ₁₆ ), Benzyldimethyldodecylbromide/ammonium chloride, benzyldimethylhexadecylbromide/ammonium chloride, benzyldimethyltetradecylbromide/ammonium chloride, cetyl Dimethylethyl bromide/ammonium chloride or cetyl bromide/pyridinium chloride.

It has been found that the size, solubility and thermal stability of the gas-filled and gas precursor vesicles used in the present invention can be controlled by selecting various additional or co-stabilizers as described herein. These stabilizers affect these parameters of the vesicles not only through their physical interaction with the lipid coating, but also through their ability to modify the viscosity and surface tension of vesicles filled with gases and gas precursors. Thus, gas-filled and gas-precursor vesicles for use in the present invention can be suitably modified and further stabilized by the addition of one or more of the following: (a) viscosity modifiers, including but not limited to carbohydrates Phosphorylated and sulfonated derivatives thereof; and polyethers, preferably polyethers having a molecular weight between 400 and 100,000, di- and tri-hydroxyalkanes and polymers thereof, preferably having a molecular weight between 200 and 50,000; ( b) Emulsifiers and/or solubilizers may also be used in combination with lipids, which give desirable modification and further stabilization, such substances include (but are not limited to) acacia, cholesterol, diethanolamine, glycerol monohard Fatty acid esters, lanolin alcohol, lecithin, mono- and di-glycerides, monoethanolamine, oleic acid, oleyl alcohol, Poloxamer (such as Poloxamer 188, Poloxamer 184 and Poloxamer 181), polyoxyethylene stearate 50, polyhydrocarbon Oxygen 35 castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60 , polysorbate 80, propylene glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitan monolauryl, sorbitan monooleate, sorbitan Monopalmitate, sorbitan monostearate, stearic acid, triethanolamine, and emulsifying waxes; (c) suspending and/or viscosity increasing agents that may be used with lipids include, but are not limited to, gum arabic , agar, alginic acid, aluminum monostearate, bentonite, magma, carbopol 934P, carboxymethylcellulose, calcium and sodium and sodium 12, carrageenan, cellulose, dextrin, gelatin, guar Gum, Locust Bean Gum, Magnesium Aluminum Silicate, Hydroxyethyl Cellulose, Hydroxypropyl Methyl Cellulose, Magnesium Aluminum Silicate, Methyl Cellulose, Pectin, Polyethylene Oxide, Povidone, Propylene Glycol Alginates, silicon dioxide, sodium alginate, tragacanth and xanthan gums, alpha-d-gluconolactone, glycerin, mannitol, (d) synthetic suspending agents such as polyethylene glycol Glycols (PEG), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polypropylene glycol, and polysorbates; and (e) may also include tonicity-enhancing agents, such agents include, but are not limited to, sorbose Alcohol, Propylene Glycol and Glycerin. aqueous diluent

As already described above, when the vesicle is a natural lipid, the specific composition of the stable vesicle is some kind of aqueous environment which induces the lipid (due to the hydrophobic/hydrophilic nature of the base) to form a vesicle (this environment the most stable configuration available). Diluents that can be used to create such an aqueous environment include (but are not limited to) water, which may be deionized or contain any amount of dissolved salts, etc., which will not interfere with the creation and maintenance of stable microspheres or affect their performance as MRI contrast agent application, as well as regular saline and normal saline. Paramagnetic and superparamagnetic contrast agents

In another embodiment of the invention, the inventive stabilized gas-filled vesicle-based contrast medium further contains other contrast agents, such as conventional contrast agents, which serve to enhance the non-invasive The role of the potency of contrast media in sexual ultrasonography. Many such contrast agents are known to those skilled in the art, and include paramagnetic and superparamagnetic contrast agents.

Examples of paramagnetic contrast agents suitable for use in the present invention include stable free radicals (e.g., stabilized nitroxides), and include transition, lanthanide, and actinide elements, which may be in salt form or combined with Complexing agents (including their lipophilic derivatives), either covalently or non-covalently bound to protein macromolecules.

Preferred are transition, lanthanide and actinide elements including Gd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II ), Ni(II), Eu(III) and Dy(III). More preferred said elements include Gd(III), Mn(II), Cu(II), Fe(II), Fe(III), Eu(III) and Dy(III), especially Mn(H) and Gd (III).

These elements may, if desired, be in the form of salts such as manganese salts such as manganese chloride, manganese carbonate, manganese acetate, and manganese organic acid salts such as manganese gluconate and manganese hydroxyapatite; Iron salts such as ferrous chloride.

If desired, these elements can be covalently or non-covalently bound to complexing agents (including their lipophilic derivatives), or to protein macromolecules. Preferred complexing agents include, for example, diethylenetriamine-valeric acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,N',N', N-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane N, N', N"-triacetic acid (DO3A), 3,6,9-triaza-12- Oxa-3,6,9-tricarboxymethylene-10-carboxy-13-phenyl-tricapric glyceric acid (B-19036), hydroxybenzyl vinyl-diamine diacetic acid (HBED), N , N'-bis(pyridoxyl-5-phosphate)ethylenediamine, N,N'-diamine (DPDP), 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane N,N',N',N'-tetraacetic acid (TETA), kryptands (ie, macrocyclic mixtures), and deferoxamine. More preferably, the complexing agent is EDTA, DTPA, DOTA, DO3A and kryptands, most preferably DTPA. Preferred lipophilic complexes include alkylated derivatives of the complexing agents EDTA, DOTA, etc., for example, EDTA-DDP, i.e. N,N'-bis-(carboxy-decylamidomethyl-N-2,3-di Hydroxypropyl)-1,2-ethylenediamine-N,N'diacetate; EDTA-ODP, that is, N,N'-bis-(carboxy-octadecylamidomethyl-N-2,3 -Dihydroxypropyl)-1,2-ethylenediamine-N,N'diacetate; EDTA-LDP, that is, N,N'-bis-(carboxy-dodecylamidomethyl-N-2, 3-dihydroxypropyl)-1,2-ethylenediamine-N,N'diacetate and the like; such as those compounding agents disclosed in U.S. Application No. 887,290 filed on May 22, 1992, which The literature is incorporated herein as a reference. Preferred proteinaceous macromolecules include albumin, collagen, polyarginine, polylysine, polyhistidine, gamma-globulin and beta-globulin. More preferably, protein macromolecules include albumin, polyarginine, polylysine, polyhistidine.

Suitable complexes include 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 iron-desferoxamine, especially Mn(II)-DTPA or Gd- (III)-DTPA.

Paramagnetic chelators include, for example, alkylated chelators of paramagnetic ions disclosed in U.S. Patent 5,312,617 (incorporated herein by reference), as disclosed in U.S. Patent 5,385,719 for binding to gas-filled liposomes and for binding to gas Paramagnetic copolymeric chelators for the surface of filled polymeric liposomes for the stabilization of phospholipids bound to gas-filled liposomes and free nitroxides bound to gas-filled liposomes (NSFRs), and hybrid complexes containing one or more paramagnetic ions closest to one or more NSFRs chelating agent components (as outlined in U.S. Patent No. 5,407,657), which can be used for the construction of paramagnetic gas-filled liposomes. These hybrid complexes greatly increase relaxation and thus greatly increase sensitivity to vesicles in magnetic resonance.

Nitroxides are paramagnetic contrast agents that increase the ratio of looseness between T1 and T2 by means of an unpaired electron of the nitro molecule. The paramagnetic effect of a given compound as an MRI contrast agent is at least in part related to the number of unpaired electrons of the paramagnetic nucleus or molecule, in particular the square of the number of unpaired electrons. For example, cadmium has seven unpaired electrons and the nitroxide molecule has only one unpaired electron; thus in general cadmium is a stronger MRI contrast agent than nitroxide, however, the effective correlation time, is used to evaluate the contrast Another parameter of agent potency may be increased looseness of the nitroxide. When the effective correlation time is very close to the proton Larmour frequency, the looseness ratio can increase dramatically. When the rate of decline is slowed, for example due to the binding of paramagnetic contrast agents to large structures, this slows down the energy transfer and facilitates the loosening of the protons of water. But for cadmium, electron spin looseness time is fast and will be limited to the point where slow spin time can improve looseness. But for nitroxides, the electron spin correlation time is better, and by making the spin correlation time of these molecules slower, the looseness tends to increase. The gas-filled vesicles of the present invention are ideal for the purpose of achieving slower spin correlation times and gaining increased bulk. While not intending to be bound by a particular theory of operation, for example by preparing its alkylated derivatives, nitroxide can coat the periphery of gas-filled vesicles so that the correlation times obtained are optimized. In addition, it can be seen that the contrast medium obtained in the present invention is a magnetic sphere, which is the most loose geometric configuration.

If desired, nitroxides can be alkylated or prepared as derivatives such as nitroxide 2,2,5,5-tetramethyl-1-pyrrolidinyloxy, free radical and 2,2 , 6,6-Tetramethyl-1-piperidinyloxy, free radical (TMPO).

Examples of superparamagnetic contrast agents suitable for use in the present invention include metal oxides metal sulfides, ferromagnetic compounds _such as pure iron, magnetite oxides (magnetite), gamma- _Fe2O3 , Fe ₃ O ₄ , iron sulfide, magnetic manganese, cobalt and nickel ferrites and ferritin filled with magnetite or other magnetically active substances such as ferromagnetic and superparamagnetic substances.

The paramagnetic and superparamagnetic contrast agents described above, for example, can be used as components within the vesicles or in the contrast medium containing the vesicles. They are used as vesicle interior components, administered as a solution with the vesicles, or incorporated into stabilizing compounds that form the vesicle walls.

Superparamagnetic agents are used as clathrates to adsorb to and stabilize vesicles. For example, emulsions of various perfluorocarbons, such as perfluorohexane or perfluorochlorocarbons mixed with irregularly shaped iron oxides. Hydrophobic cracks in the iron oxide nano-droplets of the e-liquid gas precursor adsorb to the surface of the solid substance.

Paramagnetic or superparamagnetic contrast agents can for example be incorporated into stabilizing compounds, especially the lipid walls of vesicles, in the form of alkylated or other derivatives, if desired. Specifically, by virtue of a number of different linkages such as acetoxy, nitrooxide 2,2,5,5-tetramethyl-1-pyrrolidinyloxy, free radical and 2,2,6,6- Tetramethyl-1-piperidinyloxy, free radicals can form adducts with long-chain fatty acids at positions not occupied by methyl groups on the ring. Said adducts are well suited for incorporation into stabilizing compounds, especially of lipidic nature, which can form the walls of the vesicles of the invention.

Also mixtures of one or more paramagnetic and/or superparamagnetic agents can be used for the contrast medium.

The above-mentioned paramagnetic agents and superparamagnetic agents can also be used for co-administration, respectively, if desired.

The gas-filled vesicles used in the present invention not only serve as effective carriers for superparamagnetic agents such as iron oxides, but are also shown to have the effect of increasing the magnetic susceptibility of contrast agents. Superparamagnetic contrast agents include metal oxides, especially iron oxides but including manganese oxide, and contain, as iron oxides, various amounts of manganese, cobalt and nickel that undergo a magnetic field regime. These agents are nano or microparticles with very high magnetic susceptibility and lateral bulk ratio. Larger particles, eg 100 nm diameter, have a much higher R2 bulk than R1 bulk, but smaller particles, eg 10-15 nm diameter, have lower R2 bulk but more balanced R1 and R2 values. The smallest particles such as single crystal iron oxide particles with a diameter of 3-5 nm have lower R2 bulkiness, but probably have the most balanced ratio of R1 and R2 bulkiness. It is also possible to formulate ferritin so as to coat a superparamagnetic core with a very high bulk ratio. It has been found that the gas-filled vesicles used in the present invention can improve the efficacy and safety of these conventional iron oxide based MRI contrast agents.

Iron oxide is simply incorporated into the stabilizing compound used to prepare the vesicles. In particular, iron oxides are incorporated into the walls of lipid-based vesicles, e.g., adsorbed to the surface of the vesicles, or tethered to the interior of the vesicles, as described in U.S. Patent 5,088,499, issued February 18, 1992 of.

While not intending to limit the mode of action of the invention to a particular theory, it is believed that vesicles enhance the effectiveness of superparamagnetic contrast agents through several mechanisms. First, the function of the vesicles is believed to increase the apparent magnetic concentration of iron oxide particles, and second, the vesicles are believed to increase the apparent rotational correlation time of MRI contrast agents, including paramagnetic and superparamagnetic agents. Finally, the vesicles appear to function by a novel mechanism of enhancing the magnetic domain of the apparent contrast medium, and are believed to function in the manner described immediately below.

Vesicles are considered as regions of elastic spheres with different magnetic susceptibilities of the suspending medium, i.e. aqueous contrast medium suspension, and gastrointestinal fluid for gastrointestinal administration, intravascular injection and injection into Another body space is blood or other bodily fluids. When considering ferrite or iron oxide particles, it should be noted that the contrasting effect of these contrast agents is particle size dependent, ie it depends on the particle diameter of the iron oxide particles. This phenomenon is very common and is often referred to as "long-term" looseness of water molecules. Described in a more physical way, the looseness mechanism is dependent on the effect size of the molecular complex in which a paramagnetic atom, or a paramagnetic molecule, or several paramagnetic molecules reside. A physical interpretation is described in the following Solomon Bloembergen equation, where the relationship of paramagnetism to the T1 and T2 correlation times of spin 1/2 nuclei is defined in terms of the gyromagnetic ratio g that is troubled by paramagnetic ions:

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

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

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

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

S = electron spin quantum number;

g = g factor of electron;

β = Bohr magneton;

ω _l and ω _s (=657w _l ) = Larmor angle operating frequencies for nuclear rotation and electron rotation;

γ = ion-nucleus distance;

A = very small coupling constant;

_Tc and _Te = correlation times for dipolar and ladder interactions, respectively; and

h = Planck's constant.

See eg Solomon, I. Phys. Rev. 99, 559 (1955) and Bloembergen, N. J. Chem. Phys. 27, 572, 595 (1957), incorporated herein by reference.

Usually a few large particles have a greater effect than a larger number of much smaller particles, mainly due to the larger correlation time. If the iron oxide particles were made larger, however, they would be toxic, clogging the lungs and activating a complex cascade system. Furthermore, the overall size of the particle does not matter, but the diameter of the particle especially at its edges or outer surfaces is critical. Magnetized area and magnetic susceptibility effects decrease exponentially with respect to the surface of the particle. In general, for the case of a dipolar (via surface) looseness mechanism, an exponential decrease shows a dependence on ^r6 . According to the literature, a water molecule 4 angstroms away from a paramagnetic surface is 64 times less affected than a water molecule 2 angstroms away from a paramagnetic surface. The ideal situation for maximizing the contrast effect is that the iron oxide particles are hollow, elastic, and as large as possible. Until now, this has not been possible; moreover, the benefits may have been recognized until now. By coating the interior or exterior surfaces of vesicles with contrast agent, even though individual contrast agents such as iron oxide nanoparticles or paramagnetic ions are relatively small structures, the effect of the contrast agent may be greatly enhanced. Here, the contrast agent acts as a much more potent sphere in which the effective area of magnetization is determined by the diameter of the vesicle, with the area of magnetization being greatest at the surface of the vesicle. These contrast agents are resilient, ie, compliant. Whereas rigid vesicles load the lungs or other organs and cause electrotoxic reactions, these elastic vesicles slide more easily through capillaries. Preparation

Stabilized gas and gas precursor filled vesicles for use in the present invention can be prepared by a variety of suitable methods. These are described below for the case where the vesicles are inflated and are gas precursor filled, respectively, although vesicles with gas and gas precursors are part of the invention. Applied gas

A preferred protocol comprises agitating an aqueous solution containing a stabilizing compound, preferably a lipid, in the presence of a gas at a temperature below the gel to liquid crystal phase transition temperature of the lipid to form gas and gas precursor filled vesicles. The term agitation and variations thereof as used herein means any movement of shaking an aqueous solution such that gas is introduced into the aqueous solution from the surrounding environment. Shaking must be vigorous enough to cause the formation of vesicles, especially stabilized vesicles. Shaking can be a vortex, side to side, or up and down motion. Different forms of exercise can be combined. Furthermore, shaking may be performed by shaking the container containing the aqueous lipid solution, or by shaking the aqueous solution in the container without shaking the container itself.

Also, the shaking can be manual or motorized. Useful mechanical shakers include, for example, shaker platforms such as the VWR Scientific (Cerrtos, CA) shaker platform, or the Wig- L- ^Bug® Shaker. A preferred aspect of the invention is that some type of shaking or vortexing is used to produce stable vesicles in the preferred size range. Shaking is preferred, and a Wig-L-Bug ^(R) mechanical shaker is preferred for such shaking. According to a preferred method, an oscillating motion is used to generate gas and gas precursor filled vesicles are preferred. More preferably the movement oscillates in an arc. More preferably the motion oscillates in an arc of between about 2° and 20°, still more preferably an arc of between about 5° and about 8°. It is most preferred that the movement oscillates between about 6° and about 7°, most particularly about 6.5°. It is expected that the rate of sway, and its curvature, is critical in determining the amount and size of gas- and gas-precursor-filled vesicles formed. A preferred aspect of the present invention is an oscillation value, ie, a value in the range of about 1,000 to about 20,000 vibrations per minute for a full cycle. More preferred vibration or vibration values will be between 2500 and 8000. The aforementioned Wig-L-Bug ^(R) is meant to provide 2000 impacts per 10 seconds, ie 6000 vibrations per minute. Of course, the number of vibrations depends on the content being stirred, the greater the mass, the lower the number of vibrations. Other means of producing shaking include the action of gases emitted at high velocity or pressure.

It should also be understood that, preferably, the greater the volume of the aqueous solution, the greater the amount of force. Vigorous shaking is defined as at least about 60 shaking movements per minute and is preferred. A vortex of at least 60-300 revolutions per minute is more preferred. A vortex of 300-1800 per minute is most preferred. The formation of gas and gas precursor filled vesicles based on shaking can be observed visually. The concentration of lipid required to form the desired level of stabilized vesicles will vary with the type of lipid used. and can be readily determined by routine experimentation. For example, in a preferred embodiment, the concentration of 1,2-dipalmitoylphosphatidylcholine (DPPC) used to form stabilized vesicles according to the method of the present invention is from about 0.1 mg/ml to about 30 mg/ml Saline solution, more preferably about 0.5 mg/ml to about 20 mg/ml saline solution, most preferably about 1 mg/ml to about 10 mg/ml saline solution. Distearoylphosphatidylcholine (DSPC) is used in a preferred embodiment at a concentration of about 0.1 mg/ml to about 30 mg/ml of common saline solution, more preferably about 0.5 mg/ml to about 20 mg/ml of the salt Aqueous solutions, most preferably from about 1 mg/ml to about 10 mg/ml in saline.

In addition to the simple shaking method described above, more complex but less preferred methods for this reason can also be applied, for example, the liquid crystal shaking gas instillation method, and the vacuum drying gas instillation method, as in June 11, 1993 described in filed U.S. Application No. 076,250, which is incorporated herein by reference in its entirety. When such methods are used, stabilized vesicles to be filled with gas and gas precursors can be prepared prior to gas infusion by any of the conventional liposome preparation techniques apparent to those skilled in the art. These techniques include freeze-thaw, and methods such as sonication, chelation dialysis, homogenization, solvent diffusion, microemulsion, spontaneous formation, solvent evaporation, French cell pressing, detergent dialysis, and others, each including Mode The vesicles are prepared in a solution containing the desired active ingredient so that therapeutic, cosmetic or other agents are encapsulated, netted, or tethered within the resulting polar lipid-based vesicles. See, eg, Madden et al., Chemistry and Physics of Lipids, 1990, 53, 37-46, which is incorporated herein by reference in its entirety.

Gas-filled vesicles prepared according to the method described above range in size from below 1 μ to above 100 μ. Additionally, it should be noted that following the extrusion and sterilization process, the agitation or shaking step produces gas-filled vesicles with little or no residual anhydrous lipid phase (Bangham, A.D., Standish, M.M, & Watkins, J.C. (1965) J. Mol. Biol. 13, 238-252) present in the residue of the solution. The resulting gas-filled vesicles are stable for a year or even longer when stored at room temperature.

The size of gas-filled vesicles can be adjusted, if desired, by microemulsification, vortexing, extrusion, filtration, sonication, homogenization, repeated freeze and melt cycles, pressurization through fixed sized pores, and the like. However, it is generally most desirable to use the microspheres and foams of the present invention in the formed state as described later, without any attempt to modify their size.

Gas-filled vesicles can be sized by simple extrusion through a filter; the pore size of the filter controls the size distribution of the generated gas and gas precursor-filled vesicles. By using two or more filters in series, ie superimposed, eg 10 μ followed by 8 μ, the gas-filled vesicles have a very narrow size distribution centered around 7-9 μm. After filtration, these stabilized gas-filled vesicles remained stable for more than 24 hours.

Size sieving and filtration can be accomplished by use of a filter membrane when removing the suspension from the sterile tube prior to use, preferably by introducing the filter membrane into the syringe during use. A method of screening vesicles of a certain size comprising utilizing a syringe comprising a cartridge, at least one filter and a needle, and performing an expressing step comprising squeezing said vesicles from said cartridge The vesicles are passed through a filter installed between the barrel and the needle of the syringe to select vesicles of a certain size prior to application according to the invention to patients treated with vesicles as MRI contrast agents. The expressing step also includes extruding the vesicles into the syringe, wherein the filter membrane functions in the same way, screening vesicles of a certain size based on whether they can enter the syringe. Another alternative is to fill the syringe with vesicles which have been sized in other ways, in which case the function of the filter is to ensure that only the Vesicles in the desired size range or in the desired maximum size range are used for administration.

A typical device for performing the size screening and filtration steps is the syringe and filter combination shown in Figure 2 of U.S. Patent Application No. 08/401,974 (filed March 9, 1995) (which is incorporated herein by reference) ).

In a preferred embodiment, the stabilizing compound solution or suspension is squeezed through a filter and said solution or suspension is heat sterilized prior to shaking. Once the gas-filled vesicles are formed, they will be filtered to size as previously described. These steps prior to the formation of gas- and gas-precursor-filled vesicles can bring advantages, such as reducing the amount of non-hydrated stable compounds, thus providing significantly higher yields of gas-filled vesicles, as well as providing direct use in patients. Sterile gas-filled vesicles for administration. For example, mixing containers such as vials or syringes can be filled with filtered stabilized compounds, especially lipid suspensions, and the suspensions can then be sterilized in the mixing container, eg, by autoclaving. Gas can be injected into the lipid suspension by shaking the sterile container to form gas-filled vesicles. Preferably, the sterile container is provided with a filter to allow the gas-filled vesicles to pass through the filter prior to contacting the patient.

In the first step of this preferred method, the stabilized, especially lipid solution is squeezed through a filter to reduce the amount of non-hydratable compounds by breaking up the dried compounds and exposing a larger surface area for hydration. Preferably, the filter has a pore size of about 0.1 to about 5 μm, more preferably, about 0.1 to about 4 μm, even more preferably, about 0.1 to about 2 μm, most preferably, about 1 μm. Non-hydrated compounds, especially lipids, appear as amorphous lumps of non-uniform size and are undesirable.

The second step, sterilization provides a composition that can be administered directly to a patient in need of MRI imaging. Preferably the sterilization is accomplished by heat sterilization, preferably by autoclaving at at least about 100°C, more preferably, at about 100°C to about 130°C, still more preferably at about 110°C to about 130°C, still more preferably from about 120°C to about 130°C, most preferably about 130°C. Preferably, the heating is for about at least 1 minute, more preferably for about 1 to about 30 minutes, still more preferably for about 10 to about 20 minutes, and most preferably for about 15 minutes.

If desired, the order of the first and second steps outlined above can be reversed, or only one of the two steps applied.

Sterilization may follow the formation of gas-filled vesicles and is preferred when sterilization is performed by methods other than heat sterilization at temperatures that cause gas and gas precursor-filled microspheres to burst. For example, gamma rays may be used before and/or after formation of gas-filled vesicles. Applied Gas Precursor

In addition to the aforementioned schemes, gaseous precursors contained in lipid-based vesicles, which can be activated by temperature, light or pH, or other properties of the patient's tissue to which they are administered, can also be released from entrapped lipid-based vesicles. The liquid or solid within phase changes to a gaseous state, which expands to produce the stable gas-filled vesicles used in the present invention. This technique is described in detail in Patent Application Nos. 08/160232 and 08/159687, filed November 30, 1993, both of which are incorporated herein by reference in their entirety. The technique for making gas precursor-filled vesicles is generally similar to that described herein for making gas-filled vesicles, except that a gas precursor is substituted for the gas.

A preferred method of activating the gas precursor is temperature. Activation or transition temperature, and like terms, refers to the boiling point of a gas precursor at which a phase change of the gas precursor from a liquid to a gas phase occurs. Useful gas precursors are those gases having boiling points in the range of about -100°C to 70°C. The activation temperature is specific to each gas precursor. An activation temperature of about 37°C, or human body temperature, is preferred for the gas precursors of the present invention. In this way, the liquid gas precursor is activated to become a gas at 37°C. However, gaseous precursors can be used in the method of the invention in liquid or gaseous phase. The process of preparing the MRI contrast agents for use in the present invention can be carried out below the boiling point of the gas precursors so that the liquid is incorporated into the vesicles. Alternatively, the process can be performed at the boiling point of the gas precursor so that the gas is incorporated into the vesicles. For gaseous precursors with low boiling temperatures, liquid precursors can be emulsified with a microfluidic device cooled to cryogenic temperatures. The boiling point can also be lowered with a solvent in the liquid medium to form the use precursor as a liquid. The method can also be carried out with the temperature increased throughout the process, so that the process starts with a gaseous precursor of the gas and ends with the gas.

The gas precursors can be selected to form the gas in situ in the target tissue or fluid, ie, as it enters the patient or animal body, prior to use, during storage, and during manufacture. The method of producing temperature activated gas and gas precursor filled vesicles can be performed at temperatures below the boiling point of the gas precursor. In this scheme, the gas precursors are trapped inside the vesicles so that the phase transition does not occur during production. In practice, gas and gas precursor filled vesicles are produced in the liquid phase of the gas precursor. Activation of the phase transition can be performed at any time the temperature is allowed to exceed the boiling point of the precursor. Furthermore, given the amount of liquid within the droplet of the liquid gas precursor, the size of the vesicles after reaching the gas phase can be determined.

Alternatively, gaseous precursors can be used to create stable inflated cells that are pre-formed prior to use. In this scheme, the gaseous precursors are added to a vessel containing a suspending and/or stabilizing medium at a temperature below the liquid-gas phase transition temperature of the respective gaseous precursor. If the temperature is exceeded, an emulsion forms between the gas precursor and the liquid solution, and the gas precursor undergoes a phase transition from liquid to gas. As a result of heating and gas formation, the gas displaces the air in the headspace of the liquid suspension to form a gas that traps the gas precursor, the surrounding gas (e.g. air), or an aerated lipid that co-traps the gas phase gas precursor with the surrounding air ball. This phase transition may be suitable for optimal mixing and stabilization of the MRI contrast medium. For example, the gas precursor, perfluorobutane, can be trapped in biocompatible lipids or other stable compounds, and as the temperature rises above 4 °C (the boiling point of perfluorobutane), the stable compound traps Perfluorobutane gas is produced. As an additional example, the gaseous precursor perfluorobutane, can be suspended in an aqueous suspension containing emulsifiers and stabilizers such as glycerol or propylene glycol and vortexed with a commercial vortex machine. Vortexing begins at a temperature low enough that the gas precursor is a liquid, and continues as the temperature of the sample rises past the omnidirectional phase transition temperature from liquid to gas. In such operations, the precursors are converted to a gaseous state during microemulsification. In the presence of suitable stabilizers, surprisingly, stable inflated cells are produced.

Accordingly, the gas precursor may be selected to form inflated vesicles in vivo or designed to generate in situ inflated vesicles during manufacture, or upon storage, or at some time prior to use.

As another solution of the present invention, by pre-forming the liquid phase of the gas precursor into the water emulsion and maintaining a known size, the maximum volume of the gaseous microbubble can be determined by the ideal gas law once the phase is changed to gaseous state. For the fabrication of gas-filled vesicles from gaseous precursors, the gas phase is assumed to be formed instantaneously, and no newly formed vesicle gas is consumed due to diffusion into the liquid (usually naturally aqueous). Thus, from the known volume of liquid in the emulsion it is possible to predict the upper size limit of the inflated cells.

According to the present invention, emulsions of stabilizing compounds such as lipids, gaseous precursors, and microdroplets containing a liquid of fixed size can be formulated so that at a specific temperature, boiling point of the gaseous precursor, the microdroplets will expand into gas-filled vesicles of defined size. . The determined size represents an upper limit to a practical size, since factors such as diffusion of gas into solution, loss of gas to the atmosphere, and the effect of increasing pressure are factors that cannot be accounted for by the ideal gas law.

The ideal gas law and the equation used to calculate the increase in bubble volume at the phase transition from liquid to gas are as follows:

PV＝nRT among them

P = pressure in atmospheres

V = volume in liters

n = number of moles of gas

T = temperature in °K

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

From the volume, density and temperature of the liquid in the liquid emulsion, etc., the amount (eg, number of moles) of the liquid precursor can be calculated collectively - the volume of the precursor. When converted to a gas, it will expand into a vesicle of known volume. The calculated volume will reflect an upper bound on the size of the inflated cell, assuming instantaneous expansion of the inflated cell and negligible diffusion of gas during the time of expansion.

Thus, for the stabilization of precursors in the liquid state in an emulsion, where the precursor droplets are spherical, the volume of the precursor droplets is determined by the following equation:

Volume (sphere) = 4/3πr ³ where

r = radius of the ball

In this way, once the volume is predetermined, and the density of the liquid at the desired temperature is known, the amount of liquid (gas precursor) in the droplet can be determined. For further illustration, the following formula can be used to calculate:

V gas=4/3π(r _gas ) ³

by the ideal gas law

PV＝nRT

substitute

V _gas = nRT/P _gas

or

(A) n=4/3[πr _gas ³ ]P/RT

Quantity n=4/3[πr _gas ³ P/RT] ^* MWn

back to liquid volume

(B) V _liquid =[4/3[πr _gas ³ ]P/RT] ^* MWn/D]

where D = the density of the precursor

solution liquid droplet diameter

(C) Diameter/2＝[3/4π[4/3 ^* [πr _gas ³ ]P/RT]MWn/D] ^1/3

Simplified to

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

As an additional means of preparing vesicles of the desired size for use as an MRI contrast agent in the present invention, with a stable compound/precursor liquid microvolume and a specific half-diameter, an appropriately sized filter can be used to determine the gas precursor microsphere. The size of the droplet is a sphere of appropriate radius.

Representative gas precursors can be used to form vesicles of a defined size, eg, 10 [mu] diameter. Vesicles are formed in the human bloodstream in this example, so a typical temperature is 37°C or 310°K. At 1 atmosphere and using the equation in (A), 7.54 x 10 ^-17 moles of gas precursor are required to fill the volume of a 10 μ diameter vesicle.

Using the amounts of gas precursors calculated above, and 1-fluorobutane with a molecular weight of 76.11, a boiling point of 32.5 °C and a density of ^0.7789 g/ml at 20 °C, further calculations predict that for a vesicle of 10 μ 5.74 x 10 ^-15 g of this precursor. Extrapolating further, and using Equation (B) by density, it was further predicted that 8.47 x 10 ^-16 ml of liquid precursor was required to form vesicles up to 10 μ.

Finally, using equation (C), the preparation of gas precursor-filled vesicles up to 10 μ requires the formation of droplet emulsions with a radius of 0.0272 μ or the corresponding diameter of 0.0544 μ.

An emulsion of a specific size can be easily achieved by using an appropriately sized filter. In addition, as can be seen, the size of the filter that must be used to form the gas precursor droplets of a specified size is also sufficient to remove any possible bacterial contamination, and thus, can also be used as a sterile filter.

This protocol for the preparation of inflated balloons as non-invasive ultrasound contrast agents for simultaneous magnetic resonance imaging focus in the method of the invention can be used for all gaseous precursors activated by temperature. Indeed, lowering the freezing point of the solvent system allows the use of gaseous precursors that undergo liquid-gas phase transitions at temperatures below 0 °C. The solvent system can be selected to provide a medium for suspending the gaseous precursors. For example, 20% propylene glycol that is miscible in buffered saline exhibits a lower freezing point than water alone. The freezing point can be lowered further by increasing the amount of propylene glycol or adding substances such as chlorides.

Selection of an appropriate solvent system can be determined by physical methods and the like. When a substance, solid or liquid, referred to herein as a solute, is dissolved in a solvent, such as an aqueous-based buffer, the freezing point is depressed by an amount that depends on the solution composition. Thus, as defined by Wall, the freezing point depression of a solvent can be expressed by the following equation:

Inx _a ＝In(1-x _b )＝ _ΔHmelt /R(1/T ₀ -1/T)

in:

x _a = mole fraction of solvent

x _b = mole fraction of solute

_ΔHmelt = heat of fusion of the solvent

T ₀ = normal freezing point of the solvent

The normal freezing point of the solvent is generated by solving the equation. If x _b is relatively smaller than x _a , the above equation can be written as:

x ^b ＝ _ΔHmelt /R[T-To/ToT] _{≈ΔHmeltΔT} /RT ₀ ²

The above assumption that the change of temperature ΔT is less than T ₂ , the equation can be further simplified, assuming that the concentration of solute (in moles per kilogram of solute) can be expressed by weight molar concentration m, then x _b =m/[m+1000/m _a ]≈mMa/1000 where:

Ma = molecular weight of the solvent, and

m = molarity of solute in units of moles per 1000 grams.

Substitute the fraction x _b :

ΔT＝[MaRTo ² / _1000ΔHmelt ]m

or ΔT=K _f m, where

K _f = MaRTo ² /1000ΔH _melting

_Kf refers to the molar freezing point and is equal to 1.86 degrees per unit molarity for water at 1 atmosphere. The above equation can be used to accurately measure the molar freezing point of the gas precursor-filled vesicles used in the present invention.

Thus, the above equation can be used to estimate the freezing point depression and determine the appropriate concentration of liquid or solid solute required to lower the freezing temperature of the solvent to the appropriate value.

Methods of making temperature activated gas and gas precursor filled vesicles include:

(a) Vortex the aqueous suspension of gas precursor-filled vesicles used in the present invention; variables of this method include optionally autoclaving prior to shaking, optionally heating the aqueous suspension of gas precursor and lipid liquid, selectively open the container containing the suspension, selectively shake or allow the gas precursor vesicles to form spontaneously and cool the gas precursor-filled vesicle suspension, selectively mix the gas precursor and lipid water The suspension is squeezed through a filter of about 0.22 μ, alternatively, filtration can be performed during in vivo administration of the resulting vesicles, a filter of about 0.22 μ is applied;

(b) performing microemulsification, wherein an aqueous suspension of gas and gas precursor-filled vesicles of the invention is emulsified by agitation and heating to form vesicles prior to administration to a patient; and

(c) forming a suspension of the gas precursor in the lipid by heating, and/or agitating, wherein the low-density gas and gas precursor-filled vesicles float to the bottom of the solution by expanding and converting other vesicles in the container top, and leave the container open to release air; and

(d) In any of the above methods, a closed container is used to hold the gas precursor and the aqueous suspension of the stabilizing compound, such as a biocompatible lipid, said suspension being maintained below the phase transition temperature of the gas precursor, Subsequent autoclaving to move the temperature above the phase transition temperature, selective shaking, or spontaneous formation of gaseous precursor vesicles in which expansion of the gaseous precursor in a closed container increases the pressure in said container, cools the charge The vesicle suspension can then also be shaken.

Lyophilization can be used to remove water and organics from stable compounds prior to shaking gas injection. A dry-gas injection method can be used to remove water from the vesicles. By pre-entrapping the gas precursor in the dried vesicles (ie before drying), upon warming, the gas precursor will expand to fill the vesicles. Gas precursors can also be used to fill dry vesicles after they have been emptied. If the dry vesicles are kept at a temperature below their gel state to liquid crystal temperature, the dry cavity can be slowly filled with a gaseous precursor in the gaseous state, for example, perfluorobutane can be used to fill The acylphosphatidylcholine (DPPC) composition was dried between 4°C (the boiling point of perfluorobutane) and below 4°C, the phase transition temperature of biocompatible lipids. In this case, it would be most preferred to fill the vesicles at a temperature of about 4°C to about 5°C.

A preferred method for preparing temperature-activated gas precursor-filled vesicles involves shaking in the presence of a gas precursor at temperatures below the gel-to-liquid-crystalline phase transition of lipids with stable properties such as biocompatible lipids. Aqueous solutions of compounds. The present invention also contemplates the use of a method of preparing gas precursor-filled vesicles comprising shaking an aqueous solution containing a stabilizing compound, such as a biocompatible lipid, in the presence of a gas precursor, and isolating the resulting gas precursor for MRI imaging. Body-filled vesicles. The vesicles prepared by the method described above are referred to here as gas precursor-filled vesicles prepared by the gel-state shaking gas precursor injection method.

In general, prior art water-filled liposomes are routinely formed at temperatures above the phase transition temperature of the lipids used to make them because they are more elastic and thus liquid crystalline for use in biological systems. See, eg, Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. 1978, 75, 4194-4198. In contrast, vesicles fabricated according to the preferred protocol described herein are gas precursor-filled, which is more elastic because gas precursors are more compressible and tortuous than aqueous solutions after gas formation. Thus, gas precursor-filled vesicles can be used in biological systems when formed at temperatures below the phase transition temperature of lipids, although the gel phase is more rigid.

The method contemplated by the present invention provides agitation of an aqueous solution containing a stabilizing compound, such as a biocompatible lipid, in the presence of a temperature activated gas precursor. Agitation as used herein is defined as the movement of agitating an aqueous solution such that a gaseous precursor is introduced into the aqueous solution from the surrounding environment. Any type of motion that agitates the aqueous solution and results in the introduction of the gaseous precursor can be used for shaking. Shaking must be vigorous enough to form a suitable number of vesicles after a period. Preferably, the shaking is vigorous enough that vesicles are formed within a short period of time, such as 30 minutes, preferably within 20 minutes, more preferably within 10 minutes. Agitation can be by microemulsion, by microfluidization, eg, swirling, such as a vortex, side to side, or up and down motion. In case of addition of gaseous precursors in liquid state, sonication can be applied in addition to the shaking method proposed above. Also, different types of motion can be combined, shaking can be performed by shaking the container containing the aqueous lipid solution, or by shaking the aqueous solution in the container without shaking the container itself. Further, shaking can be manual or motorized. Mechanical shakers that can be used include, for example, shaker platforms such as the VWR Scientific (Crritos, CA) shaker platform, microfluidizers, Wig-L-Bug ^™ (Crescent Dental Manufacturing, Inc., Lyons, IL) , the latter has been found to give particularly good results, and mechanical paint mixers, as well as other known machines. Other means of generating agitation include the action of gaseous precursors emitted at high velocity or pressure. It should also be understood that, preferably, for larger volumes of aqueous solution, the total amount of water increases accordingly. Vigorous shaking is defined as at least about 60 shaking movements per minute and is preferred. An example of vigorous shaking, vortexing at at least 1000 rpm is more preferred. Vortex at 1800 rpm is most preferred.

Formation of vesicles filled with agitated gas precursors can be detected by the presence of foam on top of the aqueous solution. This is accompanied by a reduction in the volume of the aqueous solution due to foam formation. Preferably the final volume of the foam is at least about twice the initial volume of the aqueous lipid solution; more preferably the final volume of the foam is at least about three times the initial volume of the aqueous solution; still more preferably the final volume of the foam is at least about four times the initial volume of the aqueous solution; Most preferably all of the aqueous lipid solution is converted to foam.

The required period of shaking can be determined by monitoring the formation of foam. For example 10ml of lipid solution is vortexed in a 50ml centrifuge tube for about 15-20 minutes until the viscosity of the gas and gas precursor filled vesicles becomes thick enough that they no longer stick to the side walls when swirled. At this point, the foam will cause the solution containing the gas and gas precursor filled vesicles to rise to a level of 30 to 35 ml.

The concentration of stabilizing compound, especially lipid, required to produce the preferred level of foam will vary with the type of stabilizing compound used, such as biocompatible lipid, and once prepared as disclosed herein, will be readily determined by the skilled artisan. Determination. For example, in a preferred version, the concentration of 1,2-dipalmitoylphosphatidylcholine (DPPC) used to form vesicles filled with gases and gas precursors according to the desired methods of the invention is from about 20 mg/ml to About 30mg/ml saline solution. Distearoylphosphatidylcholine (DSPC) is used in a preferred embodiment at a concentration of about 5 mg/ml to about 10 mg/ml saline solution.

Specifically, DPPC at a concentration of 20 mg/ml to 30 mg/ml, upon shaking, produced a total suspension and trapped gas precursor volume four times greater than the volume of the suspension alone. DSPC at a concentration of 10 mg/ml, by shaking, yields a total volume completely free of any liquid suspension volume and containing all foam.

Those skilled in the art will appreciate that lipids and other stable compounds used as starting materials, or vesicle end products, may be manipulated before or after the methods of the present invention, provided they are prepared in light of the teachings herein. For example, stable compounds such as biocompatible lipids can be hydrated and then lyophilized, subjected to freeze and thaw cycles, or simply hydrated. In a preferred embodiment, the lipid is hydrated and then lyophilized prior to forming gas and gas precursor filled vesicles.

The presence of a gas such as but not limited to air, which is desired according to the present invention, may also be provided by the ambient atmosphere. The ambient atmosphere can be the atmosphere in a closed container, or in a non-closed container, can be the external environment. Also, for example, a gas may be injected or otherwise added to the container with the aqueous lipid solution or to the aqueous lipid solution itself to provide a gas other than air. Gases that are not heavier than air are added to closed containers, while gases heavier than air are added to closed or non-airtight containers. Accordingly, the present invention includes co-trapping of air and/or other gaseous precursors together.

As already stated above in the paragraphs relating to stabilizing compounds, the preferred process contemplated by the present invention is carried out at a temperature below the phase transition temperature from the gel state to the liquid crystal state of the lipid used. By "gel state to liquid crystal phase transition temperature" is meant the temperature at which the lipid bilayer will transform from the gel state to the liquid crystal state. See, eg, Chapman et al., J. Biol. Chem. 1974, 249, 2512-2512.

Thus, the stabilized vesicle precursors described above can be used in the same manner as other stabilized vesicles of the invention, once activated by application to patient tissue, where factors such as temperature or pH can be used to induce gaseous release. produce. Preferred is the embodiment wherein the gaseous precursor undergoes a phase change from a liquid to a gas at approximately normal body temperature of said patient and is activated by the temperature of said patient's tissue to change phase to a gas. More preferably, the patient tissue in the method is human tissue having a normal temperature of about 37°C, and wherein the gas precursor is at approximately 37°C normal temperature human tissue, and wherein the gas precursor undergoes transformation from a liquid to a gaseous state at approximately 37°C phase change.

All of the above protocols involving the preparation of stabilized gas and gas precursor filled vesicles for use in the present invention can be autoclaved or sterile filtered if these processes precede the gas infusion step or prior to the temperature mediated suspension Gas conversion of medium temperature sensitive gaseous precursors. Additionally, one or more antimicrobials and/or preservatives may be included in the formulation to stabilize the foam, such as sodium benzoate, all quaternary ammonium salts, sodium azide, methyl paraben, propyl paraben, sorbic acid, ascorbic acid Palmitate, Butylated Hydroxyanisole, Butylated Hydroxytoluene, Chlorobutanol, Dehydroacetic Acid, Ethylenediamine, Monothioglycerol, Potassium Benzoate, Potassium Metabisulfite, Potassium Sorbate, Sodium bisulfite, sulfur dioxide and organic mercury salts. Such sterilization, which may also be achieved by other conventional means such as radiation, will be necessary for use of stabilized microspheres in invasive situations such as intravascular or intraperitoneal use. Suitable sterilization means will be readily apparent to those skilled in the invention to which stabilized gas and gas precursor filled vesicles and their use are concerned. Usually the contrast medium is stored as an aqueous suspension, but in the case of dried vesicles or dried lipid globules, the contrast medium can be stored as a dry powder ready to be reconstituted prior to use.

The invention is further demonstrated in Examples 1-11 shown below. However, the examples provided do not limit the scope of the invention in any way.

Example

Example 1

Transferrin was bound to dextran and added to the iron salt solution. By dissolving a mixture of ferrous and ferric ions in water and hydrochloric acid at pH 1.0 in the chamber of an anaerobic environment of a Thermal System Probe (Heat System, Farmingdale, NY) sonicator equipped with a barometric chamber, And prepare the solution of superparamagnetic oxide. The sonicator was activated at medium/high power using a standard size electrode arm to abruptly increase the 0 pH to pH = 12 as oxygen bubbles through the solution. The result is nanoparticles of iron oxide _composed of magnetite, _Fe3O4 . Nanoparticles were washed in normal saline, and nanoparticles with diameters of 20 nm and smaller were harvested using differential centrifugation. These nanoparticles were suspended at a nanoparticle concentration of 10 mg/ml in n-hexane containing 10 mg/ml dipalmitoylphosphatidylcholine. The n-hexane was evaporated and the nanoparticles of iron oxide coated with phospholipids were lyophilized. Therefore, iron oxide nanoparticles were prepared, which were coated with transferrin-bearing dextran. Superparamagnetic oxide at a concentration of 10 mg/ml was mixed with 2 mg/ml perfluoropentane and 20 mg/ml pluronic F-68 containing 10 mg/ml dioleoylphosphatidylcholine Mix in sterile water containing 5.5% by weight mannitol. This was microfluidized and resulted in a colloidal suspension of perfluoropentane coated transferrin represented by magnetite particles. It was administered intravenously (dose = 5 ml) to a 25-year-old female patient suspected of having an ectopic pregnancy. Due to the binding of transferrin to placental tissue, magnetically labeled vesicles localize to ectopic pregnancy, observed by MRI. High-energy continuous wave ultrasound, 2MHz, 2.5 watts/cm is used for uterine placental tissue, the amount of sound wave energy absorbed due to vesicles is increased, and then the ultrasonic energy destroys the placental tissue of the uterus. This avoids the use of incisional procedures such as laparotomy or more invasive procedures such as laparotomy, as ultrasound therapy under magnetic resonance guidance is usually done percutaneously and surgical access is not necessary.

Example 2

In 10 mg/ml phospholipids (82 mol% DPPC, 7 mol% DPPE-PEG 5000 and 10 mol% DPPA and 1 mol% DPPE containing 20 mg/ml Pluronic F-68 and 5.5% by weight mannitol A colloidal suspension of perfluorohexane (0.2 mg/ml) and perfluoropentane (0.2 mg/ml) was prepared in -PEG 5000 anti-fibrin antibody). To this was added iron oxide nanoparticles at 5 mg/ml and the material was microfluidized as described in previous examples. The substance is administered intravenously to patients suspected of having vascular thrombosis. The patient's body was scanned with a Superconducting Quantum INFEROMETRY Device (SQUID) magnetometer, a type of magnetic imaging, to identify areas of markedly increased magnetic susceptibility in the patient's frontal vein. The presence of clots was confirmed by ultrasound imaging. High-energy ultrasound at 500 mW/ ^cm2 is then used to bind the clotted area of the vesicle. Sonication is done under the guidance of a SQUID or a magnetometer. Install the SQUID on the ultrasonic sensor as part of the sensor. When sonication occurs, a change in magnetic susceptibility can be detected with a magnetometer. Microvesicles lead to increased absorption of sound energy; phase transition of perfluorohexane from liquid to gas in emulsions is readily seen in ultrasound or magnetic imaging, as the vesicles expand during thermal expansion, leading to clots Lysis is localized and relieved using non-invasive surgical procedures to treat thrombus.

Example 3

Gas-filled microvesicles impregnated with paramagnetic oxide particles on the microvesicle membrane were injected into the antecubital fossa. The vesicles were first taken up by lymphatic vessels, identified as a clear outline of tumor-laden lymph nodes in magnetic resonance performed with a superconducting quantum INFEROMETRY device (SQUID). Based on the identification of the lymph nodes, serial ultrasound sequencing is then used under SQUID guidance using focused ultrasound transducers for identification of tumor-laden tumors. The microvesicles are prepared to resonate and release energy in the form of heat, thereby heating and subsequently destroying the tumor. Using the SQUID, again with MRI, imaging of tumor-laden tumors in the lymphatic system is no longer identifiable, indicating that the tumor has been destroyed, thereby improving non-invasive surgical techniques.

Example 4

Disclosed in U.S. Patent 5,312,617 by N,N'-bis-(carboxy-decylamidomethyl-N-2,3-dihydroxypropyl)-1,2-ethylenediamine-N,N'diacetic acid Manganese (MN-EDTA-DDP) and Mn-EDTA-ODP, a paramagnetic complex prepared gas-filled microvesicles (this document is incorporated herein by reference) which were injected into patients with malignancies middle. MRI was used to identify the precise size and location of the tumor in the lymphatic chain, and magnetic resonance imaging and magnetic resonance imaging-compatible transducers were simultaneously performed with ultrasound techniques, operating at 1.5 watts/ ^cm2 and a frequency of 0.75 MHz ongoing. The presence of air bubbles leads to increased energy deposition, heating of the tissue and localized cavitation. The degree of tissue necrosis as well as the temperature of the tissue is non-invasively monitored using simultaneous magnetic resonance imaging on a machine equipped with echoplanar gradients, thus enabling non-invasive surgery.

Example 5

For patients with cerebral arteriovenous malformations (AVM), a cranial shield is prepared and the dura is surgically exposed. Patients were placed on MRT-0.5 Tesla, Interferometric Magnetic Resonance Imaging Technology System, (GE Medical System, Milwaukee, WI). With such a magnetic resonance system it is possible to access a patient during a surgical operation with simultaneous magnetic resonance imaging. Patients were injected with 0.2 ml/kg of Aerosomes ^™ consisting of 2 mg/ml of lipid containing 75 mol% DPPC, 8 mol% DPPE-PEG 5000 and 8 ^mol % DPPA and 9 Mole % Alabaster Alabama trapped by a mixture of platelet activating factor (PAF), Avanti Polar Lipids, air and perfluorobutane gas. The purpose of using PAF is to activate platelets after release of PAF from Aerosomes ^(TM) in order to stimulate thrombus formation of AVM. During MR imaging, a high-energy MR-compatible ultrasound transducer equipped with imaging and therapeutic functions is positioned on the AVM. After intravenous injection of the Aerosomes ^(TM) , they pass through the large catheter and carry out the microcirculation of the AVM. Microbubbles are readily visible using magnetic resonance imaging techniques and ultrasound. On magnetic resonance imaging, microbubbles are described as signal voids on gradient-echo bright blood images obtained during transfer through the AVM, and on ultrasound they appear as snowstorm-like clear spot reflections. Focus the ultrasound transducer on the AVM so that the lesion area shown on ultrasound corresponds to the lesion of the vessel. Simultaneous magnetic resonance imaging and ultrasound are performed when the power level of ultrasound is increased (eg, up to several watts). Radiation from the cavitation obstructs visualization of much of the bone during ultrasound, and magnetic resonance still shows exceptionally detailed surrounding tissue. Surgeons who abnormally perform high-energy ultrasound can better control the target, shot, and energy level of the high-energy transducer and avoid damage to critical surrounding brain vascular structures. This step results in ablation of the AVM, coagulated areas of necrosis, and thrombus in the vascular lesion. At the end of this procedure imaging of necrosis, collection of air bubbles, most of the bone is hampered during ultrasound for intra-tissue coagulation, but magnetic resonance imaging shows the entire surgical field and the surgical response to the treated and surrounding organization's impact.

Example 6

Injured victims suspected of bleeding were scanned with MRI. A scan showed bleeding in the spleen. The Aerosomes used in the above examples were injected intravenously, except that the Aerosomes also captured 1 mg/ml thrombin, while simultaneous magnetic resonance imaging and ultrasound imaging were performed. As the Aerosomes pass through the arteries of the spleen, the surgeon increases the ultrasonic power of the magnetic resonance compatible ultrasound transducer to about 1.0 watts/ ^cm2 , and the Aerosomes burst open releasing PAF and thrombin. A thrombus was obtained and bleeding was stopped. Simultaneous magnetic resonance angiography confirmed splenic artery thrombus formation. This less invasive to minimal procedure is less expensive and causes less morbidity than conventional open surgery.

Example 7

Patients with breast cancer were given intravenous injections of 100 mL of perfluoropentane vesicles made of phospholipids, 82 mole % DPPC, 8 mole % DPPE-PEG 2000 and 10 mole % DPPA (average diameter = about 1 micron) Complexes (Mn-EDTA-ODP) with 10 mol% alkylated antibodies carrying manganese, anti-breast cancer (human) CA-15-3 monoclonal antibody, anti-human breast cancer IgG1 (SIGNET LABS, Dedham , MA) coating. The vesicles also contained 10 mole % of an alkylated derivative of doxorubicin bound to the vesicle membrane. Four hours later the patient was scanned with an MRI. Enhancing lymph nodes were identified in the axilla, indicating metastatic disease. A magnetic resonance-compatible 1 MHz ultrasound probe was positioned at the lymph node, and high-energy continuous ultrasound at 200 mW/ ^cm2 was applied to the lymph node. With a commercial magnet such as 1.5 Tesla (GE SigmaMilwaukee, WI), real time-synchronized magnetic resonance imaging is performed at a sharp rotation angle of 30° using a fast pulse sequence such as Spoiled GRASS, TR = 30 ms and TE = 5 ms surgery.

When the vesicles are controlled during heating, the vesicles can be seen on MRI as areas of increased low signal intensity, which correspond to regions of magnetic susceptibility induced by the vesicles. When the vesicles burst open, a more illustrative momentary region of subintensity is seen. Vesicles disappear after vesicle bursting and clearance. The doxorubicin prodrug is released and activated in neoplastic lymph nodes due to vesicle bursting.

Example 8

by dissolving a mixture of ferrous and ferric ion salts into water and pH 1.0 hydrochloric acid in the chamber of a Thermal System Probe (Heat System, Farmingdale, NY) sonicator equipped with a barometric chamber in an anaerobic environment. , to prepare superparamagnetic oxides. The sonicator was activated at medium/high power using a standard size electrode arm to raise the pH abruptly to pH=12 as oxygen bubbles through the solution. The result is nanoparticles of iron oxide _composed of magnetite, _Fe3O4 . Nanoparticles were washed in normal saline, and nanoparticles with diameters of 20 nm and smaller were harvested using differential centrifugation. These nanoparticles were suspended at a nanoparticle concentration of 10 mg/ml in n-hexane containing 10 mg/ml dipalmitoylphosphatidylcholine. The n-hexane was evaporated and the nanoparticles of iron oxide coated with phospholipids were lyophilized. Then 10% by weight milliwatt particles of iron oxide coated with phospholipids consisting of 82 mol% dipalmitoylphosphatidylcholine (DPPC), 8 mol% di Palmitoylphosphatidylethanolamine-PEG 5000 (DPPE-PEG 5000) and 8 mole % dipalmitoylphosphatidic acid (DPPA) and 2 mole % palmitoyl diamide dichloroplatinum derivative. Suspend a mixture of phospholipids, phospholipid-coated iron oxides, and palmitoylated prodrugs at a total concentration of phospholipids of 1 mg/mL in normal saline in a sealed sterile container with perfluorobutane in the entire space gas. Shaking the material in a Wig-L-Bug ^™ at 4200 rpm for 2 hours resulted in vesicles carrying the phospholipid-coated prodrug, decorated on their surface with iron liquefied gas nanoparticles. A 20 ml dose of vesicles (average diameter = about 2 microns, bubble concentration = 1 x ¹⁰⁹ /ml) was injected intravenously into the patient, and a rapid GRASS sequence was obtained using magnetic resonance imaging. Iron oxide-labeled vesicles are readily seen in magnetic resonance imaging techniques as magnetizers that cause regions of subintensity. Prodrug vesicle-based contrast agents were demonstrated to accumulate in areas of vascularized tumors involving lymph nodes and other tissues. 1 MHz ultrasound was used under magnetic resonance guidance as described above to burst the vesicles and achieve local drug release.

Example 9

An anti-myosin antibody, Chicken Muscle, Catalog No. 476123, (CALBIOCHEM, Jolla, CA) was conjugated to dipalmitoylphosphatidylethanolamine. 0.1 mg/ml of this was added to 5.5% by weight mannitol in sterile water at a concentration of 0.1 mg/ml perfluoropentane and 5 mg/ml of phospholipids (90 mol% DPPC and 10 mol% DPPA). The mixture was bubbled with oxygen and oxygen-17, then microemulsified using a microfluidizer (Microfluidics, Newton, MA) at 16000 psi for 20 rounds, resulting in a colloid of perfluoropentane carrying the emulsified antibody. 10 ml of this material were injected into patients suspected of myocardial infarction and magnetic resonance imaging was performed. On rapid GRASS magnetic resonance imaging it was demonstrated that the infarcted myocardium was a region of increased magnetic susceptibility. An MRI-compatible continuous ultrasound transducer was positioned in the myocardium, and the myocardium was treated with 0.100 W/ ^cm2 ultrasound energy. This causes the bubbles to pop and release oxygen locally to the ischemic tissue.

Example 10

Vesicles were prepared as described in Example 9, except that the cationic phospholipid DOTMA, N-[1(-2,3-dioleoyloxy)propyl]N,N'-trimethylammonium chloride was used instead of DPPA and replacing DPPC with DPPE. Colloidal particles of antibody-bearing perfluoropentane loaded with oxygen-17 gas were then prepared as described above. Then 10 mg/ml of DNA encoding the gene for vascular epidermal growth factor (VEGF) was added and the suspension was spun for 2 minutes at 4°C, low power. Magnetic resonance imaging techniques are performed in patients with suspected myocardial infarction. An area of low signal intensity was identified in the myocardium approximately 30 minutes after contrast administration. Focusing an MRI-compatible ultrasound transducer on the ischemic/infarcted myocardium region, echo-slab imaging using gradient echo was performed on a resonant gradient-equipped, (Advanced NMR, Woburn, MA), modified 1.5 The magnetic resonance system of Tesla (GE Sigma System, Milwaukee, WI)) performs real-time simultaneous magnetic resonance imaging technology, and the perfluoropentane microbubbles "pop". This results in local integration and local gene expression of the gene encoding VEGF in the infarcted/ischemic myocardium. New blood vessels proliferate in response to VEGF, and growth of new blood vessels is enhanced. This results in a healthier regional myocardium.

Example 11

Cations were prepared by headspace shaking of lipid 82 mol% DPPC, 7 mol% DPPE-PEG 5000 and a mixture of 5 mol% DPPA and 6 mol% DOTMA with a gas prepared from a mixture of perfluorobutane and oxygen-16 vesicle. After shaking, 100 mg/ml of DNA encoding the gene for VEGF was added and the mixture was spun slowly for 1 minute as disclosed in the previous example with Wig-L-Bug ^™ . This results in DNA uptake to the surface of the microvesicles. For visualization under MR, a higher number of vesicles is more necessary than magnetic labeling of vesicles, but vesicles were still detected on T2-weighted spin echo or fast spin echo or gradient echo pulse sequences. For diabetic patients with peripheral vascular disease involving lower endpoints, Magnetic Resonance Angiography (MRA), a type of magnetic resonance imaging technique, was performed using the Flight pulse sequence of 2D Time. Significant narrowing is shown in the popliteal artery. DNA-carrying microvesicles were administered intravenously and based on vesicle signals from MRI and ultrasound, high-energy ultrasound was used under the guidance of magnetic resonance imaging of the skin surface to focus the energy on the narrowing of the popliteal artery area. This results in localized vesicle trapping and gene release at the site of arterial narrowing. Local expression of VEGF promotes the formation of collaterals and new ducts, thereby improving blood flow to the distal leg.

All patents, patent applications and publications cited and described herein are hereby incorporated by reference in their entirety.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are within the scope of the appended claims.

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.