MXPA97006402A - Stabilized gas emulsion, containing phospholipides to increase the contrast of ultrason - Google Patents

Abstract

A composition forming a gas emulsion, comprising a dry, hollow, particulate, approximately microspherical material, permeated with a gas or a mixture of gases, which, in the solution in an aqueous liquid, forms a gas emulsion comprising a plurality of bubbles surrounded by a layer of at least a first and a second surfactant, wherein the first surfactant consists essentially of a phospholipid or a mixture of phospholipids having at least one acyl chain, which comprises at least 10 atoms of carbon and comprising at least about 5%, by weight / weight, of the total surfactant, and wherein the second surfactant may or may not be a phospholipid and is more soluble in water than the first surfactant equipment for preparing these microbubbles and methods to use the micro bubbles as cons agents

Description

STABILIZED GAS EMULSION. CONTAINING FQSFQLÍPIDQS TO INCREASE THE CONTRAST OF ULTRASOUND BACKGROUND OF THE INVENTION Field of the Invention The present invention includes a method for preparing stable, long-lived gas emulsions, to increase the contrast of ultrasound and other uses, and to the compositions of gas emulsions, thus prepared. BACKGROUND ART Ultrasound technology provides an important and cheaper alternative to imaging techniques that use ionic radiation. While many conventional imaging technologies are available, for example, magnetic resonance imaging (IF) imaging, computed tomography (CT) and positron emission tomography (PET), each of these techniques use extremely expensive equipment.

Likewise, CT and PET use ionic radiation. Unlike these techniques, ultrasound imaging equipment is relatively inexpensive. Also, ultrasound imaging does not use ionizing radiation. Ultrasound imaging makes use of differences in tissue density and composition, which affect the reflection of sound waves by these tissues. The images are especially sharp where there are different variations in tissue density or compressibility, such as in the tissue interface. These interfacial zones between solid tissues, the skeletal system and various organs and / or tumors, easily image by ultrasound. Therefore, in many imaging applications, ultrasound performs adequately without using agents that increase contrast; however, for other applications, such as the visualization of flowing blood, efforts have been initiated to develop such agents to deliver increased contrast. A particularly significant application for such contrast agents is in the area of perfusion imaging. Contrast agents for ultrasound can improve the imaging of blood flowing in the heart muscle, kidneys, liver and other tissues. This, in turn, will facilitate diagnostic research, surgery and therapy related to the tissues of the image. A contrast agent of a blood pool, will also allow the formation of images based on the content of the blood (for example in tumors and inflamed tissues) and will help in the visualization of the patients and the fetus, increasing only the maternal circulation . A variety of agents that increase the contrast of ultrasound have been proposed. The most successful agents generally consist of dispersions or small gas bubbles that can be injected intravenously. More typically, the bubbles are injected into the blood stream of a living body from which the image is to be formed. The bubbles then supply a physical object in the flowing blood, that is, of a different density and much greater compressibility than the fluid tissue and blood surrounding it. As a result, these bubbles can easily form images with ultrasound. To cross the blood vessels, the bubbles must be less than 10 μm in diameter and have been called microbubbles. These microbubbles can be formed into a liquid in a variety of different ways. Simple examples are vigorous agitation or forcing a gas into a liquid through a small hole. In the absence of additional ingredients, the gas will be in direct contact with the condensed medium (ie, the pure bubbles). However, such bubbles tend to shrink rapidly, due to the diffusion of gas trapped in the liquid surrounding it. In addition, "pure" microbubbles, as shown, produce adverse responses, such as complement activation (see, for example, K. A. Shastri et al. (1981) Undersea Biomed. Res., 18, 157). Attempts to prolong the life of the microbubbles to increase their usefulness have focused on the addition of stabilizing agents which can enclose the gas bubbles, slowing the diffusion of the gas in the liquid that surrounds it. Most microbubble compositions have failed to deliver the contrast enhancement that lasts even a few seconds, let alone talk minutes. This greatly limits its usefulness. The micro-bubbles, therefore, have been "constructed" in various ways in an attempt to increase their effective life of contrast enhancement. Several routes have been attempted, such as the use of gelatins or albumin microspheres, which are initially formed in the liquid suspension, and trap the gas during solidification. However, the solid phase covers that encapsulate the gases have generally proven to be too brittle or too permeable to gas to have a satisfactory life in vivo. Also, thick covers (for example, albumin, sugar or other viscous materials) reduce the compressibility of the bubbles, thus reducing their echogenicity during the short time that may exist. The solid particles or liquid emulsion droplets that produce gas or boil when injected (as in patent PCT / US94 / 00422) of Quay) have the danger of supersaturating the blood with gas or steam. This will lead to the formation of a small number of large bubbles that produce embolisms at the few nucleation sites available, rather than the large number attempted of small bubbles. In addition, the bubbles created in vivo, in this way, will be "pure" and, consequently, will have the problem of complement activation, described above. The use of surfactants as stabilizers for gas bubble dispersions has also been explored. Surfactants are materials that tend to form an interfacial layer at the interface of the polar substance with a non-polar substance. Its "superficial active" behavior comes from the existence of both a hydrophilic region (which often includes an end which is usually referred to as the "head"), which tends to associate with the polar substance, as a hydrophobic region. (which often includes the other end, which is usually referred to as the "tail"), which tends to associate with the non-polar substance. When it stabilizes, the interfacial layer affects the characteristics of the polar / non-polar interface. When the tenso-active agents are present, the gas can be separated from the liquid by an interfacial layer, which can comprise a wide variety of surfactant materials. Some contrast-enhancing agents, which contain the surfactant, trap gas bubbles in another manner, for example, in an aqueous liposome core. These liposomes are more or less spherical "pouches" comprised of an aqueous core limited by one or more closed, concentric, bimolecular phospholipid layers. These phospholipids, which are natural components of cell membranes, are also well known for their surface-active properties. In the U. A. patent, No. 5,334,381 to Unger, liposomes containing gas bubbles are created by means of several different mechanisms. Likewise, the patent of E. U. A., No. 4,900,540 of Ryan et al. , reveals phospholipid liposomes containing a gas or a gas precursor. Presumably, gas bubbles trapped within the liposomes slowly escape outward, thus increasing the effectiveness of the contrast agent. It can be noted that this use of a surfactant does not involve the presence of an interfacial layer of surfactant at the gas / liquid interface. Rather, small gas bubbles are trapped in a larger volume of aqueous liquid, which binds itself to the uni- or multi-lamellar liposomal structure.

Contrast agents containing surfactant materials may use liposomes in other ways. For example, in the patents of E. U. A., Nos. 5,380,519 and 5,271,928 of Schneider et al. , microbubbles prepared from freeze-dried liposomes are described. According to this description, the reconstitution in water of the dry pulverulent formulation, created by lyophilization, of a liposome suspension, creates a dispersion of gas bubbles in suspension, with the liposomes filled with water. The microbubbles, thus prepared, are indicated will be surrounded by a "rather evanescent" envelope of the surfactant. Although it will generally be expected that such a layer of evanescent surfactant will not have persistence, and that these microbubbles, therefore, will not be stable for a prolonged period of time, Schneider et al. , theorizes that the surfactant laminated in or from the neighboring liposomes filled with water, stabilizes the gas present in the system in the form of microbubbles. It will be readily appreciated that a liposome-dependent, contrast-enhancing agent requires the prior formation of the liposomes, and, therefore, limits the main component of the stabilizing surfactant to a type which is capable of forming liposomes. Also, the preparation of liposomes involves sophisticated and time-consuming manufacturing. Even in the presence of stabilizing compounds or structures, the trapped gases are under an increased pressure in the bubble due to the surface tension of the surfactant that surrounds it, as described by the Laplace equation (? P = 2? / R) . This increased pressure rather facilitates the shrinkage and disappearance of the bubble as the gas moves from a high pressure area (in the bubble) to a lower pressure environment (in any surrounding liquid, which is not saturated with gas at this high pressure, or within a larger diameter bubble, of lower pressure). A purpose to deal with these problems is delineated in patent PCT / US92 / 07250 of Quay. Quay forms bubbles using selected gases, which are gaseous at body temperature (37 ° C) and have a reduced water solubility, high density and reduced gas diffusivity in solution, compared to air. Although the solubility in water and reduced diffusivity can affect the regime at which the gas leaves the bubble, numerous problems exist with the Quay bubbles. The formation of bubbles of sufficiently small diameter (for example 3-5 μm), requires a high input of energy. This is a disadvantage in that sophisticated bubble preparation systems must be supplied at the site of use. Also, the Quay criteria for gas selection are incorrect in that they fail to consider certain major causes of bubble shrinkage, ie, the effects of the surface tension of the bubbles, the effects of the surfactants and osmotic agents of the gas. , and these errors result in the inclusion of undesirable gases and the exclusion of certain optimally adequate gases. Accordingly, there is a need in the art for compositions, and a method for preparing these compositions, which provide, or utilize, a contrast-enhancing, long-lived agent that is biocompatible, is easily prepared and provides an increase in contrast superior in the formation of ultrasound images. SUMMARY OF THE INVENTION According to the present invention, a gas emulsion ultrasound contrast enhancement means is provided, which incorporates a mixture of surfactants as bubble stabilizing agents. At least one such surfactant is a hydrophobic phospholipid or a mixture of phospholipids. At least one second surfactant is supplied, which may or may not also be a phospholipid or a mixture of phospholipids, but which is more hydrophilic than the phospholipid or the combination of phospholipids provided as the first surfactant. Such a gas emulsion stabilized with phospholipids has a prolonged longevity in vivo. In an embodiment of the present invention, a gas emulsion composition is prepared by first dispersing, in an aqueous solution, a hydrophilic monomer or polymer or combinations thereof, a first and a second surfactant and an inflation agent. The first surfactant is a phospholipid or a mixture of phospholipids having at least one acyl chain comprising at least 10 carbon atoms and including at least 5% w / w of the total surfactant, and the second surfactant is more soluble in water than the first surfactant. The dispersion is then spray dried to evaporate the inflation agent and create an approximately microspherical, particulate, hollow, dry material. This dry particulate material is exposed to at least a first gas and then can be dissolved in an aqueous liquid, thus forming an aqueous gas emulsion composition, where this composition comprises gas bubbles surrounded by a layer of the first and second surfactants, whose stability is independent of liposomes. The second surfactant may be comprised of a wide variety of materials. Specific examples include fatty acids, fatty acid salts, sugar esters of fatty acids, polyoxypropylene-polyoxyethylene copolymers, non-ionic alkyl glucosides and polysorbates. Especially suitable gas emulsions are prepared when the second surfactant comprises a phospholipid or a mixture of phospholipids having one or more acyl chains, wherein each acyl chain comprises no more than 14 carbon atoms. The hydrophilic monomer or polymer or combinations thereof, may be a starch. The gas or combination of gases, which permeate the dry particulate material can also be selected from a wide variety of substances, including air, nitrogen, carbon dioxide or other gases normally present in the blood, and can also be selected from a wide variety of substances, including air, nitrogen, carbon dioxide, or other gases normally present in the blood, and may also be an organic material, such as a fluorocarbon. Preferably, one of the gases provided has a vapor pressure of less than 760 mm Hg at 37 ° C. A particularly preferred embodiment uses nitrogen saturated with perfluorohexane. The present invention also includes the containers of the gas emulsion-forming compositions, of gas-permeated dry particulate material, and methods of imaging an object or body part or body cavity, by the introduction of an emulsion composition. of gas containing a phospholipid within the object or part of the body or body cavity, and forms the image of at least a portion of the body by ultrasound. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view of a two-chamber flask containing a microbubble-forming preparation, with an aqueous solution in an upper chamber and solid and gaseous ingredients in the lower chamber. Figure 2 illustrates the bottle of Figure 1, where the aqueous solution has been mixed with the solid ingredients to form microbubbles for administration to a patient. Figure 3 is a perspective view of an inverted two chamber flask, containing a microbubble-forming preparation, with an aqueous solution in the lower chamber and solid and gaseous ingredients in the upper chamber.

Figure 4 illustrates the bottle of Figure 3, where the aqueous solution has been mixed with the solid ingredients to form microbubbles for administration to a patient. Detailed Description of the Invention As used herein, microbubbles are considered gas bubbles in an aqueous medium having a diameter between about 0.5 and 300 μm, preferably having a diameter no greater than about 200, 100 or 50 μm. The microbubbles may or may not have a layer or coating at a gas / liquid interface. If present, the coating may be one or more molecules thick. Additionally, the microbubbles can be trapped by a bimolecular layer (as in the case of uni-lamellar liposomes) or they can be trapped by several layers of bilayer type (multilamellar vesicles). These microbubbles may also be surrounded by more permanent shell-like structures, such as denatured proteins. Since emulsions are generally defined as a dispersion of two or more immiscible fluids stabilized by a surfactant interface, the gas dispersions of the present invention are, in essence, gas emulsions, with the discontinuous phase of the emulsion being a gas, rather than a liquid. Accordingly, the term gas emulsion, as used herein, means a dispersion of a plurality of gas microbubbles in an aqueous medium. For intravascular use, the optimal bubble size is determined by two competing interests. Minor bubbles are effective in circulating through small blood vessels and capillaries, but the echogenicity of ultrasound is strongly dependent on the size of the bubbles. Microbubbles suitable for increasing the contrast of the vascular ultrasound are, therefore, preferably about 1-10 μm in diameter, with 3-5 μm being preferred. The present invention provides a gas dispersion or emulsion, in which the bubbles have a prolonged longevity in vivo, and which is suitable for use as agents that increase the contrast of ultrasound or magnetic resonance imaging (MRI). Typical agents that increase the contrast of ultrasound exhibit a potential for contrast enhancement by only about one pass through the arterial system, or a few seconds to about a minute, and thus do not survive by passing the aorta in a patient, in followed by intravenous injection. In comparison, the contrast agents, prepared according to the present invention, continue to show lives of contrasts increases measured by multiple passages through the entire circulatory system of a patient, following the intravenous injection. The lives of bubbles of several minutes is easily demonstrated. Such lengthening of the contrast enhancement potential, during ultrasound is highly advantageous. In addition, agents that increase the contrast, according to the invention, provide a superior image formation; for example, clear, vivid and distinctive images of the blood that flows through the heart, liver and kidneys, are achieved. Thus, non-toxic doses can be administered in a peripheral vein and used to augment images of the entire body. While the bubbles have been shown to be the most efficient ultrasound dispersants for use in intravenous ultrasound contrast agents, their main practical drawback is the extremely short life time of small bubbles in suspension. (typically less than 5 microns in diameter), required to pass through the capillaries. This short life time is caused by the increased gas pressure inside the bubble, which results from the surface tension forces on the bubble. This elevated internal pressure increases as the diameter of the bubble is reduced. This internal pressure of the augmented gas forces the gas inside the bubbles to dissolve them, which results in the crushing of the bubbles as the gas is forced into solution. The Laplace equation,? P = 2? / R (where? P is the gas pressure increased inside the bubble,? Is the surface tension of the bubble film and r is the radius of the bubble), describes the pressure exerted on a bubble of gas by the surface or film that surrounds the bubble. The Laplace pressure is inversely proportional to the radius of the bubble; thus, as the bubble shrinks, the pressure of Laplace increases, increasing the rate of gas diffusion out of the bubble and the rate of shrinkage of the bubble. In one embodiment, the present invention contemplates gas dispersion compositions containing phospholipid surfactants having certain advantages over other surfactants and other compositions containing phospholipids. In a preferred embodiment, the composition includes two or more surfactants, which are selected to assist in the creation of a larger number of microbubbles and also to optimally reduce the surface tension at the gas / liquid interface) of the bubbles with the liquid . In addition, gases of low water solubility can advantageously constitute a part of the gas in the microbubbles. It has been found especially advantageous to use, in conjunction with a second surfactant agent of greater water solubility, as a stabilizer combination of surfactant materials to improve entrapment of the gas. The stability of a gas emulsion is highly dependent on the properties to decrease the surface tension of the surfactant used as the emulsifying agent. Phospholipids, as they are known to function as the main component of the surfactant in the lung, are extremely efficient in this regard. They also readily form lamellar structures, such as bilayer sheets and liposomes, although this feature is not necessary to stabilize the gas dispersions of the present invention. Another determinant of the stability of the gas emulsion is the gas itself and the ability to stabilize by means of an osmotic effect, as described below. This combination results in a surprisingly stable and practically useful microbubble. The gas dispersion compositions, according to the present invention, can be prepared by spray drying an aqueous dispersion of a first surfactant, comprising a phospholipid, preferably at least one additional surfactant co-agent (also referred to herein as the "second surfactant") and a hydrophilic monomer or polymer, or combinations thereof. The aqueous starting material may optionally include salts and / or an inflation agent. Spray drying of such solution, according to the present invention, results in the production of an approximately microspherical, particulate, hollow, dry material. It has been surprisingly discovered that a spherical cavity, previously formed, which includes water-soluble components (for example hydroxyethyl starch, and its salts) and a surfactant relatively soluble in water (for example Pluronic F-68, Tween 20, dioctonyl phosphotidyl choline) and a phospholipid (eg, egg yolk phospholipid) when in the physical form produced by spray drying, can form remarkably stable microbubbles when rehydrated. The surfactant does not need to be present in the liposomal or other laminar form. This can be the result of the water first making contact with the inner surface of the spherical cavity (0.5-10 microns in diameter), after filtering through the dissolving surfactants and the structural agents, which result in the formation of a bubble of the desired size (the size of the cavity) initially surrounded by a saturated surfactant solution and, therefore, having a surfactant coating packed optimally. These bubbles are remarkably stable in vivo, even when filled with water soluble gases (eg air or nitrogen). This process, like the dry and reconstituted products thus obtained, is explained and described in more detail below.

I. Preparation of a Precursor Containing Phospholipid Dispersion: For subsequent spray drying, a first aqueous solution containing a hydrophobic phospholipid was prepared as a first surfactant and at least one additional hydrophilic additional surfactant. Preferably, the hydrophobic phospholipid has at least one acyl chain with a total of at least 10 carbon atoms (for example a decanoyl phospholipid). In some embodiments, the first phospholipid surfactant has acyl chains of about 10 or 14 to about 20 or 24 carbon atoms. For example, dipalmitoylphosphatidylcholine (comprising two acyl chains, each with 16 carbon atoms) can be used. The chain of. Acyl can be hydrogenated or fluorinated. Other phospholipid head groups are also considered. For example, phosphatidylserines, phosphatidylglycerols or phosphatidylethanolamines will have suitable properties for the present invention. Combinations of these phospholipids may also comprise the "first surfactant", as may be products of naturally derived phospholipids, such as egg or soy lecithin or lung surfactants. In addition, the first phospholipid surfactant can be supplemented with other water-insoluble surfactants, such as the di-tri- and tetra-esters of sucrose. Cholesterol can also supplement the first surfactant and has been found useful in promoting stability when supplied in a range of about 0.01 to 0.5 w / w of cholesterol to the phospholipid. Preferably, the acyl chains of the phospholipid are saturated, although unsaturated acyl groups are also within the scope of the present invention. The first surfactant is preferably provided in the range of about 0.005 to 20% w / v of the solution, more preferably in the range of 0.02 to 10% w / v. The primary role of the first hydrophobic surfactant is to reduce the surface tension of the microbubbles formed below the equilibrium values. When relatively insoluble osmotic stabilizing gases are trapped (described in detail below), a first surfactant with very little water solubility is required, since the reduction of the surface tension below the equilibrium values is only possible when the surfactant agent diffuses more slowly than trapped stabilizing gas. To achieve suitably low surfactant solubilities, phospholipids with long acyl chains (ie, comprising more than 10 carbon atoms) are particularly preferred. The second surfactant is preferably more hydrophilic and diffuses faster than the long chain phospholipid comprising the first surfactant. The role of this second surfactant is that the formation of a stable gas dispersion is probably related to a faster dissolution rate in reconstitution with water and more effective entrapment of the gas, thus facilitating the creation of bubbles in the early stages of reconstitution, as also described below. In this way, the faster diffusion regime of the second surfactant helps in creating a relatively durable and continuous film, which surrounds the gas in the reconstitution. In the present invention, the second preferred surfactants can be selected from the group consisting of phospholipids, phosphocholines, lysophospholipids, nonionic surfactants, neutral or anionic surfactants, fluorinated surfactants, which may be neutral or anionic, and combinations of such emulsifying or foaming agents. Some specific examples of surfactants, which are useful as the second surfactants, include the polyoxypropylene and polyoxyethylene block copolymers (an example of such a class of compounds is Pluronic, such as Pluronic F-68), sugar esters, fatty alcohols, aliphatic amine oxides, aliphatic esters of hyaluronic acid, salts of aliphatic esters of hyaluronic acid, dodecyl-poly (ethyleneoxy) ethanol, nonylphenoxy-poly (ethyleneoxy) ethanol, derived starches, fatty acid esters of hydroxyethyl starches, salts of fatty acids, commercial food vegetable starches, dextran fatty acid esters, sorbitol fatty acid esters, gelatin, serum albumins and combinations thereof. The polyoxyethylene fatty acid esters, such as polyoxyethylene stearates, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol-polyethylene glycol oxystearate, glycerol ricinoleate, are also considered as the second surfactant. polyethylene glycol, ethoxylated soy sterols, ethoxylated castor oil and their hydrogenated derivatives. In addition, nonionic alkyl glucosides, such as Tweens®, Spans® and Brijs® are also within the scope of the present invention. Spans include sorbitan tetraoleate, sorbitan tetrastearate, sorbitan tristearate, sorbitan tripalmitate, sorbitan trioleate and sorbitan distearate. Tweens include polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan tripalmitate, polyoxyethylene sorbitan trioleate. The Brij family is another useful category of materials, which include polyoxyethylene stearyl ether 10. Anionic surfactants, particularly fatty acids (or their salts) having from 8 to 24 carbon atoms, can also be used. An example of a suitable anionic surfactant is oleic acid, or its salt, sodium oleate. Cationic surfactants and their salts, such as dodecyltrimethylammonium chloride, are also considered for use as second surfactants. It will be appreciated from the foregoing that a wide range of second surfactants can be used. In fact, virtually any surfactant (including those to be developed) of higher solubility and diffusivity in water than is typical of a longer chain phospholipid, which comprises the first surfactant, can be used in the present invention. . The optimal surfactant for a given application can be determined through empirical studies that do not require undue experimentation. Accordingly, a practice of the art of the present invention should select the surfactant based on such properties as biocompatibility. It has been found advantageous to use as a surface-active co-agent a shorter chain phospholipid, which is more hydrophilic than the first phospholipid. As a specific example, a first phospholipid having acyl chains with 12 or 14 carbon atoms, can be provided with a second phospholipid as a surfactant co-agent, having acyl chains with eight to ten carbon atoms. It has been found especially advantageous to provide a phospholipid comprising acyl chains of 12 carbon atoms as the first or second surfactants. For example, a phospholipid with acyl chains of 12 carbon atoms may comprise the first surfactant, and a sugar ester or pluronic compound, may comprise the second surfactant. As another option, a phospholipid with acyl chains of 16 carbon atoms, can comprise the first surfactant, and a phospholipid with acyl chains of 12 carbon atoms can comprise the second surfactant.

Furthermore, in order to have an excellent bubble formation and persistence quality, the microbubbles formed with the phospholipids, for both the former and the surfactant co-agent, have superior properties in the area of metabolic elimination after injection in vivo, as well as that minimizes unwanted responses in vivo, such as complement activation, which may be a problem with the microbubbles of the prior art. Gas emulsion compositions containing the phospholipid surfactants, with acyl chains of 12 or 14 carbon atoms, appear to be especially advantageous in appearance. It is believed that microbubbles containing the phospholipid are not only more biocompatible than those containing tenso-active agents without phospholipids, but are also more biocompatible than liposomes. That is, they apparently evacuate the reitculoendothelial system more effectively than liposomes, and thus are not cleared from the circulation so quickly. As noted with respect to the first surfactant, the second surfactant co-agent may comprise combinations of the surfactants described above. Preferably, prior to spray drying, the second surfactant is supplied in the range of 0.005% to 20% by weight / volume. The first surfactant is not required to predominate the mixture. Any of the first or second surfactants can be provided in higher molarity and / or weight. In general, the total surfactant in solution is approximately 0.01% to 20% by weight / volume of the solution. Following the production of the aqueous surfactant solution, as described above, an inflation agent, preferably a fluorocarbon, such as Freon 113, is added, creating a coarse suspension. This inflation agent can be any material that becomes gas during the spray drying process. This inflation agent is then dispersed through the surfactant solution, for example, a commercially available microfluidizer, at a pressure of 350 to 1050 kg / cm 2. In a preferred embodiment of the present invention, a high pressure homogenizer is used to obtain a conventional Freon 113 emulsion in a solution of the surfactant containing the phospholipid. This process forms a conventional emulsion comprising sub-micron drops of Freon not miscible with water, coated with a monomolecular layer of the surfactant. Dispersion with this and other techniques is common and well known to those skilled in the art.

The inclusion of an inflation agent in the solution to be spray dried results in a higher ultrasound signal per gram of the spray-dried powder, forming a larger number of hollow microspheres. The inflation agent nucleates the bubble formulation in the stream within the atomized droplets of the solution entering the spray dryer, as these droplets are mixed with the stream of hot air within the dryer. Suitable inflating agents are those which supersaturate the solution within the atomized droplets with gas or vapor, at the elevated temperature of the drying droplets (approximately 100 ° C). Suitable agents include: 1. Solvents of low boiling point (below 100 ° C) dissolved, with limited miscibility with aqueous solutions, such as methylene chloride, acetone and carbon disulfide, used to saturate the solution at room temperature . 2. A gas, for example CO2 or 2, used to saturate the solution at room temperature and high pressure (for example at 3 bars). The droplets are then saturated with the gas at 1 atmosphere and 100 ° C. 3. Emulsions of liquids of low boiling point (below 100 ° C) immiscible, such as Freon 113, perfluoropentane, perfluorohexane, perfluorobutane, pentane, butane FC-11, FC-11B1, FC-11B2, FC-12B2, FC-21, FC-21B1, FC-21B2, FC-31B1, FC-113A, FC-122, FC-123, FC-132, FC-133, FC-141, FC-141B, FC -142, FC-151, FC-152, FC-1112, FC-1121 and FC-1131. The blowing agents are added to the surface active agent solution in amounts of about 0.5% to 10% by volume / volume of the surfactant solution. Approximately 3% volume / volume of the inflation agent has been found to produce a spray-dried powder that forms suitable microbubbles. This inflation agent is substantially evaporated during the spray drying process and thus is not present in the final spray-dried powder in more than trace quantities. The aqueous precursor solution preferably includes a hydrophilic monomer or polymer or combinations thereof. This may be combined with the surfactant solution or, more preferably, formed as a separate solution and combined with the surfactant precursor solution just before spray drying. The hydrophilic part can, for example, be a carbohydrate, such as glucose lactose or starch. Polymers, such as PVA or PVP are also considered for use with the present invention. Various starches and derived starches have been found especially suitable. Particularly preferred starches for use in the formation of microbubbles include those with a molecular weight greater than about 500,000 daltons or an equivalent dextrose (DE) value of less than about 12. The value of the DE is a quantitative measurement of the degree of hydrolysis of the starch polymer. It is a measure of reducing energy compared to a dextrose standard of 100. The higher the ED, the greater the extent of starch hydrolysis. Such preferred starches include food grade vegetable starches, of the type commercially available in the food industry, including those sold under the N-LOK and CAPSULE trademarks by National Starch and Chemical Co., (Bridgewater, NJ); derived starches, such as hydroxyethyl starch (available under the HETASTARCH and HESPAN trademarks of Du Pont Pharmaceuticals, M-hydroxyethyl starch from Ajinomoto, Tokyo, Japan). (Note that spray-dried short-chain starches can be used to produce microbubbles, but they are not preferred, because they have a molecular weight less than about 500.00 and do not stabilize the microbubbles. in the present invention in applications where additional stabilization is not required.) The hydrophilic monomer or polymer is present in this embodiment of the precursor solution in the approximate range of 0.1 to 10% w / v of the solution, with approximately 1 at 5% weight / volume having been found especially suitable. Other optional components of this embodiment of the precursor solution are various salts or other agents within the aqueous phase. These agents may advantageously include conventional viscosity modifiers, regulators, such as phosphate regulators or other conventional biocompatible regulators or pH-adjusting agents, such as acids or bases, osmotic agents (to deliver isotonicity, hypermolarity or hypomolarity). ). Preferred solutions have a pH of about 7 and are isotonic. These additional ingredients typically each comprise less than 5% w / v of the solution. Examples of suitable salts include sodium phosphate (both monobasic and dibasic), sodium chloride, calcium phosphate and other physiologically acceptable salts.

II. Spray-drying The emulsion of the surfactant / blowing agent is preferably combined with a solution of the hydrophilic monomer / polymer and its salts, of the type described above, and spray-dried to form a powder of approximately hollow, dry microspherical structures.

Commercially available spray dryers are well known to those skilled in the art, and suitable adjustment for any particular precursor solution can be easily determined through the standard empirical test, with reference to the following examples. The "Niro Portable Spray Dryer", employed in the following Examples I-VII and IX-XII, works by atomizing a solution containing a surfactant agent with a two-fluid compressed air nozzle, which uses a high-pressure jet. compressed air velocity to break the aqueous solution of the surfactant into droplets ranging from 2 to 20 microns in diameter. These droplets are then injected into a stream of hot air (typically 200 to 375 ° C) at the top of the drying chamber. The droplets of the surfactant solution are heated almost instantaneously to their boiling point of about 100 ° C. Although the evaporative coating prevents them from rising to a higher temperature, the temperature is even higher than the glass transition temperature of the phospholipids and above the melting point of many other surfactants, such as Poloxamer 188 and the stearate saccharose. The water on the surface of the drop evaporates very quickly, causing a formation of the compounds that dissolve in the atomized solution. When the solution contains a hydrophilic polymer, such as hydroxyethyl starch (HES), a gel layer is formed on the surface. Beneath the gel layer, a vapor bubble forms that inflates the gel sphere. As briefly described above, the presence of an inflating agent comprising a volatile, sparingly soluble solvent, such as methylene chloride or a volatile non-miscible solvent, such as Freon 113, provides nucleation sites for the rapid formation of bubbles of steam, which lead to a greater inflating of steam and hollow spheres of thinner walls. During the drying process, the water or migrates through the pores in the gel layer to the surface of the sphere as a liquid, where it vaporizes or escapes from the sphere as vapor, through the same pores of the layer gel. Finally, the water trapped in the HGES gel evaporates. During this phase of the drying process, the evaporation and evaporative cooling are slower, and the sphere rises in temperature to correspond to the exit temperature of the spray-drying chamber, typically at 100 to 120 ° C. The gel sphere shrinks as it is dewatered to provide hollow porous spheres of approximately 1 to 10 μm in diameter, with coating thicknesses of approximately 0.2 μm. The exhaust air stream from the spray dryer brings the spheres to a cyclone separator, where the powder is separated from the air stream by centrifugal force and directed into the product container. For various reasons, the composite structure of the dry spherical composition of the surfactant / polymer agent is characterized as random with the substantial absence of lamellar forms. First, homogenization to form an emulsion with an inflation agent, before spray drying deposits the surfactant into a mono-molecular layer. Because the drying takes place in a small fraction of a second, the rapid trapping of the surface active agent in a concentrated polymer gel (for example the HES), keeps the surfactants approximately in the physical state they were before drying. Also, during the spray drying process, the surfactants less soluble in water (sucrose stearate, long chain phospholipids, etc.) are promoted above their melting point or glass transition temperature, in the presence of a more water-soluble surfactant, and thus incorporated into the hydrophilic polymer matrix. This leads to structures of more random surfactants. This spray-dried composition, comprising hollow microspheres filled with gas, is an important product of the present invention. This product provides significant advantages over the precursors containing the lyophilized liposome. It is believed that the spherical structure of the present microbubble precursor material serves to rapidly and uniformly form the relatively non-compressible, relatively insoluble non-Newtonian surfactant film which is characterized by the preferred microbubbles of the present invention. After completing spray drying, the microspheres are packed in a container with an appropriate gas. This gas fills the microspheres and becomes the gas trapped in the microbubbles after reconstitution. The various individual components of the microspheres preferably comprise the following proportions of the spray-dried final product, in% by weight: First surface active agent of phospholipid 0.05% to 90% Second surfactant agent 0.05% to 90% Hydrophilic structural material 1% to 99% Salts, regulator, etc. 0% to 90% In particularly preferred embodiments, the composition has the following proportions, in% by weight: First surfactant phospholipid agent 0.1% to 10% Second surfactant agent 0.1% to 10% Hydrophilic structural material 10% to 60% Salts, regulator, etc. 10% to 60% More preferably, the amount of the first tenso-active agent (advantageously a phospholipid) is at least 1%, preferably at least 3%, 4% or 5% and more preferably at least 7%, % or 10% of the total surfactant, by weight / weight. It may also constitute 25%, 50%, 75% or 95% of the total surfactant, weight / weight, and the modalities lacking the second surfactant, while not preferred, are also considered. In an alternative embodiment of the present invention, the composition of the precursor solution is such that the spray-dried powder forming the liposome is prepared. Such precursor solutions can have, for example, the composition of the U.S. Patent No. 5,380,519 to Schneider et al. , which is incorporated here as a reference. We have discovered that the spray drying technique described herein for forming the microbubble precursors of phospholipid-containing solutions is superior to the lyophilization of Schneider et al. , and that the resulting microbubbles appear to be much more stable than those formed of the lyophilized material. Thus, in this aspect of the invention, it is considered that the spray drying technique can be used to prepare precursors that form the dry liposome, which is then reconstituted to form the microbubbles, as in Schneider et al. , for use in echographic imaging techniques.

III. Reconstitution and Selection of Gas In the reconstitution in an aqueous medium, the hydrophilic monomer or polymer which, in some preferred embodiments, supplies the structure to the cover, as do the salts and any regulator, etc., which may be present, it rapidly dissolves and separates leaving behind a gas emulsion or dispersion comprising gas bubbles that are surrounded by a layer of the surfactant which is left behind. The first surfactant phospholipid and the most hydrophilic surfactant co-agent are hypothesized to perform different functions. Without limitation to any particular theory of operation, the second surfactant agent (or surfactant co-agent) is apparently useful in aiding the dissolution of water-soluble structural materials, and may also diffuse sufficient rapidly in the reconstitution to "heal" the agent surfactant free holes, which exist during the dissolution of the cover. It has been found that the reconstitution of the microspherical powder which contains, in part, a surfactant relatively soluble in water, together with a more hydrophobic surfactant, produces a much larger number of bubbles per millimeter of the intensifying agent which reconstitutes the powder containing only a single hydrophobic surfactant agent. Although the second surfactant, relatively hydrophobic, is apparently important in the transition from the dried hollow sphere to the gas bubble coated with the surfactant, it is thought that the first more hydrophobic phospholipid is the most effective stabilizing agent after forming the bubbles. The gas dispersion created thus is, therefore, fundamentally different from the previous compositions which increase the contrast, which contain the phospholipid. As described above, liposomes containing Ryan and Unger gas do not involve layers of surfactants at the gas / liquid interface of the bubbles, but essentially involve pure bubbles trapped in the aqueous nuclei of the liposomes. This can be distinguished from the gas emulsion or microbubble dispersion of the present invention, in which it seems (without being limited to any particular theory of operation) that the small gas bubbles are surrounded by a non-evanescent, relatively durable layer of surfactant with such orientation that the hydrophilic head groups are associated with the liquid aqueous, and hydrophobic tail groups are associated with dispersed gas bubbles. Also, in contrast to Schneider et al. , the gas dispersions of the present invention do not require the presence in solution of liposomes or other laminar structures of surfactants. In fact, gas dispersions with excellent in vivo stability can be prepared according to the present invention, in which the surfactants used are incapable of forming liposomes. The absence of lamellar structures of surfactants in solution does not mean that they affect the efficacy of the contrast agents of the present invention. Additionally, it has been observed that the presence of a fluorocarbon osmotic stabilizing gas in the bubbles drastically increases the stability of the gas dispersions of the present invention, while the presence of a fluorocarbon osmotic stabilized has little effect on the stability of the microbubbles produced from lyophilized liposomes. These differences in behavior further suggests ((again, without being limited to any particular theory of operation) that the gas in the bubbles of the present invention is trapped by a non-evanescent, relatively durable layer of surfactant with such orientation that the hydrophilic head groups are associated with the aqueous liquid, and the hydrophobic tail groups are associated with the dispersed gas bubbles.Appropriate bubbles containing air, nitrogen or other gases normally present in the blood can be created by reconstituting the dried microspheres by It is also found that the life of the bubbles can be improved when a gas, relatively insoluble in water, such as a fluorocarbon, is made to permeate the dried microspheres before reconstitution. In this case, the invention will use a first gas or gases (a "primary modifier gas") which, optionally, is present ordin ariously in normal blood and serum, in combination with one or more additional second gases (a "gas osmotic agent or agents" or a "secondary gas") that act to regulate the osmotic pressure within the bubble. Through the regulation of the osmotic pressure of the bubble, the osmotic agent of gas (defined here as a single entity or mixture of chemical entities) exerts pressure inside the bubble, helping to prevent deflation. Optionally, the modifier gas may be a gas that is not ordinarily present in blood or serum. However, the modifier gas must be capable of diluting and maintaining the osmotic agent or agents of the gas at a partial pressure below the vapor pressure of the gas osmotic agent or agents, while the gases in the blood or other surrounding liquid diffuse. in the bubble. In an aqueous medium, water vapor is not considered to be one of the "gases" in question. Similarly, when the microbubbles are in a non-aqueous liquid medium, the vapor of such medium is not considered to be one of the "gases". We have discovered that by adding a gas osmotic agent having, for example, a reduced membrane permeability across the surface or a reduced solubility in the continuous external liquid phase, the life of a bubble formed with it can be increased. This result is achieved through entrapment, within the chosen gas emulsion, of a combination of gases, preferably a primary modifier gas or gas mixture that will dilute a gas osmotic agent at a partial pressure less than the vapor pressure of the agent osmotic gas, until the modifying gas is exchanged with the gases normally present in the external medium. The gas osmotic agent or agents are generally relatively hydrophobic and relatively impermeable to the bubble membrane and also have the ability to develop osmotic gas pressures greater than 50., 75 or 100 Torr. In a preferred embodiment, the gas vapor pressure of the gas osmotic agent is preferably less than 760 Torr at 37 ° C, preferably less than 750, 740, 730, 720, 710 or 700 Torr, approximately, and, in some embodiments , less than 650, 600, 500 or 400 Torr. In the preferred embodiments, the vapor pressure of the primary modifier gas is at least 660 Torr at 37 ° C and the vapor pressure of the osmotic gas agent is at least 100 Torr at 37 ° C. The first gas and the second gas are respectively present in a molar ratio of about 1: 100, 1:75, 1:50, 1:30, 1:20 or 1:10, up to about 1000: 1, 500: 1, 250: 1, 100: 1, 75: 1 or 50: 1, and where the first gas has a vapor pressure of at least about (760 x) mm Hg at 37 ° C, where x is the pressure of steam from the second gas at 37 ° C and where the vapor pressure of each of the first and second gases is greater than about 75 or 100 mm Hg at 37 ° C. Gas emulsion or gas dispersion bubbles, prepared according to a preferred embodiment of the invention, may also possess appropriate additional advantages. In one embodiment, mixtures of non-osmotic gases with osmotic stabilizing gases (or gas osmotic agents) are used to stabilize the resulting distribution of the size of the bubbles during and immediately after production. In the generation of the bubbles, the greater pressure of Laplace in the smaller bubbles causes the diffusion through the liquid phase to the large bubbles of lower pressure of Laplace. This causes the average size distribution to increase above the limit of the capillary dimension of 5 microns over time. This is called disproportionation. When a mixture of a non-osmotic gas (for example air) is used with an osmotic vapor (for example CgF) _4), a slight reduction in volume of the smaller bubbles, due to the air left by the bubble, concentrates the gas osmotic and increases its osmotic pressure thus retarding further shrinkage, while large bubbles increase slightly in volume, diluting the osmotic gas and retarding further growth. An additional advantage of using a mixture of extremely soluble gases in the blood (for example, 87.5% by volume of CO2) and a mixture of osmotic gas (for example 28% of CgF vapor) _4 + 72% of air) is that which, when injected, these bubbles shrink rapidly due to the loss of CO2 in the blood. The bubbles, in the injection, will experience a decrease of 87.5% in volume due to the loss of CO2. This loss of CO2 corresponds to one-half the diameter of a bubble. Therefore, one can prepare large diameter bubbles (for example 9 μm) using simplified mechanical means that will shrink under 5 microns in the injection. In general, such a gas emulsion was initially prepared when the first gas is present in a ratio of at least 1: 1 to the second gas, preferably at least 3: 2, 2: 1, 3: 1, 4: 1 , 5: 1 or 10: 1, When the membrane of the microbubble is more permeable to the first gas than to the second gas (for example, the membrane has respective permeabilities to the gases in a ratio of at least 2: 1, 3: 1 , 4: 1, 5: 1 or 10: 1, preferably even larger, for example 10: 1, 40: 1 or 10:: 1), the bubbles advantageously shrink from their first original diameter to a second average diameter of 75. % or less of its original diameter fairly quickly (for example within one, two, four or five minutes). Then, when at least one gas relatively permeable to the membrane is present in the aqueous medium comprising the continuous phase of the gas emulsion, the bubble is preferably stabilized at or around the second diameter by at least about 1 minute, preferably 2, 3, 4 or 5 minutes. In a preferred embodiment, the bubbles maintain a size between about 5 or 6 μm and 1 μm for at least 1, 2, 3, 4 or 5 minutes, stabilized by a differential of the osmotic pressure of the gas. The tension of the gas in the external liquid is preferably at least about 700 mm Hg. Likewise, a gas relatively impermeable to the membrane is also in the microbubble to create such a differential of the osmotic pressure. As noted above, the gas osmotic agent is preferably a gas which is less permeable through the surface of the bubble than the modifier. It is also preferable that the gas osmotic agent be less soluble in blood and serum. Therefore, it will now be understood that the gas osmotic agent can be a gas at ambient or body temperature or can ordinarily be a liquid at body temperature, as long as it has sufficient partial or vapor pressure at the temperature of use to provide the desired osmotic effect. Therefore, fluorocarbons or other compounds that are not gases at ambient or body temperature can be used, provided they have sufficient vapor pressure, preferably at least about 50 or 100 Torr at body temperature or , more preferably, at least about 150 or 200 Torr. It will be noted that when the osmotic gas agent is a mixture of gases, the relevant measurement of the vapor pressure is the vapor pressure of the mixture, not necessarily the vapor pressure of the individual components of the mixed gas osmotic agent.

It is also important when a perfluorocarbon is used as the osmotic agent within a bubble, the particular perfluorocarbon does not condense at the partial pressure present in the bubble and at the body temperature. Depending on the relative concentrations of the primary modifier gas and the osmotic gas agent, the primary modifier gas can quickly leave the bubble causing it to shrink and concentrate the secondary gas osmotic agent. Such shrinkage may occur until the osmotic pressure of the gas equals the external pressure of the bubble (absolute maximum arterial pressure) plus the Laplace pressure of the bubble minus the air tension, or the saturation tension of the air, of the blood (essentially an atmosphere). Thus the partial pressure of condensation of the resulting gas mixture at 37 ° C must be above the equilibrium partial pressure, discussed above, of the osmotic agent. A list of some compounds that have suitable solubility and vapor pressure criteria is given in Table I: TABLE I perfluoro propanes, C3Fg perfluoro butanes, C4F10 perfluoro cyclobutanes, C4F8 perfluoro pentanes, C5F12 perfluoro cyclopentanes, C5H10 methylcyclobutanes perfluoro, C5F or perfluoro hexanes, CF] _4 perfluoro cyclohexanes, perfluoro CgF ^ 2 methyl-cyclopentanes, CgF] ^ perfluoro-cyclobutanes, perfluoro CgF 2 heptanes, perfluoro cycloheptans C7F, C7H14 methyl-cyclohexanes perfluoro, perfluoro-C7H14 dimethyl-cyclopentanes of perfluoro, C7F14 perfluoro trimethyl-cyclobutanes, C7F14 perfluoro triethylamines, N (C2H5) 3 As will be appreciated, one of ordinary skill in the art can readily determine other compounds that perform adequately herein invention, which do not meet the criteria of solubility and pressure described above. Rather, it will be understood that certain compounds can be considered outside the preferred range in their solubility or vapor pressure, if such compounds compensate for such aberration in the other category and provide superior insolubility or high vapor pressure.

It should also be noted that for medical purposes, the gases, both the modifier gas and the osmotic gas agent, must be biocompatible or not be physiologically harmful. Finally, the microbubbles containing the gas phase will decompose and the gas phase will be released into the blood, either as a dissolved gas or as submicrometric droplets of condensed liquid. It will be understood that the gases will be removed primarily from the body through lung breathing or through a combination of respiration and other pathways in the reticuloendothelial system. A surprising finding was that mixtures of PFCs, for example C4F10 (as a combination of the gas modifier and a gas osmotic agent) saturated with CF 4 vapor (as the main osmotic gas agent), can stabilize the bubbles for longer times than any single component. This is because C4F10 is a gas at body temperature (and, thus, it can act as both a modifier gas, and a gas osmotic agent) it has a somewhat reduced permeability of the membrane and is only slightly soluble in the CgF] ^ at body temperature. In this situation, the osmotic gas pressures of both agents are added together, leading to an increased persistence of bubbles over that of the air / CgF? 4 only in admixture. It is only possible that the condensation point of the higher molecular weight C F ^ 4 component, which persists for a longer time, is increased, allowing a higher maximum osmotic pressure of the gas to be exerted. Other mixtures of the PFCs will perform similarly. Preferred mixtures of the PFCs will have ratios of 1:10 to 10: 1 and include such mixtures as perfluorobutane / perfluorohexane and perfluorobutane / perfluoropentane. These preferred fluorine chemicals may be branched or straight chain. As discussed before, we have also discovered that mixtures of non-osmotic gases in combination with the osmotic agent of gas, act to stabilize the size distribution of the bubbles, before and after the injection. In the generation of the bubbles, the greater pressures of Laplace in the smaller bubbles cause the diffusion through the liquid phase to the larger bubbles at the lower pressure of Laplace. This causes the average size distribution to increase above the limit of the capillary dimension of 5 microns over time. This is called a disproportionation. However, when a mixture of a modifier gas (eg, air or carbon dioxide) is used with a gas osmotic agent (eg CgF ^ 4), a slight reduction in volume of the smaller bubbles, due to one of the modifying gases left by the bubble, it will concentrate the osmotic gas and increase its osmotic pressure, thus delaying the subsequent shrinkage. On the other hand, larger bubbles will increase slightly in volume, diluting the osmotic gas and also slowing down further growth. Therefore, we have discovered that through the use of a gas that is relatively hydrophobic and has a relatively low membrane permeability, the rate of bubble decomposition can be reduced. Thus, through the reduction of the bubble decomposition regime, the half-lives of the microbubbles is improved and the potential for contrast enhancement is extended. The desired gas is made permeate the dry microspheres, placing these microspheres inside a bottle, which is introduced in a vacuum chamber to evacuate the air. The air is then replaced with the desired gas or gas combination (a preferred combination of gases is nitrogen saturated with perfluorohexane, at 13 ° C). The gas will then diffuse into the hollows of the spheres. The diffusion can be aided by pressure or vacuum cycles. The bottle is then sealed by crimping and preferably sterilized with gamma radiation.

It will be appreciated that equipment for use in obtaining microbubble preparations of the present invention can be prepared. These equipment may include a container enclosing the gas or gases, described above (to form the microbubbles), the liquid and the surfactant, the container may contain all the sterile dry components, and the gas, in a chamber, with the liquid aqueous sterile in a second chamber of the same container. Suitable two-chamber bottle-type containers are available, for example, under the trademarks WHEATON RS177FLW or S-1702FL, from Wheaton Glass Co., (Millville, N.J.). Such containers are illustrated in Figures 1-4. Referring to Figures 1 and 2, the illustrated Wheaton container 5 has an upper chamber 20, which may contain an aqueous solution 25, and a lower chamber 30, which may contain the dry ingredients 35 and a desired gas. A shutter 10 is provided, which separates the upper chamber from the environment, and a seal 15 separates the upper chamber 20 from the lower chamber 30, which contains the hollow microspheres 35 (powder), spray dried, and the gas osmotic agent. . Pressing the plug 10 pressurizes the relatively incompressible liquid, which pushes the seal 15 down into the lower chamber 30. This releases the aqueous solution into the lower chamber 30, which results in the dissolution of the powder. to form the stabilized microbubbles 45, which contain the trapped gas osmotic agent. Excess of the osmotic agent 40 of the gas is released from the lower chamber 30 into the upper chamber 20. This arrangement is convenient for the user and has the unexpected additional advantage of sealing the small amount of the gas osmotic agent, impervious to water, in the lower chamber, covering the inter chamber seal with a thick layer (1.27 to 3.175 cm) of the aqueous solution and the advantage that this aqueous solution can be introduced into the lower chamber without raising the pressure in the powder chamber by more of 10%. Thus, there is no need for pressure ventilation. (In contrast, conventional reconstitution of a solute in a single-chamber bottle with a needle and syringe without ventilation can result in the production of considerable intra-chamber pressure, which could crush the microbubbles. two cameras can be used for the preparation of microbubbles With reference to Figures 3 and 4, the same bottle is used, as described above, except that the plug 50 is elongated, so that it dislodges the internal seal 15 when it is pressed In this microbubble preparation method, the hollow microspheres 35, spray dried, and the osmotic gas agent 40, are contained in the upper chamber 20. The aqueous solution 25 and the osmotic gas agent 40 are contained within the lower chamber 30. When the obturator 50 is depressed, it dislodges the seal 15, allowing the hollow microspheres, spray-dried, to be mixed with the solution a 25, in the presence of the gas osmotic agent 40. An advantage associated with this method of forming the microbubbles is that the aqueous phase can be first instilled and sterilized by means of the autoclave or other means, followed by the instillation of the spray-dried microspheres. This will prevent potential microbial growth in the aqueous phase before sterilization. Although a particular double-chamber container has been illustrated, other suitable devices are known and commercially available. For example, a two-compartment glass syringe, such as the pre-filled, double-chamber syringe system, BD HYPAK Liquid / Dry 5 + 5 ml (Becton Dickinson, Franklin Lakes, NJ, described in US Pat. 4,613,326) can be used advantageously to reconstitute the spray-dried powder. The advantages of this system include: 1. Convenience of use; 2. The gas osmotic agent, insoluble in aqueous media, is sealed by a chamber of the aqueous solution on one side and an extremely small area of the elastomer seals the needle on the other side; and 3. a filtration needle, such as the Monoject # 305 (Sherwood Medical, St. Louis MO), can be equipped in the syringe at the time of its manufacture, to ensure that no undissolved solid is injected. The use of the two chamber syringe to form the microbubbles is described in Example XIV. One of ordinary skill in the art will appreciate that other two-chamber reconstitution systems, capable of combining the spray-dried powder with the aqueous solution in a sterile manner, are also within the scope of the present invention. In such systems, it is particularly advantageous if the aqueous phase can be interposed between the osmotic gas insoluble in water and the environment, to increase the shelf life of the product. When the material necessary to form the microbubbles in the container is no longer present, it can be packaged with the other components of the equipment, preferably in the form of a container adapted to facilitate prompt combination with the other components of the equipment.

Examples of particular uses of the microbubbles of the present invention include the perfusion imaging of the heart, the tissue of the myocardium and the determination of perfusion characteristics of the heart and its tissues during stress or exercise tests, or defects or perfusion changes due to myocardial infection. Similarly, myocardial tissue can be seen after oral or venous administration of drugs designed to increase the flow of blood to a tissue. Also, visualization of changes in myocardial tissue due to or during various interventions, such as coronary tissue vein grafting, coronary angioplasty, or the use of thrombolytic agents (TPA or streptokinase), may also be increased. As these contrast agents can be conveniently administered via a peripheral vein to increase visualization of the total circulatory system, they will also aid in the diagnosis of general vascular pathologies and in the ability to monitor the viability of the placental tissue ultrasonically. However, it should be noted that these principles have application beyond the formation of ultrasound images. In fact, the present invention is broad enough to encompass the use of gas emulsions containing phospholipids in any system, including non-biological applications. It will also be understood that other components can be included in the microbubble formulations of the present invention. For example, osmotic agents, stabilizers, chelating agents, regulators, viscosity modulators, air solubility modifiers, salts and sugars can be added to modify microbubble suspensions for maximum life and contrast enhancement efficiency. Such considerations as sterility, isotonicity and biocompatibility can govern the use of such conventional additives to injectable compositions. The use of these agents, as understood by ordinary experts in the field, and the specific quantities, relationships and types of agents, can be determined empirically, without undue experimentation. Various embodiments of the present invention provide surprising advantages. Spray-dried starch formulations provide prolonged stability in the flask, particularly when the molecular weight of the starch is greater than 500,000. Fatty acid esters of sugars, such as sucrose monostearate, as well as block copolymers, such as Pluronic F-68 (with a hydrophilic / lipophilic balance (HLB) greater than 12) allow the powder to form bubbles in the instant that it rehydrates. Spray-dried formulations with a structural agent, such as starch, starch derivatives or dextrin, provide a significantly lower total dose of the surfactant than comparable sonicated formulations. The use of two-chamber water bottles, which provide an additional seal of the osmotic gas agent, provide increased shelf life and increased convenience of use. Spray-dried formulations with a structural agent (such as a starch or dextrin), a hydrophobic phospholipid and a more water-soluble surfactant coagent, provide gas emulsions with greatly increased in vivo half-lives. Any of the microbubble preparations of the present invention can be administered to a vertebrate animal, such as a bird or a mammal, as a contrast agent for ultrasonically imaging portions of this vertebrate animal. Preferably the vertebrate is a human being and the portion from which the image is formed is the vasculature of the vertebrate. In this embodiment, a small amount of microbubbles (for example from 0.1 ml / kg to 12 mg / kg of the spray-dried powder), based on the vertebrate body weight) is introduced intravascu larly into the animal. Other amounts of microbubbles, such as from about 0.005 ml / kg to 1.0 ml / kg, can be used. Imaging of the heart, arteries, veins and organs rich in blood, such as the liver and kidneys, can be done ultrasonically with this technique. The following description will be more fully understood with reference to the following Examples. However, these examples are illustrative of the preferred methods of practicing the present invention and do not limit the scope of the invention or the appended claims.

EXAMPLE I Spray Drying of a Solution Containing Phospholipids One liter of the following solution was prepared in water for injection: 2.0% w / v Maltodextrin Maltrin M-100, (Grain Processing Corp. Muscatine, IA), 0.95 % w / v sodium chloride (Mallinckrodt, St. Louis, MO), 1.0% Superonic F-68 (Serva, Heidelberg, Germany), 1.0% w / v Ryoto Sucroise Stearate S-1670 (Mitsubishi-Kasei Food Corp., Tokyo Japan) and 0.5% of the hydrogenated phospholipid, Lipoid E-100-3, (Lipoid, Ludwigshafen, Germany). This solution was then spray dried on the Niro Atomizer Portable Spray Dryer, equipped with a two fluid atomizer (Niro Atomizer, Copenhagen, Denmark), which uses the following settings: hot air flow rate 1,119 m3 / min inlet air temperature 245 ° C exit air temperature 100 ° C Atomizer air flow 350 liters / min liquid feed rate 1 liter / hour The hollow, dry spherical product had a diameter between about 1 μM and 15 μM and was collected in a cyclone separator, as is normal for this dryer. Aliquots of powder (250 mg) were weighed into 10 ml tubular flasks, evacuated and sprayed with nitrogen saturated with perfluorohexane, at 13 ° C, and sealed. The nitrogen was saturated with perfluorohexane, passing it through three gas washing bottles filled with perfluorohexane, submerged in a water bath at 13 ° C. In the reconstitution with 5 ml of water for injection, numerous bubbles were observed by a light microscope, which vary in size from 1 to 20 microns. The fact that many bubbles of approximately 1 miera could be observed for a measurable time, shows that the aggregate stability gained by the inclusion of a phospholipid in the formula, as an additional non-Newtonian viscoelastic surfactant.

Example II Comparison of phospholipid emulsions vs. sucrose ester gas emulsions One liter of each of the following four solutions was prepared with water for injection: Solution 1: 3.9% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.25% w / v of sodium chloride (Mallinckrodt, St. Louis, MO) 2.83% sodium phosphate, dibasic (Mallinckrodt, St. Louis, MO) 0.42% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) Solution 2: 2.11% w / v of Poloxamer 188 (BASF, Parsipany, NJ) 0.32% p / v of Ryoto Sucrose Stearate S-1670 (Mitsubishi-Kasei Food Corp., Tokyo, Japan) 0.16% p / v of Ryoto Sucrose Stearate S-570 (Mitsubishi-Kasei Food Corp., Tokyo, Japan) Solution 3 3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% sodium phosphate, dibasic (Mallinckrodt. Louis, MO) 0.39% w / v of sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) Solution 4: 0.15% w / v of Poloxamer 188 (BASF, Parsipany, NJ) 0.45% w / v phosphotidylcholine egg, hydrogenated, EPC-3 (Lipoid, Ludwigshafen, Germany) Solutions 2 and 4 were added to a high shear mixer and cooled in an ice bath. A coarse suspension of 3.0% v / v of 1,1,1-trichlorotrifluoroethane (Freon 113, EM Science, Gibbstown, NJ) was obtained in the liter of solutions 2 and 4. These suspensions were emulsified using a microfluidizer (Microfluidics Corporation, Newton, MA, model M-110F) at a pressure of 700 kg / cm2, a temperature of 5 ° C, for 5 passes. The resulting emulsion 4 was added to solution 3 and the resulting emulsion 2 was added to solution 1. The mixture of Formulas 1 and 2 (containing the sucrose ester surfactant) and the mixture of Formulas 3 and 4 (containing the phospholipid surfactant) were then spray-dried in a Niro Atomizer Portable Spray Dryer, equipped with a two fluid atomizer (Niro Atomizer, Copenhagen, Denmark), which uses the following settings: Mixture of Formulas 1 and 2: flow regime of the hot air 878 liters / min inlet air temperature 370 ° C outlet air temperature 120 ° C Atomizer air flow 290 liters / min liquid feed rate 1.5 liters /hour Mixture of Formulas 3 and 4 hot air flow rate 878 liters / min inlet air temperature 325 ° C outlet air temperature 120 ° C Atomizer air flow 290 liters / min liquid feed rate 1.5 liters / hour The spherical, hollow, dry product had a diameter between about 1 μM and about 15 μM, and was collected in a cyclone separator as is common for this dryer. Aliquots of powder (250 mg) were weighed into 10 ml tube vials, sprayed with perfluorohexane-saturated nitrogen at 13 ° C and sealed. The nitrogen was saturated with perfluorohexane by passing it through three gas washing bottles filled with perfluorohexane, submerged in a water bath at 13 ° C. The bottles were reconstituted with 5 ml of water for injection, after inserting an 18 gauge needle as a ventilation to relieve the pressure as the water was injected. One ml of the resulting microbubble suspension was injected intravenously into a rabbit of approximately 3 kg in weight, instrumented to monitor the Doppler ultrasound signal of his carotid artery. A 10 MHz flow cuff (Triton Technology, Inc., San Diego, CA, model ES-10-20) connected to a System 6 Doppler flow module (Triton Technology Inc.) fed the RF Doppler signal to a Lecroy 9410 oscilloscope (LeCroy, Chestnut Ridge, NY). The voltage of the mean square root (RMS) of the signal computed by the oscilloscope was transferred to a computer and the resulting curve was adjusted to obtain the intensity of the echogenic peak signal and the half-life of the micro-bubbles in the blood . The signals before the contrast were less than 0.1 volts of RMS. While the sucrose ester formulation produced an initial ultrasound dispersion signal 29% greater than the signal of the phospholipid formulation due to the higher concentration of microbubbles, surprisingly, the persistence of the phospholipid formulation was substantially higher. The signal of the sucrose ester formula decreased to 30% of its original signal in 140 seconds, while the phospholipid formula lasted 550 seconds before decreasing to the 30% signal level, demonstrating a superior persistence of a formula that employs a phospholipid as the viscoelastic surfactant does not Newtonian.

Example III Comparison of the microbubbles of the water-insoluble phospholipid formulation vs. microbubbles the water-insoluble phosphoglide / water-soluble surfactant formulation (Poloxamer 188) One liter of each of the following emulsions was prepared by spray drying, as described in Example II: Formulation A: Formulation of Phospholipid, Insoluble in Water 3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% p / v Sodium phosphate, dibasic (Mallinckrodt, St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.45% w / v egg phospholipids, hydrogenated E PC 3 (Lipoid, Ludwigshafen, Germany) 3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113, EM Science, Gibbstown, NJ).

Formulation B: Formulation Soluble in Phospholipid / Water, Insoluble in Water (Poloxamer 188) 3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis , MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt, St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.45% w / v phospholipids egg, hydrogenated E PC 3 (Lipoid, Ludwigshafen, Germany) 0.45% w / v of Poloxamer 188 (BASF, Parsipany, NJ) 3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown , NJ) In the reconstitution of 100 mg of the spray-dried powder of Formulation A with 5 ml of water, approximately 20 million bubbles per ml were observed, ranging in size from 1 to 20 μm. In the reconstitution of 100 mg of the spray-dried powder of Formulation B, with 5 ml of water, approximately 315 million bubbles (1575% more bubbles than in Formulation A9 per ml were observed, ranging in size from 1 to 20 μm.) The addition of a surfactant relatively soluble in water [HLB ( Poloxamer 188) = 29-0] to a water-insoluble surfactant in the microbubble formulations, significantly increased the concentration of the bubbles formed, leading to a more effective ultrasound contrast agent.The (HLB) is a number between 0 and assigned to emulsified agents and emulsifying substances HLB is indicative of the emulsification behavior and is related to the balance between the hydrophilic and lipophilic portions of the molecule (Rosen, M., (1989), Surfactants and Interfacial Phenomena, Second Edition, John Wiley &; Sons, New York, pages 326-329).

Example IV Comparison of microbubbles of the water-insoluble phospholipid formulation vs. the microbubbles of the water soluble insoluble water soluble formulation (Polysorbate 20) One liter of each of the following emulsions was prepared for spray drying, as described in Example II: Formulation A: Formulation of water-insoluble phospholipid 3. 6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.45% w / v hydrogenated egg phospholipids E PC 3 (Lipoid, Ludwigshafen, Germany) 3.0% v / v 1.1, 2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) Formulation B: Formulation soluble in phosph olipid / water, insoluble in water (Polysorbate 20) 3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt, St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.45% w / v hydrogenated egg phospholipids E PC 3 (Lipoid, Ludwigshafen, Germany) 0.15% w / v Polysorbate 20 (ICI, Wilmington, DE) 3.0% v / v 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown , NJ) In the reconstitution of 100 mg of the spray-dried powder of Formulation A with 5 ml of water, approximately 20 million bubbles per ml were observed, ranging in size from 1 to 20 μm. In the reconstitution of 100 mg of the spray-dried powder of Formulation B, with 5 ml of water, approximately 250 million bubbles were observed per ml (1150% more bubbles than in Formulation A), which vary in size of 1 at 20 μm. In conclusion, the addition of the relatively water-soluble surfactant, the polysorbate 20 [HLB = 16.7] to the water-insoluble surfactant, the hydrogenated phosphatidylcholine in the microbubble formulations significantly increased the concentration of the bubbles formed, leading to an more effective ultrasound contrast.

Example V Gas emulsion prepared with the phospholipid combination One liter of the following emulsion was prepared for spray drying, as described in Example II: 3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan ) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt, St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.22% w / v of dipalmitoylphosphatidyl choline (Genzyme, Cambridge, MA) 0.31% w / v of dioctanoylphosphatidyl choline (Avanti Polar Lipids, Albaster, AL) 3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) In the reconstitution with 5 ml of water, approximately 51 million bubbles per ml were observed, which vary in size from 1 to 20 microns. The constant decrease of the echogenic signal for this microbubble formulation was determined to be 0.0029 (1 / sec). One milliliter of this formulation was injected into the ear vein of a New Zealand White Rabbit 2.5 kg rabbit. An image of the rabbit was subsequently taken with a Acuson 128xP-5 ultrasound scanner, equipped with a 5MHz transducer. In the infusion, the echogenicity of the blood vessels and chambers of the heart was intense and persisted for several minutes. In addition, the echogenicity of the myocardium and a solid organ, such as the liver and kidney, was homogeneously intense and persisted for several minutes. Notably, the echogenicity of the portal and hepatic veins were isointense, indicating minimal uptake by the reticuloendothelial phagocytic cells of the liver, resulting in prolonged vascular persistence.

Example VI Biocompatibility of Gas Emulsions Prepared from Long Chain / Short Chain Mixed Phospholipids One liter of the following emulsion was prepared for spray drying, as described in Example II. 3. 6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.22% w / v dipalmitoylphosphatidyl-choline (Syngene Ltd., Cambridge, MA) 0.31% w / v dioctanoylphosphatidyl choline (Avanti Polar Lipids Inc., Albaster, AL) 3.0% v / v 1, 1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) In these ratios of dipalmitoylphosphatidyl choline to dioctanoylphosphatidyl choline, the surfactants formed mixed micelles only. In the reconstitution with 5 ml of water, approximately 51 million droplets of gas emulsion per ml were observed, ranging in size from 1 to 20 microns. The first order decrease constant of the echogenic signal of the gas emulsion in rabbits at a dose of 5 mg / kg was determined to be 0.0029 sec. This corresponds to an intravascular half-life of 4 minutes. The gas emulsion was tested for complement activation using a C3a in vi tro diagnostic kit, supplied by Quidel Corp. (San Diego, CA) No difference was observed between the gas emulsion and the negative control (saline), which indicates that the gas emulsion does not activate the complement. It is well known that the complement activates the microbubbles with the naked eye.

Proved sample [C3a] (ng / ml) Zymosan (positive control) 43403 Saline solution (negative control) 604 Gas emulsion 412 The gas emulsion was also tested for changes in hemodynamics in anesthetized dogs at a dose of 20 mg / kg. No change in mean arterial pressure or pulmonary artery pressure was observed. These results indicate that there are no hemodynamic effects with the gas emulsion at 10,100 times the clinically relevant dose.

Arterial ion Pressure of i (mm Hg) Pulmonary Artery (mm of Hg) 0 109.4 13.3 1 109.2 14.2 2 110.4 14.1 5 115.0 14.3 10 117.9 15.7 60 111.0 13.2 90 120.9 13.6 Thus, excellent efficiency and biocompatibility are provided in the same gas emulsion formulation.

EXAMPLE VII Gas emulsion containing phospholipids supplemented with cholesterol Half a liter of each of the following solutions was prepared in water for injection. Solution 1 contains the starch and salts and Solution 2 contains the phospholipids and cholesterol dissolved in a mixture of Freon 113 and ethanol. Solution 2 was added to a high shear mixer and cooled in an ice bath. A thick suspension of 1,1-trichlorotrifluoroethane (Freon 113) was obtained by adding half a liter of water with vigorous stirring. This suspension was emulsified with Solution 2, as described in Example II. The resulting emulsion was added to the Solution 1 to produce the following formula for spray drying: 3. 6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.22% w / v dipalmitoylphosphatidy-lcholine (Genzyme., Cambridge, MA) 0.31% w / v dioctanoylphosphatidyl -lcholine (Avanti Polar Lipids Inc., Albaster, AL) 0.05% w / v cholesterol (Sigma, St. Louis, MO) 2.4% v / v 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown , NJ) 0.6% v / v ethanol (Spectrum Chemical, Gardena, CA).

This emulsion was then spray dried on a Niro Atomizer Portable Spray Dryer, equipped with a two fluid atomizer (Niro Atomizer, Copenhagen, Denmark), which employs the following approximate settings: hot air flow rate 878 liters / min inlet air temperature 325 ° C outlet air temperature 120 ° C atomizer air flow 200 liters / minute emulsion feed rate 1.5 liters / hour.

The spherical, hollow product, dry, had a diameter between about 1 μM and 15 μM and was collected in the cyclone separator as is normal for this dryer. Aliquots (100 mg) were weighed into 10 ml tube flasks, sprayed with perfluorohexane-saturated nitrogen at 13 ° C and sealed as in Example II. The bottles were reconstituted with 5 ml of water for injection, after inserting an 18-gauge needle as ventilation to relieve the pressure as the water was injected. A 0.25 ml / kg dose of the resulting microbubble suspension was injected intravenously into a rabbit of approximately 3 kg, instrumented to inspect the Doppler ultrasound signal from his carotid artery, again, as in Example II. The signal, 1 minute after the injection, was 0.71 volt with a decrease constant of 0.010 sec. The hematology samples were taken during the first 60 minutes after the injection. There was no detectable drop in platelet count or detectable complement activation according to Example VI.

EXAMPLE VIII In Vivo Efficacy of Reconstituted Lyophilized Liposomes A liposome-forming solution, with a total lipid concentration of 50 mg / ml, was prepared with hydrogenated soy lecithin (S PC-3, Lipoid, Ludwigshafen, Germany), and dicetyl phosphate (Sigma, St. Louis, MO) in a molar ratio of 9: 1. Following the Inverse Phase Evaporation Method of Szoka and Papahadjopoulos in Proc. Na t. Acad. , Sci. USA (1978), 4194, the surfactants were dissolved in 120 ml of a 1/1 v / v solution of diethyl ether / chloroform. 40 ml of deionized water were added. The mixture is sonicated for 10 minutes at 0-4 ° C with a 3 mm probe sonicator (50 W, Vibra Cell, Sonics &Materials Inc., Danbury CT) to form an emulsion. A liposome dispersion was formed by removing the solvent under reduced pressure by rotary evaporation and filtration of the solution through a 1.0 μm polycarbonate filter at 65 ° C. Fractions of 1 ml of the liposome solution were then mixed with 4 ml of a 15% w / v maltose solution (Sigma, St. Louis) in 10 ml ultrasound bottles, frozen at -30 ° C and lyophilized ( FTS Systems, Stone Ridge, NY). The flasks were gasified with nitrogen or nitrogen saturated with perfluorohexane at 13 ° C. The lyophilized powder was reconstituted in 5 ml of water at the following concentrations: 12. 0% w / v Maltose (Sigma, St. Louis, MO) 0.926% w / v of hydrogenated soy lecithin S PC-3 (Lipoid, Ludwigshafen, Germany) 0. 072% w / v of dicetyl phosphate (Sigma, St. Louis, MO) One ml of the resulting suspension of microbubbles was injected intravenously into a rabbit of approximately 3 kg, instrumented to inspect the Doppler ultrasound signal from his carotid artery. A 10 MHz flow cuff (Triton Technology Inc., San Diego, CA, model ES-10-20) connected to a Doppler flow module, System 6 (Triton Technology Inc.) fed the RF Doppler signal to an oscilloscope LeCroy 9410 (LeCroy, Chestnut Ridge, NY). The voltage of the mean square root (RMS) of the signal computed by the oscilloscope was transferred to a computer and the resulting curve was adjusted to obtain the peak intensity of the echogenic signal and the half-life of the micro-bubbles in the blood . The signals before the contrast were less than 0.1 volts of RMS. Neither nitrogen nor formulations containing liposomes gasified with perfluorohexane showed significant or lasting echogenicity in the rabbit model.

Example IX Effect of gasification of perfluorohexane on the efficacy of ultrasound of lyophilized liposome formulations vs. spray-dried gas emulsion formulations One liter of each of the following emulsions was prepared for spray drying, as described in Example II: Formulation A: Sucrose Ester Microbubble Formulation 3. 6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.45% w / v sucrose ester 11025003 (Alliance Pharmaceutical Corp., San Diego, CA) 1.95% p / v from Poloxamer 188 (BASF, Parsipany, NJ) 3.0% v / v from 1, 1, 2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) Formulation B: Formulation of Phospholipid Microbubbles 3. 6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.45% w / v dipalmitoyl-phosphatidylcholine (Genzyme, Cambridge Corp., MA) 0.15% w / v Poloxamer 188 (BASF, Parsipany, NJ) 3.0% v / v of 1, 1, 2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) Formulation C: Formulation of Lyophilized Liposomes Approximately 40 ml of a dispersion of liposomes containing hydrogenated soy lecithin and dicetyl phosphate (molar ratio of 9: 1) at a total lipid concentration of 50 mg / ml in water, were prepared using the Reverse Phase Evaporation Method (REV), described by Szoka and Papahadjopoulos ( see Example VIII) The formulation is summarized as follows: Formula of Reconstituted Dry Powder for Injection (% P / P) (%, w / v) Hydrogenated Soy Lecithin (Lipoid S PC-3, Lipoid, 7, 14, 918 Ludwigshafen, Germany) Dicetyl Phosphate (Sigma, St. Louis, MO) 0. 55 0. 072 Maltose (Sigma, St. Louis, MO) 92. 3 12. 0 Water for injection 5. 0 ml Two bottles of each of the formulations described above were prepared; one was gassed with a perfluorohexane-nitrogen mixture, the other contained only nitrogen. Samples (6 in total) were reconstituted with 5 ml of water and evaluated for efficacy using a Rabbit Model Enhanced Pulsed Doppler Signal apparatus. The doses administered to the rabbit were 5 mg of dry powder per kg of rabbit, for formulations A, B and C, respectively. The echogenic signals in 60 sec for the formulations that do not contain the perfluorohexane, A, B and C, were 0.040, 0.142 and 0.005 V, respectively, the echogenic signals in 60 sec. for their respective formulations containing the perfluorohexane were 1,232, 0.826 and 0 V. In conclusion, the addition of a gasification stage of perfluorohexane did not significantly increase the efficiency of the ultrasound (defined here as the echogenic signal in 60 sec.) of the formulation of lyophilized liposomes. In both the efficacy of spray-dried sucrose ester microbubble formulations and phospholipid microbubbles containing perfluorohexane, was increased by 2980 and 482%, respectively. Thus, fundamental differences in structure and behavior exist between the gas emulsions of the present invention and the microbubble preparations obtained from the lyophilized liposomes.

EXAMPLE X Efficacy of the spray-dried dispersion containing the water-insoluble phospholipid, described in Example 4, of U.A. Patent No. 5,380,519 to Schneider, et al. A formulation containing the proportions of phospholipid and dicetyl phosphate, as described in Example 4 of the U.S. Patent No. 5,380,519 to Schneider et al. , was prepared by spray drying the following emulsion. The surfactants were not laminated (converted into liposomes) as in the Schneider example. One liter of each of the following solutions was prepared with water for injection: Solution 1, which contains the starch and salts and Solution 2 containing the surfactants. Solution 2 was added to a high shear mixer and cooled in an ice bath. A coarse suspension of 1,2-trichlorotrifluoroethane (Freon 113) was obtained in 1 liter of Solution 2. This suspension was emulsified using a Microfluidizer (Microfluidics Corporation, Newton, MA; model M-110F) at 700 kg / cm 2 , 5 ° C for 5 passes. The resulting emulsion was added to Solution 1 to produce the following formula for spray drying: 3. 6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.058% w / v dicetyl phosphate (Sigma, St. Louis, MO) 0.742% w / v Phospholipid PC-3 (Lipoid, Ludwigshafen, Germany) 3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) This emulsion was then spray dried on a Niro Atomizer Portable Spray Dryer , equipped with a two fluid atomizer (Niro Atomizer, Copenhagen, Denmark), which employs the following approximate adjustments: hot air flow rate = 878 liters / min inlet air temperature = 325 ° C outlet air temperature = 120 ° C atomizer air flow = 290 liters / min emulsion feed rate = 1.5 liters / hour The spherical, hollow, dry product had an approximate diameter of 1 μM to 15 μM and was collected in the cyclone separator as is normal for this dryer, aliquots of powder (100 mg) were weighed into 10 ml tube jars, sprayed with nitrogen only or with nitrogen plus perfluorohexane (PFH) and sealed as in the previous examples. The bottles were reconstituted with 5 ml of water for injection, after inserting an 18-gauge needle as a vent to relieve the pressure as the water was injected. One ml of the resulting suspension of microbubbles was injected intravenously into a rabbit of approximately 3 kg, instrumented to inspect the Doppler ultrasound signal from his carotid artery, as in the previous examples. Signals were observed with both the PFH and without it. The agent containing the PFH produced a 0.08 volt signal in 60 seconds, with a 0.01 volt signal in 200 seconds. The nitrogen-only agent produced a 0.2 volt signal in 60 seconds with a 0.4 volt signal in 200 seconds. As this formula does not contain a more water-soluble surfactant, the signals are much lower than in the previous examples. The spray drying process, however, presents this mixture of non-lamellar surfactants in a physical state, which produced detectable signals, unlike the laminarized formula of Schneider et al. in Example 4, as demonstrated in Example VIII above. This formula also differs from the other examples of this application in that the addition of perfluorohexane reduced the resulting signal rather than greatly increasing it.

Example XI Microbubbles that do not contain fluorocarbon Two formulations (A, mixed phospholipids and B phospholipid + Poloxamer 188) were prepared by spray drying the following emulsions with a similar process.

One liter of each of the following solutions prepared with water for injection: Solution 1 containing starch and salts and Solution 2 containing the surfactants. Solution 2 was added to a high shear mixer and cooled in an ice bath. A coarse slurry of 1, 1, 2-trichlorotrifluoroethane (Freon 113) was obtained in 1 liter of Solution 2. This suspension was emulsified using a Microfluidizer (Microfluidics Corporation, Newton, MA; model M-110F) at 700 kg / cm 2 , 5 ° C for 5 passes. The resulting emulsion was added to Solution 1 to produce the following formula for spray drying: Formula A (Mixed Phospholipids) 3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v phosphate sodium, dibasic (Mallinckrodt, St. Louis, MO) 0.39% w / v sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.22% w / v dipalmitoylphosphotidalkoline (Genzyme., Cambridge, MA) 0.31% p / v of dioctinoyl-phosphothyl-choline (Genzyme, Cambridge, MA) 3.0% v / v of 1, 1, 2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) Formula B (Phospholipid + Poloxamer 188) 3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt, St. Louis, MO) 0.39% w / v of sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.15 w / v of Poloxamer 188 (BASF, Parsipany, NJ) 0.45% p / v from Phospholipid PC-3 (Lipoid, Ludwigshafen, Germany) 3.0% v / v from 1, 1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) This emulsion was then spray-dried on a Niro Atomizar Portable Spray Dryer, equipped with a two fluid atomizer (Niro Atomizer, Copenhagen, Denmark), which employs the following approximate settings: hot air flow rate = 878 liters / min inlet air temperature = 325 ° C outlet air temperature = 120 ° C atomizer air flow = 290 liters / min emulsion feed rate = 1.5 liters / hour The spherical, hollow, dry product had a diameter between about 1 and 15 μM and was collected in the cyclone separator as is usual for this dryer. Aliquots of powder (100 mg) were weighed into tube bottles of ml, they were sprayed with nitrogen only and sealed. The bottles were reconstituted with 5 ml of water for injection, after inserting an 18 gauge needle as a ventilation to relieve the pressure as the water was injected. One ml of the resulting suspension of microbubbles was injected intravenously into a rabbit of approximately 3 kg, instrumented to inspect the Doppler ultrasound signal of his carotid artery. A 10 MHz flow cuff (Triton Technology Inc., San Diego, CA, model ES-10-20) connected to a System 6 Doppler flow module (Triton Technology Inc.) fed the RF Doppler signal to a LeCroy oscilloscope 9410 (LeCroy, Chestnut Ridge, NY). The voltage of the mean square root (RMS) of the signal computed by the oscilloscope was transferred to a computer and the resulting curve was adjusted to obtain the intensity of the echogenic signal and the average life of the microbubbles in the blood. Significant signs were observed with both formulas. Formula A produced a 0.25 volt signal in 60 seconds with a 0.13 volt signal in 200 seconds. Formula B produced a 0.3 volt signal in 60 seconds with a 0.2 vol signal in 200 seconds. Formulas without phospholipids produced only background signals when treated in the same way. As described above, this may be the result of the water first coming into contact with the inner surface of the spherical cavity (0.5 - 10 microns in diameter), after filtering through the dissolving surfactants and the structural agents that result in the formation of a bubble of the desired size (the size of the cavity) that is initially surrounded by the saturated surfactant solution and, therefore, , it has a maximum coating of the surfactant package optimally, increasing the trapped gas. These bubbles are remarkably stable in vivo even when filled with water-soluble gases (for example air or nitrogen).

Example XII Effect of Acyl Chain Length of Phospholipid on the Echogenic Efficiency of Ultrasound One liter of each of the following emulsions was prepared for spray drying, as described in Example II: Formulation A: Dimiristoil-Phosphatidylcholine Formulation 3. 6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt. St. Louis, MO) 0.39% w / v of sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.45 w / v of dimyristoyl-phosphatidylcholine (Genzyme Corp., Cambridge, MA) 0.15% w / v of Poloxamer 188 (BASF, Parsipany, NJ) 3.0% v / v 1, 1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ) Formulation B: Formulation of Distearoyl-Phosphatidylcholine 3.6% w / v m-HES, hydroxyethyl starch (Ajinimoto, Tokyo, Japan) 3.0% w / v sodium chloride (Mallinckrodt, St. Louis, MO) 2.6% w / v sodium phosphate, dibasic (Mallinckrodt, St. Louis, MO) 0.39% p / v of sodium phosphate, monobasic (Mallinckrodt, St. Louis, MO) 0.45% w / v of distearoyl-phosphatidyl-choline (Genzyme Corp., Cambridge, MA) 0.15% w / v of Poloxamer 188 (BASF, Parsipany, NJ ) 3.0% v / v of 1,1,2-trichlorotrifluoroethane (Freon 113; EM Science, Gibbstown, NJ ) After reconstitution with 5 ml of water, the two formulations were evaluated for efficacy, using a Rabbit Model apparatus to Increase the Pulsed Doppler Signal, as in Example II, and the echogenic signal measured as a function of time. The doses administered to the rabbit were 5 mg of the dry powder per kg of the rabbit.

Time (seconds) 20 60 100 200 300 400 500 600 Formulation A - Signal 0.8 0.6 0.5 0.4 0.4 0.4 0.3 0.2 Ecogenic (V) Formulation B - Signal 0.5 0.4 0.2 0.2 0.1 0.1 0.1 0.1 0.1 Ecogenic (V) The echogenic signal as a function of time was on average higher for the formulation containing the dimyristoyl-phosphatidyl choline (DMPC) than for the formulation containing the distearoyl-phosphatidyl-choline (DSPC). Both chains of fatty acid esters of the DMPC contain 14 carbon atoms, while the chains of esters of fatty acids are 18 carbon atoms in length for the DSPC. This difference in the chain length between the two phospholipid compounds results in a gel different from the glass-liquid phase transition temperature. At temperatures above this transition temperature, the hydrocarbon chains are in the molten state and the phospholipids form a liquid crystal phase. This transition temperature is 55.5 ° C for the DSPC and 23.5 ° C for the DMPC in water. Therefore, the use of a first phospholipid surfactant which is in the liquid crystal state after injection (rabbit body temperature = about 37.5 ° C) can be advantageous.

Example XIII Formation of Microbubbles with the Use of a Two-Chamber Flask 800 mg of the spray-dried powder was weighed in the lower chamber of a 20 ml two-chamber flask Wheaton RS-177FLW (Figure 1). The bottle was filled with nitrogen saturated with perfluorohexane at 13 ° C, before inserting the seal of the internal chamber. The upper chamber was filled with 10 ml of sterile water for injection. The shutter of the upper chamber was inserted to eliminate all air bubbles in the upper chamber. By pressing the top shutter, the seal between the chambers was forced into the lower chamber, allowing water to flow into the lower chamber and reconstitute the powder (Figure 2). Many stable microbubbles were formed, as demonstrated by the light microscope. This procedure demonstrated the convenience of this form of packaging and the elimination of the need to provide ventilation to eliminate pressure build-up, when the aqueous phase is added to the powder.

EXAMPLE XIV Formation of microbubbles with the use of a two-chamber syringe One hundred mg of the spray-dried powder were weighed in a double-chamber syringe of 5 ml + 5 ml HYPAK Liquid / Dry (Becton Dickinson, Franklin Lakes, NJ) and shook into the dust chamber (end of the needle). The seal between cameras was then placed just above the diversion channel. A needle, containing a 5 μM filter, was then mounted in the syringe. The chamber containing dust was then filled with the osmotic gas agent, placing the assembly in a vacuum chamber, evacuating and filling the chamber with the gas osmotic agent, the nitrogen saturated with per-fluorohexane, at 13 ° C. The filter needle allowed the evacuation and filling of the atmosphere in the chamber containing the powder. A cover of the sealing needle was then placed on the needle. The liquid chamber was then filled with 4 ml of water for injection and the plunger was seated using temporary ventilation (a wire inserted between the cylinder of the glass syringe and the plunger, so as to eliminate all air bubbles. To reconstitute, the needle sealing cover was removed to eliminate buildup of pressure in the powder chamber.The plunger was then squeezed, forcing the seal of the inner chamber to the deviation position, which allowed the water flow around the seal of the internal chamber inside the chamber containing the powder .. The movement of the plunger stopped when all the water was inside the dust chamber.The syringe was stirred to dissolve the powder.The excess gas and any large bubble was ejected by holding the syringe with the end of the needle facing upwards and also depressing the plunger.The solution containing numerous stabilized microbubbles (as noted by The light microscope was then ejected from the syringe, pressing the plunger to its limit. The foregoing description details certain preferred embodiments of the present invention and describes the best mode considered. However, it will be appreciated that regardless of how detailed the preceding text may appear, the invention can be practiced in many ways and it must be interpreted in accordance with the appended claims, and any equivalent thereof.

Claims (49)

1. CLAIMS 1. A method for forming a gas emulsion, this method comprises the steps of: supplying a container, having within it a dry, hollow, particulate, approximately microspherical material, which comprises at least one first and one second agent surfactants, a hydrophilic monomer or polymer or combinations thereof, and a gas or a mixture of gases, this first surfactant includes a phospholipid or a mixture of phospholipids, having at least one acyl chain, which comprises at least 10 carbon atoms, carbon, and comprises at least 5% w / w of the total surfactant, the second surfactant is more water soluble than the first surfactant; add a watery liquid to the container; Substantially dissolving the microspherical material in the aqueous liquid, thus forming a gas emulsion within the container, this gas emulsion comprises gas bubbles or the mixture of gases surrounded by a layer of the first and second surfactants. The method of claim 1, wherein the second surfactant comprises a fatty acid, a salt of a fatty acid, a sugar ester of one or more fatty acids, a copolymer of polyoxypropylene and polyoxyethylene, a nonionic alkyl glucoside, a polysorbate or a combination thereof. The method of claim 1, wherein the second surfactant comprises a phospholipid, a mixture of phospholipids, a phosphocholine or a lysophospholipid wherein the acyl chain comprises no more than 14 carbon atoms. The method of claim 1, wherein the first surfactant comprises a phosphatidylcholine with one or more acyl chains, at least one chain includes 12 to 18 carbon atoms, and the second surfactant comprises a phosphatidylcholine with one or more acyl chains, at least one chain comprises from 6 to 12 carbon atoms. The method of claim 1, wherein the hydrophilic monomer or polymer or a combination thereof, comprises a starch or a derived starch. 6. The method of claim 1, wherein the gas or gas mixture comprises a fluorocarbon or a mixture of fluorocarbons. The method of claim 1, wherein the gas or gas mixture comprises a first non-fluorocarbon gas in admixture with a second gas, this second gas has a vapor pressure of less than 760 mm Hg at a temperature of 37 ° C. The method of claim 7 wherein the first gas consists essentially of nitrogen or air, and the second gas consists essentially of perfluorohexane. 9. A method for forming a precursor composition of a gas emulsion, this method comprises the steps of: dispersing an aqueous solution, including a hydrophilic monomer or polymer, or a combination thereof, and a first and second surfactant, wherein the first surfactant consists essentially of a phospholipid or a mixture of phospholipids, having at least one acyl chain, which includes at least 10 carbon atoms, and comprises at least about 5% w / w of the agent total surfactant, and the second surfactant is more water soluble than the first surfactant; and spraying the dispersion, to create a dry, hollow, particulate, approximately micro-spherical material, which, when combined with an aqueous medium, forms an emulsion of echogenic gas, which includes bubbles of a gas surrounded by a layer of the first and second surfactants. 10. The method of claim 9, further comprising the step of enclosing the microspherical material in a container, with a gas or a mixture of gases, in which this gas or mixture of gases comprises a fluorocarbon or a mixture of fluorocarbons. The method of claim 9, wherein the hydrophilic monomer or polymer comprises from about 1 to 99% w / w of the dry, microspherical material, and the total surfactant includes from about 0.05 to 90% w / w of the dry material, microspherical. The method of claim 9, wherein the aqueous dispersion further comprises an inflation agent. The method of claim 12, wherein the inflation agent comprises a substance selected from the group consisting of methylene chloride, Freon 113, perfluorohexane and carbon dioxide. The method of claim 9, wherein the second surfactant comprises a fatty acid, a salt of a fatty acid, a sugar ester of one or more fatty acids, a copolymer of polyoxypropylene and polyoxyethylene, a nonionic alkyl glucoside, a polysorbate, or a combination thereof. 15. The method of claim 9, wherein the second surfactant comprises a phospholipid, a mixture of phospholipids, a phosphocholine, or a lysophospho-lipid, wherein each acyl chain includes no more than 14 carbon atoms. 16. The method of claim 9, wherein the first surfactant comprises a phosphatidylcholine with one or more acyl chains, at least one chain includes 12 to 18 carbon atoms, and the second surfactant comprises a phosphatidylcholine with one or more acyl chains, at least one chain includes 6 to 12 carbon atoms. 17. The method of claim 9, wherein the hydrophilic monomer or polymer, or a combination thereof, comprises a starch or a derived starch. 18. The method of claim 10, wherein the gas or gas mixture comprises a first non-fluorocarbon gas in admixture with a second gas, this second gas has a vapor pressure of less than 760 mm Hg at a temperature of 37 ° C. The method of claim 18, wherein the first gas consists essentially of nitrogen or air, and the second gas consists essentially of perfluorohexane. 20. A composition forming a gas emulsion, this composition comprises: a container; a dry, hollow, particulate, approximately microspherical material, within the container, which includes at least a first and a second surfactant, and a sufficient amount of a hydrophilic monomer or polymer or a combination thereof, to impart structural integrity to the microspherical material; wherein the first surfactant consists essentially of a phospholipid or a mixture of phospholipids, which has at least one acyl chain, which comprises at least 10 carbon atoms, and which includes at least 5% w / w of the agent total surfactant, and wherein the second surface active agent is more soluble in water than the first surfactant; and a gas or a mixture of gases within the container, impregnated within the microspherical material. The composition of claim 20, wherein the second surfactant comprises a fatty acid, a salt of a fatty acid, a sugar ester of one or more fatty acids, a copolymer of polyoxypropylene and polyoxyethylene, a nonionic alkyl glucoside, a polysorbate, or a combination thereof. 22. The composition of claim 20, wherein the second surfactant comprises a phospholipid, a mixture of phospholipids, a phosphocholine or a lysophospholipid, wherein each acyl chain includes no more than 14 carbon atoms. The composition of claim 20, wherein the first surfactant comprises a phosphatidylcholine with one or more acyl chains, at least one chain comprises 12 to 18 carbon atoms, and the second surfactant comprises a phosphatidylcholine with one or more acyl chains, at least one chain comprises from 6 to 12 carbon atoms. The composition of claim 20, wherein the gas or gas mixture comprises a fluorocarbon or a mixture of fluorocarbons. The composition of claim 20, wherein the gas or gas mixture comprises a first non-fluorocarbon gas in admixture with a second gas, this second gas has a vapor pressure less than 760 mm Hg, at a temperature of 37 ° C. 26. The composition of claim 25, wherein the first gas consists essentially of nitrogen or air, and the second gas consists essentially of perfluorohexane. 27. The composition of claim 20, wherein the hydrophilic monomer or polymer comprises a starch or a derived starch. 28. An ultrasound contrast medium for gas emulsion, this means comprises: gas bubbles or a mixture of gases, comprising a fluorocarbon or a mixture of fluorocarbons, having at least four carbon atoms surrounded by a layer of surfactant agent, which includes at least a first and a second surfactant, this first surfactant consists essentially of a phospholipid or a mixture of phospholipids, which has at least one acyl chain, which comprises at least 10 carbon atoms, and which includes at least 5% w / w of the surfactant total, and in that the second surfactant is more soluble in water than the first surfactant. 29. The contrast medium of claim 28, wherein the second surfactant comprises a fatty acid, a n-fatty acid salt, a sugar ester of one or more fatty acids, a polyoxypropylene and polyoxyethylene copolymer, an alkyl glucoside not ion, a polysorbate, or a combination thereof. 30. The contrast medium of claim 28, wherein the second surfactant comprises a phospho-lipid, a mixture of phospholipids, a phosphocholine or a lysophospholipid, wherein each acyl chain includes no more than 14 carbon atoms. The contrast medium of claim 28, wherein the first surfactant comprises a phosphatidylcholine with one or more acyl chains, at least one chain includes 12 to 18 carbon atoms, and the second surfactant comprises a phosphatidylcholine with one or more acyl chains, at least one chain includes 6 to 12 carbon atoms. 32. The contrast medium of claim 28, wherein the gas or mixture of gases comprises a first non-fluorocarbon gas in admixture with a second gas, this second gas having a vapor pressure less than 760 mm Hg, a a temperature of 37 ° C. 33. The contrast medium of claim 32, wherein the first gas consists essentially of nitrogen or air, and the second gas consists essentially of perfluorohexane. 34. A composition that forms a gas emulsion, this composition comprises: a container; a dispersion, spray dried, of a hydrophilic monomer or polymer or a combination thereof, and a first and second surfactant, wherein the first surfactant consists essentially of a phospholipid or a mixture of phospholipids, having at least one acyl chain, which comprises at least 10 carbon atoms and includes at least 5% w / w of the total tension-active agent, the second surfactant is more soluble in water than the first surfactant; and a gas or a mixture of gases within the container, impregnated into the spray dried dispersion. 35. The composition of claim 34, wherein the second surfactant comprises a fatty acid, a salt of a fatty acid, a sugar ester of one or more fatty acids, a copolymer of polyoxypropylene and polyoxyethylene, a nonionic alkyl glucoside, a polysorbate, or a combination thereof. 36. The composition of claim 34, wherein the second surfactant comprises a phospholipid, a mixture of phospholipids, a phosphocholine or a lysophospholipid, wherein each acyl chain comprises no more than 14 carbon atoms. 37. The composition of claim 34, wherein the first surfactant comprises a phosphatidylcholine with one or more acyl chains, at least one chain comprises 12 to 18 carbon atoms, and the second surfactant comprises a phosphatidylcholine, with a or more acyl chains, at least one chain comprises from 6 to 12 carbon atoms. 38. The composition of claim 34, wherein the gas or gas mixture comprises a fluorocarbon or a mixture of fluorocarbons. 39. The composition of claim 34, wherein the gas or gas mixture comprises a first non-fluorocarbon gas, in admixture with a second gas, this second gas has a vapor pressure less than 760 mm Hg at a temperature of 37 ° C. 40. The composition of claim 39, wherein the first gas consists essentially of nitrogen or air, and the second gas consists essentially of perfluorohexane. 41. The method of claim 34, wherein the aqueous dispersion further comprises an inflation agent. 42. The method of claim 41, wherein the inflation agent comprises a substance selected from the group consisting of methylene chloride, Freon 113, perfluorohexane and carbon dioxide. 43. A method for obtaining an ultrasound contrast medium, which comprises substantially dissolving the composition of claim 34 in an aqueous liquid, thereby forming a gas emulsion within the container, this gas emulsion includes bubbles containing the gas or the Gas mixture surrounded by a layer of the first and second surfactants. 44. A method for obtaining an ultrasound contrast medium, comprising substantially dissolving the composition of claim 40 in an aqueous liquid, thus forming a gas emulsion within the container, this gas emulsion includes bubbles containing at least nitrogen or air and perfluorohexane, surrounded by a layer of the first and second surfactants. 45. A method for forming images of an object or body, this method comprising the steps of: substantially dissolving the composition forming the gas emulsion, according to claim 20, in an aqueous liquid, to produce a gas emulsion; introduce the gas emulsion inside the object or body; and forming the image ultrasonically of at least a portion of the object or body. 46. A method for forming images of an object or body, this method comprises the steps of: introducing the ultrasound contrast medium, according to claim 28, into the object or body; and forming the image ultrasonically of at least a portion of the object or body. 47. A method for forming images of an object or body, this method comprises the steps of: introducing the ultrasound contrast medium, according to claim 33, into the object or body; and forming the image ultrasonically of at least a portion of the object or body. 48. A method for forming images of an object or body, this method comprising the steps of: substantially dissolving the composition forming the gas emulsion, according to claim 34, in an aqueous liquid, to produce a gas emulsion; introduce the gas emulsion inside the object or body; and forming the image ultrasonically of at least a portion of the object or body. 49. A method for forming images of an object or body, this method comprises the steps of: introducing the ultrasound contrast medium, according to claim 44, into the object or body; and forming the image ultrasonically of at least a portion of the object or body.