MXPA96003330A - Method of storage of suspensions gaseosasultrasoni - Google Patents

Abstract

The present invention discloses suspensions of immobilized gas microbubbles within a frozen aqueous carrier liquid constituted of customary additives and stabilizers, in which the carrier liquid is physiologically acceptable, the immobilized gas microbubbles are microbubbles bound by an evanescent or ephemeral envelope, or a tangible membrane. Suspensions, when in liquid form, are injectable and useful as a contrast agent in the formation of ultrasonic images of the blood and tissue accumulation of living beings. The gas microbubbles are immobilized inside the carrier by freezing a suspension of microbubbles with size

Description

METHOD OF ATTACHMENT OF SUSPI ULTRASÓNICAS TECHNICAL FIELD The invention relates to suspensions of gas bubbles immobilized within a frozen aqueous carrier medium. The invention also relates to a method of cold storage of gas bubble suspensions, and their use as contrast agents for the ultrasonic imaging of the human and animal body.

ANTECEDENTS OF THE TECHNIQUE The rapid development of contrast agents for ultrasound in recent years has generated many different formulations which are useful in ultrasound imaging of the organs and tissues of the human body or of animals. These agents are designed to be used mainly as injectable intravenous or intraarterial substances together with the use of medical ultrasound equipment. These instruments usually group the formation of images in the mode B (based on the spatial distribution of tissue backscattering properties) and signal processing REF: 22845 Doppler (based on continuous wave or pulsed Doppler processing of ultrasonic echoes to determine blood or fluid flow parameters) . Other ultrasound imaging methods may also benefit from these agents in the future, such as the Ultrasound Computed Tomography (which measures the attenuation in transmission) or the diffraction computed tomography (which measures the dispersion and attenuation parameters in the angular reflex). Based on the suspensions of gas microbubbles in aqueous liquid carriers, these injectable formulations can be basically divided into two categories: Aqueous suspensions in which the gas microbubbles are surrounded by a gas / liquid interface, or an evanescent or ephemeral envelope that it involves liquid molecules and a surfactant loosely attached at the gas-to-liquid interface, and suspensions in which the microbubbles have a material boundary or a tangible covering formed of natural or synthetic polymers. In the latter case, it is said that the microbubbles are microspheres or microballoons. There is another additional class of contrast agents for ultrasound: suspensions of porous particles of polymers or other solids which carry gas microbubbles trapped within the pores of the microparticles. These contrast agents are considered in this document as a variant of the class of microspheres or microballoons. Although physically different, both kinds of gas microbubbles, when in suspension, are useful as contrast agents for ultrasound. More of these different formulations can be found in EP-A-0 077 752 (Schering), EP-A-0 123 235 (Schering), EP-A-0 324 938 (Widder et al.,), EP-A -0 474 833 (Schneider et al.), EP-A-0 458 745 (Bichon et al.), US-A-4, 900, 540 (Ryan), US-A-5,230,882 (Unger), etc. Some of the contrast agents for ultrasound mentioned in the above have been developed and are commercially available, however others are in different stages of clinical trials. However, whether they are available commercially or in clinical trials, all of these products suffer from problems related to storage. Storage problems are intrinsic to suspensions which, due to their very nature, undergo separation of phases or segregation, aggregation of gas bubbles, diffusion of gas and, after prolonged periods, even present precipitation of the various additives. The segregation of the microbubbles or gas microspheres stems from the fact that the suspensions are usually made of uncalibrated microbubbles whose sizes vary from about 1 μm to about 50 μm. The vast majority of gas bubbles in known suspensions are between 1 μm and about 10 μm. Due to the size distribution of the microbubbles, these suspensions undergo segregation during storage, in which the larger microbubbles migrate to the top while the smaller microbubbles are concentrated in the lower parts, which often provides complete phase separation. Attempts to solve this problem by using agents that increase viscosity have shown that the rate of segregation can be reduced, but can not be eliminated. The aggregation of gas microbubbles is a process during which a large bubble absorbs the smallest, which increases its size. With the separation of phases, this process is accelerated and the suspensions with microbubbles having an average size of, for example, between 2 μm and 8 μm, after a certain time, can be developed in suspensions with microbubbles having a size between, for example, example, 5 μm to 12 μm or even higher. This is particularly undesirable in cases where suspensions of calibrated microbubbles and suspensions designed for opacification of the left part of the heart are related. The change in size not only alters the echogenic properties of the contrast agent, but also makes people inapplicable for certain applications such as those based on the passage of microbubbles through the lungs. It is unlikely that microbubbles with sizes greater than 10 μm pass through the capillaries of the lungs and therefore, in addition to generating dangerous conditions, such suspensions are less suitable for imaging the left side of the heart. Another problem with gaseous suspensions and their storage comes from the diffusion of gas, which occurs at relatively slow speeds, but which accelerates with phase separation. The inevitable escape of gas from the microbubble suspensions is further aggravated and in extreme cases can lead to a total decrease of the gas in the medium. Therefore, the combined effect of the various mechanisms involved in the destruction of gaseous suspensions results in a very rapid degradation of the agent. In some study methods in which ultrasound images are formed, one of the desirable aspects of these contrast agents is that the microbubbles or particles containing gas are distributed within a narrow size range. The reason is given in the following. The effectiveness of these agents in increasing the contrast in the images produced by medical ultrasonographic equipment is mainly based on the dispersion intensified greatly by the ultrasonic energy that enters, and secondly by the modified attenuation properties of the tissues that contain these agents. By contrast, it is meant a measure of the relative signal amplitude obtained from regions to be irrigated or perfused by the contrast agent, as compared to the signal amplitude of regions that do not receive the contrast agent. By "intensification" is meant an increase in the contrast value observed after the administration of the contrast agent, in comparison with the contrast observed before administration. As mentioned before, the type of imaging equipment that benefits most directly with these agents is the family of ultrasound instruments (B-mode or Doppler). The different attenuation properties of the tissues containing the agent as compared to those that do not contain the agent can also be exploited to improve the diagnostic value of the imaging procedure. In addition, the dependence of the ultrasonic frequency on both the dispersion and attenuation properties of the agent can be used to further increase spatial tissue differentiation. In these cases, the physical laws that govern this frequency dependence systematically depend on the size of the microbubble or the particle. Thus, the algorithms used are more efficient when they act on echoes that originate from microbubbles or particles with a narrow size distribution. As an example, one such approach exploits the non-linear oscillation of the microbubbles to detect the echo-frequency components in the second harmonic of the fundamental excitation frequency. Since the tissues that do not contain the contrast agent do not show the same non-linear behavior as the microbubbles, this method is able to significantly improve the contrast between the regions that contain and those that do not contain the contrast agent. This intensification is more pronounced, for a given particle count per unit volume, when the sizes are narrowly distributed. However, preparing products with such narrow-sized distributions takes time; providing an easy supply of these calibrated suspensions would greatly facilitate the further development and use of this technique. Reliable storage of such preparations with size distributions that do not show changes would also be of great interest. Additional difficulties are experienced with the storage of aqueous gaseous suspensions, with ultrasound contrast agents which contain phospholipids as stabilizers of the gas microbubbles. Due to the hydrolysis of the phospholipids, the concentration of the stabilizer (surfactant) during storage, decreases constantly, causing losses in the content of microbubbles and degradation of the echogenic properties of the suspension. Therefore, the storage problem of ultrasound contrast agents consisting of suspensions of gas microbubbles remains open to date. The cold storage of aqueous gas suspensions by freezing has been known for some time in the food industry. For example, US-A-4,347, 707 (General Foods Corp.) describes the storage of gasified ice with high gas content and good storage stability by a method in which gasified ice is prepared by contacting a liquid aqueous with hydrate-forming gases under pressure at a temperature such that the gas hydrate complex suspended in the liquid is formed, and the temperature and pressure are controllably decreased to produce, for example, carbonated ice with 85-110 ml of C02 / g. According to the document, the gasified ice has a high gas content, an extended storage stability, is suitable for commercial distribution in the frozen state and, when placed in water, provides vigorous effervescence. It follows that the freezing of gaseous suspensions in order to store them for extended periods of time and their reuse of the preserved suspensions when necessary is not suitable for contrast agents for ultrasound because the suspended gas has a tendency to escape from the half carrier during thawing. Additional difficulties are encountered with the suspensions of frozen microbubble gas by the fact that the expansion of the carrier medium during freezing generates internal forces which simply destroy or oppress the microbubble cover which releases the trapped gas and allows it to escape either during storage or later during the unfreezing of the suspension. This problem is particularly serious for suspensions of microbubbles that have material or tangible covers.

BRIEF DESCRIPTION OF THE INVENTION In brief, the invention relates to frozen suspensions of gas bubbles immobilized within a frozen aqueous carrier medium in which the carrier medium consists of gas bubbles with the usual additives and which is a physiologically acceptable carrier. The immobilized gas bubbles are microbubbles linked by an evanescent or ephemeral cover or by a tangible membrane and the suspensions, when in liquid form, are injectable in living beings and are useful as contrast agents for the formation of ultrasonic images of the accumulation of blood and tissues of human and animal patients. According to the invention, the temperatures of the frozen suspensions are between -1 ° C and -196 ° C, preferably between -10 ° C and -76 ° C, and the size of the gas microbubbles is less than 50 μm, and preferably less than 10 μm. Particularly useful are suspensions in which the size of the microbubbles is between 2 μm and 9 μm, and microbubble suspensions with sizes between 3 μm and 5 μm are even more useful. In view of the difference in biodegradability between suspensions comprising gas microbubbles or microbubbles with a tangible shell in which the membrane is made of a synthetic or natural polymer, or a protein, and suspensions constituted with gas microbubbles with evanescent covers, the frozen suspensions of the latter will be more advantageous, particularly when lamellar phospholipids are used in the form of mono or plurimolecular membrane layers. The invention also relates to a method for storing suspensions of microbubbles in which the suspension is placed in a cooling device, the microbubbles are immobilized within the carrier medium upon cooling to a temperature below the freezing point of the suspension, so preferable a temperature between -1 ° C and -196 ° C, and even more preferably between -10 ° C and -76 ° C, and when these freezing conditions are maintained for prolonged periods of time. Optionally, the frozen suspension can be maintained in an atmosphere of an inert gas or in a gas mixture, at least one of which is gas trapped in the microbubbles. Preferably the gas is selected from gases containing halogen, air, oxygen, nitrogen, carbon dioxide or mixtures thereof. The use of injectable suspensions of the invention for the ultrasonic ultrasound imaging of organs and tissues, as well as the manufacture of contrast agents for ultrasound are also described.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph of the change in the concentration of bubbles (counts) in defrosted suspensions of SF6 and air containing 5% microbubbles C5F12 in an aqueous carrier medium, as a function of storage temperature. Figure 2 is a graph of the change in total gas volume of thawed suspensions of SF6 and air containing 5% C5F12 microbubbles in an aqueous carrier medium as a function of storage temperature. Figure 3 is a graphical representation of the change in absorbance of thawed microbubble suspensions as a function of the applied external pressure and storage temperature. Figure 4 is a diagram showing the change in relative absorbance as a function of the external pressure applied to the thawed suspensions after storage at different temperatures. Figure 5 is a diagram showing the size distribution of microbubbles, in number (Figure 5A) and gas volume (Figure 5B) of calibrated microbubbles, with an average size of 3 μm, 4 μm and 6 μm.

Figure 6 is a diagram showing the microbubble size distribution, in number (Figure 6A) and in gas volume (Figure 6B) for an uncalibrated microbubble sample.

PKffrFTPH T pgTT.TATiA DP! T.A INVENTION According to the invention, a frozen suspension of microbubbles of gas is provided in which the microbubbles are immobilized within a frozen aqueous carrier medium which, in addition to the microbubbles, includes the usual additives. The aqueous carrier is physiologically acceptable and the suspension, when in liquid form, is injectable in living beings and is useful as an ultrasound contrast agent for imaging the accumulation of blood and tissues in humans and patients animals. The immobilized gas microbubbles bound by an evanescent cover or a tangible membrane are trapped between molecules of the frozen carrier medium whose temperature is between -1 ° C and -196 ° C, preferably between -10 ° C and -76 ° C. The exact temperature range will depend on the choice of gas in the microbubbles but also on the kind and amount of additives used. Thus, for example, in the case of air or nitrogen, the temperature can be anywhere in the range between -1 ° C and -196 ° C, while in the case of C4F8, the temperature will be between -1 ° C and -5 ° C. When polyoxypropylene / polyoxyethylene or polyethylene glycol copolymers are used as additives, based on the total amount present in the suspension, the highest acceptable temperature can be -5 ° C or even -10 ° C, instead of -1 ° C . The size of most of the microbubbles in the suspension is usually less than 50 μm, but for injectable intravenous contrast agents, the microbubble size will preferably be less than 10 μm. For most applications, suspensions with microbubbles having a size distribution between 2 μm and 9 μm satisfy the requirements, however, when working with calibrated microbubble suspensions, sizes can be anywhere in that range . For example, between 2-4 μm, 3-5 μm, 4-6 μm, 5-7 μm, 6-8 μm, 7-9 μm, however, microbubbles with a size range between 3 μm and 5 μm they are the ones that are preferred. Therefore, the invention also provides frozen suspensions of microbubbles with a very narrow size distribution. The typical microbubble size distributions of the calibrated microbubble suspensions will be as illustrated in Figure 5, in which the distributions are indicated in terms of the microbubble number distribution (determined by a Coulter counter) Figure 5A and in terms of of the distribution of microbubble volumes (determined by a Coulter counter) figure 5B. In contrast to suspensions containing calibrated microbubbles, suspensions containing uncalibrated gas bubbles will usually have size distribution patterns, in terms of number and volume, similar to those shown in Figures 6A and 6B. From these figures, it can be readily appreciated that the sonographic responses of calibrated gas microbubbles suspensions will be more uniform, provide less dispersion and consequently generate sharper images compared to suspensions with uncalibrated microbubbles. As mentioned, these calibrated suspensions are very desirable, but their use so far has been very limited. Therefore, these desirable suspensions are now readily available through a simple conversion of the frozen suspensions which can be stored at low temperatures for prolonged periods of time without undue loss of their initial echogenic capacity. A further advantage of the invention stems from the fact that, in order to generate echo signal components at twice the fundamental frequency, the second harmonic image formations require a non-linear oscillation of the contrast agent. Such behavior imposes that the level of excitation of ultrasound exceeds a certain acoustic threshold at a certain depth in the tissue. During the non-linear oscillation, a frequency conversion takes place which causes the conversion of the acoustic energy of the fundamental excitation frequency above its second harmonic. Therefore, the significant energies transmitted to the microbubbles during this type of imaging require microbubbles strong enough to survive these conditions. The invention provides easy and convenient access to suspensions having microbubbles with good resistance to pressure variations so that they can now be prepared in advance, stored and used when required. As already mentioned, in addition to the gas microbubbles, the frozen suspensions of the invention contain additives which include various surfactants, viscosity improving agents, stabilizers, etc. Surfactants which may include surfactants that form films or that do not form films include phospholipids in the form of lamellas or sheets which are known to stabilize the evacuated gas / liquid cover of microbubbles. Lamellar phospholipids may be in the form of monomolecular or plurimolecular membrane layers or liposomes. Based on the type of microbubbles, ie with evanescent cover or a tangible membrane, the carrier medium may include as additives moisturizing agents and / or hydrophilic stabilizing compounds such as polyethylene glycol, carbohydrates such as galactose, lactose or sucrose, dextran, starch, and other polysaccharides or other conventional additives such as polyoxypropylene glycol and polyoxyethylene glycol, and their copolymers; ethers of fatty alcohols with polyoxyalkylene glycols; esters of fatty acids with polyoxyalkylated sorbitan; soaps; polyalkylene glycerol stearate; polyoxyethylene glycerol ricinooleate; homopolymers and copolymers of polyalkylene glycols; soybean oil and polyethoxylated castor oil as well as hydrogenated derivatives; ethers and esters of sucrose or other carbohydrates with fatty acids, fatty alcohols, these may optionally be polyoxyalkylated; monoglycerides, diglycerides and triglycerides of saturated or unsaturated fatty acids; glycerides of soybean oil and sucrose can also be used. The surfactants may be film-forming or non-film-forming surfactants and may include polymerizable amphiphilic compounds of the linoleyl-lecithin or polyethylene dodecanoate type. Preferably, the surfactants are film-forming surfactants and more preferably are phospholipids selected from phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin, sphingomyelin and mixtures thereof. In addition to the mentioned film-forming surfactants, the suspensions may additionally include up to 50% by weight of non-lamellar surfactants selected from fatty acids, esters and ethers of fatty acids and alcohols with polyols such as polyalkylene glycols, polyalkylenated sugars and other carbohydrates, and polyalkylenated glycerol. Particularly suitable substances include dicetyl phosphate, cholesterol, ergosterol, photosterol, sitosterol, lanosterol, tocopherol, propyl gallate, ascorbyl palmitate and butylated hydroxytoluene. It should be understood that the invention is not limited solely to suspensions of gas microbubbles with an evanescent cover. Any suitable particle filled with gases such as porous particles, liposomes or microballoons having a casing produced from phospholipids, synthetic or natural polymers or proteins can be used conveniently. It has been established that microballoons or microspheres prepared with albumin, or liposome vesicles, can be stored successfully for extended periods of time in the frozen state. The thawed suspensions containing these microballoons or microspheres have demonstrated acceptable echogenic capacities that show a relatively small loss of microbubbles. Suspensions in which the microbubbles are stabilized with sorbitol or with nonionic surfactants such as polyoxyethylene / polyoxypropylene copolymers (commercially known as Pluronic) have shown equally good capabilities in imaging after storage.Additives used for suspensions of the invention can additionally include viscosity-improving substances and / or stabilizers selected from linear and cross-linked polysaccharides and oligosaccharides, sugars, hydrophilic polymers and iodinated compounds, in which case the weight ratio of these compounds to the surfactants is constituted between about 1: 5 to 100: 1 The suspensions according to the invention prepared with various gases or gas mixtures usually contain 107-108 microbubbles / ml, 108-109 microbubbles / ml or 109-1010 microbubbles / ml. concentrations remain virtually the same after s for prolonged storage, ie for several months, and if suspensions are made with SF6, C3F8 or with their mixtures or with air mixtures with SFS or with C5F12, they do not change even after repeated cycles of freezing and heating. When the microbubbles in the suspension have a tangible membrane, the membrane is made of a synthetic or natural polymer or a protein. The polymer which forms the shell or boundary membrane of the injectable microballoons can be selected from most hydrophilic polymers, biodegradable, physiologically compatible. Among such polymers there may be mentioned polysaccharides of low solubility in water, polylactides and polyglycolides and their copolymers, copolymers of lactides and lactones such as e-caprolactone, 6-valerolactone and polypeptides. The great versatility in the selection of synthetic polymers is of great advantage since, with allergic patients, one wishes to avoid using microballoons made of natural proteins (albumin, gelatin). Other suitable polymers include poly- (ortho) esters, copolymers of lactic acid and glycolide, poly (DL-lactide-co-d-caprolactone), poly (DL-lactide-co-d-valerolactone), poly (DL-lactide- co-g-butyro-lactone), polyalkyl cyanoacrylates; polyamides, polyhydroxybutyrate; polydioxanone; poly-ß-amino ketones, polyphosphazenes and polyanhydrides. Polyamino acids such as polyglutamic polyaspartic acids can also be used as well as their derivatives, i.e. partial esters with lower alcohols or glycols. A useful example of such polymers is poly- (tertbutyl glutamate). When the membrane is made of proteinaceous material, the protein is albumin. Although in conjunction with suitable surfactants and stabilizers, gases containing halogen, air, oxygen, nitrogen or carbon dioxide can be used alone, recently, it has also been proposed to use mixtures of halogen-containing gases with air, oxygen, nitrogen and carbon dioxide. Gases containing halogen are selected from sulfur hexafluoride gas, tetrafluoromethane, chlorotrifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, bromochloro-difluoromethane, dibromodifluoromethane, dichlorotetra-fluroetano, chloropentafluoroethane, hexafluoroethane, hexafluoropropylene, octafluoropropane, hexafluorobutadieno, octafluoro-2-butene, octafluorocyclobutane, I decafluoro -butane, perfluorocyclopentane, dodecafluoropentane or mixtures thereof, and preferably sulfur hexafluoride, tetrafluoromethane, hexafluoroethane, hexafluoropropylene, octafluoropropane, hexafluorobutadiene, octafluoro-2-butene, octafluorocyclobutane, decafluorobutane, perfluorocyclopentane, dodecafluoropentane.

The invention also relates to a method for storing microbubble suspensions in which the suspension is placed in a cooling device such as a refrigerator, the microbubbles are immobilized within the carrier medium upon cooling to a temperature below the freezing point of the microbubbles. the suspension, preferably a temperature between -1 ° C and -196 ° C, and more preferably between -10 ° C and -76 ° C, the freezing conditions are maintained for prolonged periods of time. Optionally, the frozen suspension is maintained in an inert gas atmosphere or in a gas mixture at least one of which is the gas encapsulated in the microbubbles. Preferably, the gas is selected from gases containing halogen, air, oxygen, nitrogen, carbon dioxide or mixtures thereof. It has been established that the gases or gas mixtures used for the frozen suspension of the invention must have boiling points which are lower than -18 ° C. This means that suspensions produced with halogenated gases such as C4F8 and CSF10 alone will have very poor storage stability and will lose practically all of their echogenic capacity after freezing. This is particularly surprising since all other halogenated gases produce highly stable frozen suspensions which survive several freeze / heating cycles without significant loss of echogenic capacity. Even "adding an additive" to these gases with small amounts of other halogenated gases or even vapors of a halogenated substance which at room temperature are liquids such as C5F12 has produced mixtures which can not be stored frozen. On the other hand, when mixing air with certain quantities of these gases, it has produced suspensions which have presented very good storage stability and very good echogenic capacity after several cycles of freezing / heating. As mentioned above, when in the liquid state, the injectable suspensions of the invention are useful as contrast agents for ultrasound imaging of organs and tissues. Clearly, before use, the suspensions are thawed and optionally kept at room temperature for a period of time and subsequently administered to the patient. Afterwards, the patient undergoes exploration with an ultrasonic probe and an image of the scanned region is produced. A method for the manufacture of ultrasonic ultrasound contrast agents from the frozen injectable suspensions of the invention is also within the scope of the invention. By "manufacture" it is meant that highly concentrated suspensions (eg, OO-1111 microbubbles / ml or more) of calibrated or uncalibrated microbubbles are stored in a frozen state for a period of time and when required, the reheated suspensions are, optionally , diluted to the desired concentration by addition of the same carrier or a different physiologically acceptable liquid carrier. It is also considered that additional additives or conditioners can be added at this point. The invention is further illustrated by the following examples: EXAMPLE 1 In a round-bottomed glass flask, at 50 ° C in tert-butanol (20 ml), fifty-eight milligrams of diarachyidoylphosphatidylcholine (DAPC), 2.4 mg of dipalmitoylphosphatidic acid (DPPA), both of Avanti Polar Lipids ( United States) and 3.94 g of polyethylene glycol (PEG 4000 from Siegfried). The clear solution is rapidly cooled to -45 ° C and lyophilized. Aliquots (25 mg) of the white cake obtained are filled into 10 ml glass jars. The bottles are closed with rubber stoppers, purged and filled with the selected gases or gas mixtures (see Table 1). Subsequently saline solution (0.9% NaCl) is injected through the plugs (5 ml per bottle) and the lyophilized ones are dissolved with vigorous agitation. The suspensions of microbubbles are introduced in a cold storage room (-18 ° C). Three days later they are heated to room temperature (23 ° C) and subjected to analysis to determine the bubble concentration (by using a Coulter Multisizer) and the absorbance at 700 nm of a 1/50 dilution. The absorbance at 700 nm, is a measure of the total turbidity of the bubble suspension. TABLE 1 expressed as the Bunsen coefficient "" "The results given in Table 1 show that the freezing stability depends on the boiling point and the water solubility of the gas, less bubbles are recovered in the case of gases with 5 boiling points higher than -18 ° C. With regard to gases with a boiling point lower than -18 ° C, the lower the solubility in water, the greater the recovery of bubbles.It is also interesting to note that the addition of a small amount of a high molecular weight gas with Low solubility in water such as dodecafluoropentane (C5F12) improves the recovery of bubbles after reheating, in the case of gases with low boiling points, but not for gases with boiling points above -18 ° C. Finally, the microbubbles filled with C4F8 and C4F10 by themselves, or in mixtures with small amounts of dodecafluoropentane have very little stability to the freezing / heating process. However, when these gases are used in combination with air suspensions of microbubbles containing the mixtures are observed best results in terms of echogenic capacity or loss of microbubbles compared to suspensions containing microbubbles of air.

EXAMPLE 2 Suspensions of SF6 microbubbles are prepared as described in Example 1, which are frozen slowly (approximately 30 minutes) to -18 ° C, or rapidly (in one minute) at -45 ° C. Other suspensions of SF6 microbubbles are first kept at room temperature until all the bubbles have reached the surface, before freezing. Others freeze while the bubbles are distributed homogeneously in the solution. The frozen suspensions are stored at -18 ° C and -45 ° C for one month and then heated.

TABLE 2 The results in Table 2 show that the freezing speed has relatively little influence on the final result. Recovery in the case of homogeneous suspensions is better than in the case of suspensions of decanted microbubbles.

EXAMPLE 3 Ultilamellar liposomes (MLV) are prepared by dissolving 4.5 g of hydrogenated soy phosphatidylcholine (HSPC from Nattermann) and 0.5 g of dicetylphosphate (Fluka, Switzerland) in chloroform-methanol (2/1) and then evaporating the solvents to dryness in a Round bottom flask using a rotary evaporator. The residual lipid film is dried in a vacuum desiccator. After the addition of 100 ml of distilled water, the suspension is incubated at 70 ° C for 30 minutes under agitation. The liposome suspension is adjusted to a final lipid concentration of 25 mg per ml by the addition of distilled water. An aliquot of the liposome suspension (100 ml) is introduced into a gas-tight glass reactor equipped with a high speed mechanical emulsifier (Polytron). The gas phase in the reactor is air containing 5% C5F12 (determined by density measurement). After homogenization (1000 rpm, 1 min), the milky suspension obtained is introduced into a separating funnel. After 6 hours, a white layer of bubbles can be seen at the top of the solution. The lower phase (containing liposomes) is separated, a small amount of fresh water is added and the microbubble layer is homogenized again. The procedure is repeated (decantation) and four samples with different sizes of microbubbles are prepared (see O94 / 09829). The samples are frozen at -18 ° C and 24 hours later they are heated to room temperature.

TABLE 3 From the results obtained (Table 3) it is evident that the freezing / heating treatment has no influence on the average diameter of the microbubbles. From the experiments, it is clear that microbubbles with larger diameter (> 2.5 μm) are better at resisting the freezing / heating treatment compared to microbubbles with a smaller diameter. It is further noted that in certain cases, the defrosting speed can influence the final concentration of the microbubbles present in the sample. However, the exact relationship is not yet clear, as there are indications that the loss of microbubbles is inversely proportional to the rate of thawing. Preliminary results indicate that better results are obtained in terms of microbubble concentration (count) and the total volume of gas in the suspensions with an accelerated thawing, that is, a thawing carried out in a 25 ° water bath C, compared to what is observed when the samples are left at 5 ° C or at 20 ° C so that they are thawed more gradually. The greatest losses of gas volume are observed for samples thawed at 5 ° C EXAMPLE 4 The experiment described in Example 3 is repeated using a mixture of surfactants instead of MLV. The mixture of surfactants is obtained by dissolving 1 g of dipalmitoylphosphatidylglycerol (Na salt of DPPG, from Avanti Polar Lipids, EU), and 3 g of Pluronic® FS8 (a copolymer of polyoxyethylene-polyoxypropylene with a molecular weight of 8400) in water distilled (80 ml). After heating to approximately 70 ° C a clear solution is obtained. This solution is cooled to room temperature and the volume is accorded 100 ml with glycerol. The surfactant solution is introduced into a sealed glass reactor equipped with a Polytron emulsifier. After homogenization (10'000 rpm, 1 min) a milky suspension is obtained with a layer on top of foam. The foam is discarded and the lower phase containing 109 microbubbles per ml is recovered. This phase is left for several hours before collecting the white layer of microbubbles. The microbubbles collected are homogenized in distilled water, decanted a second time and frozen / heated as previously described. When comparing the characteristics of the bubbles before and after freezing, no significant variation is observed in the total bubble concentration (before the test, 1.3 x 106 bubbles per ml, after the test, 1.25 x 108), or the diameter Dn medium (before the test, 4.0 μm, after test 3.9).

EXAMPLE SF6 bubbles are prepared as described in Example 1, and decanted as described in Example 3. During the process of removing the lower aqueous phase, an equivalent volume of SF6 is introduced into the funnel. The bubble layer is resuspended in distilled water, 0.9% NaCl, 3% glycerol aqueous solution and 100 mg / ml trehalose solution. Bubble concentrations and absorbance at 700 nm (after dilution) are determined before and after the freeze-heating treatment. The results in Table 4 show an excellent recovery of the bubbles independently of the suspension medium. It is important to note that the recovery of microbubbles in this test is between 66 and 96%, which can be considered a contradiction with the results presented previously in Tables 1 and 2. However in this case, first, the microbubbles are calibrated, that is, they are more stable than those not calibrated, and secondly, the size of the bubbles used here is greater than 4 μm. TABLE 4 EXAMPLE 6 SF6 microbubbles decanted are obtained as described in Example 5. The microbubble layer is suspended at different concentrations of bubbles in 0.9% NaCl, then frozen at -18 ° C, stored for approximately 4 months and heated to temperature ambient .

TABLE 5 * between pairs, resumes or after the second freezing / heating treatment.

The results indicated in Table 5 do not show a significant effect of the concentration of bubbles on the resistance to freezing / heating.

Even a second treatment of freezing / heating, which is carried out after storage, has no effect on the final concentration, which shows that these suspensions are resistant to repeated cycles of freezing and heating even after storage, without great loss in the count of bubbles and without damage to the echogenic capacity of the samples.

EXAMPLE 7 Sonicated albumin microspheres are prepared by using the method described in EP-A-0 324 928 (idder). Briefly, 5 ml of 5% sterile human albumin solution (from the Blood Transfusion Service of the Swiss Red Cross, Bern, Switzerland) is placed in a 10 ml calibrated syringe. . A sonicator probe (Model 250 from Branson Ultrasonic Corp. USA) is placed inside the solution, up to the 4 mi mark. The sonication is carried out for 30 seconds at an energy set at 7. Subsequently the sonicator probe is raised to the 6 mi mark (ie, above the solution level) and sonication is continued in the pulse mode (0.3 s / cycle) for 40 seconds. After removing the foam layer, a suspension of albumin microspheres containing 1.5 x 108 bubbles per ml is obtained, with a mean diameter number of 3.6 μm. This suspension is frozen at -18 ° C and left at this temperature overnight. The day after, the suspension is heated to room temperature. The suspension analyzed before and after freezing / heating has the following characteristics.

TABLE 6 The results in Table 6 show some degree of destruction of the microbubbles containing air by subjecting them to the cycle of freezing and heating. When the bubbles are prepared with air mixtures containing approximately 5% by volume of C5F12 or C3F8 instead of pure air, the stability of the microspheres against the loss of the bubble count by freezing / heating is greatly improved (see Table 7). From the results obtained with albumin microsphere, it can also be seen that the freezing / heating cycles in the case of air microbubbles are harmful to the lower end of the population sizes of microbubbles, ie, in that microbubbles with an average size of approximately 5 μm or more have a better resistance and chance of survival compared to smaller microbubbles.

TABLE 7 EXAMPLE 8 When 3 g of saccharide microparticles SHU-454, a commercial echo contrast agent (Echovist ", Schering, Germany) is reconstituted in a galactose solution (8.5 ml, 200 mg / ml), as recommended by the manufacturer and submitted to a freezing / heating treatment, only a few bubbles are present in the solution after heating If 10 mg per ml of a mixture of dipalmitoylphosphatidyl glycerol and Pluronic * ® F68 (1: 5, p: p) is added to the galactose solution before the addition of the saccharide microparticles, stable microbubbles can be obtained at a concentration of 2.5 x 108 bubbles per ml After freezing and heating the concentration of bubbles decreases, approximately three orders of magnitude, to 2.3 x 105 per me, which indicates a considerable loss of microbubbles during treatment, however if, before reconstitution, the air phase in the gas is replaced by a mixture of air containing 10% C5F12, the bubbles are more stable and also more resistant to the treatment of freezing / heating. The loss of microbubbles in the latter case is only 2.3 x 10a and the concentration decreases from 3.9 x 108 to 1.6 x 108 bubbles per ml.

EXAMPLE 9 Microballoons or polymeric microspheres are prepared by using the technique described in Example 4 of EP-A-0 458 745 (Braceo International). The polymer used in this preparation is poly-POMEG, which is described in the North American patent 4 '888' 398. The microballoons are suspended at a concentration of 1.8 x 108 particles per ml in distilled water, 0.9% NaCl, 5% dextrose and 3% glycerol. Each of these suspensions is frozen and then heated. No detectable changes are observed after heating, neither in the total particle concentration nor in the average particle diameter.

EXAMPLE 10 An amount of 126 mg of egg lecithin and 27 mg of cholesterol is dissolved in 9 ml of diethyl ether in a 200 ml round bottom flask. To the solution is added 3 ml of a 0.2 molar aqueous solution of sodium bicarbonate with ionophore A23187, and the resulting two-phase system is subjected to sonication until it becomes homogeneous. The mixture is evaporated to dryness in a Rotavapor and 3 ml of a 0.2 molar aqueous solution of bicarbonate are added to the flask containing the lipid deposit. After allowing a period of time to stand, the resulting liposome suspension is dialyzed against saline to remove the bicarbonate that has not been trapped and to acidify. After a certain time it is washed with distilled water and then passed several times through a cascade of carbonate filters.

Subsequently an aliquot of the liposome suspension with a size smaller than 50 μm is frozen at -18 ° and stored at this temperature. Two weeks later, the suspension is heated to room temperature. The suspension analyzed before and after the freezing / heating process shows an acceptably low loss of its echogenic capacity and its microbubble count.

The procedure of Example 3 is repeated using SF6 and air containing 5% S5F12 as the gas phase, and the samples of the resulting suspension are frozen at -18 ° C, -45 ° C, -76 ° C and -196 ° C. After 30 days of storage at the corresponding freezing temperatures, the samples are heated and the suspensions are analyzed. The results obtained are presented in Table 8. The concentration of microbubbles in the samples before freezing is 11.6 x 107 / ml for SF6 and 9.8 x 107 / ml for the air / C5F12 mixture at 5%, while the The respective volumes of the gases in the samples before storage are 2.9 μl / ml and 5.9 μl / ml. The average diameter of the microbubbles for the two gases is 2.3 μm and 4.2 μm.

From the results it is deduced that the lower the storage temperature the greater the loss in the concentration of microbubbles and the total amount of gas recovered when heating. The results also show that heated samples of SF5 suspensions average diameter first increases and then, as the temperature drops, it decreases significantly. For the 5% air / C5F12 mixture, there is a relatively small change in the diameter of the microbubbles. The results also show that the average diameter of the microbubbles decreases with decreasing storage temperature. As shown in Figure 1 for suspensions with SFS and with the air / C5F12 mixture, only 51.7% and 52% of the initial microbubble population is recovered after storage at -196 ° C. However, as seen in Figure 2, the volume of total gas recovered after storage for SF6 is 21.1% of the initial volume while for the air / C5F12 mixture, the volume recovered is 46.5%. This indicates that the size of the microbubbles in the case of the suspensions prepared with SF6 has changed, that is, that the recovered microbubbles have reduced diameters.

TABLE 8 Figures 3 and 4 illustrate the effect of freezing or storage temperature on the resistance to pressure changes of microbubble suspensions containing a mixture of air with 5% C5F12. In the diagram in Figure 3, the absorbance (measured at 700 nm) is presented as a function of the pressure applied to the suspensions after storage for 3 days at different temperatures. From this diagram it follows that the decrease in storage temperature has an impact on the properties of the suspensions so that the changes in the properties of the suspensions stored at temperatures between -18 ° C and -76 ° C is significant , while the change for samples stored between -76 ° C and -196 ° C is relatively small. Therefore, keeping storage temperatures below -76 ° C does not seem to provide additional benefits. In view of the costs involved in choosing temperatures below -76 ° C, it can only be justified in exceptional cases. On the other hand, Figure 4 shows that if the suspensions of microbubbles containing the 5% air / C5F12 mixture are stored for three days at low temperatures, their resistance to pressure variation remains relatively unchanged while the same suspension maintained during the same period at 25 ° C loses approximately 10% of its initial resistance to pressure variations. The exact procedure and importance of critical pressure and absorbance measurements have already been explained in EP-A-0 554 213 which is incorporated herein by reference.

EXAMPLE 12 Two-dimensional echocardiography is performed using a 128 XP 5 ACUSON equipment (Acusón Corp. USA) with the preparations of Examples 1, 3 and 7 in experimental miniature pigs after peripheral vein injection of 0.04 ml / kg body weight. The intensification of the left ventricular echo contrast is determined in the case of original samples as well as frozen / heated. No significant differences are observed even in the cases of samples such as those described in Example 7 for alumina microspheres, which indicates that the residual bubbles are large enough to produce strong and long lasting echo intensification. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates. Having described the invention as above, property is claimed as contained in the following:

Claims (25)

1. A frozen suspension of gas bubbles immobilized within a frozen aqueous carrier medium, the suspension is characterized in that the carrier medium is constituted by the gas bubbles and the usual additives, which is a physiologically acceptable carrier, the immobilized gas bubbles are microbubbles attached to an evanescent or ephemeral cover or a tangible membrane, the suspension, when in liquid form, is injectable and is useful as a contrast agent in the formation of ultrasonic images of the accumulation of blood and tissues of beings alive

2. The suspension according to claim 1, characterized in that the temperature of the frozen medium is between -1 ° C and -196 ° C, preferably between -10 ° C and -76 ° C.

3. The suspension according to claim 1 or 2, characterized in that the size of most of the microbubbles is less than 50 μm, preferably less than 10 μm.

The suspension according to claim 3, characterized in that the size of the microbubbles is between 2 μm and 9 μm, preferably between 3 μm and 5 μm

5. The suspension according to claim 1 or 2, characterized in that the additives include, as surfactants, lamellar or lamellar phospholipids in the form of monomolecular membrane layers or 10 plurimolecular.

6. The suspension according to claim 5, characterized in that the lamellar phospholipids are in the form of unilamellar liposomes or 15 multilaminares.

7. The suspension according to claim 5, characterized in that the lamellar phospholipids are saturated phospholipids.

8. The suspension according to claim 6 or 7, characterized in that the phospholipids are selected from phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidyl-glycerol, phosphatidylinositol, cardiolipin and sphingomyelin, or mixtures thereof.

9. The suspension according to claim 1 or 2, characterized in that the additives include substances selected from dicetyl phosphate, cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate, ascorbyl palmitate and butylated hydroxytoluene.

10. The suspension according to claim 1 or 2, characterized in that the additives include sorbitol or nonionic surfactants such as polyoxyethylene / polyoxypropylene copolymers.

11. The suspension according to any of the preceding claims, characterized in that the additives include viscosity-increasing substances and / or stabilizers selected from polysaccharides and linear and cross-linked oligosaccharides, sugars, hydrophilic polymers and iodinated compounds, in a proportion by weight with respect to surfactant that is between approximately 1: 5 and 100: 1.

~ 12. The suspension according to any of the preceding claims, characterized in that the additives additionally comprise up to 50% by weight of non-lamellar surfactants selected from fatty acids, esters and ethers of fatty acids and alcohols with polyols.

13. The suspension according to claim 12, characterized in that the polyols are ± 0 polyalkylene glycols, polyalkylenated sugars and other carbohydrates, and polyalkylenated glycerol.

14. The suspension according to claim 1 or 2, characterized in that it contains 107-108 15 microbubbles / ml, 108-109 microbubbles / ml or 109-1010 microbubbles / ml.

15. The suspension according to claim 1 or 2, characterized in that the membrane is 20 manufactures a natural or synthetic polymer, or protein.

16. The suspension according to claim 15, characterized in that the polymer is selected from polysaccharides, polyamino acids and their esters, 25 polylactides and polyglycolides and their copolymers, copolymers of lactones and lactones, polypeptides, poly- (ortho) esters, polydioxanone, poly-ß-amino ketones, polyphosphazenes, polyanhydrides, polyalkyl- (cyano) -acrylates, polyolefins, polyacrylates, polyacrylonitrile, polyesters non-hydrolysable, polyurethanes and polyureas, polyglutamic or polyaspartic acid derivatives and their copolymers with other amino acids.

17. The suspension according to claim 15, characterized in that the protein is albumin.

18. The suspension according to claim 1, characterized in that the gas is selected from gases containing halogen, air, oxygen, nitrogen, carbon dioxide or mixtures thereof.

19. The suspension according to claim 18, characterized in that the halogen-containing gas is selected from the group consisting of SF6, CF4, C2Fβ, C2F8, C3F6, C3F8, C4F6, C4F8, C4F10, C5F10, C5F12 and mixtures thereof.

20. A method for storing microbubble suspensions, according to claims 1 to 19, the method is characterized by the steps of: a) placing the liquid suspension in a cooling device, b) immobilizing the microbubbles on cooling to a temperature below the freezing point of the suspension, preferably at a temperature between -1 ° C and -196 ° C, so more preferable, at a temperature between -10 ° C and -76 ° C, and c) maintaining freezing conditions for extended periods of time.

21. The method according to claim 20, characterized in that the frozen suspension is maintained in an atmosphere of an inert gas or a mixture of gases in which at least one of which is the gas encapsulated in the microbubbles.

22. The method according to claim 20, characterized in that the microbubbles contain a gas or a mixture of gases in which at least one of which has a boiling point lower than -18 ° C.

23. The method according to claim 20, characterized in that before use, the suspension is thawed and maintained at room temperature for a period of time.

24. Injectable suspensions according to claims 1 to 19, characterized in that they are for use in ultrasound imaging of organs and tissues.

25. The use of injectable suspensions according to claims 1 to 19 for the manufacture of ultrasonic ultrasound contrast agents.