RU2344833C2 - Contrast medium for ultrasonic imaging - Google Patents

Abstract

FIELD: medicine; ultrasonic diagnostics.

SUBSTANCE: clinical research is conducted to develop specific medicine forms using polymer microspheres with inserted perfluorocarbon. Medicine forms include microspheres of biologically compatible polymer, preferably with lipids incorporated in polymer, and containing perfluorocarbon, which transits to gaseous form at human body temperature. Patient receives microspheres in effective amount for improvement of ultrasonic imaging of ventricle chambers for longer than 5 minutes, dosage varying from 0.025 to 8.0 mg of microspheres per kg of body weight. Preferable dosage varies from 0.05 to 4.0 mg of microspheres per kg of body weight. Medicine form is usually stored in ampoule. Medicine in dry powder form is recovered with sterile water before application by adding water to ampoule or syringe and shaking it to obtain isosmotic or isotonic microsphere suspension.

EFFECT: improved image of ultrasonic imaging of cardiovascular area for long time period.

33 cl, 5 tbl, 5 ex

Description

BACKGROUND OF THE INVENTION

The present invention relates to the field of tools for diagnostic imaging and especially relates to specific tools for ultrasound diagnostics, providing improved images and long-term images.

When using ultrasound to obtain images of the internal organs and structures of a person or animal, ultrasonic vibrations, vibrations of sound waves in frequency higher than those that the human ear perceives, are reflected, as they pass through the body. Different types of body tissues reflect ultrasonic vibrations in different ways, and reflections that are caused by ultrasonic vibrations reflected from various internal structures are detected and automatically converted into a visible image.

For some medical indications, obtaining the desired image of an organ or structure of interest is especially difficult, since the details of the structure are not sufficiently distinguishable from the surrounding tissue in an ultrasound image obtained by reflecting ultrasonic vibrations in the absence of a contrast enhancing substance. The detection and observation of certain physiological and pathological conditions can be significantly improved by increasing the contrast of the ultrasound image by introducing into the organ or other structure of interest an ultrasound contrast medium. In other cases, it is especially important to detect the movement of the ultrasound contrast medium. For example, a certain model of blood flow, which is known to be the result of only specific cardiovascular disorders, may be more visible with the introduction of an ultrasound contrast medium into the bloodstream and further monitoring of the blood flow or blood volume.

Substances that are effective as contrast agents for ultrasound act by influencing ultrasonic vibrations as they pass through the body and reflect, forming an image that makes a medical diagnosis. Different types of substances affect ultrasonic vibrations in various ways and to different degrees. In addition, some of the effects caused by contrast enhancing agents are more easily evaluated and observed than others. The substance presented in the selected ideal composition of the contrast medium for ultrasound probably has the most powerful effect on ultrasonic vibrations, as it penetrates the body. Also, the effect on ultrasonic vibrations should be easily measurable. Gases are the preferred medium for use as a contrast medium for ultrasound. Before use in the form of surfactant-stabilized vesicles or by encapsulation in liposomes or microparticles, the gas must be stabilized. There are three main contrast enhancing effects that can be observed with an ultrasound image: backscattering, beam attenuation, and sound velocity difference.

To encapsulate contrast media for ultrasound imaging, various natural and synthetic polymers, such as air, were used during injection to produce an ultrasound contrast agent that lasts longer after administration. In Schneider et al., Invest. Radiol., Vol. 27, pp. 134-139 (1992) describe three-micron air-filled synthetic polymer particles. As reported, these particles are stable in plasma and under external pressure. However, at 2.5 MHz their echogenicity was reduced. Another type of suspension of microbubbles was obtained from albumin subjected to ultrasound. Feinstein et al., J. Am. Coll. Cardiol., Vol. 11, pp. 59-65 (1988). Feinstein describes the preparation of microbubbles with sizes suitable for transpulmonary passage with excellent in vitro stability. However, these microbubbles are short-lived in vivo , having a half-life of the order of several seconds (which is approximately equal to one circuit) due to their instability under pressure. Gottlieb, S. et al., J. Am. Soc. Echo., Vol. 3, pp. 328 (1990), Abstract; and Shapiro, JR et al., J. Am. Coll. Cardiol. Vol. 16, pp. 1603-1607 (1990).

Gelatin-enclosed microbubbles have also been described by Rasor Associates, Inc. in WO 80/02365. They are formed as a result of gelatin sticking together. The gas microbubbles enclosed within a fluorine-containing shell are described by Molecular Biosystems, Inc. in WO 96/04018.

In addition, microbubbles stabilized with galactose microcrystals (SHU 454 and SHU 508) were reported by Fritzch et al. Fritzsch, T. et al., Invest. Radiol. Vol. 23 (Suppl 1), pp. 302-305 (1988); and Fritzsch, T. et al., Invest. Radiol., Vol. 25 (Suppl 1), 160-161 (1990). Microbubbles persist for up to 15 min in vitro , but less than 20 sec in vivo . Rovai, D. et al., J. Am. Coll. Cardiol., Vol. 10, pp. 125-134 (1987); and Smith, M. et al., J. Am. Coll. Cardiol., Vol. 13, pp. 1622-1628 (1989). EP 398935 Schering Aktiengesellschaft discloses the preparation and use of microencapsulated gas or volatile liquids for ultrasound imaging in which microcapsules are formed by synthetic polymers or polysaccharides. In European patent 458745, Sintetica showed air or gas beads surrounded by a border deposited polymer membrane that can be dispersed in an aqueous vehicle for administration to an animal host or for oral, rectal or urethral administration, for therapeutic or diagnostic purposes.

WO 92/18164 Delta Biotechnology Limited describes the preparation of microparticles by spray drying of an aqueous protein solution to form hollow spherical shells containing gas inside for use in imaging. WO 93/25242 describes the synthesis of microparticles for ultrasound imaging, consisting of a gas that is contained within a shell of polycyanoacrylate or polyester. WO 92/21382 shows the production of microparticles of contrast agents, which include a covalently bonded base containing a gas in which the base is a carbohydrate. U.S. Patent Nos. 5,334,381, 5,234,414 and 5,354,435 Unger describe liposomes for use as ultrasound contrast agents, which include gases, gas precursors such as pH or light activated gaseous precursors, and other liquid or solid contrast enhancing agents.

The effect of the gas encapsulated in the capsule was observed and the use of fluorine-containing gas was proposed to improve the image compared to air. US Pat. No. 5,393,524 Quay discloses the use of perfluorocarbon-containing agents to improve contrast in ultrasound imaging. Composed of small vials or microbubbles of selected gases, which demonstrate long-term viability, fill the solution and are quite small for penetration through the lungs, facilitating their use in ultrasound imaging of the cardiovascular system and other critical organs. In European patent 554213 Bracco shows the use of fluorinated hydrocarbon gases to prevent the collapse of microvesicles after applying pressure to the vascular bed. WO 95/23615 Nycomed shows microcapsules for visualization that are formed by donor-acceptor type complexes from a solution, for example, a protein solution containing perfluorocarbon. WO 95/03357 of the Massachusetts Institute of Technology shows microparticles formed by a polyethylene glycol block copolymer — lactide-glycolide copolymers containing encapsulated radioactive preparations including gases such as air and perfluorocarbons. As described in WO 94/16739, Sonus Pharmaceuticals, Inc., although solids and liquids reflect the signal to the same extent, gases are known to be more effective and preferred media for use as ultrasound contrast media. Indeed, as shown in WO 94/16739 in Example 12, protein microcapsules were rejected as a risk factor (and also due to performance problems) when administered to dwarf pigs. US Pat. Nos. 6,132,699 and 5,611,344 describe methods for enhancing contrast using perfluorocarbon in synthetic polymer shells. US Pat. No. 5,837,221 describes a method for producing a permeable polymer microparticle containing a hydrophobic substance incorporated into a polymer to enhance echogenicity.

Some ultrasound contrast media have been approved in the United States or Europe for severely limited cardiac applications. OPTISON ^® (Amersham, Mallinkrodt) consists of heat-denatured human albumin microcapsules that contain octafluoropropane gas. Each milliliter of a suspension of microspheres contains 5-8 · 10 ⁸ microspheres with an average diameter in the range of 2-4.5 microns and 220 μg of octafluoropropane. These microspheres were not approved for assessing coronary blood flow, but were approved solely for isolating the contours of the ventricular chamber. At high shock doses (5 ml of suspension or 1100 μg of octafluoropropane), the ventricular chamber contours continue to last up to 5 minutes.

DEFINITY ^® (Bristol Myers Medical Imaging) consists of octafluoropropane containing lipid microspheres, in which the lipid membrane consists of phospholipids DPPA, DPPC and mPEG-DPPE. Each ml of suspension contains 1.2 · 10 ¹⁰ microparticles having an average diameter in the range of 1.1-3.3 microns and 1100 μg of octafluoropropane. The tool was tested solely for isolating the contours of the ventricular chamber, and not for assessing coronary blood flow. At a loading dose of 700 μl (per 70 kg of a person’s weight) or 5133 μg of gas, the agent increased the operating time in the ventricular chambers to approximately 3.4 minutes.

IMAGENT ^® (Photogen Inc.) consists of lipid microspheres containing perfluorohexane, the lipid membrane of which is composed of phospholipid DMPC. Each ml of suspension contains 1.4 · 10 ⁹ microparticles having an average diameter of less than 3 microns and 92 μg of perfluorohexane. The tool was tested solely for isolating the contours of the ventricular chamber, and not for assessing coronary blood flow. At a shock dose of 0.43 ml (per 70 kg of a person’s weight) or 40 μg of gas, the substance has an average increase in operating time in the ventricular chambers up to approximately 2.6 minutes.

In all cases, these commercial products are of limited use and are not approved for use, except for highlighting the contours of the ventricular chamber and providing an average duration of image quality improvement in the ventricular chambers lasting for 5 minutes or less. There is a shortage of commercial ultrasound contrast agents that provide long-term enhanced images of the cardiovascular system, especially the myocardium and ventricular chambers. The agents described in the prior art, when administered in the form of a shock dose or a short infusion, result in an image of the myocardium, which lasts for a significantly shorter time than the total time required for a complete heart examination. Typically, means of the prior art provide images that last significantly less than 1 minute for the myocardium. A tool that can provide enhanced image durations greater than 1 minute in the myocardium and / or higher than 5 minutes in the ventricular chambers is preferred.

Therefore, the aim of the invention is to create a formulation containing microparticles, which allow you to get enhanced images and long-term images, especially for cardiac applications.

Another objective of the invention is to provide a kit for administration of a formulation containing microparticles for use in ultrasound imaging methods.

SUMMARY OF THE INVENTION

Clinical studies have been conducted and specific dosage forms have been developed using polymer microparticles containing perfluorocarbon gas inside, which provide long-term significant image enhancement. A formulation typically includes one, two, or up to five doses, most preferably one or two doses of microparticles formed by biocompatible polymers, preferably including lipids introduced inside, and containing perfluorocarbon, which is a gas at body temperature. The microparticles are administered to the patient in a dose effective to enhance ultrasound imaging of the ventricular chambers for more than 5 min and / or myocardium for more than a minute, and within the dose range from 0.025 to 8.0 mg microparticles / kg body weight. Preferably, the administered dose for the patient ranged from 0.05 to 4.0 mg microparticles / kg body weight. In a preferred embodiment, the ultrasound image is amplified in the chambers of the ventricle for 9 minutes and / or in the myocardium for 2 minutes.

The formulation is usually in an ampoule or in a syringe.

The standard formulation is in the form of a dry powder, which is reconstituted with sterile water before use by adding water to an ampoule or syringe with dry powder and shaking until an isosmotic or isotonic suspension of microparticles forms. In a preferred embodiment of this formulation, the suspension contains 1.0-3.5 · 10 ⁹ microparticles / ml suspension or 25-50 mg microparticles / ml suspension with the most preferred concentration of the resulting suspension containing 1.5-2.8 · 10 ⁹ microparticles / ml suspension or 30-45 mg microparticles / ml suspension. In a preferred embodiment, the microparticles have an average particle size of less than 8 microns, most preferably an average particle size of 1.8-3.0 microns.

In a most preferred embodiment, the gas is CF ₄ , C ₂ F ₄ , C ₂ F ₆ , C ₃ F ₆ , C ₃ F ₈ , C ₄ F ₈ , C ₄ F ₁₀ , or SF ₆ . In a preferred embodiment, the gas is n-perfluorobutane (C ₄ F ₁₀ ), prepared in an amount of 75-500 μg / ml of the introduced volume of a suspension of microparticles; preferably n-perfluorobutane is prepared in an amount of 100-400 μg / ml of the introduced volume of the suspension of microparticles and most preferably 150-350 μg / ml of the introduced volume of the suspension of microparticles; or the gas is n-octafluoropropane, prepared in an amount of 75-375 μg / ml of the introduced volume of the suspension of microparticles, most preferably 120-300 μg / ml of the introduced volume of the suspension of microparticles.

In a most preferred embodiment, the microparticle is formed by a synthetic polymer, such as polyoxyacids, which includes polylactic acid, polyglycolic acid and a copolymer of lactic acid and glycolic acid, polyglycolides, polylactides and a lactide-glycolide copolymer, polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyethylene and polypropylene, polyalkylene glycols such as polyethylene glycol, polyalkylene oxides such as polyethylene oxide polyvinyl alcohols, polyvalent ianoic acid and a copolymer of lactide with caprolactone, derivatives, copolymers and mixtures thereof both comprise a hydrophobic compound incorporated into the polymer in a ratio of from 0.01 to 30% by weight of the hydrophobic compound to the polymer, most preferably a lipid incorporated into the polymer a ratio of from 0.01 to 30% (mass of lipid / mass of polymer). In a particularly preferred embodiment, the lipid is represented dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadekanoilfosfatidilholinom (DPDPC), dilauroylphosphatidylcholine (DLPC), dipalmitoyl phosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarahidoilfosfatidilholinom (DAPC), dibehenoilfosfatidilholinom (DBPC), ditrikozanoilfosfatidilholinom (DTPC ), dilignoceroylphatidylcholine (DLGPC); or phosphatidylethanolamine.

The most preferred synthetic polymer in microparticles is a lactide-glycolide copolymer with a lactide to glycolide ratio of 50:50 (i.e. 1: 1) and a weight average molecular weight in the range of 20000-40000 Daltons, and the hydrophobic compound in the microparticles is DAPC, in relation to from 5 to 6.6% (DAPC mass / polymer mass).

The preparative dosage form may be in the form of a dry powder in an ampoule or in a syringe containing microparticles, or in a kit including a solution for resuspension of microparticles. Typically, a dry powder in an ampoule or syringe also contains carriers such as sugar and salt to make the solution is isosmotic or isotonic after reconstitution. This preparative dosage form is administered to the patient to be visualized by injection, or as a bolus or injection over a period of time up to 30 minutes.

Microparticles are used in various diagnostic imaging procedures, including ultrasound imaging, magnetic resonance imaging, fluoroscopy, x-ray and computed tomography. The microparticles were investigated in clinical trials for use in cardiology, such as assessing coronary blood flow and isolating the contours of the ventricular chambers.

DETAILED DESCRIPTION OF THE INVENTION

Improved methods, microparticles, kits and formulations for ultrasound imaging are described. Microparticles are used in various fields of diagnostic ultrasound imaging, especially in ultrasound methods such as blood vessel imaging and ultrasound cardiography, such as coronary blood flow estimation, myocardial blood volume estimation, and ventricular chamber outlines.

I. Definitions

As a rule, in the framework of this application, the term "microparticle" includes "microspheres" and "microcapsules", as well as other microparticles, except as otherwise indicated. Microparticles may or may not be spherical in shape. "Microcapsules" are defined herein as microparticles having an outer polymer shell surrounding a core of gas. "Microspheres", as defined here, can be solid polymer spheres or porous spheres with a cellular structure or sponge-like structure formed by pores in the entire thickness of the polymer, which are filled with gas. Some microspheres may contain an outer polymer shell with a cellular structure or a sponge-like structure formed by pores throughout the thickness of the polymer shell, and the pores are filled with gas. For this type of microsphere, this outer polymer shell surrounds the core of gas.

As a rule, in the framework of this application, the terms "dosage" and "dose" are used as synonyms to refer to the amount of substance that is given simultaneously, or the amount of substance that is necessary to cause the desired diagnostic or contrasting effect.

As used herein, the term “formulation” refers to an ampoule or other standard packaging, such as a syringe, containing one or more doses of a substance necessary to cause the desired diagnostic or contrast effect.

As a rule, in the framework of this application, the "patient area" refers to a particular site or organ of the patient. In some cases, the "patient area" refers entirely to the entire body of the patient. Examples of such areas are the lung region, the gastrointestinal region, the cardiovascular region (including myocardial tissue and myocardium (i.e. heart muscle), ventricular chambers, atrial chambers, valve function), the renal region, and other areas bodies, tissues, organs, and the like, including the vasculature and circulatory system, as well as diseased tissue, including malignant tissues. For example, in the "patient area" include areas for diagnostic imaging. The "patient area" is predominantly internal, although it may be external.

As a rule, in the framework of this application, "vascular network" means blood vessels (including arteries, veins, capillaries, etc.).

As a rule, in the framework of this application, the "gastrointestinal region" includes the region defined by the esophagus, stomach, small and large intestine, and rectum.

As a rule, in the framework of this application, the "renal region" refers to the region defined by the kidney and vascular network, which directly leads to and from the kidney, and includes the abdominal aorta.

As a rule, within the framework of this application, the "targeting area" and the "target organ" are used interchangeably to refer to a site in the patient's body to which delivery of the drug is desired.

Typically, within the scope of this application, “imaging area” and “imaging area” are used interchangeably to refer to a patient area in which imaging is desired.

Typically, as used herein, “ventricular blood flow or ventricular chamber outlines” refer to blood flow through the ventricles of the heart in one or more cardiac cycles.

As a rule, in the framework of this application, "atrial blood flow" refers to blood flow to the atrium of the heart in one or more cardiac cycles.

As a rule, in the framework of this application, "myocardial blood flow" refers to blood flow in the vasculature of the heart muscle or myocardium, including the blood vessels of the heart in one or more heart cycles.

Typically, as used herein, “myocardial blood volume” refers to the volume of blood in the vasculature of the heart muscle or myocardium.

As a rule, in the framework of this application, the "cardiac cycle" refers to the implementation of the period of contraction of the heart and includes both diastolic and systolic periods.

As a rule, in the framework of the present application, “increased brightness” refers to an increase in the brightness of an image compared to an image obtained without contrast means for ultrasound.

Typically, as used herein, an “enhanced image” refers to an image with enhanced brightness compared to an image obtained in the absence of contrast means for ultrasound.

Typically, as used herein, “duration” refers to the total time at which the occurring increase in image brightness could be determined.

As a rule, within the framework of this application, a “coronary vasodilator” refers to a bioactive agent, such as dipyridamole or adenosine, which, when administered to a patient, causes vasoconstriction in the cardiovascular region.

II. Microparticles.

In a preferred embodiment, the microparticles comprise a polymer, a lipid and perfluorocarbon gas. Microparticles can consist of both microspheres and microcapsules, or only microspheres, or microcapsules.

Polymers

In a preferred embodiment, the microparticles are formed from synthetic polymers. Microparticles are produced from synthetic polymers that are biocompatible and do not pollute biomaterials.

In addition, synthetic polymers are preferred due to their more reproductive synthesis and degradation both in vitro and in vivo. The polymer is selected taking into account the necessary time for in vivo stability, i.e. this is the necessary time for distribution in the area in which visualization is necessary, and the necessary time for visualization. Synthetic polymers can be modified to produce microparticles with various properties (e.g., changes in molecular weight and / or functional groups).

Typical synthetic polymers are polyhydroxy acids, such as polylactic acid, polyglycolic acid and a copolymer of lactic acid and glycolic acid, polyglycolides, polylactides, a lactide-glycolide copolymer and mixtures thereof, polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylene and polyethylene such as polyethylene polyalkylene glycols such as polyethylene glycol, polyalkylene oxides such as polyethylene oxide polyvinyl alcohols, polyvaleric acid, and a copolymer of lactide with caprolactone m derived substances, copolymers and mixtures thereof. In the framework of this application, “derivatives” include polymers containing substitutions, additions of chemical groups, for example, an aliphatic radical, alkylene, hydroxylation, oxidation, and other modifications, as is customary for specialists in this field.

Examples of preferred biodegradable polymers include hydroxy acid polymers such as lactic acid and glycolic acid, polylactide, polyglycolide, lactide-glycolide copolymer, PEG copolymers, polyanhydrides, polyorthoesters, polyurethanes, polybutyric acid, polyvaleric acid, lactic copolymer mixtures thereof and copolymers. The most preferred polymer is a lactide-glycolide copolymer with a lactide to glycolide ratio of 50:50 (i.e. 1: 1) and a polymer having a weight average molecular weight in the range of 20,000-40000 Daltons. The weight average molecular weight (M _w ) of the polymer is the average molecular weight based on the calculation of the mass of the molecules with a given molecular weight distribution of the individual polymer chains. M _w can be determined using gel permeation chromatography (GPC).

Hydrophobic compounds

In a preferred embodiment, the polymer contains a hydrophobic compound, as described in US Pat. No. 5,837,221. Typically, the inclusion of compounds, such as lipids, which are hydrophobic and in an effective amount in polymers, limits the penetration and / or absorption of water by microparticles and therefore limits gas leakage from microparticles. This is effective by increasing the duration of the enhanced image provided by microparticles that contain a lipid, a synthetic polymer and a gas encapsulated therein, in particular fluorine-containing gases such as perfluorocarbon. Lipids that can be used to ensure gas stability inside polymer microparticles include, but are not limited to, the following classes of lipids: fatty acids and derivatives, mono-, di- and triglycerides, phospholipids, sphingolipids, cholesterol and steroid derivatives, terpenes and vitamins.

Fatty acids and their derivatives may include, but are not limited to, saturated and unsaturated fatty acids, even or odd amounts of fatty acids, cis and trans isomers, and derivatives of fatty acids including alcohols, esters, anhydrides, fatty hydroxy acids and prostaglandins. Saturated and unsaturated fatty acids that can be used include, but are not limited to, molecules that contain from 12 carbon atoms to 22 carbon atoms in both unbranched and branched forms. Examples of saturated fatty acids that may be used include, but are not limited to, lauric, myristic, palmitic and stearic acids. Examples of unsaturated fatty acids that may be used include, but are not limited to, lauric, physoteric, myristoleic, palmitoleic, petroselinic and oleic acids. Examples of branched fatty acids that may be used include, but are not limited to, isolauric, isomyristic, isopalmitic and isostearic acids and isoprenoids. Derivatives of fatty acids include 12 - (((7'-diethylaminocoumarin-3-yl) carbonyl) methylamino) octadecanoic acid; N- [12 - (((7'-diethylaminocoumarin-3-yl) carbonyl) methylamino) octadecanoyl] -2-aminopalmitic acid, N-succinyldioleoylphosphatidylethanolamine and palmitoyl homocysteine; and / or combinations thereof. Mono-, di- and triglycerides or their derivatives that can be used include, but are not limited to, molecules that contain fatty acids or mixtures of fatty acids containing from 6 to 24 carbon atoms, digalactosyl diglyceride, 1,2- dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol; and 1,3-dipalmitoyl-2-succinylglycerol.

Phospholipids that can be used include, but are not limited to, phosphatidic acids, phosphatidylcholines with both saturated and unsaturated lipids, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines, phosphatidylinosites, lysophosphatidyl-hydroxy-derivatives, cardiolipids, and cardiolipids, Examples of phospholipids include, but are not limited to, phosphatidylcholines such as dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadekanoilfosfatidilholin (DPDPC), dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarahidoilfosfatidilholin (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC); and phosphatidylethanolamines such as dioleoylphosphatidylethanolamine or 1-hexadecyl-2-palmitoylglycerophosphoethanolamine. Synthetic phospholipids with asymmetric acyl chains (for example, with one acyl chain of 6 carbons and another acyl chain of 12 carbons) can also be used.

Sphingolipids that may be used include ceramides, sphingomyelins, cerebrosides, gangliosides, sulfatides and lysosulfatides. Examples of sphingolipids include, but are not limited to, gangliosides GM1 and GM2.

Steroids that can be used include, but are not limited to, cholesterol, cholesterol sulfate, cholesterol hemisuccinate, 6- (5-cholesterol-3b-yloxy) hexyl-6-amino-6-deoxy-1-thio-b-D -galactopyranoside, 6- (5-cholesten-3b-yloxy) hexyl-6-amino-6-deoxyl-l-thio-b-D-mannopyranoside and cholesteryl) -4'-trimethyl-35-ammonio) butanoate.

Auxiliary lipid compounds that can be used include tocopherol and derivatives and oils and derivatives of oils, such as stearylamine.

Various cationic lipids can be used, such as DOTMA, N- [1- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium chloride; DOTAP, 1,2-dioleoyloxy-3- (trimethylammonium) propane; and DOTB, 1,2-dioleoyl-3- (4'-trimethylammonium) butanoyl-sn-glycerol.

Most preferred lipids are lipids and phospholipids, preferably DPPC, DAPC, DSPC, DTPC, DBPC, DLPC, and most preferably DPPC, DSPC, DAPC and DBPC.

The lipid content varies from 0.01 to 30% (lipid mass / polymer mass); preferably from 0.1 to 20% (mass of lipid / mass of polymer) and most preferably 1-12% (mass of lipid / mass of polymer).

Upon receipt of the described methods, the size of the microparticles is constantly reproducible. For the purposes of this application, the terms "size" or "diameter" in relation to particles refer to the number average particle size, unless otherwise indicated. The following is an example of a formula that can be used to determine the amount of average particle size (X _n ):

where n _i = the number of particles of a given diameter (d _i ).

In the framework of this application, the term "volume of average diameter" refers to the average value of the volumetric mass diameter. The following is an example of a formula that can be used to determine the diameter of the average volume (X _v ):

where n _i = the number of particles of a given diameter (d _i ).

Particle size analysis can be performed on a Coulter counter, light microscopy, scanning electron microscopy, transmission electron microscopy, laser diffraction methods, such as methods using the Malvern Mastersizer, light scattering methods or particle time-of-flight methods. As used herein, a “Coulter method” refers to a method in which a powder is dispersed in an electrolyte, and the resulting suspension is analyzed using a Coulter Multisizer II equipped with a 50 μm diameter tube lens. In this method, measurements of particle sizes and concentrations are provided.

In a preferred embodiment, to obtain injectable, capable of penetrating through the capillary layer of light microparticles, the microparticles have a diameter of less than 8 microns. Larger microparticles can clog the pulmonary lining, and smaller particles may not provide sufficient contrasting action. A preferred microparticle size for intravenous administration of an ultrasound contrast medium is from 0.75 to 5 microns, and most preferably from 1.8 to 3.0 microns.

In a preferred embodiment, the microparticles are a cellular structure or a sponge-like structure formed by pores throughout the polymer, or the microparticles have a polymeric shell with a cellular or spongy, porous structure. In both cases, the pores are filled with gas. These microparticles are formed by spray drying a polymer solution containing a void forming agent, such as ammonium carbonate, as described below.

Contrast media for ultrasound imaging

Examples of fluorine-containing gases include CF ₄ , C ₂ F ₄ , C ₂ F ₆ , C ₃ F ₆ , C ₃ F ₈ , C ₄ F ₈ , C ₄ F ₁₀ and SF ₆ . n-Perfluorobutane (C ₄ F ₁₀ ) is particularly preferred since it provides insolubility of the gas, which will not dissolve at the temperature of use, and is pharmacologically acceptable.

The amount of gas contained in the microparticles will depend on the type of gas, but is usually 75-500 μg / ml of the introduced volume of the suspension of microparticles. For n-perfluorobutane, the preferred gas content is 100-400 μg / ml of the introduced volume of the suspension of microparticles, and most preferably is 150-350 μg / ml of the introduced volume of the suspension of microparticles. For n-octafluoropropane, the preferred gas content is 75-375 μg / ml of the injected volume of the microparticle suspension, and most preferably 120-300 μg / ml of the injected volume of the microparticle suspension.

III. Methods for producing microparticles

Microparticles can be obtained in various ways, and preferably obtained by spray drying. The main evaluation method is that the polymer should be dissolved or melted with a hydrophobic compound or lipid before microparticle formation.

Solvents

During formation, the polymer is usually dissolved in a solvent. In the framework of the present application, the polymer solvent is an organic solvent that readily evaporates or has a relatively low boiling point, or which can be removed by vacuum, and which is suitable for administration to people in micro amounts such as dichloromethane. Other solvents may also be used, such as ethyl acetate, ethyl formate, ethanol, methanol, dimethylformamide (DMF), acetone, acetonitrile, tetrahydrofuran (THF), formamide, acetic acid, dimethyl sulfoxide (DMSO) and chloroform, or combinations thereof. Typically, the polymer is dissolved in a solvent to form a polymer solution having a concentration of from 0.1 to 60% by weight (w / v), more preferably from 0.25 to 30% (w / v) and most preferably 0.5-10% (w / v).

Spray drying

It is preferable to obtain microparticles by spray drying by dissolving the biocompatible polymer and the lipid in a suitable solvent, dispersing the pores of the forming agent as solid or as a solution inside the polymer solution, and then spray drying the polymer solution and the pores of the forming agent to form microparticles. In the framework of the present application, the spray drying method of the polymer solution and the pore forming agent are referred to the method in which the polymer solution and the pore forming agent are finely ground to form a fine mist and dried by direct contact with hot carrier gases. In the application of spray dryers in the art, the solution and the pore forming agent can be finely ground at the inlet of the spray dryer chamber, passing through at least one drying chamber, and then collected in powder form. The temperature may vary depending on the gas or polymer used. The temperature at the inlet and outlet of the chamber can be controlled to obtain the desired products.

The size and morphology of the microparticles obtained during spray drying depends on the atomizer used to atomize the polymer solution and the pore forming agent, the pressure of the atomizer, the volumetric rate of the polymer solution with the pore forming agent, the polymer used, the polymer concentration in the solution, the type of polymer solvent, type and amount pore forming agent, atomization temperature (both inlet and outlet temperature) and polymer molecular weight. Usually, with a higher molecular weight of the polymer, with a larger particle size, the resulting concentration of the polymer solution coincides.

Typically, the process parameters for spray drying are as follows: inlet temperature = 30-200 ° C, outlet temperature = 5-100 ° C and polymer bulk velocity = 10-5000 ml / min.

A gaseous diagnostic agent can be encapsulated with an emulsified gas with a polymer solution and a pore-forming agent prior to spray drying. Alternatively, microparticles filled with air can be obtained during the spray drying step, and subsequently the air is replaced with perfluorocarbon gas by directing the flow of this gas into microparticles, or by pulling vacuum onto microparticles to remove encapsulated air, then filling with perfluorocarbon with this gas. A lyophilizer or a vacuum chamber can be used to replace gas during the vacuum phase.

Additives for simplified microparticle formation

Various surfactants can be added during the formation of microparticles. Typical emulsifying agents (emulsifiers) or surfactants that can be used (0.1-15% w / w polymer) include the most physiologically acceptable emulsifying agents. Examples include natural and synthetic forms of salts of bile acids or bile acids, both associated with amino acids and unbound, such as taurodeoxycholate and cholic acid.

The pore forming agents are contained in the polymer solution in an amount of from 0.01% to 90% w / v. polymer solution to increase pore formation. For example, in spray drying, a pore forming agent such as ammonium carbonate, for example ammonium bicarbonate, ammonium acetate, ammonium carbonate, ammonium chloride or ammonium benzoate or other ammonium carbonate in solid form or in solution with a solvent such as water, can be used . The solid pore forming agent or solution containing the pore forming agent is then emulsified in the polymer solution to create dispersion or droplets of the pore forming agent in the polymer. This dispersion or emulsification is then spray dried to eliminate both the polymer solution and the pore-forming agent. After the polymer is precipitated, the hardened microparticles can be hardened and lyophilized to remove any pore-forming agents that were not removed during the polymer deposition step.

The isolated microparticle is created using a polymer, a lactide-glycolide copolymer with a 50:50 ratio of lactide and glycolide and having a weight average molecular weight in the range of 20000-40000 Daltons and phospholipids, diarachidoylphosphatidylcholine ((1,2-diarachidoyl-sn-glycerol-3-phosph DAPC)) in a ratio of 5-6.6% (wt. DAPC / wt. Polymer). Next, the microparticles are formed in a solution of mannitol and TWEEN ^® 80 and subjected to processing of the obtained dry powder of microparticles, which are filled on a n-perfluorobutane lyophilizer. Dry powder restore 5 ml of sterile water before use by adding water to the ampoule with a dry powder and shaking the resulting suspension of microparticles in isosmotic mannitol. Properties of the isolated microparticles of the suspension are a gas content of 150-350 μg / ml n-perfluorobutane for each volume of suspension of microparticles introduced, 1.5-2.8 · 10 ⁹ microparticles / ml of the introduced volume of the suspension of microparticles, 30-45 mg of microparticles / ml of the introduced volume of the suspension of microparticles and an average particle size in the range of 1.8-3.0 microns.

IV. Microparticle applications

1. Formulations for administration to a patient

The microparticles can then be further processed with fillers to obtain a dry powder. Excipients provide a concentration or osmotic concentration or attenuation of the bearing capacity of the microparticles after reconstitution with a pharmaceutically acceptable carrier prior to administration to a patient. Suitable excipients for imparting osmolarity or concentration are sugars, which include, but are not limited to, mannitol, dextrose or glucose, and salts, which include, but are not limited to, sodium chloride and sodium phosphate. Suitable excipients for impairing the microsphere loadability include any pharmaceutically acceptable humectant or surfactant, including, but not limited to, Polysorbate 80 (TWEEN ^® 80), Polysorbate 20 (TWEEN ^® 20), Pluronic or polyethylene glycol. Excipients or excipients suitable for imparting osmolarity or tonicity that can be used as humectants are described in references such as Handbook of Pharmaceutical Excipients (Fourth Edition, Royal Pharmaceutical Society of Great Britain, Science & Practice Publishers) or Remingtons: The Science and Practice of Pharmacy (Nineteenth Edition, Mack Publishing Company). Dry microparticle powder and fillers are formed by suspending microparticles in a filler solution. If necessary, you can use the stage of further fractionation by size. Ampoules or syringes are filled with microparticles in a solution of excipients, frozen and lyophilized to obtain a dry powder formulation. At the end of the lyophilization step, the microparticles are filled with perfluorocarbon gas, filling the lyophilizer with perfluorocarbon gas. The ampoules or syringes are then closed or covered and, in the case of ampoules, pressed. This leads to the accumulation of perfluorocarbon in the free space of the ampoule or syringe.

Alternatively, the microparticles may be a dry mixture with pharmaceutical excipients, and then ampoules or syringes are filled with them. Microparticles can be filled with perfluorocarbon gas, using vacuum after filling ampoules and syringes in a lyophilizer or vacuum chamber. The ampoules or syringes are then closed or covered and, in the case of ampoules, compressed. This leads to the accumulation of perfluorocarbon in the free space of the ampoule or syringe.

2. Dose units

You can use different values of dose units. For example, small doses may contain 25-75 mg of microparticles. Medium doses may contain 75-150 mg. Large doses may contain 150-250 mg of microparticles. Very large doses may contain 250-1000 mg of microparticles.

When a suspension of microparticles is formed after reconstitution, the mass concentration of microspheres in the suspension usually varies from 20 to 60 mg / ml. The preferred mass concentration of microspheres in suspension is 25-50 mg / ml, and the most preferred mass concentration of microspheres in suspension is from 30 to 45 mg / ml. The preferred concentration of microparticles in the suspension is 1.0-3.5 · 10 ⁹ microparticles / ml of the suspension, and the most preferred concentration of microparticles in the suspension is 1.5-2.8 · 10 ⁹ microparticles / ml. The microparticles have a preferred average particle size of less than 8 microns, most preferably in the range of 1.8-3.0 microns.

Pharmaceutically acceptable carriers may contain water for injection, sterile water, saline, saline with glycerin, saline with TWEEN ^® 20, saline with TWEEN ^® 80, isoosmotic dextrose (5%), isosmotic dextrose (2.5%), isoosmotic mannitol (5%), isosmotic mannitol (2.5%), isotonic mannitol with TWEEN ^® 20 and isotonic mannitol with TWEEN ^® 80.

3. Sets

Kits for parenteral administration of microparticles containing perfluorocarbon gas may be provided. The kit contains at least 2 components. One component contains a unit dose of a dry contrast agent powder in an ampoule or syringe, and the other component contains a pharmaceutically acceptable carrier in an ampoule or syringe. Prior to administration to a patient, a pharmaceutically acceptable carrier is added to a unit dose of a dry powder of contrast medium to form a suspension of gas-filled microparticles, which are used as a contrast medium for ultrasound imaging for diagnostic imaging by any route of administration.

4. Ampoules or containers for microparticles

Kits do not require any specific ampoule or syringe or contact systems; conventional ampoules, syringes and tips can be used with microparticles. Therefore, the quality of sealing has become a matter of particular importance; any destruction of hermetic integrity can make it possible for undesirable substances to enter the ampoule or allow gas to escape. In addition to ensuring sterility, maintaining a vacuum space is important for closed products under reduced pressure to ensure safety and proper recovery. As for plugs, they can be a compound or a multicomponent compound based on an elastic polymer, such as polyisobutylene or "butyl rubber" and must be impermeable to the gas used. The size of the ampoule is selected depending on the total dose of dry powder in the ampoule. Ampoule volumes of 5 ml, 10 ml, 20 ml and 30 ml are preferred. The size of the syringe is selected depending on the total dose of dry powder in the syringe. Preferred syringe sizes are 5 ml, 10 ml, 20 ml and 50 ml.

5. Diagnostic applications

Microparticle compositions can be used in many diagnostic applications, including ultrasound imaging, magnetic resonance imaging, fluoroscopy, x-ray and computed tomography.

In a preferred embodiment, the microparticles are used for ultrasound procedures such as blood vessel imaging and echocardiography, including, but not limited to, imaging of the ventricular chambers, estimation of coronary blood flow, estimation of blood volume in the myocardium, diagnosis of coronary artery disease, and evaluation of the ejection fraction.

Microparticles can be used for vascular imaging, as well as for the detection of diseases of the liver and kidneys, for the detection and characterization of tumor formations and tissues, and for assessing the speed of peripheral blood. Microparticles can also bind to ligands that reduce tissue adhesion or that direct microparticles to specific areas of the body in vivo .

Common image acquisition method

Prior to administration, the dry particulate microparticles are reconstituted with pharmaceutically acceptable carriers, then an effective amount for detection is administered to the patient using a suitable route by injection into a blood vessel (such as intravenously (i.v.) or intraarterially (i.a.)) or orally. The composition of the microparticles can be administered to the patient intravenously, for example, by shock infusion or short infusion (less than 30 minutes). Preferably, administering the injection over a period of time in the range of 15 seconds to 20 minutes, most preferably in the range of 30 seconds to 15 minutes. Typically, a dose in the range from 0.025 to 8 mg / kg of body weight per injection is administered intravenously to the patient, preferably the dose ranges from 0.05 to 4 mg / kg.

For diagnostic ultrasound applications, energy is applied to at least a specific area of the patient to image the target tissue. A visual image of the patient’s inner region is then obtained in such a way that the presence or absence of damaged tissue can be established. Ultrasonic imaging techniques including second harmonic imaging and synchronized imaging are well known in the art and are described, for example, in Uhlendorf, IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, 14 (1): 70-79 (1994) and Sutherland , et al., Journal of the American Society of Echocardiography, 7 (5): 441-458 (1994), so a demonstration of each is included in this document.

Ultrasonic vibrations are used with a measuring transducer. If desired, the ultrasound may be pulsed or continuous. Thus, ultrasound diagnostics usually cause a superposition of reflected sounds, after which the ultrasound transducer receives reflected signals during the listening period. You can use harmonics, ultraharmonics or subharmonics. It is possible to advantageously use a different mode of operation at harmonics at which a 2x frequency is perceived, where x represents a random frequency. This can help reduce the signal from the base material and amplify the signal from a transducer using radiopharmaceuticals that can be sent to a desired location, such as blood clots. Other harmonic signals, such as odd harmonic signals, such as 3x or 5x, would similarly represent the use of this method. Subharmonic signals, for example, x / 2 and x / 3, can also be represented and processed in order to form an image.

In addition, you can apply energy Doppler study or color Doppler study. In the case of energetic Doppler studies, the relatively high energy energies of Doppler studies can resonate cavities. This can cause sound emissions, which can be in the range of subharmonics and ultraharmonics or, in some cases, at the same frequency, since ultrasound is used.

Specific Image Applications

The microparticles described herein can be used in both cardiological and radiological applications. For cardiological applications, the microparticle compositions are administered to the patient and the patient is scanned using an ultrasound device to obtain visual images of the cardiovascular region. If desired, the compound of microparticles is administered in combination with pharmacological or physiological agents that cause stress. Suitable pharmacological agents that cause stress include a coronary vasodilator, such as dipyridamole or adenosine, an inotropic agent (i.e., increases cardiac contraction), such as dobutamine or a chronotropic drug (i.e., increases the frequency of contractions), such like dobutamine. Suitable physiological stressors include physical exercise, such as using a treadmill and a bicycle ergometer.

For radiological applications, the microparticle formulations are administered to the patient and the patient is scanned using an ultrasound device to obtain visual images of the patient area for examination.

Microparticles can be used to evaluate cardiovascular function, as well as to evaluate coronary blood flow or blood volume in the myocardium or to diagnose coronary heart disease (coronary artery disease). For example, microparticles can enhance the images of the ventricular chambers and thus contribute to the study of the regional function of the heart, by studying the movement of the walls, and the assessment of the overall function of the heart, by measuring the ejection fraction. Microparticles can also be used to assess coronary blood flow to establish differences in the functioning of cardiac tissue from either ischemic cardiac tissue (insufficient blood flow) or infarction (dead) cardiac tissue. Myocardial contrast signals can be used as an estimate of myocardial blood volume with ultrasound contrast media located intravascularly after intravenous administration. The absence or decrease in the intensity of contrast or brightness of the image in a particular myocardial region over time indicates a decreased blood flow (i.e., impairment).

Most often, if the patient has severe coronary artery disease, blood flow in various areas of the heart when assessed by methods such as ultrasound contrast will look normal. In order to detect blood flow disorders in a patient without severe heart disease or to detect minor damage to coronary blood flow, it is necessary to increase the heart's need for blood flow, including stress. Stress can be caused by an exercise by the patient or by administering a pharmacological compound, such as a vasodilator, inotropic, or chronotropic. During exercise or pharmacological stress, blood flow disorders can be easily detected, since the ability to increase blood flow in the area supplied with coronary arteries with stenosis is reduced. Comparison of ultrasound images of the myocardium after administration of an ultrasound contrast medium can be done both in the pre-stress state (i.e., the resting state) and under stress. A myocardial region without increased brightness, detected during the image of stress, but not during rest, indicates ischemia. A myocardial region without increased brightness, detected during the image of stress and during the image of rest, indicates a heart attack.

In one embodiment, coronary blood flow can be measured (1) by administering the first injection of the microparticle composition to the patient, (2) scanning the patient using an ultrasound imaging device to obtain a visible image of the cardiovascular region, (3) turning on the patient’s stress state using a pharmacological agent causing stress or exercise, (4) by administering a second injection of the microparticle composition and ongoing scanning, and (5) assessing differences in images obtained in stages (2) and (4), either visually or using quantitative image analysis.

For radiological applications, microparticles can be used to improve the capabilities of ultrasound imaging for radiological indications, including the image of a kidney, liver, peripheral vascular disease, increasing the visibility of blood flow and blood circulation, and improving the definition of small lesions or deep structures within the body. Microparticles can be used for both macrovascular and microvascular indications. In macrovascular indications (diagnosis of disease states and conditions of the main arteries and veins of an organism), microparticles can help to determine stroke and pre-stroke conditions by visualizing intracranial blood vessels, determining atherosclerosis in large vessels such as carotid arteries, assessing the level of carotid stenosis, patency of the vascular graft and vascular graft and peripheral vascular thrombosis. In microvascular indications (diagnosis of disease conditions and analysis of blood flow in small vessels), microparticles can help identify damage, tumors, or other diseases in the liver (e.g., adenoma or hemangioma), kidneys, spleen (e.g., splenic artery aneurysms), chest and ovaries, and in other tissues and organs.

Affected tissues in a patient can be diagnosed by administering microparticle compositions to the patient and scanning the patient using ultrasound imaging to obtain visible images of any affected tissues in the patient. Affected tissues can be identified as an area of increased brightness or an area that is not indicated by increased brightness.

Enhanced Images Using Microparticle Compositions

After the introduction of the microparticles create an improved image. You can identify improved images by increasing the brightness in the image compared to those when no ultrasound contrast medium is introduced or when the image distortions are significantly removed from the body. Thus, in connection with ultrasound imaging of the cardiovascular region, which includes cardiac tissue and the associated vasculature, an amplified image can be detected, for example, by an increase in brightness in the image of the cardiovascular region and / or enhanced purification in the occurrence of distortions in image of the cardiovascular region. After a single injection, the image is stored for 10 seconds to 60 minutes. Preferably, the images are stored from 20 seconds to 30 minutes, and most preferably are stored from 30 seconds to 20 minutes. In a preferred embodiment, ultrasound imaging is enhanced in the chambers of the ventricles in more than 5 minutes or in the myocardium in more than 1 minute. Image brightness enhancement can be evaluated either visually with the naked eye or using quantitative image analysis.

With specific reference to the gray scale (from 0 to 255 VDU or gray levels) set earlier, there is preferably an increase in the brightness level at least at 10 VDU (gray levels). Most preferably, when the image has an increased brightness higher than at 10 VDU, for example, at 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 VDU. In some embodiments, the increased brightness is higher than at 100 VDUs, for example, at 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 VDUs. In other embodiments, the increased brightness is higher than at 150 VDUs, for example, at 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 VDUs. Alternatively, the increased brightness is higher than at 200 VDUs, for example, at about 205, 210, 215, 220, 225, 230, 235, 240, 245, 250 or 255 VDUs. The above methods and compositions will now be presented based on the following, but not limited to, examples.

Examples

Materials

Acetic acid, ammonium bicarbonate, USP mannitol, and polysorbate 80 (no animal-derived components) were purchased from Spectrum Chemicals, Gardena, CA. The polymer (lactide-glycolide copolymer (PLGA) (50:50)) and diarachidoylphosphatidylcholine (1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC)) were obtained from Boehringer Ingelheim (Ingelheim, Germany) and Avanti (Alabaster, AL), respectively. Dichloromethane was purchased from EM Science (EMD Chemicals, Gibbstown, NJ). Ampoules (30 ml tubular ampoules) and plugs (20 mm, gray, with a single hole, Fluro-Tec) were obtained from West Pharmaceutical Services (Lionville, PA). N-perfluorobutane gas (DFB) was purchased from F2 Chemicals Ltd, Lancashire, UK.

Analytical methods

Quantification of the mass concentration of microparticles

The mass concentration of microparticles in the ampoules was quantified using ICP-MS (inductively coupled plasma mass spectrometry). The amount of polymer in the microparticles was determined based on tin ICP-MS. The amount of polymer present in the microparticles was determined based on a comparison of the amount of tin found in the microparticles to the amount of tin found in the specific polymer set used to produce the microparticles. The amount of phospholipids in the microparticles was determined by analysis of phosphorus on ICP-MS. The amount of phosphorus in the microparticles was determined based on the amount of phosphorus found in the phospholipids. The mass of microparticles per ml of suspension was calculated by adding the amount of polymer and phospholipid to the ampoule, and then dispersing this association with a recovery volume (5 ml).

Particle size analysis

A sample of recovered microparticles was added to the electrolyte solution, and the resulting suspension was analyzed by particle size and microparticle concentration using a Coulter Multisizer II equipped with a 50 mm diameter tube opening.

Microparticle Gas Content

The dry powder in ampoules was reconstituted with 5 ml of water and shaken to form a suspension of microparticles. The resulting suspension was analyzed for DFB content by extracting a series of 0.3 ml aliquots through the lid using a needle or syringe. These aliquots were introduced into the sealed ampoules airspace. The airspace of the ampoules was balanced for at least 10 hours at room temperature. The samples were then heated to 45 ° C for 20 min in the free air space of the dryer. Gas above the suspension was analyzed by gas chromatography using a plug-free inlet and a flame ionization detector. Quantification was performed using a single point calibration based area.

GC system parameters and temperature program are given in table 1 and 2.

Table 1 GC system parameters Sample Free space, 1 ml closed sample Detector Fid Speaker Supelco 60/80 Carbopack B 5%
Fluorocol Inlet temperature 150 ° C Detectable temperature 325 ° C Carrier gas Helium (25 ml / min) Fid gases Hydrogen (60 ml / min) Air (350 ml / min) Nitrogen (5 ml / min)

table 2 GC temperature program Initial temperature Speed Final temperature Hold Time Initial state 40 ° C N / a N / a 2.0 min First cycle 40 ° C 5 ° C / min 65 ° C 0.0 min Second cycle 65 ° C 10 ° C / min 130 ° C 0.0 min Third cycle 130 ° C 50 ° C / min 200 ° C 0.0 min Final state 200 ° C N / a N / a 3.1 min

Example 1: Obtaining microparticles for use as a contrast medium for ultrasound

An organic solution was obtained by dissolving 176 g of PLGA, 10.6 g of diarachidoylphosphatidylcholine (1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC)) and 2.26 g of acetic acid in 5.88 L of dichloromethane at 25 ° C. An aqueous solution consisting of 68.5 g of ammonium bicarbonate dissolved in 338 ml of water for injection was added to the organic solution and homogenized for 10 minutes at 4310 rpm in a 10-liter homogenization vessel using a rotor-emulsifier mixer stator.

The resulting emulsion was spray dried using nitrogen both as a spray and as a drying gas. The emulsions were spray dried on a work surface, a spray dryer using a pneumatic spray nozzle spray systems (Wheaton, IL) and a glass drying chamber / cyclone system Buchi (Brinkmann, Westbury, NY). Spray drying conditions were as follows: 40 ml / min — volumetric flow rate of the emulsion, 30 l / min — gas spray rate, 46 kg / h — drying gas flow rate and 12 ° C — exit temperature.

The spray dried product is further processed using the steps of dispersion, solidification and lyophilization. An aqueous solvent was obtained by dissolving 140 g of mannitol and 4.10 g of polysorbate 80 in 5.0 l of water. Spray dried microparticles were dispersed in a solvent at a concentration of 25 mg / ml. Scattered particles were collected using stainless steel, 800 series, a flow-through electrolyte ultrasonic apparatus Misonix Incorporated (Farmingdale, NY) and sifted through a 10 "diameter (RBF-10) Vorti-Siv vibrating sieve (Salem, OH). The ultrasound apparatus was enclosed in jacket with 4 ° C to prevent the dispersed particles from heating up. The dispersed particles were sifted through 25 μm and 20 μm sieve cells in series at 150 ml / min. The ampoules were filled with sifted dispersed particles (10 ml filled with 30 ml ampoules), partially corked and frozen immersion in liquids nitrogen.

After freezing, the ampoules were lyophilized. After lyophilization, the chamber was isolated and the ampoules were filled with n-perfluorobutane (DFB) at a pressure of 5 kilopascals until clogging.

Dry powder was reconstituted with 5 ml of sterile water until water was added to the ampoules with dry powder and shaken to obtain a suspension of microparticles in isosmotic mannitol. The suspension contained 2.2 × 10 ⁹ microparticles / ml of suspension and 37 mg of microparticles / ml of suspension, and the microparticles had an average size of 2.2 microns.

Example 2: Microparticle Gas Leak Rate

The gas leakage rate from two separated batches (batch 1 and batch 2) of microparticles, as obtained in the methods of example 1, was estimated using gas chromatography (GC), as described in the analytical methods section. A third batch of microspheres (batch 3) was obtained in a manner similar to Example 1, however, a phospholipid, diarachidoylphosphatidylcholine (1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC)) was isolated during the preparation of microparticles.

Table 3 Gas content and gas leakage rate from microparticles Gas content (μg / ml suspension) immediately after vial reconstitution Gas Content (μg / ml suspension)
70 min after ampoule recovery Gas content in%
after 70 min Party 1 341 312 9% Party 2 259 232 10% Party 3 139 eighteen 87%

DAPC-containing microparticles lost about 10% of the starting gas content after 70 minutes, while microparticles that did not contain DAPC lost 87% of the starting gas content. In addition, microparticles that contain DAPC have a higher starting gas content than microparticles without DAPC. This means that the inclusion of DAPC is important for the formation of the internal pore structure of microparticles during spray drying, as well as while maintaining gas inside the microparticles.

The total duration of the intended use of the contrast medium for ultrasound after administration of the substance is usually from 30 seconds to 60 minutes, depending on the type of cardiology or radiology of the ultrasound examination. In this way, gas leakage from microparticles containing DAPC lipid is estimated to be insignificant throughout the ultrasound study.

Example 3: Improving the heart image depending on the dose of microparticles

Microparticles, as obtained by the methods of example 1, were studied in healthy adults. The dry powder was reconstituted prior to use by adding 5 ml of sterile water to the ampoule and shaking the ampoule ten times. The final concentration of microspheres in the resulting suspension was approximately 37 mg / ml. The subjects received one of the doses of 0.5, 2.0, or 4.0 mg / kg body weight. The subjects underwent transthoracic ultrasound imaging using a continuous harmonic image (frame rate of 15 Hz and transducer frequency of 2.1 / 4.2 MHz). Images were evaluated visually by the intensity and duration of optimization.

The duration of ventricular chamber improvement exceeded 9 min at doses of both 2 mg / kg and 4 mg / kg. The contrasting effect was still noticeable in 13 out of 15 subjects at these two doses, when the subjects were re-visualized in 30 minutes, indicating a longer duration of improvement due to microparticles.

Data on the duration of improvement of the ventricular chamber are summarized in table 4.

Table 4 Duration of left ventricular image optimization Dose (mg / kg body weight) The average duration of the improvement of the ventricular chamber (min) 0.5 2.6 2.0 > 9.6 4.0 > 9.6

Example 4: Comparison of microparticles in a commercial product for the evaluation of images of the heart

A comparative study of images with echocardiography was conducted in two adults, selected by body weight and heart function. The first subject received a single injection of microparticles, as obtained by the method in Example 1. The dry powder was reconstituted prior to use by introducing 5 ml of sterile water into the ampoule and shaking the ampoule 10 times. The final concentration of microspheres in the resulting suspension was approximately 37 mg / ml, and the gas content in the suspension was approximately 250 μg / ml of the suspension. The first subject received a dose of 4 mg microparticles / kg, which corresponds to a gas dose of 27 μg / kg body weight. The second subject received a single dose of a marketed ultrasound contrast medium, OPTISON ^® (Amersham Health), which contains perfluoropropane with albumin microspheres. Two subjects received the same total amount of gas (27 μg / kg body weight), which is an acoustically active component. Two subjects underwent transthoracic ultrasound imaging using a continuous harmonic image (frame rate of 15 Hz and a transducer frequency of 2.1 / 4.2 MHz). Images were evaluated visually by the intensity and duration of optimization.

The duration of the optimization of the ventricular chamber is summarized in table 5.

Table 5 Image Optimization Duration
with various contrast media for ultrasound Contrast media administered and dose of gas (mcg / kg) Duration
ventricular chamber optimization (min) Duration of myocardial optimization
(sec) Example 1
Microparticles
(27 mcg / kg body weight) > 9 160 OPTISON ^®
(27 mcg / kg body weight) one 10

The microparticles obtained using the method described in Example 1 provide optimized images of both the ventricular and myocardial chambers, which are significantly longer than OPTISON ^® , and which are of adequate duration to accompany the completion of ultrasound examination of the heart.

Example 5: Assessment of coronary blood flow to assess ischemia using microparticle preparations

The microparticles obtained, as in the method of example 1, were introduced to the individual to be evaluated for coronary heart disease. Subject received two injections of microparticles separated by 60 minutes. The first injection of microparticles ("injection at rest", 1.7 mg / kg) was used to assess myocardium at rest. Prior to the second injection of microparticles, the subject was subjected to pharmacological stress using a coronary vasodilator, dipyridamole (0.56 mg / kg). After inducing stress, the subject received a second injection of microparticles ("injection under stress" 1.3 mg / kg) to assess myocardium after stress.

Comparison of images during rest and stress in time over time after administration of microparticles to a subject denotes a region of the myocardium that has amplification in image optimization, and this region becomes larger in size upon subsequent induction of stress. This indicates a zone of myocardial tissue that has both a heart attack and ischemic components. The detection of ischemia was confirmed using an alternative diagnostic method, radionuclide imaging. Radionuclide perfusion at rest and under stress was performed after administration of 99Tc (MIBI) and an image of the test subject was obtained using a commercial gamma counter. Damage observed during ultrasound imaging at rest and stress was confirmed by images of radionuclide perfusion at rest and under stress.

Claims (33)

1. A dosage form providing improved ultrasound contrast images containing microspheres containing a biocompatible synthetic polymer and containing perfluorocarbon introduced therein, which is gas at body temperature, where the microspheres are porous spheres with a cellular structure or sponge-like structure, where the dosage preparative the form contains a dose of microspheres effective to provide improved ultrasound images for more than 5 min in the chambers ventricles or more than 1 min in the myocardium when the microspheres are administered intravenously, and where the dose is from 0.025 to 8.0 mg microspheres / kg body weight. 2. The dosage form according to claim 1, containing a dose in the range from 0.05 to 4.0 mg microspheres / kg body weight. 3. The formulation according to claim 1 in the form of a dry microsphere powder, which can be dissolved before use with sterile water, adding water to an ampoule or syringe with dry powder and shaking to obtain an isosmotic suspension of microspheres. 4. A formulation providing improved ultrasound contrast images comprising microspheres, containing a biocompatible synthetic polymer, and containing perfluorocarbon introduced therein, which is gas at body temperature, where the microspheres are porous spheres with a cellular structure or sponge-like structure, where the formulation contains a dose of microspheres effective to provide improved ultrasound images in the chambers of the ventricles for more than 5 minutes or more than 1 minute in the myocardium, when the microspheres are administered intravenously, and where the microspheres are dissolved with sterile water before use to form a suspension with a concentration of microspheres ranging from 1.0 · 10 ⁹ to 3.5 · 10 ⁹ microspheres / ml suspension or a mass concentration of microspheres ranging from 25 up to 50 mg microspheres / ml suspension. 5. The formulation according to claim 1 or 4, providing improved ultrasound images in the chambers of the ventricles for more than 9 minutes or for myocardium for more than 2 minutes. 6. The formulation of claim 1 or 4, providing improved ultrasound images in the chambers of the ventricles for at least 30 minutes. 7. The formulation according to claim 4, forming a suspension having a microsphere concentration in the range of 1.5 · 10 ⁹ to 2.8 · 10 ⁹ microspheres / ml of a suspension or a mass concentration of microspheres in the range of 30 to 45 mg of microspheres / ml of a suspension . 8. The formulation according to claim 1 or 4, wherein the microspheres have an average size of less than 8 microns. 9. The formulation of claim 8, wherein the microspheres have an average size in the range of 1.9 to 2.6 microns. 10. The dosage form according to claim 1, containing a dose in the range from 0.5 to 4.0 mg microspheres / kg body weight. 11. The formulation of claim 10, wherein the dose is selected from the group consisting of 0.5 mg microspheres / kg body weight, 2.0 mg microspheres / kg body weight and 4.0 mg microspheres / kg body weight. 12. The formulation of claim 1 or 4, wherein the gas is selected from the group consisting of CF ₄ , C ₂ F ₄ , C ₂ F ₆ , C ₃ F ₆ , C ₃ F ₈ , C ₄ F ₈ and C ₄ F ₁₀ . 13. The formulation of claim 12, wherein the gas is n-perfluorobutane (C ₄ F ₁₀ ), which is contained in an amount of 75 to 500 μg / ml of the injected volume of the microsphere suspension. 14. The formulation of claim 13, wherein n-perfluorobutane (C ₄ F ₁₀ ) is provided in an amount of from 100 to 400 μg / ml of the injected volume of the microsphere suspension. 15. The formulation of claim 14, wherein n-perfluorobutane (C ₄ F ₁₀ ) is provided in an amount of 150 to 350 μg / ml of the administered volume of the microsphere suspension. 16. The formulation of claim 12, wherein the gas is n-octafluoropropane (C ₃ F ₈ ), provided in an amount of 75 to 375 μg / ml of the injected volume of the microsphere suspension. 17. The dosage form according to clause 16, where n-octafluoropropane (C ₃ F ₈ ) provide in an amount of from 120 to 300 μg / ml of the introduced volume of the suspension of microspheres. 18. The formulation according to claim 1 or 4, wherein the microsphere is formed from a synthetic polymer selected from the group consisting of polyhydroxy acids, polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyvaleric acid, a lactide copolymer of caprolactone, and and mixtures thereof. 19. The formulation according to claim 1 or 4, further comprising a hydrophobic compound additionally included in the polymer in a ratio of from 0.01 to 30% (weight of the hydrophobic compound / polymer weight). 20. The formulation according to claim 19, wherein the hydrophobic compound is a lipid incorporated into the polymer in a ratio of from 0.01 to 30% (lipid weight / polymer weight). 21. The formulation according to claim 20, wherein the lipid is a phospholipid selected from the group consisting of dioleoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine (DPDPC), dilauroylphosphatidylcholine (DL), dilmitroylpholine doline (DL), , diarachidoylphosphatidylcholine (DAPC), dibechenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLGPC) and phosphatidylethanolamines. 22. The formulation of claim 21, wherein the polymer is a lactide-glycolide copolymer with a lactide to glycolide ratio of 1: 1 and the weight average molecular weight varies from 20 to 40 kDa, and where the lipid is diarachidoylphosphatidylcholine incorporated into the polymer in a ratio of from 5 up to 6.6% (lipid weight / polymer weight). 23. The formulation of claim 1 or 4 in an ampoule or syringe containing dry powder from microspheres. 24. The formulation of claim 23, wherein the ampoule or syringe further comprises one or more excipients selected from the group consisting of sugars, salts, and surfactants. 25. The formulation according to claim 1 or 4 in a kit containing an ampoule or syringe with dry microspheres and an ampoule or syringe with a solution for resuspension of microspheres. 26. The formulation according to claim 1 or 4, consisting essentially of one or two doses. 27. The formulation according to claim 1 or 6, consisting essentially of up to five doses. 28. A method for providing improved ultrasound images, comprising administering to a patient a formulation according to any one of claims 1 to 27, and then visualizing the patient area to obtain an improved image compared to when no contrast agent is present. 29. The method of claim 28, further comprising administering to the patient an agent that causes stress to the patient’s cardiovascular system and re-visualizing the patient. 30. The method according to p, where the region for visualization is selected from the group consisting of cardiovascular region, liver, kidneys, spleen, breast and ovaries. 31. A kit containing a formulation according to any one of claims 1 to 27 and a solution for dissolving the formulation. 32. A method for preparing a formulation for ultrasound contrast imaging, comprising suspending microspheres containing a biocompatible polymer and a hydrophobic compound in a solution, optionally including excipients, placing the suspension in an ampoule or syringe, freezing the suspension, lyophilization of the ampoule to obtain a dry powder in an ampoule or a syringe, and filling the lyophilizer with perfluorocarbon gas, to obtain a formulation according to any one of claims 1 to 27. 33. A method of manufacturing a dosage form for ultrasonic contrast imaging, including dry mixing microspheres, including a biocompatible polymer and a hydrophobic compound, optionally including excipients, placing the mixture in an ampoule or syringe, evacuating the ampoule to remove encapsulated air, and filling the ampoule or syringe with perfluorocarbon gas obtaining a formulation according to any one of claims 1 to 27.