HK1172830B - Ultrasound contrast agent dosage formulation - Google Patents

Abstract

Clinical studies have been conducted and specific dosage formulations developed using polymeric microparticles having incorporated therein perfluorocarbon gases that provide significantly enhanced images of long duration. The dosage formulation includes microparticles formed of a biocompatible polymer, preferably including a lipid incorporated therein, and containing a perfluorocarbon that is a gas at body temperature. The microparticles are provided to a patient in an amount effective to enhance ultrasound imaging in the ventricular chambers for more than 5 minutes or in the mycocardium for more than a minute, in a dose ranging from 0.025 to 8.0 mg microparticles/kg body weight. Preferably the dose ranges from 0.05 to 4.0 mg microparticles/kg body weight. The dosage formulation typically is provided in a vial. A typical formulation is in the form of a dry powder that is reconstituted with sterile water prior to use by adding the water to the vial or syringe of the dry powder and shaking to yield an isosmotic or isotonic suspension of microparticles.

Description

Ultrasound contrast agent dosage formulation

The present application is a divisional application of an invention patent application having an application date of 2004, 6/4, application No. 200480043713.0(PCT/US2004/017813), entitled "ultrasound contrast agent dosage formulation".

Technical Field

The present invention is in the field of diagnostic imaging agents in general and in particular relates to an ultrasound contrast agent (ultrasound contrast agent) dosage formulation that provides enhanced images and images of long duration.

Background

When ultrasound is used to obtain images of internal organs and structures of a human or animal, ultrasound, sound energy waves are reflected as they pass through the human body at a certain frequency (above which they are perceived by the human ear). Different types of body tissue reflect different ultrasound waves, and reflections resulting from the ultrasound waves reflecting different internal structures can be detected and electronically converted into a visual image.

For some medical situations, obtaining a useful image of an organ or structure of interest is particularly difficult because the details of the structure cannot be adequately discerned from the surrounding tissue in an ultrasound image produced by reflection of ultrasound waves in the absence of a contrast-enhancing agent. The detection and observation of certain physiological and pathological conditions is essentially improved by increasing the contrast in ultrasound images by administering ultrasound contrast agents into the organ or other structure of interest. In some cases, detection of the motion of the ultrasound contrast agent itself is important. For example, unique blood flow patterns caused by particular cardiovascular abnormalities can only be identified by administering ultrasound contrast agents to the blood flow and observing the blood flow or blood volume.

Materials used as ultrasound contrast agents function by affecting ultrasound waves as they pass through the body and are reflected to produce images from which medical diagnoses can be made.

Different types of substances affect the ultrasound in different ways and to different extents. In addition, certain effects caused by contrast-enhancing agents are more easily measured and observed than other agents. In selecting an ideal ultrasound contrast agent composition, one generally prefers those substances that have the most significant effect on ultrasound waves as they pass through the body. Furthermore, the effect on the ultrasound should be easy to measure. Gas is a preferred medium for use as an ultrasound contrast agent. The gas must be immobilized prior to use, either as a surfactant to immobilize the gas bubbles or by encapsulation in liposomes or microparticles. Three major contrast enhancement effects can be seen in ultrasound images, namely back reflection, beam attenuation, and sound velocity difference.

A variety of natural and synthetic polymers have been used to encapsulate ultrasound contrast agents such as air, and these polymers have helped to produce ultrasound contrast agents that last longer after administration. Schneider et al inInvest.Radiol.Three micron-sized, air-filled, synthetic, polymer particles are described in Vol 27, pp.134-139 (1992). These particles are reported to be stable in plasma and under pressure of use. However, the device is not suitable for use in a kitchenWhile their echogenicity is low at 2.5 MHz. Another microvesicle suspension has been obtained from sonicated protein endosperm. Feinstein, and the like,J.Am.Coll.Cardiol.vol.11, pp.59-65 (1988). Feinstein describes the preparation of microvesicles with good in vitro stability, sized to fit the channel between the lung and the lung lumen (transmucon clearance). However, since these microbubbles are unstable under pressure, they have a short lifetime in vivo, with a half-life of about a few seconds (approximately equal to a cyclic process). Gottlieb, S, etc.,J.Am.Soc.Echo.vol.3, pp.328(1990), abstract; and Shapiro, J.R. et al,J.Am.Coll.Cardiol.Vol.16，pp.1603-1607(1990)。

gelatin encapsulated microbubbles are also described in WO 80/02365 by Rasor Associates. The gelatin-encapsulated microbubbles are formed by "coalescing" gelatin. Gas microbubbles encapsulated inside a shell of a fluorine-containing material are described in WO96/04018 by Molecular Biosystems, Inc.

Fritzch et al reported that microbubbles were immobilized with galactose microcrystals (SHU454 and SHU 508). Fritzsch, T, et al,Invest.Radiol.vol.23(Suppl 1), pp.302-305 (1988); and Fritzseh T, et al,Invest.Radiol.vol.25(Suppl1) 160-. The microbubbles last 15 minutes in vitro but less than 20 seconds in vivo. The number of changes in the shape of Rovai, D, etc.,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). The preparation and use of microencapsulated gases or volatile liquids for ultrasound imaging is disclosed by Schering Aktiengesellschaft in european patent EP 398935, wherein the microcapsules are formed from synthetic polymers or polysaccharides. Sinertica in European patent 458745 discloses air or gas microspheres for therapeutic or diagnostic purposes, which are encapsulated by an interfacially deposited polymer film capable of being dispersed in an aqueous carrier for injection into a host animal or for buccal, rectal or urethral administration.

Delta bioengineering, Inc. in WO 92/18164 describes a method of preparing microparticles for imaging by spray drying an aqueous protein solution to form hollow spheres with gas content. WO 93/25242 describes a method for the synthesis of microparticles for ultrasound imaging, the microparticles having a structure that contains a gas inside a shell of polycyanoacrylate or polyester. WO 92/21382 discloses the manufacture of a particulate contrast agent comprising a covalently bonded matrix containing a gas, wherein the matrix is a carbohydrate. Unger, in U.S. patent nos. 5,334,381 and 5,123,414 and 5,352,435, describes liposomes for use as ultrasound contrast agents that include a gas, a gaseous precursor, such as a pH activated or light activated gaseous precursor, and other liquid or solid contrast enhancing agents.

Others have also proposed the use of fluorinated gases to enhance imaging compared to air in view of the effects of the encapsulated gas. Quay, in U.S. Pat. No.5,393,524, discloses the use of various agents, including perfluorocarbons, to enhance contrast in ultrasound imaging. The agent consists of small bubbles or microbubbles of selected gases that exhibit a long life in solution and are small enough to pass through the lungs, which enables its use in ultrasound imaging of the cardiovascular system and other vital organs. Bracco in European patent 554213 discloses the effect of fluorinated hydrocarbon gases on preventing the collapse of microbubbles when exposed to blood pressure. Nycomed in WO95/23615 discloses microcapsules for imaging that are formed by coagulation of a perfluorocarbon containing solution, such as a protein solution or the like. The Massachusetts Institute of technology, WO95/03357, discloses microparticles formed from polyethylene glycol-poly (lactide-co-glycolide) bulk polymers, which microparticles encapsulate an imaging agent, including a gas such as air and perfluorocarbon. As described in WO94/16739 by Sonus pharmaceuticals, when solids and liquids reflect sound waves to a similar extent, it is well known that gases are the more effective and popular medium for ultrasound contrast agents. In fact, as shown in example 12 of WO94/16739, protein microcapsules were not considered when administered to mini-pigs due to safety issues (and efficacy issues). U.S. Pat. nos. 6,132,699 and 5,611,344 both describe methods of using perfluorocarbons in synthetic polymer shells to enhance contrast. U.S. patent 5,837,221 describes a method of producing porous polymeric microparticles that incorporates a hydrophobic agent into the polymer to increase echogenicity.

Several ultrasound contrast agents are approved in both the united states and europe for very limited cardiotonic administration.(Amersham, Mallinkrodt) comprises heat denatured human albumin microcapsules containing octafluoropropane gas. Microsphere suspensions of 5-8X 10/ml⁸A particle size range of 2-4.5 microns in average diameter and 220 μ g of octafluoropropane. These microspheres have not been approved for myocardial blood flow assessment, but only for ventricular enhancement. At higher bolus doses (5mL suspension or 1100 μ g octafluoropropane), ventricular potentiation persists for up to 5 minutes.

(Bristol Myers medical imaging) comprises octafluoropropane containing lipid microspheres in which the lipid shell is composed of the phospholipids DPPA, DPPC, and mPEG-DPPE. 1.2X 10 per ml of suspension¹⁰Particles having an average diameter in the size range of 1.1-3.3 microns and having 1100 μ g octafluoropropane. This agent is only approved for ventricular enhancement and not for myocardial blood flow assessment. The duration of the potentiating effect of the agent in the ventricle was approximately 3.4 minutes at a bolus dose of 700 μ L (for a 70kg person) or at 5133 μ g of gas.

(Photogen Co.) containing lipid microspheres containing perfluorohexane, whichIn (1), the lipid shell consists of the phospholipid DMPC. 1.4X 10 per ml of suspension⁹A microparticle and 92 μ g of perfluorohexane, wherein the microparticle has an average diameter of less than 3 microns. This agent is only approved for ventricular enhancement and not for myocardial blood flow assessment. The mean duration of potentiation of the agent in the ventricle was approximately 2.6 minutes at a bolus dose of 0.43 ml (for a 70kg person) or 40 μ g of gas.

In any event, these commercial agents have only limited application and are not approved for use in applications other than ventricular augmentation, where the average imaging enhancement duration of these agents in the ventricles is 5 minutes or less. Commercial ultrasound contrast agents capable of enhancing the imaging of the cardiovascular system, in particular the myocardium and the ventricles, and having a longer duration are still lacking. The agents described in the prior art, when administered as a bolus or short infusion, produce myocardial images of significantly shorter duration than the time required to process a complete cardiac test. Typically, the prior art agents provide images that are less than a minute in duration when applied to the myocardium. It would be desirable to have agents that provide enhanced imaging durations of more than one minute in the myocardium and/or more than 5 minutes in the ventricles.

It is therefore an object of the present invention to provide a dosage formulation particularly suitable for cardiotonic agents, comprising microparticles, wherein the microparticles are capable of providing enhanced images and images of long duration.

It is another object of the present invention to provide a kit for the administration of a dosage formulation comprising microparticles in a method of ultrasound imaging.

Disclosure of Invention

Specific dosage formulations have been developed and used in clinical studies that employ polymeric microparticles incorporating perfluorocarbon gases that provide significant and long-lasting enhanced imaging. The dosage formulation typically comprises one, two or up to five doses, preferably one or two doses, of microparticles formed from a biocompatible polymer, preferably containing lipids, which contain a perfluorocarbon that is gaseous at body temperature. The microparticles are administered to the patient at a dose effective to enhance ultrasound imaging, for a duration of greater than five minutes in the ventricle and/or greater than one minute in the myocardium, ranging from 0.025 to 8.0mg microparticles/kg body weight. Preferably, the dose range administered to the patient is from 0.05 to 4.0mg microparticles/kg body weight. In a preferred embodiment, the enhancement of ultrasound imaging lasts more than 9 minutes in the ventricle and/or more than 2 minutes in the myocardium.

Dosage formulations are typically provided in vials or syringes. In a typical formulation, the dosage formulation is in the form of a dry powder that is reconstituted with sterile water prior to use, specifically: water is added to a vial or syringe containing the dry powder and then shaken to produce an isotonic or isostatically pressed suspension of the microparticles. In a preferred embodiment of these dosage formulations, the suspension comprises 1.0 to 3.5X 10⁹microparticles/mL, or 25-50mg microparticles/mL, the most preferred concentration being a suspension comprising 1.5-2.8X 10⁹microparticle/mL or 30-45mg microparticle/mL. In a preferred embodiment, the particles have an average particle size of less than 8 microns, and most preferably an average particle size of 1.8 to 3.0 microns.

In the most preferred embodiment, the gas is CF₄、C₂F₄、C₂F₆、C₃F₆、C₃F₈、C₄F₈、C₄F₁₀Or SF₆. In a preferred embodiment, the gas is n-perfluorobutane (C)₄F₁₀) Is provided in an amount of 75 to 500. mu.g/mL of the microparticle suspension to be administered; preferably, n-perfluorobutane is provided in an amount of 100-400 μ g/mL of the particulate suspension administered; preferably n-perfluorobutane is provided in an amount to administer a suspension of particles of 150-; or the gas is octafluoro-n-propane and is provided at an amount of 75-375 μ g/mL of the particulate suspension, preferably at an amount of 120-300u g/mL of the particulate suspension.

In a most preferred embodiment, the microparticles are formed from synthetic polymers such as poly (alkyd) acids including polylactic acid, polyglycolic acid, and poly (lactic-co-glycolic acid), polyglycolide, polylactide and poly (lactide-co-glycolide), polyorthoesters, polyamides, polycarbonates, polyolefins such as polyethylene and polypropylene, polyalkylene glycols such as polyethylene glycol, polyalkylene oxides such as polyethylene oxide, polyvinyl alcohol, poly (valeric acid), and poly (lactide-co-caprolactone), derivatives, copolymers and mixtures thereof, and polymers containing a hydrophobic compound in a proportion of 0.01 to 30 wt% relative to the weight of the polymer, preferably polymers containing lipids in a proportion of 0.01 to 30 wt% (weight lipid/weight polymer). In a particularly preferred embodiment, the lipid is Dioleoylphosphatidylcholine (DOPC), Dimyristoylphosphatidylcholine (DMPC), dipentadecylphosphatidylcholine (DPDPC), Dilauroylphosphatidylcholine (DLPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Diarachioylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (didaxylphosphatidylcholine, DAPC), ditolylphosphatidylcholine (dibehenylphosphatidylcholine), Ditosylphosphatidylcholine (DBPC), Ditridecylphosphatidylcholine (DTPC), ditridecylphosphatidylcholine (ditridecylphosphatidylcholine), Ditridecylphosphatidylcholine (DLGPC); or phosphatidylethanolamine.

Preferably, the synthetic polymer in the microparticles is poly (lactide-co-glycolide), the ratio of lactide to glycolide is 50: 50 (i.e., 1: 1), the weight average molecular weight is in the range of 20,000-40,000 daltons, and the hydrophobic compound in the microparticles is DAPC in a ratio of 5: 6.6% (DAPC weight/polymer weight).

The dosage formulation may be provided in the form of a vial or syringe containing the particulate dry powder, or in the form of a kit containing a solution of resuspended particles. Typically, the vials or syringes of dry powders also contain excipients such as sugar or salt to render the solution isotonic, or isotonic after reconstitution. This dosage formulation is then administered to the patient by injection as a bolus or injection and imaging is performed over a period of 30 minutes.

Microparticles are useful in a variety of diagnostic imaging procedures, including ultrasound imaging, magnetic resonance imaging, fluoroscopy, X-ray, and computerized tomography. Microparticles were tested in clinical trials for cardiology applications such as myocardial blood flow assessment and ventricular enhancement.

Detailed Description

Improved methods, microparticles, kits, and dosage formulations for ultrasound imaging are described herein. Microparticles are useful in a variety of diagnostic ultrasound imaging applications, particularly in ultrasound procedures such as vascular imaging and echocardiography such as myocardial blood flow assessment, myocardial blood volume assessment and ventricular reinforcement.

I. Definition of

Unless otherwise specified, the term "microparticles" includes "microspheres" and "microcapsules", as well as other microparticles, as commonly used. The microparticles may be spherical or non-spherical in shape.

"microcapsules" are generally defined herein as microparticles having an outer polymeric shell surrounding a gas core. "microspheres" are defined as porous spheres that may be solid spheres, or cellular structures or sponge-like structures formed by pores throughout a gas-filled polymer. Some microspheres may comprise an outer polymeric shell having a honeycomb-like configuration or a sponge-like structure formed by gas-filled pores throughout the polymeric shell. For such microspheres, the outer polymeric shell surrounds an inner gas core.

As used generally, the terms "dose" (dosage) "and" dose "(dose)" are synonymous and refer to the amount of substance administered at one time, or the amount of substance required to produce a desired diagnostic or comparative effect.

The term "dosage formulation" as used herein refers to a vial or other container, such as a syringe, containing one or more doses of a substance required to produce a desired diagnostic or contrast effect.

As is conventional, the term "region of a patient" as used herein refers to a specific area or portion of the patient. In some instances, a "site of a patient" refers to a site throughout the entire body of the patient. Examples of such sites are the lungs, the gastrointestinal tract, the cardiovascular site (including myocardial tissue or muscle (e.g. heart muscle), the chambers of the ventricles, the gill cavity, valve function), the renal site and other parts of the body, tissues, organs, etc., including vascular tissue and circulatory system, and diseased tissue, including cancerous tissue. The "region of the patient" includes, for example, a region to be visualized by a diagnostic image. Preferably, the site of the patient is in vivo, although it may also be in vitro.

As is customary, the term "vascular tissue" herein denotes blood vessels (including arteries, veins, capillaries, etc.).

As is customary, the term "gastrointestinal site" as used herein includes the sites defined by the esophagus, stomach, small and large intestines, and rectum.

As is customary, the term "renal site" as used herein refers to the kidney and the vascular tissue directly connecting the kidney, including the abdominal aorta.

As is customary, the terms "target site" and "target area" are used interchangeably herein to refer to a site in a patient to which a pharmaceutical agent is intended to reach.

As is customary, the terms "site to be imaged" and "imaging site" are used interchangeably herein to refer to the intended imaging site of a patient.

As is customary, herein "ventricular blood flow or ventricular enhancement" refers to the circulation of blood through the ventricles of the heart in one or more hearts.

As is customary, by "atrial blood flow" is meant blood flow that circulates through the atria of the heart in one or more cycles.

As is customary, the term "myocardial blood flow" here refers to the flow of blood in the heart muscle or the vascular tissue of the myocardium (including in the blood vessels of the heart) in one or more cycles.

As is customary, the term "myocardial blood volume" here refers to the amount of blood in the heart muscle or the vascular tissue of the myocardium.

As is conventional, the term "cardiac cycle" herein refers to a complete systolic cycle of the heart, including the diastolic and systolic cycles of the heart.

As is customary, herein "increased brightness" refers to an increase in brightness of an image as compared to an image obtained without an ultrasound contrast agent.

As is customary, herein "enhanced visualization" refers to an image having a relatively increased brightness compared to an image obtained without an ultrasound contrast agent.

As is customary, the "duration" here refers to the total time during which an increased brightness of the visualization can be detected.

As is customary, by "coronary vasodilator" herein is meant a physiologically active substance such as dipyridamole or adenosine which, when administered to a patient, causes dilation of vascular tissue in the cardiovascular region.

Particles of II

In a preferred embodiment, the microparticles comprise a polymer, a lipid, and a perfluorocarbon gas. The microparticles may comprise both microspheres and microcapsules, or only microspheres or microcapsules.

Polymer and method of making same

In a preferred embodiment, the microparticles are formed from a synthetic polymer. Synthetic polymers produce microparticles that are biocompatible and free of biomaterial contamination.

In addition, synthetic polymers are preferred because synthetic polymers can be reproducibly synthesized and degraded in vitro and in vivo. The polymer is selected based on the time required for in vivo stability, e.g., the time required for distribution to the site to be imaged, and the time required for imaging. Synthetic polymers may be modified to produce microparticles of different properties (e.g., changing molecular weight and/or functional groups).

Typical synthetic polymers are: polyglycolic acids such as polylactic acid, polyglycolic acid, and poly (lactic-co-glycolic acid), polyglycolides, polylactides, poly (lactide-co-glycolide) and mixtures thereof, polyanhydrides, polyorthoesters (polyorthoesters), polyamides, polycarbonates, polyolefins such as polyethylene and polypropylene, polyglycols such as polyethylene glycol, oxidized polyolefins such as polyethylene oxide polyvinyl alcohol, poly valeric acid, and poly (lactide-co-caprolactone), and derivatives, copolymers, and mixtures thereof. "derivatives" as used herein include polymers where chemical groups are substituted or added, such as alkyl, alkylene, hydroxylation, oxidation and other modifications commonly used by those skilled in the art.

Specific examples of preferred biodegradable polymers include: alkyds such as polymers of lactic and glycolic acid, polylactides, polyglycolides, poly (lactide-co-glycolide), copolymers with PEG, polyanhydrides, poly (ortho) esters, polyurethanes, poly (butyric acid), poly (valeric acid), poly (lactide-co-caprolactone) and copolymers thereof. Most preferably the polymer is poly (lactide-co-glycolide) wherein the ratio of lactide to glycolide is 50: 50 (i.e. 1: 1) and the weight average molecular weight of the polymer is in the range of 20,000-40,000 daltons. The weight average molecular weight (Mw) is the average molecular weight calculated based on the mass of molecules having a given molecular weight within a distribution of individual polymer chains. Mw can be determined by Gel Permeation Chromatography (GPC).

Hydrophobic compounds

In a preferred embodiment, the polymer comprises a hydrophobic compound, as described in U.S. patent No.5,837,221. Typically, for example, lipids, which are hydrophobic and are present in an effective amount within the polymer, in combination with such compounds, limit water permeation and/or absorption through the microparticles and thus limit gas loss from the microparticles.

This is effective to increase the duration of enhanced imaging provided by microparticles comprising lipids, synthetic polymers and encapsulated gases, especially fluorinated gases such as perfluorocarbons.

Lipids that can be used to immobilize the gas within the polymer particles include, but are not limited to, the following classes of lipids: fatty acids and their derivatives, monoglycerides, diglycerides and triglycerides, phospholipids, sphingolipids, cholesterol and steroid derivatives, terpenes and vitamins.

Fatty acids and derivatives thereof may include, but are not limited to, saturated and unsaturated fatty acids, odd and even numbered fatty acids, cis-trans isomers, and fatty acid derivatives including alcohols, esters, anhydrides, hydroxy fatty acids, and prostaglandins. Saturated and unsaturated fatty acids that may be used include, but are not limited to: straight or branched chain molecules having 12 to 22 carbon atoms. Examples of saturated fatty acids that may be used include, but are not limited to: lauric acid, myristic acid, palmitic acid and stearic acid. Examples of unsaturated fatty acids that may be used include, but are not limited to: lauric acid, sperm whale acid, myristoleic acid, palmitoleic acid, petroselinic acid, and oleic acid. Examples of branched chain fatty acids that may be used include, but are not limited to: isolauric acid, isomyristic acid, isopalmitic acid, isostearic acid and isoprenes. Fatty acid derivatives include 12- (((7' -diethylaminocoumarin-3-) carbonyl) methylamino) -stearic acid; n- [12- (((7' diethylaminocoumarin-3-) carbonyl) methylamino) octadecanoyl ] -2-aminopalmitic acid, N-succinyl-dioleoylphosphatidylethanolamine and palmitoyl-homocysteine; and/or combinations thereof. Monoglycerides, diglycerides, triglycerides and derivatives thereof that may be used include, but are not limited to: fatty acids or mixtures of fatty acids with a number of carbon atoms in the molecule between 6 and 24, digalactosyldiglyceride, 1, 2-dioleoyl-sn-glycerol; 1, 2-dipalmitoyl-sn-3-succinyl glycerol; and 1, 3-dipalmitoyl-2-succinyl glycerol.

Phospholipids that can be used include, but are not limited to: phosphatidic acid, lecithin with saturated and unsaturated lipids, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, lysophosphatidyl derivatives, cardiolipin, and β -acylalkylphospholipids. Examples of phospholipids include, but are not limited to: dioleoylphosphatidylcholine (DOPC), Dimyristoylphosphatidylcholine (DMPC), Dipentacrylphosphatidylcholine (DPPC), Dilauroylphosphatidylcholine (DLPC), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), diarachioylphosphatidylcholine (diarachioylphosphatidylcholine, DADADAPC), Didodecanoylphosphatidylcholine (DBHOLIPE), Didodecanoylphosphatidylcholine (DTPC), ditereoylphosphatidylcholine (DBDLPC), Didodecanoylphosphatidylcholine (DTPCPC), didodecanoylphosphatidylcholine (DTDP), Diglylphosphatidylcholine (DTPC), Ditridecylphosphatidylcholine (DLPC); and phosphatidylethanolamines such as dioleoylphosphatidylethanolamine (dioleylphosphatidylethanolamine) or 1-hexadecyl-2-palmitoyl glycerophosphoethanolamine. Synthetic phospholipids with asymmetric acyl chains (e.g. with one acyl chain of 6 carbon atoms and another acyl chain of 12 carbon atoms) can also be used.

Sphingolipids which may be used include ceramides, sphingomyelins, cerebrosides, gangliosides, brain sulfatides and lysobrain sulfatides. Examples of sphingolipids include, but are not limited to: gangliosides GM and GM 2.

Steroids that may be used include, but are not limited to: cholesterol, cholesterol sulfate, cholesterol hemisuccinate, 6- (5-cholesterol-3 β -oxy) hexyl-6-amino-6-deoxy-1-thio- α D-lactopyranoside, 6- (5-cholesten-3 β -oxy) hexyl 6-amino 6-deoxy-1-thio- α D-mannopyranoside and (cholestenyl-4' -trimethyl-35-amino) butyrate.

Other lipid compounds that may be used include tocopherols and derivatives thereof, as well as oils and derived oils, such as stearamide.

Many cationic lipids can be used, for example DOTMA, N- [1- (2, 3-dioxaoleoyl) propyl-N, N, N-trimethyl chloride; DOTAP, 1, 2-dioxetyl-3- (trimethylamino) propane; and DOTB, 1, 2-dioleoyl-3- (4' -trimethyl) butanoyl-sn-glycerol.

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

The lipid content is in the range of 0.01-30% (weight lipid/weight polymer); preferably 0.1-20% (weight of lipid/weight of polymer), most preferably 1-12% (weight of lipid/weight of polymer).

When the particles are formed by the process of the present invention, their size can be reproduced well. Herein, unless otherwise specified, the term "size" or "diameter" of a particle refers to the number average particle size. An example of an equation that can be used to define the number average particle size (Xn) is listed below.

Where ni is the number of particles of a given diameter (di)

Here, the term "volume average diameter" means volume weight diameter average. An example of an equation that can be used to define the volume mean diameter (Xv) is listed below:

here, ni is the number of particles of a given diameter (di).

Particle size analysis can be carried out using a coulter suspension analyzer by optical microscopy, scanning electron microscopy, transmission microscopy, laser diffraction methods, e.g. using a Malvern Mastersizer, light scattering or time-of-flight. The "Coulter method" here means: the powder was dispersed in an electrolytic solution, and the resulting suspension was analyzed by a Coulter multisizer II equipped with a 50 μm-diameter tube. The method can provide size measurements and particle concentrations.

In a preferred embodiment for preparing injectable microparticles capable of passing through the pulmonary capillary bed, the microparticles are less than 8 microns in diameter. Large particles may clog the lung bed and small particles may not provide sufficient contrast effect. The particle size of the ultrasonic contrast agent for intravenous administration is preferably 0.75-5 microns, and more preferably 1.8-3.0 microns.

In a preferred embodiment, the microparticles have a honeycomb-like structure or a sponge-like structure, formed by pores throughout the polymer, or the microparticles have a polymer shell with a honeycomb or sponge-like porous structure. In both cases, the pores are filled with gas. These particles are formed by spray drying a polymer solution containing a pore former such as ammonium carbonate as described below.

Ultrasound contrast agent

Examples of the fluorinated gas include CF₄、C₂F₄、C₂F₆、C₃F₆、C₃F₈、C₄F₈、C₄F₁₀And SF₆. Preferably perfluoron-butane (C)₄F₁₀) Because it provides an insoluble gas that does not condense at the use temperature and is pharmaceutically acceptable.

The amount of gas containing microparticles depends on the type of gas, but is generally administered in an amount of 75-500. mu.g/mL of microparticle suspension. For perfluoron-butane, the preferred gas content is a particulate suspension administered in an amount of 100-. For octafluoro-n-propane, the preferred gas content is a particulate suspension administered in an amount of 75-375 μ g/mL, most preferably in an amount of 120-300 μ g/mL.

III Process for producing microparticles

Microparticles can be made by a number of methods, preferably by spray drying. The main criterion is that the polymer must be dissolved or melted in a hydrophobic compound or lipid before the microparticles are formed.

Solvent(s)

During formation, the polymer is typically dissolved in a solvent. Herein, polymer solvent means a volatile organic solvent or has a relatively low boiling point or can be removed under vacuum and is acceptable when administered in trace amounts to humans, for example, methylene chloride. Other solvents such as ethyl acetate, ethyl formate, ethanol, methanol, Dimethylformamide (DMF), acetone, acetonitrile, Tetrahydrofuran (THF), formamide, acetic acid, dimethyl sulfoxide (DMSO), and chloroform, or mixtures thereof, may also be used. Typically, the polymer is dissolved in the solvent to form a polymer solution having a concentration of 0.1 to 60% (w/v), preferably 0.25 to 30% (w/v), more preferably 0.5 to 10% (w/v).

Spray drying

The particles are preferably produced by spray drying, dissolving the biocompatible polymer and lipid in a suitable solvent, dispersing the pore-forming agent as a solid or solution into the polymer solution, and then spray drying the polymer solution and pore-forming agent to form the particles. Here, the "spray-drying" of the polymer solution and the pore-forming agent refers to a process of atomizing the polymer solution and the pore-forming agent to form a mist, and directly contacting with a hot carrier gas to dry them. The polymer solution and the pore-forming agent are atomized at the air inlet of the spray dryer using a spray dryer commonly used in the art, pass through at least one drying chamber, and then are collected in the form of powder. The temperature may vary depending on the gas or polymer used. The desired product can be produced by controlling the inlet and outlet temperatures.

The size and morphology of the microparticles formed during spray drying is affected by the following factors: the nozzle function used to spray the polymer solution and pore-forming agent, the pressure of the nozzle, the flow rate of the polymer solution with the pore-forming agent, the polymer used, the concentration of the polymer in the solution, the type of polymer solvent, the type and amount of pore-forming agent, the spray temperature (inlet and outlet temperatures), and the molecular weight of the polymer. Generally, the higher the molecular weight of the polymer, the larger the particle size, assuming the same concentration of polymer solution.

The general process parameters for spray drying were as follows: the inlet temperature is 30-200 deg.C, the outlet temperature is 5-100 deg.C, and the polymer flow rate is 10-5,000 ml/min.

The polymer solution and pore former may be emulsified with the gas prior to spray drying to encapsulate the gaseous diagnostic agent. Alternatively, the aerated microparticles can be produced in a spray drying step, followed by applying a perfluorocarbon gas to the microparticles, replacing the air with the desired gas, or by evacuating the microparticles to remove the encapsulated air and then filling with the desired perfluorocarbon gas. If a vacuum step is used to exchange gases, a freeze dryer or vacuum box may be used.

Additive for promoting the formation of microparticles

Various surfactants may be added during the formation of the microparticles. Examples of emulsifiers or surfactants (0.1-15% w/w polymer) that may be used include the most biologically acceptable emulsifiers. Examples include natural and synthetic bile salts or bile acids in combination with amino acids, and unconjugated deoxycholic acid and cholic acid.

The pore-forming agent is included in the polymer solution in an amount of 0.01 to 90 wt% with respect to the volume of the polymer solution to increase the formation of pores. For example, in spray drying, a pore-forming agent, which may be exemplified by volatile salts such as ammonium bicarbonate, ammonium acetate, ammonium carbonate, ammonium chloride or ammonium benzoate or other volatile salts, may be used in the form of a solid or in the form of a solution dissolved in a solvent such as water. The solid pore former or solution containing the pore former is then emulsified with the polymer solution to produce a dispersion or microdroplets of the pore former in the polymer. These dispersions or emulsions are then spray dried to remove the polymer solvent and pore former. After polymer precipitation, the hardened microparticles may be frozen and lyophilized to remove any pore formers that were not removed during the polymer precipitation step.

It is preferred to form microparticles using polymers, such as poly (lactide-co-glycolide) with a lactide-to-glycolide ratio of 50: 50 and a weight average molecular weight in the range of 20,000-40,000 daltons, and phospholipids, 5-6.6% (weight of DAPC/weight of polymer) of diacylphosphatidyl choline ((1, 2-diacyl-sn-glycero-3-phosphocholine) (DAPC)) ((1, 2-diacyl-sn-glycero-3-phosphocholine)Further formulated to produce a particulate dry powder that is reinflated with perfluoron-butane on a freeze dryer. The dry powder was reconstituted with 5ml of sterile water prior to use, and water was added to a vial of the dry powder followed by shaking to produce a suspension of microparticles in isotonic mannitol. The properties of the preferred suspension are: each dosage of the particle suspension contains perfluoron-butane with gas content of 150-⁹The amount of the microparticles per ml, the microparticle suspension administered is 30-45mg microparticles per ml, and the average particle size is in the range of 1.8-3.0 μm.

Use of IV microparticles

1. Formulations for administration to patients

The microparticles may be further processed with excipients to produce a dry powder. The excipient provides tonicity or permeability or reduces the suspensibility of the microparticles when reconstituted with a pharmaceutically acceptable carrier prior to administration to a patient. Is suitable forSuitable excipients that provide osmotic or tonicity properties are sugars including, without limitation, mannitol, dextrose (dextrose), or glucose, and salts including, without limitation, sodium chloride or sodium phosphate. Excipients suitable for providing relief from microsphere suspension include any pharmaceutically acceptable wetting agent or surfactant, including without limitation polysorbate 80 (Tween)) Polysorbate 20 (Tween)) Pluronic (Pluronic) or polyethylene glycol. For reference, some Excipients suitable for providing permeability or tonicity or that can be used as wetting agents are described herein, for example, Handbook of Pharmaceutical Excipients (fourth edition, royal Pharmaceutical association, scientific and practice publishers) or ramington: pharmaceutical science and practice (nineteenth edition, Mack publishing company). Dry powders of microparticles and excipients are produced by suspending microparticles in a solution of the excipient. A size fractionation step may be further used if necessary. The microparticles in the excipient solution are filled into vials or syringes, frozen, lyophilized, and produced into a dry powder formulation. At the end of the lyophilization step, the microparticles are filled with perfluorocarbon gas by refilling the freeze-dryer with perfluorocarbon gas. The vial or syringe is then stoppered or capped with a stopper, which in the case of a vial is crimped down. This results in the headspace in the vial or syringe being filled with perfluorocarbon.

Alternatively, the microparticles may be dry mixed with the pharmaceutical excipient and then filled into vials or syringes. The vial syringe is placed on a freeze dryer or in a vacuum chamber and evacuated, and the microparticles are then filled with perfluorocarbon gas. The vial or syringe is then stoppered or capped with a stopper, which in the case of a vial is crimped down. This results in the headspace in the vial or syringe being filled with perfluorocarbon.

2. Dosage unit

Different sized dose units of microparticles may be used. For example, a small dosage unit may contain 25-75mg of microparticles. A medium dosage unit may contain 75-150 mg. A large dosage unit may contain 150-250mg of microparticles. An oversized dosage unit may contain 150-1000mg of microparticles.

When a suspension of microparticles is formed after reconstitution, the mass concentration of microspheres in the suspension typically varies from 20 to 60 mg/mL. The mass concentration of microspheres in the suspension is preferably 25-50mg/mL, and the mass concentration of microspheres in the suspension is most preferably 30-45 mg/mL. The concentration of the microparticles in the suspension is preferably 1.0-3.5X 10⁹microparticle/mL, the concentration of microparticles in the suspension is most preferably 1.5-2.8X 10⁹Microparticles/ml. Preferably the particles have an average particle size of less than 8 microns, most preferably in the range of 1.8 to 3.0 microns.

The pharmaceutically acceptable carrier includes water for injection, sterile water, saline containing glycerol, and TweenSaline containing TweenSaline, isotonic glucose (5%), 1/2 isotonic dextrose (2.5%), isotonic mannitol (5%), 1/2 isotonic mannitol (2.5%), containing tweenIsotonic mannitol, containing TweenIsotonic mannitol.

3. Reagent kit

Kits can be provided for parenteral administration of microparticles comprising a perfluorocarbon gas. The kit comprises at least two components. One component comprises a dose unit of dry powder contrast agent in a vial or syringe and the other component comprises a pharmaceutically acceptable carrier in a vial or syringe. Prior to administration to a patient, a pharmaceutically acceptable carrier is added to the dosage unit of dry powder contrast agent to form a gas-filled microparticle suspension that can be used as an ultrasound contrast agent in diagnostic imaging of any route of administration.

4. Vials or containers for microparticles

For the kit, no special vial or syringe or adapter is required, and ordinary vials, syringes and adapters can be used for microparticles. The only requirement for the vial is a good seal between the stopper and the container. Thus, the quality of the seal is a major concern and any loss of seal integrity will result in unwanted material entering the vial or gas leaking out. In addition to ensuring sterility, maintenance of vacuum is essential for products stoppered under reduced pressure to ensure safe and correct reconstitution. With regard to the stopper, it may be a single compound or multicomponent formulation based on an elastomer, such as poly (isobutylene) or "butyl rubber", and must not be permeable to the gas used. The size of the vial is selected based on the total dose of dry powder in the vial. The vial size is preferably 5mL, 10mL, 20mL and 30 mL. The size of the syringe is selected based on the total dose of dry powder in the syringe. The syringe size is preferably 5mL, 10mL, 20mL and 50 mL.

5. Diagnostic applications

The microparticle compositions can be used in a number of different diagnostic applications, including ultrasound imaging, magnetic resonance imaging, fluoroscopy, X-ray, and computerized tomography.

In a preferred embodiment, the microparticles are used in ultrasound procedures such as vascular imaging and echocardiography, including, without limitation, ventricular imaging, myocardial blood flow assessment, myocardial blood volume assessment, diagnosis of coronary artery disease, and ejection fraction assessment.

The microparticles are useful for vascular imaging, for detecting liver and kidney disease, for detecting and characterizing tumor masses and tissues, and for measuring peripheral blood velocity. The microparticles may also be associated with ligands that minimize tissue adhesion or target the particles to specific sites in the body.

General method of obtaining an image

The dry powder particles are reconstituted with a pharmaceutically acceptable carrier prior to use and then administered to a patient in an effective amount for testing by an appropriate route, such as injection into a blood vessel (e.g., intravenous (i.v.) or intra-arterial (i.a)) or oral administration. The microparticle composition may be administered intravenously to the patient as a bolus or as a short infusion (less than 30 minutes). Preferably, the injection is controlled to be within a time range of 15 seconds to 20 minutes, preferably 30 seconds to 15 minutes. Generally, for patients, each intravenous injection is usually controlled in a dosage range of from 0.025 to 8mg/kg body weight, preferably in a dosage range of 0.05 to 4 mg/kg.

For diagnostic ultrasound applications, energy is applied to at least a portion of the patient's body to visualize the target tissue. A visible image of the internal region of the patient is then obtained from which the presence or absence of diseased tissue can be determined. Ultrasound imaging techniques including second harmonic imaging and gated imaging are well known in the art and have been described, for example, Uhlendorf,IEEE Transactions on Ultrasonics，Ferroelectrics，and Frequency Control,14(1): 70-79 (1994); and Sutherland, et al,Journal ofthe American Society ofEchocardiography,7(5): 441-458(1994), which is hereby incorporated by reference in its entirety.

The ultrasound may be applied with a transducer. The ultrasound may be pulsed or may be continuous, if desired. Thus, diagnostic ultrasound typically involves the application of echoes, followed by the reception of reflected signals by an ultrasound transducer during a listening period. Harmonics, higher harmonics or subharmonics may be used. A second harmonic mode that accepts 2x frequencies, where x is the non-dominant frequency, may be used effectively. This can be used to reduce the signal from background material, enhancing the signal from the sensor with an imaging agent that targets the desired location, such as a blood clot. Other harmonic signals, such as odd harmonic signals, e.g. 3x or 5x, may likewise be received in this way. Subharmonic signals, such as x/2 and x/3, may also be received and processed simultaneously to form an image.

In addition, Power Doppler (Power Doppler) or Color Doppler (Color Doppler) may be applied. In the case of power doppler, the relatively high energy of power doppler can cause the pores to resonate. This may result in the radiation of acoustic waves in the range of subharmonics or ultraharmonics, etc., as well as the applied ultrasound frequency.

Specific imaging applications

The microparticles described herein may be used for cardiology and radiology applications. For cardiology applications, the microparticle composition is administered to a patient, and the patient is scanned with an ultrasound machine to obtain a visual image of the cardiovascular site. Alternatively, the microparticle composition may be administered in combination with a pharmaceutical or physical stressor. Suitable pharmacological stressors include vasodilators of the coronary arteries such as dipyridamole or adenosine, inotropic agents such as dobutamine (e.g. to increase cardiac contractility), or myocardial rate-shifting agents such as dobutamine (e.g. to increase the frequency of contractions). Suitable physical stressors include physical exercise, for example using a treadmill or stationary bicycle.

For radiological applications, the microparticle composition is administered to a patient, and the patient is scanned with an ultrasound machine to obtain a visual image of the patient at the site to be examined.

The microparticles can be used to assess the function of the cardiovascular system and to assess myocardial blood flow or myocardial blood volume or to diagnose coronary heart disease (coronary artery disease). For example, microparticles may enhance ventricular imaging, and thus may facilitate local cardiac function analysis through vessel wall motion analysis, and comprehensive cardiac function analysis through ejection fraction measurement. The microparticles may also be used to assess myocardial blood flow to distinguish functional heart tissue from ischemic (underflowing) heart tissue or infarcted (dead) heart tissue. The contrast signal detected in the myocardium can be used as an estimate of myocardial blood volume, since the ultrasound contrast agent resides intravenously. Over time, the absence or reduction of contrast intensity or image intensity at a particular myocardial site is indicative of a decrease (i.e., insufficiency) in blood flow.

Generally, unless the patient has severe coronary heart disease, blood flow in various parts of the heart should appear normal as assessed, for example, by ultrasound contrast. In order to detect blood flow abnormalities in patients without severe heart disease, or to detect small myocardial insufficiency, the need for blood flow to the heart must be increased by inducing a stress response. Stress may be induced by exercising the patient or administering a pharmaceutical compound such as a vasodilator, a inotropic agent or a myocardial rate-changing agent. Insufficient blood flow can be more easily detected during exercise or drug stress because the ability to increase blood flow is reduced at the site where blood is supplied by the coronary artery with stenosis. Contrast of the myocardial ultrasound image after administration of the ultrasound contrast agent can be made both before the pre-stressed state (i.e., resting state) and under stress. The myocardial sites that did not require brightness enhancement were found during stress imaging and were not found during quiet imaging, which is indicative of ischemia. Myocardial sites that do not require intensity enhancement are found during both stress imaging and during quiet imaging and are indicative of infarction.

In one embodiment, myocardial blood flow may be determined by: (1) administering the microparticle composition to a patient in a first injection, (2) imaging the patient with an ultrasound scanner to obtain a visual image of the cardiovascular site, (3) inducing stress in the patient with a drug stressor or exercise, (4) administering the microparticle composition in a second injection and continuing the scan, and (5) evaluating the difference in the images obtained in steps (2) and (4) by direct visual observation or by quantitative image analysis.

For radiological applications, microparticles may be used to improve the ability to ultrasonically image for radiological indications, including imaging of renal, hepatic, and peripheral vascular diseases, to increase visualization of blood flow and blood flow patterns, and to improve detection of subtle lesions or structures deep within the body. Microparticles can be used for indication of large (macrovascular) and small (microvascular) vessels. In the case of indications of large blood vessels (diagnosis of disease states and conditions of the body's aorta and veins), microparticles may help to detect stroke and pre-stroke conditions by visual inspection of intracranial vessels, and atherosclerosis in large vessels (e.g., carotid arteries) by assessing the extent of carotid stenosis, vascular graft patency, and peripheral vascular thrombosis. For microvascular indications (disease state and diagnosis by analysis of microvascular blood flow patterns), microparticles may help to identify lesions, tumors or other diseases in the liver (e.g., adenoma or hemangioma), and lesions, tumors or other diseases in the kidney, spleen (e.g., aneurysms of the splenic artery), as well as lesions, tumors or other diseases of the breast, ovary, other tissues and organs.

The diseased tissue of the patient may be diagnosed by administering the particulate composition to the patient and then ultrasonically scanning the patient for imaging to obtain a visual image of any diseased tissue on the patient. The diseased tissue may appear as a region of increased brightness or as a region that does not exhibit increased brightness.

Enhanced images obtained with microparticle compositions

The microparticles produce an enhanced image after administration. The enhanced image may be represented by an increase in brightness of the image as compared to when no ultrasound contrast agent is administered, or by a substantial elimination of an artificial image in the image. Thus, in connection with ultrasound imaging of cardiovascular sites including heart tissue and connected vascular tissue, enhanced images may be rendered by, for example, an increase in brightness of the cardiovascular site image and/or a substantial elimination of artificial images appearing in the cardiovascular site image. The images last from 10 seconds to 60 minutes after the administration of a single agent. The image preferably lasts for 20 seconds to 30 minutes, and most preferably for 30 seconds to 20 minutes. In a preferred embodiment, the ultrasound imaging enhancement lasts greater than 5 minutes in the ventricle and/or greater than 1 minute in the myocardium.

The increase in image brightness can be assessed by naked eye observation or using quantitative imaging analysis. As identified above with reference to a particular gray scale (from about 0 to about 255VDUs or color scale), it is preferred that the level of brightness increase be at least about 10VDUs (gray scale). More preferably, the level of brightness increase is greater than about 10VDUs, for example, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 VDUs. In some embodiments, the increase in brightness is greater than about 100VDUs, for example, about 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 VDUs. In other embodiments, the brightness increase is greater than about 150VDUs, for example, about 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 VDUs. Alternatively, the brightness increase is greater than about 200VDUs, for example, about 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, or 255 VDUs.

The above methods and compositions may be further understood by reference to the following non-limiting examples.

Examples

Material

Acetic acid, ammonium bicarbonate, mannitol USP, and polysorbate 80 (without animal derived ingredients) were purchased from SpectrumChemicals, Gardena, CA. Polymers (poly (lactide-co-glycolide) (PLGA) (50: 50)) and di-arachidoylphosphatidylcholine (1, 2-di-arachidoyl-sn-glycero-3-phosphocholine (DAPC)) were purchased from Boringer Invitrogen (Ingelheim, Germany) and Avanti (Albabaster, AL), respectively. Methylene chloride was purchased from EM Science (EMDCchemicals, Gibbstown, NJ). Vials (30 ml tubular vials) and stoppers (20 mm, grey, single vent, Fluro-Tec corporation) were from West Pharmaceutical Services (Lionville, PA). Perfluoro-n-butane (DFB) gas was purchased from F2 Chemicals, Inc. (Lancashire, UK).

Analytical method

Quantitative analysis of particulate mass concentration

The mass concentration of the microparticles in the vial was quantified by ICP-MS (inductively coupled plasma mass spectrometry). The amount of polymer in the microparticles was determined by analyzing tin by ICP-MS. The amount of polymer present in the microparticles is determined based on the amount of tin present in the microparticles compared to the amount of tin present in the particular portion of polymer used to make the microparticles. The amount of phospholipids in the microparticles was determined by analysis of phosphorus by ICP-MS. The amount of phosphorus present in the microparticles is determined by comparing the amount of phosphorus present in the microparticles with the amount of phosphorus present in the phospholipid itself. The mass of particles per ml of suspension is calculated as: the amount of polymer and phospholipid in each vial was added and then divided by the reconstituted volume (5 mL).

Particle size analysis

The reconstituted microparticle sample was added to the electrolyte solution and the resulting suspension was analyzed for particle size and microparticle concentration using a Coulter Multisizer II equipped with a 50 μm port tube.

Gas content of the particles

The dry powder vial was reconstituted with 5mL of water and shaken to produce a suspension of microparticles. A set of 0.3 ml aliquots were removed through the stoppers using a needle and syringe and the resulting suspensions were analyzed for DFB content. These aliquots were filled into sealed-mouth headspace vials. The headspace vials were equilibrated at room temperature for at least 10 hours. The sample was then heated to 45 ℃ and held in a headspace sampler incubator for 20 minutes. The headspace above the suspension was analyzed by gas chromatography using a gas-blown wrapped inlet and a flame ion detector. Quantification was performed using an area single point calibration method.

The gas chromatography system parameters and temperature program are listed in tables 1 and 2.

Table 1: GC System parameters

Sample preparation:	headspace, 1mL sample introduction ring
		Detector	FID
Chromatographic column	Supelco 60/80Carbopack B 5％Fluorocol
		Temperature at sample inlet	150℃

Temperature of detector	325℃
		Carrier gas	Helium (25mL/min)
FID gas	Hydrogen (60mL/min)
			Air (350mL/min)
	Nitrogen (5mL/min)

Table 2: GC temperature program

	Initial temperature	Rate of speed	Final temperature	Retention time
					Initial conditions	40℃	N/A	N/A	2.0min
First gradient	40℃	5℃/min	65℃	0.0min
					Second oneGradient of gradient	65℃	10℃/min	130℃	0.0min
Third gradient	130℃	50℃/min	200℃	0.0min
					Final conditions	200℃	N/A	N/A	3.1min

Example 1: microparticle fabrication as ultrasound contrast agents

An organic solution was prepared by dissolving 176g of PLGA, 10.6g of diacylphosphatidylcholine (1, 2-diacyl-sn-glycero-3-phosphocholine (DAPC)), and 2.26g of acetic acid in 5.88L of dichloromethane at 25 ℃. An aqueous solution of 68.5g of ammonium bicarbonate dissolved in 338ml of water for injection was added to the above organic solution, and homogenized for 10 minutes in a 10L homogenizing tank at 4310RPM using a rotor-stator emulsion mixer.

The resulting emulsion was spray dried using nitrogen as the atomizing gas and the drying gas. The emulsion was spray dried on the top of the bench top using an air spray nozzle from Spraying Systems, Inc. (Wheaton, Ill.) and a glass drying chamber/cyclone from Buchi, Brinkmann, Westbury, NY). The spray drying conditions were as follows: the emulsion flow rate was 40ml/min, the atomizing gas flow rate was 30L/min, the drying gas flow rate was 46kg/hr, and the exit temperature was 12 ℃.

The spray dried product is further processed by dispersion, freezing, and lyophilization steps. An aqueous medium was prepared by dissolving 140g of mannitol and 4.10g of polysorbate 80 in 5.0L of water. The spray-dried microparticles were dispersed in the above medium at a concentration of 25 mg/ml. The dispersion was deagglomerated and sieved through a 10 "diameter vibrating screen (RBF-10) (available from Vorti-Siv (Salem, OH)) using a stainless steel, grade 800, flow cell sonicator from Misonix (Farmingdale, new york). The sonicator was protected with a 4 ℃ jacket to prevent the dispersion from heating. The dispersion was sieved through a 25 μm and 20 μm sieve in this order at a rate of 150 mL/min. The sieved dispersion was filled into vials (10ml dispersion in 30ml vials), partially stoppered and frozen by immersion in liquid nitrogen.

After freezing, the vials were lyophilized. After lyophilization was complete, the container was isolated and n-perfluorobutane (DFB) was reinflated into the vial to bring the pressure to 5 kPa prior to stoppering.

The dry powder was reconstituted with 5ml of sterile water before use, and water was added to a vial of the dry powder followed by shaking to give a suspension of microparticles suspended in isotonic mannitol. The suspension contained 2.2X 10⁹microparticle/mL, 37mg microparticle/mL, average particle size of microparticle was 2.2 microns.

Example 2: gas leakage rate of particles

The gas leak rates of each of the two batches of microparticles (batch 1 and batch 2) produced according to the method of example 1 were evaluated by Gas Chromatography (GC), as detailed in the analytical methods section above. A third batch of microspheres (batch 3) was produced in a similar manner to example 1, except that the phospholipids, the di-arachidoylphosphate choline (1, 2-di-arachidoyl-sn-glycero-3-phosphocholine (DAPC)) were omitted during the microparticle production.

Table 3: gas content of particles and gas leakage rate

Particles containing DAPC lost about 10% of the initial gas content after 70 minutes, while particles without DAPC lost 87% of the initial gas content. In addition, particles containing DAPC have a higher initial gas content relative to particles without DAPC. This indicates that the inclusion of DAPC is important for the formation of a porous structure inside the particles and for maintaining the gas inside the particles during the spray drying process.

The total duration of ultrasound contrast agent that is desirably used after administration to a patient will vary depending on the type of cardiology or radiology ultrasound examination being used, and is typically about 30 seconds to 60 minutes. Therefore, if the period of the ultrasonic examination is exceeded, the determination of the loss of particulate gas containing the lipid DAPC is of no significance.

Example 3: cardiac image enhancement as a function of microparticle dose

The microparticles produced according to the method described in example 1 were studied in healthy adults. The dry powder was reconstituted before use, 5mL of sterile water was added to the vial and the vial was shaken 10 times. The final concentration of microspheres in the resulting suspension was approximately 37 mg/mL. The subject received a single dose of 0.5mg/kg, 2.0mg/kg or 4.0mg/kg body weight. Subjects were subjected to transthoracic ultrasound imaging with continuous harmonic imaging (frame frequency 15Hz, transducer frequency 2.1/4.2 MHz). The images were visually evaluated for the intensity and duration of enhancement.

At both 2mg/kg and 4mg/kg, the duration of ventricular potentiation exceeded 9 minutes. At both doses, when the subject was imaged again after 30 minutes, a contrast effect was still visible in 13 of 15, indicating that the microparticles provided a prolonged enhancement.

The duration of ventricular potentiation is summarized in table 4.

Table 4: duration of left ventricular image enhancement

Dosage (mg/kg body weight)	Average duration of ventricular potentiation (min)
		0.5	2.6
2.0	＞9.6
		4.0	＞9.6

Example 4: comparison of microparticles and commercial products in evaluating cardiac images

A comparative study of cardiac sonography was performed on two adults of matched body weight and cardiac function. A first subject is administered a single formulation of microparticles produced by the method of example 1. The dry powder was reconstituted before use by adding 5ml of sterile water to the vial and then shaking the vial 10 times. The final concentration of microspheres in the resulting suspension was approximately 37mg/mL and the gas content of the suspension was approximately 250 g/mL. The first subject received a dose of 4mg microparticles/kg, which corresponds to 27g/kgas dose in g body weight. The second subject received a single dose of a commercially available ultrasound contrast agent(Amersham health) which contains perfluoropropane comprising protein endosperm microspheres. Both subjects received the same total amount of gas (27 μ g/kg body weight), which is the acoustically active component. Two subjects were subjected to transthoracic ultrasound imaging with continuous harmonic imaging (frame frequency 15Hz, transducer frequency 2.1/4.2 MHz). The images were visually evaluated for the intensity and duration of enhancement.

The duration of the ventricular and myocardial potentiation is summarized in table 5.

Table 5: duration of different ultrasound contrast agent image enhancements

The microparticles produced by the method described in example 1 provided enhanced images in both the ventricle and myocardium that were significantly longer in duration thanWith appropriate duration for performing a complete cardiac test with ultrasound.

Example 5: assessment of myocardial blood flow with microparticle formulations to assess ischemia

The microparticles produced by the method of example 1 are administered to a subject diagnosed with coronary heart disease. Subjects received two injections of microparticles within 60 minutes. The first microparticle injection ("quiet injection", 1.7mg/kg) was used to evaluate the myocardium in a quiet state. Prior to the second microparticle injection, the subject was pharmacologically stressed with the vasodilator dipyridamole (0.56 mg/kg). After induction of the stress, subjects received a second microparticle injection ("stress injection", 1.3mg/kg) to evaluate the myocardium under stress.

Comparison of the resting and stressed state images of the subject following microparticle administration showed the myocardial site with the least increase in image enhancement, and this site had a greater area following stress induction. This indicates that a region of myocardial tissue has both infarcted and ischemic portions. Detection of ischemia is confirmed by nuclear imaging, another diagnostic technique. Quiet and stress responsive nuclear perfusion (null perfusion) was performed after administration of 99tc (mibi), and subjects were imaged with a commercially available gamma particle counting tube. Lesions recorded on the quiet and stress-responsive ultrasound images were identified in the quiet and stress-responsive nuclear perfusion images.

Claims (33)

1. A kit comprising a dosage formulation and a solution for reconstitution of the dosage formulation,

wherein the dosage formulation comprises microspheres comprising a polyolic acid copolymer, and a hydrophobic compound, and the microspheres encapsulate a perfluorocarbon gas that is gaseous at body temperature,

wherein the poly (alkyd acid) copolymer is poly (lactide-co-glycolide), the hydrophobic compound is a dianhydroacyl phosphatidylcholine, and the perfluorocarbon gas is perfluoro-n-butane, and

wherein the microspheres are porous spheres having a honeycomb-like structure or a sponge-like structure, and

wherein the microspheres are dry powders, and

wherein the microspheres have an average particle size of 1.8 to 3.0 microns and

the microspheres are reconstituted with a solution to form an isotonic suspension having a microsphere concentration of 1.0X 10⁹To 3.5X 10⁹microspheres/mL suspension or a suspension having a microsphere mass concentration of 25-50mg microspheres/mL,

wherein the amount of perfluoron-butane gas is 75 to 500 μ g/mL of suspension, and

wherein the microspheres are dosed in a dosage formulation in the range of 0.5 to 4.0mg microspheres/kg body weight, thereby being adapted to provide enhanced ultrasound imaging in the myocardium for more than 1 minute compared to when no contrast agent is administered.

2. The kit of claim 1, wherein the concentration of microspheres in said suspension is in the range of 1.5 x 10⁹To 2.8X 10⁹A suspension of microspheres/mL or a suspension of microspheres having a mass concentration of 30 to 45mg microspheres/mL.

3. The kit of claim 1, wherein the microspheres have an average particle size of 1.9 to 2.6 microns.

4. The kit of claim 1, wherein said dose is selected from 0.5mg microspheres/kg body weight, 2.0mg microspheres/kg body weight, and 4.0mg microspheres/kg body weight.

5. The kit of claim 1, wherein perfluoron-butane is provided in a microsphere suspension administered in an amount of 100 to 400 μ g/mL.

6. The kit of claim 5, wherein perfluoron-butane is provided in a microsphere suspension administered in an amount of 150 to 350 μ g/mL.

7. The kit of claim 1, wherein the hydrophobic compound is mixed with the poly (alkyd) copolymer at a ratio of 0.01 to 30% (weight hydrophobic compound/weight poly (alkyd) copolymer).

8. The kit of claim 1, wherein the poly (lactide-co-glycolide) has a lactide to glycolide ratio of 1:1 and a weight average molecular weight of from 20 to 40kDa, and wherein the dioarachidylphosphatidylcholine is mixed with the poly (ol acid copolymer) at a ratio of from 5 to 6.6% (weight of dioarachidylphosphatidylcholine/weight of poly (ol acid copolymer).

9. The kit of claim 1, wherein the suspension further comprises one or more excipients selected from the group consisting of sugars, salts, and surfactants.

10. A method of making a composition for ultrasound contrast imaging comprising:

suspending microspheres comprising a polyolic acid copolymer and a hydrophobic compound in a solution;

placing the suspension into a vial or syringe;

freezing the suspension;

lyophilizing the vial to produce a dry powder in a vial or syringe; and

backfilling the freeze-dryer with a perfluorocarbon gas that is gaseous at a gaseous temperature to obtain a composition,

wherein the composition comprises microspheres and the microspheres encapsulate a perfluorocarbon gas, and

wherein the microspheres are porous spheres having a honeycomb-like structure or a sponge-like structure, and

wherein the microspheres are dry powders, and

wherein the microspheres have an average particle size of 1.8 to 3.0 microns,

wherein the poly (alkyd acid) copolymer is poly (lactide-co-glycolide), the hydrophobic compound is a dianhydroacyl phosphatidylcholine, and the perfluorocarbon gas is perfluoro-n-butane, and

when the microspheres are reconstituted with a pharmaceutically acceptable carrier prior to use, they may form a second suspension which is an isotonic suspension, wherein the concentration of microspheres is 1.0X 10⁹To 3.5X 10⁹microspheres/mL of the second suspension or microspheres having a mass concentration of 25 to 50mg microspheres/mL of the second suspension,

wherein the amount of perfluoron-butane gas is 75 to 500 μ g/mL of suspension, and

wherein the microspheres are dosed in a dosage formulation in the range of 0.5 to 4.0mg microspheres/kg body weight, thereby being adapted to provide enhanced ultrasound imaging in the myocardium for more than 1 minute compared to when no contrast agent is administered.

11. The method of claim 10, wherein said suspension further comprises an excipient.

12. A method of making a composition for ultrasound contrast imaging comprising:

dry mixing microspheres in a solution containing a polyolic acid copolymer and a hydrophobic compound;

placing the mixture into a vial or syringe;

after evacuation, the vial or syringe is filled with a perfluorocarbon gas which is gaseous at a gaseous temperature to obtain a composition,

wherein the composition comprises microspheres, the microspheres encapsulating a perfluorocarbon gas, and

wherein the microspheres are porous spheres having a honeycomb-like structure or a sponge-like structure, and

wherein the microspheres are dry powders, and

wherein the microspheres have an average particle size of 1.8 to 3.0 microns,

wherein the poly (alkyd acid) copolymer is poly (lactide-co-glycolide), the hydrophobic compound is a dianhydroacyl phosphatidylcholine, and the perfluorocarbon gas is perfluoro-n-butane, and

when the microspheres are reconstituted with a pharmaceutically acceptable carrier prior to use, they may be formed into an isotonic suspension having a microsphere concentration of 1.0X 10⁹To 3.5X 10⁹microspheres/mL suspension or a suspension having a microsphere mass concentration of 25-50mg microspheres/mL,

wherein the amount of perfluoron-butane gas is 75 to 500 μ g/mL of suspension, and

wherein the microspheres are dosed in a dosage formulation in the range of 0.5 to 4.0mg microspheres/kg body weight, thereby being adapted to provide enhanced ultrasound imaging in the myocardium for more than 1 minute compared to when no contrast agent is administered.

13. The method of claim 12, wherein the mixture further comprises an excipient.

14. A dosage formulation in the form of a dry powder for use as an injectable contrast enhanced ultrasound imaging agent when reconstituted with a pharmaceutically acceptable carrier, said dosage formulation comprising

(a) A container for placing the dry powder is arranged in the container,

(b) the dry powder comprises dry powder particles in an amount selected from the group consisting of 150-250mg and 250-1000mg, the dry powder particles comprising a polyalcohol acid copolymer and a hydrophobic compound, wherein the particles are porous spheres having a honeycomb structure or a sponge structure, and the average particle size of the particles is 1.8-3.0 μm, and

(c) n-perfluorobutane (C)₄F₁₀) The gas is a mixture of a gas and a water,

the poly (alkyd) copolymer is poly (lactide-co-glycolide), the hydrophobic compound is a di-arachidoylphosphatidylcholine,

wherein the dry powder in the container is reconstituted by adding a prescribed amount of a pharmaceutically acceptable carrier suitable for injection to form a particulate concentration of 1.5X 10⁹-2.8×10⁹MicroparticlesA suspension of/mL or particles having a mass concentration of 25-50mg of particles/mL,

wherein the amount of perfluoron-butane gas is 75 to 500 μ g/mL of suspension, and

wherein the microparticle suspension provides enhanced ultrasound imaging of the myocardium for greater than 1 minute.

15. The dosage formulation of claim 14, which provides enhanced ultrasound imaging of the ventricle for at least 30 minutes.

16. The dosage formulation of claim 14, comprising sterile water as a pharmaceutically acceptable carrier suitable for injection.

17. The dosage formulation of claim 16, which forms a suspension having a microparticle mass concentration of 30-45mg microparticles/mL.

18. The dosage formulation of claim 14, wherein the microparticles have an average particle size of 1.9-2.6 microns.

19. The dosage formulation of claim 14, comprising a dose of 0.5-4.0mg microparticles/kg body weight.

20. The dosage formulation of claim 19, wherein said dose is selected from the group consisting of 0.5mg microparticles/kg body weight, 2.0mg microparticles/kg body weight, and 4.0mg microparticles/kg body weight.

21. The dosage formulation of claim 14, wherein n-perfluorobutane (C)₄F₁₀) Is provided in an amount of 100-.

22. The dosage formulation of claim 21, wherein perfluoro-n-butane (C)₄F₁₀) Reconstituted microparticle suspension at 150-The dosage of the liquid is provided.

23. The dosage formulation of claim 14, wherein the hydrophobic compound is present in a ratio of 1-12% (weight hydrophobic compound/weight polymer) relative to the polyglycolic acid or copolymer thereof or mixture thereof.

24. The dosage formulation of claim 14, wherein said poly (lactide-co-glycolide) has a lactide-to-glycolide ratio of 1:1 and a polymer weight average molecular weight in the range of 20,000-40,000 daltons, and said proportion of di-arachidoylphosphatidylcholine is 5-6.6% (weight of di-arachidoylphosphatidylcholine/weight of polymer).

25. The dosage formulation of claim 14, wherein said container is a vial or a syringe.

26. The dosage formulation of claim 25, wherein said vial or syringe further comprises one or more excipients selected from the group consisting of sugars, salts, and surfactants.

27. The dosage formulation of claim 14, consisting essentially of 1 or 2 doses.

28. The dosage formulation of claim 14, consisting essentially of up to 5 doses.

29. An enhanced ultrasound contrast imaging kit comprising

(i) Dry powder dosage formulations comprising

(a) The container is a container, and the container is a container,

(b) a dry powder particle in an amount selected from the group consisting of 150-250mg and 250-1000mg, the dry powder particle comprising a poly (alkyd) copolymer, which is a poly (lactide-co-glycolide), and a hydrophobic compound, which is a diacylphosphatidylcholine,

wherein the fine particles are porous spheres having a honeycomb structure or a sponge structure, and the average particle size of the fine particles is 1.8 to 3.0 μm, and

(c) n-perfluorobutane (C)₄F₁₀) A gas, and

(ii) pharmaceutically acceptable carriers suitable for injection, for reconstitution of dosage formulations,

wherein the dry powder dosage formulation in the container is reconstituted with a pharmaceutically acceptable carrier to form a microparticle concentration of 1.5 x 10⁹-2.8×10⁹microparticle/mL or a microparticle suspension having a microparticle mass concentration of 25-50mg microparticle/mL,

wherein the amount of the perfluoro-n-butane gas is 75-500 mu g/mL of the suspension,

wherein the microparticles provide enhanced ultrasound imaging of the myocardium for greater than 1 minute when the microparticles are administered intravenously.

30. The kit of claim 29, wherein the container is a vial or syringe and the kit further comprises a second vial or syringe having a defined amount of a pharmaceutically acceptable carrier for suspending microparticles.

31. The kit of claim 29, wherein the pharmaceutically acceptable carrier is selected from the group consisting of: water for injection, sterile water, saline containing glycerin, and TweenSaline containing TweenSaline, isotonic glucose (5%), 1/2 isotonic dextrose (2.5%), isotonic mannitol (5%), 1/2 isotonic mannitol (2.5%), containing tweenIsotonic mannitol, containing TweenIsotonic mannitol.

32. The kit of claim 31, wherein the pharmaceutically acceptable carrier is water for injection.

33. The dosage formulation of claim 14, wherein the concentration of the suspension of microparticles reconstituted with the carrier is suitable for intravenous administration as a bolus injection.