JP4808214B2 - Dosage formulation for ultrasound contrast agent - Google Patents

Description

(Background of the Invention)
The present invention is in the general field of diagnostic imaging agents and, in particular, relates to specific ultrasound contrast agent dosage formulations that provide enhanced and long duration images.

  When using ultrasound to obtain images of human or animal organs and internal structures, ultrasound waves (acoustic energy waves with frequencies above those recognizable by the human ear) Reflected when passing through. Different types of body tissue reflect ultrasound waves differently, and reflections caused by ultrasound waves reflected by different internal structures are detected and electronically converted into a visual display.

  For some medical conditions, obtaining a useful image of the organ or structure of interest is particularly difficult. This is because the details of this structure cannot be properly distinguished from the surrounding tissue in the ultrasound image produced by the reflection of ultrasound waves without the contrast enhancing agent. Detection and observation of certain physiological and pathological conditions can be significantly improved by administering an ultrasound contrast agent to the organ or other structure of interest to enhance the contrast of the ultrasound image. In other cases, detection of the motion of the ultrasound contrast agent itself is particularly important. For example, a well-defined blood flow pattern known to arise from a particular cardiovascular abnormality is the administration of an ultrasound contrast agent into the blood stream and the blood flow or volume Can be identified only by observing any of the following.

  Materials useful as ultrasound contrast agents have an effect on ultrasound waves when the ultrasound waves pass through the body and are reflected to produce an image that is medically diagnosed. Function by. Different types of materials have different degrees of influence on the ultrasonic wave in different ways. Furthermore, certain effects produced by contrast enhancing agents are more easily measured and observed than other effects. In selecting an ideal composition for an ultrasound contrast agent, a material that has the most dramatic effect on the ultrasound wave as it passes through the body is preferred. Also, the effect on the ultrasonic wave should be easily measured. Gas is a preferred medium for use as an ultrasound contrast agent. This gas needs to be stabilized prior to use, either as bubbles stabilized by a surfactant or encapsulated in liposomes or microparticles. There are three main contrast enhancement effects that can be seen in ultrasound images: backscatter, beam attenuation, and acoustic velocity difference.

  A variety of natural and synthetic polymers have been used to encapsulate ultrasound contrast agents such as air in order to create longer lasting ultrasound contrast agents after administration. Non-Patent Document 1 describes 3 micron synthetic polymer particles filled with air. These particles were reported to be stable in plasma and under applied pressure. However, at 2.5 MHz, their echo brightness was low. Another type of microbubble suspension was obtained from sonicated albumin. Non-Patent Document 2. Feinstein describes the preparation of microbubbles that have excellent stability in vitro and are sized for passage through the lungs. However, these microbubbles are short-lived in vivo and have a half-life of approximately a few seconds (approximately equal to one cycle of circulation) due to their instability under pressure. Non-Patent Document 3; and Non-Patent Document 4.

  Microbubbles encapsulated in gelatin have also been described in US Pat. No. 5,637,097 by Rasor Associates, Inc. These are formed by “coalsing” the gelatin. Gaseous microbubbles enclosed in a shell of fluorine-containing material are described in US Pat.

  Microbubbles (SHU 454 and SHU 508) stabilized by galactose microcrystals were also reported by Fritzch et al. Non-Patent Document 5; and Non-Patent Document 6. These microbubbles last up to 15 minutes in vitro but last less than 20 seconds in vitro. Non-Patent Document 7; and Non-Patent Document 8. U.S. Pat. No. 6,057,037 to Schering Aktiengesellschaft discloses the preparation and use of microencapsulated gases or microencapsulated volatile liquids for ultrasound imaging methods, these microcapsules being made from synthetic polymers or polysaccharides. It is formed. Sintetica, US Pat. No. 6,057,028 discloses an air microballoon or a gas microballoon surrounded by a polymer film deposited at the interface for therapeutic or diagnostic purposes, which polymer is injected into a host animal. Or can be dispersed in an aqueous carrier for oral, rectal, or urethral administration.

  U.S. Patent No. 6,057,049 by Delta Biotechnology Limited describes the preparation of microparticles for use in imaging by spray drying an aqueous protein solution to form hollow spheres that trap gas in the spheres. U.S. Patent No. 6,057,031 describes the synthesis of microparticles for ultrasound imaging composed of a gas contained in a polycyanoacrylate or polyester shell. U.S. Patent No. 6,057,031 discloses the preparation of a particulate contrast agent containing a covalently bonded matrix containing a gas, where the matrix is a carbohydrate. U.S. Pat. Nos. 5,099,637 and 5,037 to Unger describe liposomes for use as ultrasound contrast agents, which are activated by gases, gaseous precursor materials (e.g., pH, Or a gaseous precursor that is activated by light), and other contrast enhancing agents that are liquids or solids.

  Others have examined the effects of encapsulated gas and have suggested the use of fluorinated gases to enhance imaging compared to air. U.S. Patent No. 6,053,836 to Quay discloses the use of agents containing perfluorocarbons to enhance contrast in ultrasound images. These drugs are composed of small bubbles or microbubbles of selected gases that are long-lived in solution and small enough to cross the lungs, and these drugs are necessary for cardiovascular and life support Enabling their use in ultrasound imaging of other organs. U.S. Patent No. 6,057,017 to Bracco discloses the use of fluorinated hydrocarbon gas to prevent microvesicles from collapsing upon exposure to pressure in the bloodstream. Nycomed, US Pat. No. 6,057,031, discloses microcapsules for imaging containing perfluorocarbons formed by coacervation of solutions (eg, protein solutions). U.S. Patent No. 6,057,049 by Massachusetts Institute of Technology discloses microparticles formed from a polyethylene glycol-poly (lactide-co-glycolide) block polymer in which an imaging agent is encapsulated, wherein these microparticles are gaseous (e.g., Air and perfluorocarbon). Sonus Pharmaceuticals, Inc. While solids and liquids reflect sound waves to a similar extent, gas is known to be more efficient and is preferred for use as an ultrasound contrast agent, as described in US Pat. It is a medium. In fact, as shown in Example 12 of Patent Document 15, protein microcapsules are rejected as causing safety problems (and efficacy problems) when administered to mini-pigs. It was done. Both U.S. Pat. Nos. 6,057,086 and 5,047, describe methods for enhancing contrast using perfluorocarbon gas in a synthetic polymer shell. U.S. Patent No. 6,057,034 describes a method of making porous polymer microparticles, which have a hydrophobic factor incorporated into the polymer to increase echo brightness.

Several ultrasound contrast agents have been approved for very limited cardiac applications in either the US or Europe. OPTSON® (Amersham, Mallinkrodt) is composed of microcapsules of heat-denatured human albumin containing gaseous octafluoropropane. Each 1 mL microsphere suspension contains 5 × 10 ⁸ to 8 × 10 ⁸ microspheres with an average diameter in the 2 micron to 4.5 micron size range and 220 μg octafluoropropane. These microspheres were not approved for myocardial blood flow assessment and were only approved for ventricular augmentation. At high bolus doses (5 mL suspension or 1100 μg octafluoropropane) ventricular enhancement lasts up to 5 minutes.

DEFINITY® (Bristol Myers Medical Imaging) consists of lipid microspheres containing octafluoropropane, the lipid shell being composed of phospholipids DPPA, DPPC, and mPEG-DPPE. Each 1 mL suspension contains 1.2 × 10 ¹⁰ microparticles with an average diameter in the 1.1 micron to 3.3 micron size range and 1100 μg octafluoropropane. This drug is only approved for ventricular augmentation and not for myocardial blood flow assessment. At a bolus dose of 700 μL (for a 70 kg person) or 5133 μg of gas, the drug has a duration of augmentation in the ventricle of about 3.4 minutes.

IMAGEENT® (Photogen Inc.) consists of lipid microspheres containing perfluorohexane, and the lipid shell is composed of phospholipids (DMPC). Each 1 mL suspension contains 1.4 × 10 ⁹ microparticles with an average diameter of less than 3 microns and 92 μg perfluorohexane. This drug is only approved for ventricular augmentation and not for myocardial blood flow assessment. At a bolus dose of 0.43 mL (for a 70 kg person) or 40 μg of gas, the drug has an average duration of enhancement in the ventricle of about 2.6 minutes.

In all cases, these over-the-counter drugs have limited utility, are not approved for applications other than ventricular augmentation, and the average duration of image enhancement in the ventricle lasting for a period of 5 minutes or less I will provide a. There are no commercially available ultrasound contrast agents that allow enhanced images of the cardiovascular system (particularly the myocardium and ventricles) over a long duration. When administered as a bolus or as a short infusion, the drugs described in the prior art produce an image of the myocardium that lasts much shorter than the amount of time required to perform a complete examination of the heart. Typically, prior art agents provide images that last much less than a minute for the myocardium. Agents that can provide enhanced image duration in the myocardium for more than 1 minute and / or longer in the ventricle for more than 5 minutes are desired.
International Publication No. 80/02365 Pamphlet International Publication No. 96/04018 Pamphlet European Patent No. 398935 European Patent No. 458745 International Publication No. 92/18164 Pamphlet International Publication No. 93/25242 Pamphlet International Publication No. 92/21382 Pamphlet US Pat. No. 5,334,381 US Pat. No. 5,123,414 US Pat. No. 5,352,435 US Pat. No. 5,393,524 EP 554213 International Publication No. 95/23615 Pamphlet International Publication No. 95/03357 Pamphlet WO94 / 16739 pamphlet US Pat. No. 6,132,699 US Pat. No. 5,611,344 US Pat. No. 5,837,221 Schneider et al., “Invest. Radiol.”, 1992, 27, p. 134-139 Feinstein et al., “J. Am. Coll. Cardiol.”, 1988, Volume 11, p. 59-65 Gottlieb, S.M. Et al., “J. Am. Soc. Echo.”, 1990, Volume 3, p. 328, summary Shapiro, J. et al. R. Et al., “J. Am. Coll. Cardiol.”, 1990, Vol. 16, p. 1603-1607 Fritzsch, T.W. Et al., “Invest. Radiol.”, 1988, Vol. 23, First Addendum, p. 302-305 Fritzsch, T.W. “Invest. Radiol.”, 1990, Vol. 25, 1st Addendum, 160-161. Rovai, D.M. Et al., “J. Am. Coll. Cardiol.”, 1987, Volume 10, p. 125-134 Smith, M.M. Et al., “J. Am. Coll. Cardiol.”, 1989, Vol. 13, p. 1622-2628

  Accordingly, it is an object of the present invention to provide a dosage formulation comprising microparticles that provides enhanced and long duration images, particularly for cardiac applications.

  Another object of the present invention is to provide a kit for administering a dosage formulation containing microparticles for use in ultrasound imaging techniques.

(Summary of the Invention)
Clinical studies have been conducted and specific dosage formulations have been developed that provide a significantly enhanced long duration image using polymer microparticles. The polymer particulate has a perfluorocarbon gas incorporated therein. This dosage formulation typically contains 1 dose, 2 doses, or up to 5 doses (most preferably 1 dose or 2 doses) of microparticles formed from a biocompatible polymer, and these The microparticles preferably contain lipids incorporated into the polymer and include perfluorocarbons that are gaseous at body temperature. These microparticles are doses effective to enhance ultrasound imaging for longer than 5 minutes in the ventricle and / or longer than 1 minute in the myocardium, and range from 0.025 mg to 8.0 mg per kg body weight. Are administered to the patient at a dose of microparticles. Preferably, the dose administered to a patient is microparticles ranging from 0.05 mg to 4.0 mg per kg body weight. In a preferred embodiment, the ultrasound imaging method is enhanced for more than 9 minutes in the ventricle and / or is enhanced for more than 2 minutes in the myocardium.

Typically, the dosage formulation is provided in a vial or syringe. In a typical formulation, the dosage formulation is in the form of a dry powder that is reconstituted with sterile water prior to use, the dosage formulation comprising an isotonic suspension or the like of the microparticles. To obtain a tonic suspension, sterile water is added to the dry powder vial or syringe and reconstituted by shaking. In a preferred embodiment of this dosage formulation, the suspension is from 1.0 × 10 ⁹ to 3.5 × 10 ⁹ microparticles per mL of suspension, or 25 mg to 50 mg per mL of suspension. Of microparticles, most preferably 1.5 × 10 ⁹ to 2.8 × 10 ⁹ microparticles per mL of suspension, or a suspension containing 30 mg to 45 mg microparticles per mL of suspension Has a concentration that produces In preferred embodiments, these microparticles have an average particle size of less than 8 microns, and most preferably have an average particle size of 1.8 microns to 3.0 microns.

In the 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 ) provided in an amount between 75 μg and 500 μg per mL of dosage volume of the microparticle suspension; Perfluorobutane is provided in an amount between 100 μg and 400 μg per mL of dosage volume of the microparticle suspension, and most preferably in an amount between 150 μg and 350 μg per mL of dosage volume of the microparticle suspension. Or the gas is present in an amount between 75 μg and 375 μg per mL of dosage volume of the microparticle suspension (most preferably 120 μg and 300 μg per mL of dosage volume of the microparticle suspension. N-octafluoropropane provided in an amount between

  In most preferred embodiments, the microparticles are synthetic polymers (eg, poly (lactic acid), poly (glycolic acid), and poly (lactic acid-co-glycolic acid), polyglycolide, polylactide, and poly (lactide-co-glycolide). Of poly (hydroxy acids), polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly (ethylene glycol), poly (ethylene oxide) polyvinyl alcohol Such as polyalkylene oxides, poly (valeric acid), and poly (lactide-co-caprolactone), and their derivatives, copolymers and blends) and a proportion between 0.01% and 30% (fat (Weight of polymer) / hydrophobic compound incorporated into the polymer (most preferably, lipid incorporated into the polymer at a ratio between 0.01% and 30% (weight of lipid / weight of polymer)) including. In particularly preferred embodiments, the lipid comprises dioleoylphosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipentadecanoyl phosphatidylcholine (DPDPC), dilauroyl phosphatidylcholine (DLPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl. Phosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignocelloylfatidylcholine (dilignphosine) It is Jill ethanol amine.

  Most preferably, the synthetic polymer in the microparticles is a poly (lactide-co-) having a 50:50 (ie, 1: 1) lactide to glycolide ratio and a weight average molecular weight in the range of 20,000 Da to 4,000 Da. Glycolide) and the hydrophobic compound in the microparticle is DAPC present in a proportion between 5% and 6.6% (DAPC weight / polymer weight).

  The dosage formulation may be provided as a dry powder vial or syringe containing the microparticles or in a kit with a solution for resuspending the microparticles. Typically, dry powder vials or syringes also include excipients (eg, sugars or salts) to make the solution isotonic or isotonic after reconstitution. This dosage formulation is then administered to the patient to be imaged by injection, either as a bolus or an injection over a period of up to 30 minutes.

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

(Detailed description of the invention)
Improved methods, microparticles, kits, and dosage formulations for ultrasound imaging methods are described herein. The microparticles can be used in various diagnostic ultrasound imaging applications (especially ultrasound procedures such as blood vessel imaging and myocardial blood flow assessment, myocardial blood volume assessment and ventricular enhancement). Useful in inspection)).

(I. Definition)
In general, as used herein, the term “microparticle” includes “microspheres” and “microcapsules”, as well as other microparticles, unless otherwise specified. The fine particles may be spherical or non-spherical. A “microcapsule” is defined herein as a microparticle having an outer polymer shell surrounding a gas core. As defined herein, “microspheres” are porous spheres having a honeycomb or sponge-like structure formed by solid polymer spheres or pores throughout the polymer filled with gas. It can be. Some microspheres may include an outer polymer shell having a honeycomb or sponge-like structure formed by pores throughout the polymer shell, and these pores are filled with a gas. For this type of microsphere, this outer polymer shell surrounds the gas inner core.

  In general, as used herein, the terms “dosage” and “dose” refer to the amount of a substance given at one time or the amount of substance required to produce the desired diagnostic or contrast effect. It is used as a synonym for that purpose.

  As used herein, the term “dosage formulation” refers to a vial or other container that contains one or more dosages of a substance required to produce the desired diagnostic or contrast effect. (For example, syringe).

  In general, as used herein, “patient area” refers to a specific area or portion of a patient. In some examples, “patient area” refers to a range that spans the entire patient. Examples of such regions include lung region, gastrointestinal region, cardiovascular region (including myocardial tissue or myocardium (ie, heart muscle, ventricle, atrium, valve function), kidney region and others Body regions, tissues, organs, and the like, including vascular and circulatory systems, and affected tissues including cancerous tissues. Examples of the “patient region” include a region imaged by diagnostic imaging. This “patient area” is preferably internal, but may be external.

  In general, as used herein, “vasculature” refers to blood vessels, including arteries, veins, capillaries and the like.

  In general, as used herein, “gastrointestinal region” includes the region defined by the esophagus, stomach, small and large intestines, and rectum.

  In general, as used herein, “renal region” refers to the region defined by the vasculature that directly leads to and is derived from the kidney and directly from the kidney, and this region is the abdominal region. Including the aorta.

  In general, as used herein, “targeted area” and “target area” are used interchangeably to refer to an area of a patient where delivery of a drug is desired.

  In general, as used herein, “region to be imaged” and “imaging region” are used interchangeably to refer to a region of a patient in which imaging is desired.

  In general, as used herein, “enhancing ventricular blood flow or ventricular chamber” refers to the flow of blood through the ventricles in one or more cardiac cycles.

  In general, as used herein, “atrial blood flow” refers to the flow of blood through the atrium in one or more cardiac cycles.

  In general, as used herein, “myocardial blood flow” means in the vasculature of the heart muscle or myocardium, including the blood vessels of the heart, in one or more cardiac cycles. Refers to blood flow.

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

  In general, as used herein, the “cardiac cycle” refers to the complete contractile period of the heart, and this period includes both diastole and systole.

  In general, as used herein, “increased brightness” refers to an increase in brightness of an image compared to an image obtained without using an ultrasound contrast agent.

  In general, as used herein, “enhanced image” refers to an image having increased brightness relative to an image obtained without the use of an ultrasound contrast agent.

  In general, as used herein, “duration” refers to the total time during which an image with increased brightness can be detected.

  In general, as used herein, a “coronary vasodilator” refers to a bioactive factor (eg, dipyridamole or adenosine) that, when administered to a patient, causes dilation of the cardiovascular vasculature.

(II. Fine particles)
In a preferred embodiment, the microparticle includes a polymer, a lipid, and a perfluorocarbon. The microparticles may be composed of both microspheres and microcapsules, or may be composed of only microspheres or microcapsules.

(polymer)
In a preferred embodiment, the microparticles are formed from a synthetic polymer. Synthetic polymers result in microparticles that are biocompatible and not contaminated with biological material. In addition, synthetic polymers are preferred due to more reproducible synthesis and degradation both in vitro and in vivo. This polymer is based on the time required for in vivo stability (ie, the time required to distribute to the site where imaging is desired) and the time required to image. Selected. Synthetic polymers can be modified to produce microparticles with different properties (eg, changing molecular weight and / or functional groups).

  Exemplary synthetic polymers are: poly (lactic acid), poly (glycolic acid), and poly (lactic acid-co-glycolic acid), polyglycolide, polylactide, and poly (lactide-co-glycolide) copolymers Poly (hydroxy acids) and blends, polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly (ethylene glycol), poly (ethylene oxide) polyvinyl alcohol and the like Polyalkylene oxides, poly (valeric acid), and poly (lactide-co-caprolactone), and their derivatives, copolymers and blends. As used herein, a “derivative” refers to a polymer having substitution and addition of chemical groups (eg, alkyl, alkylene), hydroxylation, oxidation, and other modifications routinely made by those skilled in the art. Including.

Examples of preferred biodegradable polymers include polymers of hydroxy acids (eg, lactic acid and glycolic acid), polylactide, polyglycolide, poly (lactide-co-glycolide), copolymers with PEG, polyanhydrides, poly (ortho) Esters, polyurethanes, poly (butyric acid), poly (valeric acid), poly (lactide-co-caprolactone), and blends and copolymers thereof. The most preferred polymer is poly (lactide-co-glycolide) in a 50:50 (ie, 1: 1) lactide to glycolide ratio, and the polymer has a weight average in the range of 20,000 Da to 4,000 Da. Has a molecular weight. The weight average molecular weight (M _w ) of this polymer is an average molecular weight calculated based on the mass of molecules having a predetermined molecular weight within the distribution range of individual polymer chains. M _w can be determined using gel filtration chromatography (GPC).

(Hydrophobic compound)
In a preferred embodiment, the polymer comprises a hydrophobic compound as described in US Pat. No. 5,837,221. In general, incorporating a compound such as a lipid that is hydrophobic in an effective amount within a polymer limits water permeation and / or uptake by the microparticles, and thus limits gas loss from the microparticles. This is effective to increase the duration of enhanced imaging provided by microparticles containing lipids, synthetic polymers and gases encapsulated therein (especially fluorinated gases such as perfluorocarbons). It is. Lipids that can be used to stabilize the gas inside the polymer microparticles include, but are not limited to, the following classifications of lipids: fatty acids and derivatives, monoglycerides, diglycerides and triglycerides, phospholipids, sphingolipids , Cholesterol and steroid derivatives, terpenes and vitamins.

  Fatty acids and derivatives thereof include saturated and unsaturated fatty acids, odd and even chain fatty acids, cis and trans isomers, and fatty acid derivatives including alcohols, esters, anhydrides, hydroxy fatty acids and prostaglandins. Can be, but is not limited to. Saturated and unsaturated fatty acids that may be used include, but are not limited to, molecules having either 12 or 22 carbon atoms, either in linear or branched form. Examples of saturated fatty acids that can be used include, but are not limited to, lauric acid, myristic acid, palmitic acid, and stearic acid. Examples of unsaturated fatty acids that can be used include, but are not limited to, lauric acid, physeteric acid, myristoleic acid, palmitoleic acid, petroceric acid, and oleic acid. Examples of branched fatty acids that can be used include, but are not limited to, isolauric acid, isomyristinic acid, isopalmitic acid, and isostearic acid and isoprenoids. Examples of fatty acid derivatives include 12-(((7′-diethylaminocoumarin-3-yl) carbonyl) methylamino) -octadecanoic acid; N- [12-(((7′diethylaminocoumarin-3-yl) carbonyl) methyl- Amino) octadecanoyl] -2-aminopalmitic acid, N-succinyl-dioleoylphosphatidylethanolamine and palmitoyl-homocysteine; and / or combinations thereof. Monoglycerides, diglycerides and triglycerides which can be used or their derivatives include molecules having fatty acids or fatty acid mixtures between between 6 and 24 carbon atoms, digalactosyl diglycerides, 1,2-dioleoyl-sn-glycerol 1,2-dipalmitoyl-sn-3 succinylglycerol; and 1,3-dipalmitoyl-2-succinylglycerol; but are not limited to these.

  Phospholipids that can be used include phosphatidic acid, phosphatidylcholine including both saturated and unsaturated lipids, phosphatidylethanolamine, phosphatidylserine, phosphatidylserine, phosphatidylinositol, lysophosphatidyl derivatives, cardiolipin, and β-acyl-alkylphospholipids However, it is not limited to these. Examples of phospholipids include phosphatidylcholines (eg, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine (DPDPC), dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), Stearoyl phosphatidylcholine (DSPC), diarachidoyl phosphatidylcholine (DAPC), dibehenoyl phosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignocelloyl phatidylcholine (DLPC)); Oleoylphosphatidylethanolamine or 1-hexadecyl-2-palmitoylglycol Rojo Suho ethanolamine) include, but are not limited to. Synthetic phospholipids with asymmetric acyl chains (eg, having one acyl chain of 6 carbons and another acyl chain of 12 carbons) can also be used.

  Sphingolipids that can be used include ceramide, sphingomyelin, cerebroside, ganglioside, sulfatide and lysosulfatide. Examples of sphingolipids include, but are not limited to, ganglioside GM1 and ganglioside GM2.

  Steroids that can be used include cholesterol, cholesterol sulfate, cholesterol hemisuccinate, 6- (5-cholesterol 3β-yloxy) hexyl-6-amino-6-deoxy-1-thio-α-D-galactopyranoside, 6- (5-Cholesten-3β-yloxy) hexyl-6-amino-6-deoxyl-1-thio-α-D mannopyranoside and cholesteryl (4′-trimethyl 35 ammonio) butanoate include It is not limited.

  Additional lipid compounds that can be used include tocopherols and derivatives, as well as derivatized oils such as oils and stearylamine.

  Various cationic lipids (eg, DOTMA, N- [1- (2,3-dioleyloxy) propyl-N, N, N-trimethylammonium chloride; DOTAP, 1,2-dioleoyloxy-3- ( Trimethylammonio) propane; and DOTB, 1,2-dioleoyl-3- (4′-trimethyl-ammonio) butanoyl-sn-glycerol) can be used.

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

  The lipid content ranges from 0.01% to 30% (lipid w / polymer w); preferably from 0.1% to 20% (lipid w / polymer w); and Most preferably, it is in the range of 1% to 12% (w of lipid / w of polymer).

When formed by the methods described herein, the size of the microparticles is consistently reproducible. As used herein, with respect to particles, the term “size” or “diameter” refers to the number average particle size unless otherwise specified. Examples of equations that can be used to define the number average particle size (X _n ) are shown below:

ｎ_ｉ＝所定の直径（ｄ_ｉ）の粒子の数。

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

As used herein, the term “volume average diameter” refers to a volume weighted diameter average. Examples of equations that can be used to define the volume average diameter (X _v ) are shown below:

ｎ_ｉ＝所定の直径（ｄ_ｉ）の粒子の数。

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

  Particle size analysis is performed using a Coulter counter by optical microscopy, scanning electron microscopy or transmission electron microscopy, laser diffraction, such as using a Malvern Mastersizer, light scattering or time-of-flight. obtain. As used herein, “Cluter 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 fitted with a 50 μm open tube. . This method provides size and particle concentration.

  In a preferred embodiment for preparing injectable microparticles that can pass through a pulmonary capillary bed, the microparticles have a diameter of less than 8 microns. Larger particles can clog the lung bed and smaller particles cannot provide sufficient contrast effects. Preferred microparticle sizes for ultrasound contrast agents administered intravenously are between 0.75 and 5 microns, and most preferably between 1.8 and 3.0 microns.

  In preferred embodiments, the microparticles have a honeycomb or sponge-like structure formed by pores throughout the polymer, or the microparticles have a polymer shell with a honeycomb-like or sponge-like porous structure. In both cases, these pores are filled with gas. These microparticles are formed by spray drying a polymer solution containing a pore former (eg, a volatile salt as described below).

(Contrast ultrasound imaging agent)
Examples of fluorinated 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)} provides a gas insoluble not condense at the temperature of use, and because drugs are pharmacologically acceptable, particularly preferred.

  The amount of gas contained in the microparticles depends on the type of gas, but is typically between 75 μg and 500 μg per mL of dosage volume of the microparticle suspension. For n-perfluorobutane, the preferred gas content is between 100 μg and 400 μg per mL of microparticle suspension dosage volume, and most preferably 150 μg to 350 μg per mL of microparticle suspension dosage volume. Between. For n-octafluoropropane, the preferred gas content is between 75 μg and 375 μg per mL of microparticulate suspension dosage volume, and most preferably 120 μg to 300 μg per mL of microparticulate suspension dosage volume. Between.

(III. Method for producing fine particles)
The microparticles can be produced by various methods and are preferably produced by spray drying. The main condition is that the polymer needs to be dissolved or melted with the hydrophobic compound or lipid before forming the microparticles.

(solvent)
During formation, the polymer is generally dissolved in a solvent. As defined herein, the solvent for the polymer is an organic solvent, which is volatile or has a relatively low boiling point or is removed under vacuum. And acceptable for minor doses to humans (eg, methylene chloride). Can other solvents such as ethyl acetate, ethyl formate, ethanol, methanol, dimethylformamide (DMF), acetone, acetonitonyl, tetrahydrofuran (THF), formamide, acetic acid, dimethyl sulfoxide (DMSO) and chloroform also be utilized? , Or a combination thereof may be utilized. Generally, the polymer is dissolved in the solvent to form a polymer solution, the polymer solution having a concentration between 0.1% (w / v) and 60% (w / v) Preferably a concentration between 0.25% (w / v) and 30% (w / v), and most preferably a concentration between 0.5% (w / v) and 10% (w / v) Have

(Spray drying)
The microparticles are preferably produced by spray drying, which dissolves the biocompatible polymer and lipid in a suitable solvent and disperses the pore former in the polymer solution as a solid or as a solution. The polymer solution and the pore former are then spray dried to form microparticles. As defined herein, the process of “spray drying” a polymer solution and pore former is the atomization of the polymer solution and pore former to form a fine mist. , And the process by which the mist is dried by direct contact with a hot carrier gas. Using dry sprayers available in the art, the polymer solution and pore former can be atomized at the inlet port of the dry sprayer, passed at least once through the drying chamber, and then recovered as a powder. The temperature can be varied depending on the gas or polymer used. The inlet and outlet port temperatures can be controlled to produce the desired product.

  The size and form of the microparticles formed during spray drying are the nozzle used to spray the polymer solution and pore former, the pressure of the nozzle, the flow rate of the polymer solution containing the pore former, the polymer used, It depends on the concentration of the polymer in the solution, the type of solvent for the polymer, the type and amount of pore former, the spraying temperature (both inlet and outlet temperature) and the molecular weight of the polymer. In general, assuming that the concentration of the polymer solution is the same, the particle size increases as the molecular weight of the polymer increases.

  Typical process parameters for spray drying are as follows: inlet temperature = 30 ° C.-200 ° C., outlet temperature = 5 ° C.-100 ° C., and polymer flow rate = 10 ml / min-5, 000 ml / min.

  The gaseous diagnostic agent can be encapsulated by emulsification with the polymer solution and pore former prior to spray drying. Alternatively, air-filled microparticles can be produced during the spray drying process, after which the air applies the desired gas flow to the microparticles or pulls the microparticles to a vacuum and encloses the air Can then be replaced with perfluorocarbon gas by filling with desired perfluorocarbon gas. If a vacuum process is used to exchange the gas, a lyophilizer or a vacuum chamber can be used.

(Additives for promoting fine particle formation)
Various surfactants can be added during the formation of the microparticles. Exemplary emulsifiers or surfactants (0.1% w / w polymer to 15% w / w polymer) that may be used include most physiologically acceptable emulsifiers. Examples include natural forms of bile salts conjugated to amino acids or synthetic forms of bile salts conjugated to amino acids, and non-conjugated forms of bile salts or unconjugated forms of natural forms. Of bile salts such as taurodeoxycholate and cholate.

  A pore-forming agent is included in the polymer solution in an amount from 0.01% to 90% by weight of the polymer solution to increase pore formation. For example, in spray drying, the pore former (eg, volatile salt (eg, ammonium bicarbonate, ammonium acetate, ammonium carbonate, ammonium chloride, or ammonium benzoate, or other volatile salt)) Or it can be used as a solution in a solvent (eg water). Thereafter, a solid pore-forming agent or a solution containing the pore-forming agent is emulsified with the polymer solution to form a dispersion or droplet of pore-forming agent in the polymer. The dispersion or emulsion is then spray dried to remove both the polymer solvent and the pore former. After the polymer has precipitated, the cured microparticles can be frozen and lyophilized to remove any pore former that was not removed during the polymer precipitation step.

Preferred microparticles are the above polymers (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 to 40,000 daltons)) and the above Phospholipid (formed by using diarachidoyl phosphatidylcholine ((1,2-dialachidoyl-sn-glycero-3-phosphocholine (DAPC))) at 5% to 6.6% (DPAC weight / polymer weight) The microparticles are further formulated in a solution of mannitol and TWEEN® 80 to be processed to yield a microparticle dry powder that is lyophilized with n-perfluorobutane. The dry powder is made isotonic by adding 5 ml of sterile water to the dry powder vial and shaking. Reconstituted with this sterile water prior to use by producing a microparticle suspension in mannitol, the preferred properties of which are 150 μg n-perfluorobutane per mL microparticle suspension volume administered. Gas content of ˜350 μg, 1.5 × 10 ^{9 to} 2.8 × 10 ⁹ microparticles per mL of microparticle suspension administered, 30 mg of microparticles per mL of microparticle suspension administered 45 mg and an average particle size in the range of 1.8 microns to 3.0 microns.

(IV. Application of the fine particles)
(1. Formulation for administration to patients)
The microparticles are further processed with excipients to produce a dry powder. This excipient provides tonicity or osmolarity or ease of suspension of the microparticles after reconstitution with a pharmaceutically acceptable carrier prior to administration to a patient. Suitable excipients for providing osmolarity or tonicity include sugars (including but not limited to mannitol, dextrose, or glucose) and salts (sodium chloride or sodium phosphate). But are not limited to these). Suitable excipients to provide ease of suspending the microparticles include pharmaceutically acceptable wetting agents or pharmaceutically acceptable surfactants (Polysorbate 80 (TWEEN® 80), Polysorbate 20 (TWEEN® 20), Pluronic, or Polyethylene Glycol include, but are not limited to. Excipients that are suitable to provide osmolarity or tonicity, or that can be used as wetting agents, can be found in the references (eg, the Handbook of Pharmaceutical Excipients (Fourth Edition, Royal Pharmaceutical Society of Science , Science & Practice Publishers), or Remingtons: The Science and Practice of Pharmacy (Nineteenth Edition, Mack Publishing Company)). A dry powder of microparticles and excipients is produced by suspending the microparticles in an excipient solution. Additional size fractionation steps can be used if necessary. The microparticles in the excipient solution are filled into vials or syringes, frozen and lyophilized to produce a dry powder formulation. At the end of the freeze-drying process, the fine particles are filled with perfluorocarbon gas by filling the freeze-dryer with perfluorocarbon gas. The vial or syringe is then closed or capped and crimped in the case of a vial. This creates a perfluorocarbon headspace in the vial or syringe.

  Alternatively, the microparticles can be dry mixed with the pharmaceutical excipient and then filled into a vial or syringe. The microparticles can be filled with perfluorocarbon gas by applying a vacuum after filling the vial or syringe in a lyophilizer or vacuum chamber. The vial or syringe is then closed or capped and crimped in the case of a vial. This creates a perfluorocarbon headspace in the vial or syringe.

(2. Dosage unit)
Various sizes of particulate dosage units can be used. For example, a small dosage unit can contain 25 mg to 75 mg of microparticles. An intermediate dosage unit may comprise 75 mg to 150 mg. A large dosage unit may contain 150 mg to 250 mg of microparticles. A very large dosage unit may contain between 250 mg and 1000 mg of microparticles.

When a microparticle suspension is formed after reconstitution, the mass concentration of microparticles in the suspension is typically in the range of 20 mg / mL to 60 mg / mL. The preferred mass concentration of the microparticles in this suspension is 25 mg / mL to 50 mg / mL. The most preferred mass concentration of the microparticles in this suspension is 30 mg / mL to 45 mg / mL. The preferred concentration of microparticles in this suspension is 1.0 × 10 ⁹ microparticles to 3.5 × 10 ⁹ microparticles per mL of suspension, and the most preferred concentration of microparticles in this suspension is per mL. 1.5 × 10 ⁹ fine particles to 2.8 × 10 ⁹ fine particles. The microparticles have a preferred average particle size of less than 8 microns, and most preferably have an average particle size in the range of 1.8 microns to 3.0 microns.

  Examples of pharmaceutically acceptable carriers include water for injection, sterile water, physiological saline, physiological saline containing glycerol, physiological saline containing TWEEN (registered trademark) 20, physiological saline containing TWEEN (registered trademark) 80, etc. Pressure dextrose (5%), 1/2 isotonic dextrose (2.5%), isotonic mannitol (5%), 1/2 isotonic mannitol (2.5%), TWEEN® 20 Mention may be made of isotonic mannitol containing, and isotonic mannitol containing TWEEN® 80.

(3. Kit)
A kit for parenteral administration of the microparticles containing perfluorocarbon gas can be provided. The kit includes at least two components. One component includes a dosage unit of dry powder contrast agent in a vial or syringe, and the other component includes a pharmaceutically acceptable carrier in a vial or syringe. Prior to administration to a patient, the pharmaceutically acceptable carrier is added to the dry powder contrast agent of the dosage unit to form a gas-filled particulate suspension. The gas-filled microparticles can be used as an ultrasound imaging contrast agent in diagnostic imaging by any route of administration.

(4. Vial or container for fine particles)
No special vials, syringes or connection systems are required for this kit. Conventional vials, syringes, and adapters can be used with the microparticles. The only requirement for the vial is a good seal between the stopper and the container. Therefore, the quality of this seal is of primary concern. Any reduction in the integrity of the seal can also allow undesirable materials to enter the vial or allow gas to flow out. In addition to ensuring sterility, vacuum retention is required for products that are closed at reduced pressure to ensure safe and proper reconstitution. With respect to the plug, the plug can be an elastomer-based compound or other component formulation (eg, poly (isobutylene) or “butyl rubber”), and the plug must be impermeable to the gas used. The size of the vial is selected depending on the total dose of dry powder in the vial. Preferred vial sizes are 5 mL, 10 mL, 20 mL, and 30 mL. The syringe size is selected depending on the total dose of dry powder in the syringe. Preferred syringe sizes are 5 mL syringe, 10 mL syringe, 20 mL syringe, and 50 mL syringe.

(5. Diagnosis application)
The microparticle composition can be used in many different diagnostic applications, including ultrasound imaging, magnetic resonance imaging, fluoroscopy, x-ray, and computed tomography.

  In a preferred embodiment, the microparticles include ultrasound procedures (eg, vascular imaging and echocardiography (ventricular imaging, myocardial blood flow assessment, myocardial blood volume assessment, coronary artery disease diagnosis, and ejection fraction assessment). Used in, but not limited to).

  The microparticles can be used in vascular imaging, and in applications for detecting liver and kidney disease, in detecting and characterizing tumor masses and tumor tissues, and in measuring peripheral blood velocity. . The microparticles can also be linked with a ligand that minimizes tissue adhesion or a ligand that targets the microparticle in vivo to a specific region of the body.

(General method for obtaining images)
The microparticles in dry powder form are reconstituted with a pharmaceutically acceptable carrier prior to administration. Thereafter, an effective amount for detection may be using an appropriate route, or by injection into a blood vessel (eg, intravenous (iv) injection or intraarterial (ia) injection). Or orally to the patient. The microparticle composition can be administered intravenously to the patient as a bolus injection or as a short-term infusion (less than 30 minutes). Preferably, the injection is administered over a period ranging from 15 seconds to 20 minutes, most preferably over a period ranging from 30 seconds to 15 minutes. Typically, a dose in the range of 0.025 mg to 8 mg per kg body weight per injection is administered intravenously to the patient, preferably the dose is 0.05 mg / kg to 4 mg / kg. Is in range.

  For diagnostic ultrasound applications, energy is applied to at least a portion of the patient to image the target tissue. Thereafter, a visible image of the patient's internal region is obtained so that the presence or absence of affected tissue can be confirmed. Ultrasound imaging techniques (including second harmonic imaging and gated imaging) are well known in the art and are described, for example, in Uhlendorf, IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control: 1 (1). 70-79 (1994) and Sutherland et al., Journal of the American Society of Echocardiography, 7 (5): 441-458 (1994), the disclosures of each of which are incorporated herein by reference in their entirety. It is described in.

  Ultrasound can be applied using a transducer. This ultrasonic wave may be a pulse wave or, if desired, a continuous wave. Thus, diagnostic ultrasound generally involves the application of echoes, and then during the listening period, the ultrasound transducer receives the reflected signal. Harmonics, ultraharmonics, or subharmonics can be used. The second harmonic mode can be beneficially used, in which case a 2 × frequency is received. X is an incidental frequency. This can act to reduce the signal from the background material and to enhance the signal from the transducer using an imaging agent. The imaging agent can be targeted to a desired site (eg, a blood clot). Other harmonic signals (eg, odd harmonic signals (eg, 3x or 5x) are similarly received using this method. Subharmonic signals (eg, x / 2 and x / 3) Can also be received and processed to form an image.

  In addition, power Doppler or color Doppler can be applied. In the case of a power Doppler, the relatively high energy of the power Doppler can cause the vesicle to resonate. This can cause acoustic emission. This acoustic emission can be in the subharmonic or harmonic range, and in some cases can be at the same frequency as the applied ultrasound.

(Specific imaging applied)
The microparticles described herein can be used in both cardiology and radiology applications. For cardiological applications, the particulate composition is administered to a patient, and the patient is scanned using an ultrasound device to obtain a visible image of the cardiovascular region. If desired, the microparticle composition is administered in combination with a pharmacological or physical stressor. Suitable pharmacological stressors include coronary vasodilators (eg, dipyridamole or adenosine), cardiotonic agents (ie, factors that increase cardiac contractile strength) (eg, dobutamine), or chronotropic agents (ie, , A factor that increases contraction frequency) (for example, dobutamine). Suitable physical stressors include physical exercise (eg, by using a treadmill or stationary bicycle).

  For radiological applications, the microparticle composition is administered to a patient, and the patient is scanned using an ultrasound instrument to obtain a visible image of the patient area to be examined.

  The microparticles can be used to assess cardiovascular function and to assess myocardial blood flow or volume, or to diagnose coronary heart disease (coronary artery disease). For example, the microparticles can enhance images of the ventricles and can assist regional cardiac function analysis via wall motion analysis and can assist global cardiac function via ejection fraction measurement. The microparticles can also be used to assess myocardial blood flow to distinguish functional heart tissue from either ischemic (blood flow deficient) heart tissue or infarcted (dead) heart tissue. The contrast signal detected in the myocardium can be used as an estimate of myocardial blood volume. This is because the ultrasound contrast agent is present in the vein after intravenous administration. Absence of contrast intensity or image brightness in a particular myocardial region or a decrease over time is an indicator of decreased blood flow (ie, a defect).

  In most cases, unless the patient has severe coronary heart disease, the blood flow to the various heart regions assessed by techniques such as sonography appears normal. To detect blood flow abnormalities in patients without severe heart disease or to detect relatively small myocardial blood flow defects, increasing blood flow to the heart by inducing stress conditions is necessary. Stress can be induced by exercising the patient or by administering a pharmacological compound (eg, a vasodilator, a cardiotonic agent, or a chronotropic agent). Blood flow defects can be more easily detected during exercise or during pharmacological stress. This is because the ability to increase blood flow diminishes in areas that are blood-fed by constricted coronary arteries. Comparison of myocardial ultrasound images after administration of the ultrasound contrast agent can be made both in the pre-stress state (ie, resting state) and in the stress state. A myocardial region with no enhanced brightness, found during stress imaging but not during rest imaging, is an indicator of ischemia. The myocardial region with no enhanced brightness found during stress imaging and during rest imaging is an indication of infarction.

  In one embodiment, myocardial blood flow uses (1) a first microparticle composition injection to a patient, and (2) uses ultrasound instrument imaging to obtain a visible image of the cardiovascular region. Scanning the patient, (3) inducing a stress state in the patient using pharmacological stress or exercise, (4) administering the second injection of the microparticle composition and continuing the scan. As well as (5) evaluating the difference between the images obtained in steps (2) and (4) either visually or by using quantitative image analysis.

  For radiological applications, the microparticles are used to improve ultrasound imaging capabilities (including renal disease imaging, liver disease imaging, and peripheral vascular disease imaging) for radiological applications Can increase blood flow visibility and improve detection of small lesions or structures deep in the body. The microparticles can be used for both macrovascular and microvascular applications. In macrovascular applications (diagnosis of disease states and conditions in the body's main arteries and veins), the microparticles can assist in the detection of stroke and prestroke conditions via visualization of intracranial blood vessels, such as large blood vessels (e.g., Atherosclerosis in the carotid artery) is detected by assessing the extent of carotid atherosclerosis, vascular graft patency, and peripheral vascular thrombosis. In microvascular applications (diagnosing disease states through pattern analysis of small vessel flow), the microparticles can be found in the liver (eg adenoma or hemangioma), kidney, spleen (eg splenic aneurysm), breast and ovary and other It may assist in identifying lesions, tumors, or other diseases in tissues and organs.

  Affected tissue in a patient is diagnosed by administering the particulate composition to the patient and scanning the patient using ultrasound imaging to obtain a visible image of any affected tissue in the patient Can be done. Affected tissue can be shown as an area with enhanced brightness or as an area that does not show enhanced brightness.

(Enhanced image obtained using fine particle composition)
The microparticles produce an enhanced image after administration. An enhanced image may be indicated by an increase in the brightness of the image compared to when no ultrasound contrast agent is administered or by a substantial elimination of artifacts in the image. Thus, with respect to ultrasound imaging of the cardiovascular region (cardiac tissue and associated vasculature), the augmented image is, for example, due to increased brightness in the cardiovascular region image and / or artifact generation in the cardiovascular region image Can be indicated by the substantial exclusion of. The image after a single dose of the drug lasts from 10 seconds to 60 minutes. This image preferably lasts from 20 seconds to 30 minutes, most preferably from 30 seconds to 20 minutes. In a preferred embodiment, ultrasound imaging is enhanced for more than 5 minutes in the ventricle and for more than 1 minute in the myocardium.

  This increase in brightness in the image can be assessed visually by the naked eye or using quantitative image analysis. There is preferably an increase in brightness level (gray level) of at least about 10 VDU, particularly for the gray scale identified above (about 0 VDU to about 255 VDU, or gray level). More preferably, the image is larger than about 10 VDU (e.g., about 15 VDU, about 20 VDU, about 25 VDU, about 30 VDU, about 35 VDU, about 40 VDU, about 45 VDU, about 50 VDU, about 55 VDU, about 60 VDU, about 65 VDU, about 70VDU, about 75VDU, about 80VDU, about 85VDU, about 90VDU, about 95VDU, or 100VDU). In some embodiments, the brightness increase is greater than about 100 VDU (eg, about 105 VDU, about 110 VDU, about 120 VDU, about 125 VDU, about 130 VDU, about 135 VDU, about 140 VDU, about 145 VDU, or 150 VDU). In other embodiments, the brightness increase is greater than about 150 VDU (eg, about 155 VDU, about 160 VDU, about 165 VDU, about 170 VDU, about 175 VDU, about 180 VDU, about 185 VDU, about 190 VDU, about 195 VDU, or about 200VDU). Alternatively, the brightness increase is greater than about 200 VDU (eg, about 205 VDU, about 210 VDU, about 215 VDU, about 220 VDU, about 225 VDU, about 230 VDU, about 240 VDU, about 245 VDU, about 250 VDU, or about 255 VDU).

  The above methods and compositions are further understood with reference to the following non-limiting examples.

(material)
Acetic acid, ammonium bicarbonate, mannitol (USP), and polysorbate 80 (no animal-derived ingredients) were purchased from Spectrum Chemicals, Gardena, Calif.). Polymers (poly (lactide-co-glycolide) (PLGA) (50:50)) and diarachidoylphosphatidylcholine (1,2-dialachidoyl-sn-glycero-3-phosphocholine (DAPC)), respectively, Boehringer Ingelheim (Ingelheim) , Germany) and Avanti (Alabaster, AL). Methylene chloride was purchased from EM Science (EMD Chemicals, Gibbstown, NJ). Vials (30 ml tubular vials) and stoppers (20 mm, grey, one vent, Fluro-Tec) were purchased from West Pharmaceutical Services (Lionville, PA). n-Perfluorobutane (DFB) gas was purchased from F2 Chemicals Ltd, Lanciashire, UK.

(Analysis method)
(Quantification of mass concentration of fine particles)
The mass concentration of the microparticles in the vial was quantified using ICP-MS (Inductively Coupled Plasma-Mass Spectrometry). The amount of polymer in the microparticles was determined by analyzing for tin by ICP-MS. The amount of polymer present in the microparticle is determined based on a comparison of the amount of tin found in the microparticle and the amount of tin found in the specific lot of polymer used to produce the microparticle. did. The amount of phospholipid in the microparticles was determined by analyzing for phosphorus by ICP-MS. The amount of phosphorus present in the microparticles was determined based on the amount of phosphorus found in the microparticles compared to the amount of phosphorus in the phospholipid itself. The mass of microparticles per mL of suspension was calculated by adding the amount of polymer and phospholipid per vial and then dividing the sum by the reconstituted volume (5 mL).

(Particle size analysis)
A sample of reconstituted microparticles was added to the electrolyte solution. The resulting suspension was analyzed for particle size and microparticle concentration using a Coulter Multisizer II fitted with a 50 μm aperture tube.

(Gas content of fine particles)
The dry powder vial was reconstituted with 5 mL of water and shaken to produce a fine particle suspension. The resulting suspension was analyzed for DFB content by withdrawing a set of 0.3 mL aliquot groups through the stopper using a needle and syringe. These aliquots were injected into sealed headspace vials. The headspace vial was equilibrated at room temperature for at least 10 hours. The sample was then heated and then heated to 45 ° C. for 20 minutes in a headspace sampler oven. The headspace gas above the suspension was analyzed by gas chromatography using a purged filled inlet and a flame ionization detector. Quantification was performed using an area-based single point calibration. The GC system parameters and temperature program are listed in Tables 1 and 2.

  (Table 1: GC system parameters)

（表２：ＧＣ温度プログラム）

(Table 2: GC temperature program)

（実施例１：超音波造影剤として使用するための微粒子の調製）
有機溶液を、１７６ｇのＰＬＧＡ、１０．６ｇのジアラキドイルホスファチジルコリン（１，２−ジアラキドイル−ｓｎ−グリセロ−３−ホスホコリン（ＤＡＰＣ））、および２．２６ｇの酢酸を５．８８Ｌの塩化メチレン中に２５℃にて溶解することによって調製した。３３８ｍｌの注射用水中に溶解した６８．５ｇの炭酸水素アンモニウムから構成される水溶液を、この有機溶液に添加し、ローター−ステーター乳化混合機を使用して１０Ｌホモジナイゼーションタンク中にて４３１０ＲＰＭで１０分間ホモジナイズした。

Example 1: Preparation of microparticles for use as an ultrasound contrast agent
The organic solution was mixed with 176 g PLGA, 10.6 g diarachidoylphosphatidylcholine (1,2-dialachidoyl-sn-glycero-3-phosphocholine (DAPC)), and 2.26 g acetic acid in 5.88 L methylene chloride. Prepared by dissolving at 25 ° C. An aqueous solution composed of 68.5 g of ammonium bicarbonate dissolved in 338 ml of water for injection is added to this organic solution and 10 at 4310 RPM in a 10 L homogenization tank using a rotor-stator emulsification mixer. Homogenized for minutes.

  The resulting emulsion was spray dried using nitrogen gas as the spray and dry gas. The emulsion was spray dried in a bench top spray dryer using an air spray nozzle obtained from Spraying Systems (Wheaton, IL) and a glass drying chamber / cyclone system obtained from Buchi (Brinkmann, Westbury, NY). . The spray drying conditions were as follows: emulsion flow rate of 40 ml / min, spray gas rate of 30 L / min, dry gas rate of 46 kg / hr, and outlet temperature of 12 ° C.

  The spray dried product was further processed through a dispersion process, a freezing process, and a freeze drying process. An aqueous vehicle was prepared by dissolving 140 g mannitol and 4.10 g polysorbate 80 in 5.0 L water. The spray-dried microparticles were dispersed in the vehicle at a concentration of 25 mg / ml. This dispersion was deagglomerated using a stainless steel 800 series flow cell sonicator obtained from Misonix Incorporated (Farmingdale, NY), and a 10 ″ diameter vibration obtained from Vorti-Siv (Salem, OH). Sieving through a sieve (RBF-10). The sonicator was covered at 4 ° C. to prevent heating of the dispersion. This dispersion was sieved through a 25 μm screen and a 20 μm screen in succession at 150 mL / min. The sieved dispersion was filled into vials (10 ml filling in 30 ml vials), partially capped, and frozen by immersion in liquid nitrogen.

  After freezing, the vial was lyophilized. At the end of lyophilization, the chamber was separated and n-perfluorobutane (DFB) was filled into the vial to a pressure of -5 kPa and then capped.

The dry powder was reconstituted with 5 mL of sterile water before use. This reconstitution was by adding the water to the dry powder vial and shaking to produce a fine particle dispersion in isotonic mannitol. This suspension contained 2.2 × 10 ⁹ microparticles per mL suspension and 37 mg microparticles per mL suspension. The microparticles had an average particle size of 2.2 microns.

(Example 2: Gas leakage rate from the fine particles)
Gas leak rates from two separate batches of microparticles produced by the method of Example 1 (Batch 1 and Batch 2) were analyzed using gas chromatography (GC) as described in the analytical methods section above. And evaluated. A third lot of microspheres (batch 3) was produced in the same manner as in the example method except that the above phospholipid (diarachidyl phosphatidylcholine (1,2-diarachidyl-sn-glycero-3-phosphocholine (DAPC)) was prepared. ) Was omitted during this fine particle formation.

  (Table 3: Gas content and gas leakage rate for fine particles)

ＤＰＡＣを含む上記微粒子は、７０分間後に出発ガス含有量のうちの約１０％を失った。一方、ＤＰＡＣを含まない上記微粒子は、出発ガス含有量のうちの８７％を失った。さらに、ＤＰＡＣを含む上記微粒子は、ＤＡＰＣを含まない上記微粒子と比較して、高い初期出発ガス含有量を有した。このことは、ＤＡＰＣを含むことが、噴霧乾燥の間の上記微粒子の内部多孔性構造の形成および上記微粒子内でのガスの保持にとって重要であることを、示す。

The microparticles containing DPAC lost about 10% of the starting gas content after 70 minutes. On the other hand, the fine particles without DPAC lost 87% of the starting gas content. Furthermore, the microparticles containing DPAC had a higher initial starting gas content compared to the microparticles not containing DAPC. This indicates that inclusion of DAPC is important for the formation of the internal porous structure of the microparticles and the retention of gas within the microparticles during spray drying.

  The total duration of intentional use of an ultrasound contrast agent after administration to a subject is generally about, depending on the type of cardiological or radiological ultrasound test being performed. It is about 30 seconds to about 60 minutes. Therefore, it is estimated that the loss of gas from the microparticles containing liquid DAPC is not a problem during the ultrasonic test period.

Example 3: Cardiac image enhancement as a function of particulate dose
The microparticles produced by the method in Example 1 were studied in healthy human adults. The dry powder was reconstituted before use by adding 5 ml of sterile water to the vial and shaking the vial 10 times. The final concentration of microspheres in the resulting suspension was approximately 37 mg / mL. Subjects were given a single dose of either 0.5 mg / kg body weight, 2.0 mg / kg body weight, or 4.0 mg / kg body weight. Subjects were subjected to transthoracic ultrasound imaging using continuous harmonic imaging (frame rate 15 Hz and transducer frequency 2.1 / 4.2 MHz). Images were visually evaluated for intensity and enhancement duration.

  The augmentation duration in the ventricle was longer than 9 minutes at both 2 mg / kg and 4 mg / kg doses. Contrast effects were still apparent in 13 of the 15 subjects at these two doses when subjects were re-imaged at 30 minutes. This indicates the long duration of enhancement provided by the microparticles.

  The duration of ventricular augmentation is summarized in Table 4.

  (Table 4: Duration of left ventricular image enhancement)

（実施例４：心臓画像評価用の市販製品に対する微粒子の比較）
比較心臓超音波画像研究を、体重および心機能について一致させた２人の成人男性において実行した。第一の被験体には、実施例１の方法により生成した微粒子の単回投与を与えた。その乾燥粉末は、バイアルに５ｍＬの滅菌水を添加してそのバイアルを１０回振盪することによって、使用前に再構成した。生じた懸濁物中のミクロスフェアの最終濃度は、約３７ｍｇ／ｍＬであり、その懸濁物のガス含有量は、懸濁物１ｍＬ当たり約２５０μｇであった。この第一の被験体には、４ｍｇ微粒子／ｋｇの用量を与えた。これは、体重１ｋｇ当たり２７μｇのガス用量に対応する。第二の被験体には、アルブミンミクロスフェアを含むペルフルオロプロパンを含む市販の超音波造影剤であるＯＰＴＩＳＯＮ（登録商標）（Ａｍｅｒｓｈａｍ Ｈｅａｌｔｈ）の単回投与を与えた。この２人の被験体に、音響的に活性な成分であるガスを同じ合計量（２７μｇ／体重ｋｇ）与えた。この２人の被験体に対して、連続的ハーモニックイメージング（フレームレート１５Ｈｚおよび変換器周波数２．１／４．２ＭＨｚ）を使用して、経胸壁超音波画像化を行った。画像を、強度および増強持続時間について視覚的に評価した。

(Example 4: Comparison of fine particles with a commercial product for cardiac image evaluation)
A comparative cardiac ultrasound imaging study was performed in two adult males matched for weight and cardiac function. The first subject was given a single dose of microparticles produced by the method of Example 1. The dry powder was reconstituted before use by adding 5 mL of sterile water to the vial and shaking the vial 10 times. The final concentration of microspheres in the resulting suspension was about 37 mg / mL, and the gas content of the suspension was about 250 μg per mL of suspension. This first subject was given a dose of 4 mg microparticles / kg. This corresponds to a gas dose of 27 μg / kg body weight. The second subject received a single dose of OPTISON® (Amersham Health), a commercially available ultrasound contrast agent containing perfluoropropane containing albumin microspheres. The two subjects were given the same total amount (27 μg / kg body weight) of gas, an acoustically active ingredient. Transthoracic ultrasound imaging was performed on these two subjects using continuous harmonic imaging (frame rate 15 Hz and transducer frequency 2.1 / 4.2 MHz). Images were visually evaluated for intensity and enhancement duration.

  The duration of ventricular and myocardial enhancement is summarized in Table 5.

  (Table 5: Duration of image enhancement using a separate ultrasound contrast agent)

実施例１において記載される方法を使用して生成した微粒子は、心室および心筋の両方の画像増強を提供する。この画像増強は、ＯＰＴＩＳＯＮ（登録商標）よりもかなり長く、そしてこれは、超音波による完全心臓試験を実行するために適切な持続時間である。

Microparticles generated using the method described in Example 1 provide both ventricular and myocardial image enhancement. This image enhancement is much longer than OPTISON®, and this is a suitable duration for performing a complete heart test with ultrasound.

Example 5: Evaluation of myocardial blood flow to assess ischemia using a microparticle formulation
The microparticles produced according to the method in Example 1 were administered to a subject to be evaluated for coronary heart disease. The subject was given two injections of the microparticles at 60 minute intervals. The first microparticle injection (“rest injection”, 1.7 mg / kg) was used to assess resting myocardium. Prior to the second microparticle injection, the subject was pharmacologically stressed using the coronary vasodilator dipyridamole (0.56 mg / kg). After stress induction, the subject was given a second microparticle injection (“stress injection”, 1.3 mg / kg) to ignite the myocardium under stress.

  A comparison of the resting image and the stress image over time after the microparticle administration for the subject shows a myocardial region with minimal increase in image enhancement. This region increased in size after induction of the stress. This indicates that the area of myocardial tissue has both an infarcted component and an ischemic component. The detection of ischemia was confirmed using nuclear imaging, an alternative diagnostic technique. Resting nuclear stress and stress nuclear perfusion were performed after 99Tc (MIBI) administration. The subject was imaged using a commercially available gamma counter. Defects noticed in the ultrasound resting image and the ultrasound stress image were confirmed in the resting nuclear perfusion image and the stressed nuclear perfusion image.

Claims (22)

A dosage formulation comprising microspheres and providing enhanced contrast-enhanced ultrasound images, the microspheres comprising a poly (lactide-co-glycolide) polymer and diarachidoylphosphatidylcholine , the microspheres comprising a honeycomb a porous spheres having a structure or sponge-like structure, and is incorporated into the microspheres have a n- perfluoro butane,
The dosage formulation is an isotonic suspension prepared from a dry powder by reconstitution with a pharmaceutically acceptable carrier;
The dosage formulation comprises a dose of the microsphere effective to provide an enhanced ultrasound image in the myocardium for longer than 1 minute when the microsphere is administered intravenously;
The dose range is microspheres ranging from 0.5 mg to 4.0 mg per kg body weight, and the formulation is reconstituted from 1.0 × 10 ⁹ to 3 per mL of suspension. A dosage formulation that can form a suspension with a concentration of microspheres in the range of 5 × 10 ⁹ or a mass concentration of microspheres in the range of 25 mg to 50 mg per mL of suspension. 2. A dosage formulation according to claim 1, wherein the microspheres are in the form of a dry powder and the microspheres are reconstituted with sterile water prior to use to form a suspension. The dosage formulation of claim 1, which provides an enhanced ultrasound image in the myocardium for more than 2 minutes. 2. The dosage formulation of claim 1 in the form of a dry powder of microspheres that can be reconstituted with sterile water prior to use, wherein the dosage formulation comprises an isosmotic suspension of the microspheres. A dosage formulation that can be reconstituted by adding sterile water to the dry powder vial or syringe and shaking to obtain a product. Suspensions having a concentration of microspheres ranging from 1.5 × 10 ⁹ to 2.8 × 10 ⁹ microspheres per mL of suspension or a mass concentration of microspheres ranging from 30 mg to 45 mg per mL of suspension. 3. A dosage formulation according to claim 2 which forms a product. The dosage formulation of claim 1, wherein the microsphere has an average particle size of less than 8 microns. 7. The dosage formulation of claim 6, wherein the microsphere has an average particle size in the range of 1.9 microns to 2.6 microns. The dosage formulation of claim 1, wherein the dose is selected from the group consisting of 0.5 mg microspheres per kg body weight, 2.0 mg microspheres per kg body weight, and 4.0 mg microspheres per kg body weight. . Before Symbol n - perfluorobutane per 1mL of administered volume of the microspheres suspension is provided in an amount between 75μg and 500 [mu] g, the dosage formulation of claim 1. The n- perfluoroalkyl pig down the per 1mL of administered volume of the microspheres suspension is provided in an amount between 100μg and 400 [mu] g, the dosage formulation of claim 9. The n- perfluoroalkyl pig down the per 1mL of administered volume of the microspheres suspension is provided in an amount between 150μg and 350 [mu] g, the dosage formulation of claim 10. Wherein the poly (lactide -co- glycolide) polymer is 1: 1 lactide to have a weight average molecular weight in the range of ratios and 20kDa~40kDa glycolide, and the di-Araki Doyle phosphatidylcholine, 5% and 6.6% Ru is incorporated seen set at a ratio (weight / weight polymer lipid) between, the dosage formulation of claim 1. The dosage formulation of claim 1 in a vial or syringe with a dry powder of microspheres. 14. The dosage formulation of claim 13 , wherein the vial or syringe further comprises one or more excipients selected from the group consisting of sugars, salts, and surfactants. 3. A dosage formulation according to claim 1 or 2 in a kit comprising a vial or syringe of dry microspheres and a vial or syringe of solution for resuspending the microspheres. 3. A dosage formulation according to claim 1 or 2 consisting essentially of one or two doses. 3. A dosage formulation according to claim 1 or 2, consisting essentially of a maximum of 5 doses. 18. A dosage formulation according to any of claims 1 to 17 , suitable for intravenous administration to a patient in order to provide an enhanced ultrasound image in the myocardium for longer than 1 minute, Dosing formulation that produces an enhanced image in the myocardium for more than 1 minute by imaging the area compared to the absence of contrast agent. Further containing a pharmacological or physical stressor to load the patient's cardiovascular system and re-image the patient , the pharmaceutical stressor comprising a coronary vasodilator, a cardiotonic agent and an inotropic is selected from the group consisting of factor (chronotropic agent), the physical stressor, Ru physical exercise der, dosage formulation of claim 18. A kit comprising a dosage formulation according to any of claims 1 to 17 and a solution for reconstituting the dosage formulation. A method of making a dosage formulation for imaging ultrasound imaging methods, the method, in order to produce a dosage formulation as defined by any of claims 1 to 17, the following:
Suspending microspheres comprising poly (lactide-co-glycolide) polymer and diarachidoylphosphatidylcholine in a solution containing excipients as required;
Placing the suspension in a vial or syringe;
Freezing the suspension;
Lyophilizing the vial to form a dry powder formulation in the vial or syringe; and
step to re-fill the lyophilizer with n- perfluoro butane gas,
Including the method. A method of making a dosage formulation for imaging ultrasound imaging methods, the method, in order to produce a dosage formulation as defined by any of claims 1 to 17, the following:
Poly step of dry-blending the di Araki Doyle phosphatidyl choline comprising a microsphere containing (lactide -co- glycolide) polymer, an excipient if required;
Placing the mixture in a vial or syringe;
Thereafter, the step of applying a vacuum to the vial to fill the vial or syringe with n- perfluoro butane gas,
Including the method.