Note: Descriptions are shown in the official language in which they were submitted.
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ULTRASOUND CONTRAST AGENT DOSAGE FORMULATION
Background of the Invention
The present invention is in the general field of diagnostic imaging
agents, and is particularly directed to specific ultrasound contrast agent
dosage formulations that provide enhanced images and images of long
duration.
When using ultrasound to obtain an image of the internal organs and
structures of a human or animal, ultrasound waves, waves of sound energy at
a frequency above that discernable by the human ear, are reflected as they
pass through the body. Different types of body tissue reflect the ultrasound
waves differently and the reflections that are produced by the ultrasound
waves reflecting off different internal structures are detected and converted
electronically into a visual display.
For some medical conditions, obtaining a useful image of the organ
or structure of interest is especially difficult because the details of the
structure are not adequately discernible from the surrounding tissue in an
ultrasound image produced by the reflection of ultrasound waves absent a
contrast-enhancing agent. Detection and observation of certain physiological
and pathological conditions may be substantially improved by enhancing the
contrast in an ultrasound image by administering an ultrasound contrast
agent to an organ or other structure of interest. In other cases, detection of
the movement of the ultrasound contrast agent itself is particularly
important.
For example, a distinct blood flow pattern that is known to result from=
particular cardiovascular abnormalities may only be discernible by
administering the ultrasound contrast agent to the bloodstream and observing
either blood flow or blood volume.
Materials that are useful as ultrasound contrast agents operate by
having an effect on ultrasound waves as they pass through the body and are
reflected to create the image from which a medical diagnosis is made.
Different types of substances affect ultrasound waves in different ways and
to varying degrees. Moreover, certain of the effects caused by contrast-
enhancing agents are more readily measured and observed than others. In
selecting an ideal composition for an ultrasound contrast agent, one would
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prefer the substance that has the most dramatic effect on the ultrasound wave
as it passes through the body. Also, the effect on the ultrasound wave should
be easily measured. Gases are the preferred media for use as ultrasound
contrast agents. The gas must be stabilized prior to usage as either
surfactant
stabilized bubbles or by encapsulating in liposomes or microparticles. There
are three main contrast-enhancing effects which can be seen in an ultrasound
image: backscatter, beam attenuation, and speed of sound differential.
A variety of natural and synthetic polymers have been used to
encapsulate ultrasound contrast agents, such as air, in an effort to make an
ultrasound contrast agent that lasts longer following administration.
Schneider et al., Invest. Radiol., Vol. 27, pp. 134-139 (1992) describes three
micron, air-filled, synthetic, polymer particles. These particles were
reported
to be stable in plasma and under applied pressure. However, at 2.5 MHz,
their echogenicity was low. Another type of microbubble suspension has
been obtained from sonicated albumin. Feinstein et al., J. Am. Coll.
Cardiol., Vol. 11, pp. 59-65 (1988). Feinstein describes the preparation of
microbubbles that are appropriately sized for transpulmonary passage with
excellent stability in vitro. However, these microbubbles are short-lived in
vivo, having a half-life on the order of a few seconds (which is
approximately equal to one circulation pass) because of their instability
under pressure. Gottlieb, S. et al., 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 have also been described in WO
80/02365 by Rasor Associates, Inc. These are formed by "coalescing" the
gelatin. Gas microbubbles encapsulated within a shell of a fluorine-
containing material are described in WO 96/04018 by Molecular Biosystems,
Inc.
Microbubbles stabilized by microcrystals of galactose (SHU 454 and
SHU 508) have also been reported by Fritzch et al. Fritzsch, T. et al.,
Invest.
Radiol. Vol. 23 (Suppl 1), pp. 302-305 (1988); and Fritzsch, T. et al.,
Invest.
Radiol., Vol. 25 (Suppl 1), 160-161 (1990). The microbubbles last up to 15
minutes in vitro but less than 20 seconds in vivo. Rovai, D. et al., J. Am.
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Coll. Cardiol., Vol. 10, pp. 125-134 (1987); and Smith, M. et al., J. Am.
Coll. Cardiol., Vol. 13, pp. 1622-1628 (1989). EP 398 935 by Schering
Aktiengesellschaft discloses the preparation and use of microencapsulated
gas or volatile liquids for ultrasound imaging, where the microcapsules are
formed of synthetic polymers or polysaccharides. European Patent 458 745
by Sintetica discloses air or gas microballoons bounded by an interfacially
deposited polymer membrane that can be dispersed in an aqueous carrier for
injection into a host animal or for oral, rectal, or urethral administration,
for
therapeutic or diagnostic purposes.
WO 92/18164 by Delta Biotechnology Limited describes the
preparation of microparticles by spray drying an aqueous protein solution to
form hollow spheres having gas entrapped therein, for use in imaging. WO
93/25242 describes the synthesis of microparticles for ultrasonic imaging
consisting of a gas contained within a shell of polycyanoacrylate or
polyester. WO 92/21382 discloses the fabrication of microparticle contrast
agents which include a covalently bonded matrix containing a gas, wherein
the matrix is a carbohydrate. U.S. Patent Nos. 5,334,381, 5,123,414 and
5,352,435 to Unger describe liposomes for use as ultrasound contrast agents,
which include gases, gas precursors, such as a pH activated or photo-
activated gaseous precursor, as well as other liquid or solid contrast
enhancing agents.
Others have looked at the effect of the gas which is encapsulated, and
suggested the use of fluorinated gases to enhance imaging as compared to
air. U.S. Patent No. 5,393,524 to Quay discloses the use of agents, including
perfluorocarbons, for enhancing the contrast in an ultrasound image. The
agents consist of small bubbles, or microbubbles, of selected gases, which
exhibit long life spans in solution and are small enough to traverse the
lungs,
enabling their use in ultrasound imaging of the cardiovascular system and
other vital organs. EP 554213 by Bracco discloses the use of fluorinated
hydrocarbon gases to prevent collapse of microvesicles upon exposure to
pressure in the bloodstream. WO 95/23615 by Nycomed discloses
microcapsules for imaging which are formed by coacervation of a solution,
for example, a protein solution, containing a perfluorocarbon. WO 95/03357
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by Massachusetts Institute of Technology discloses microparticles formed of
polyethylene glycol-poly(lactide-co-glycolide) block polymers having
imaging agents encapsulated therein, including gases such as air and
perfluorocarbons. As described in WO 94/16739 by Sonus Pharmaceuticals,
Inc., while solids and liquids reflect sound to a similar degree, gases are
known to be more efficient and are the preferred media for use as ultrasound
contrast agents. In fact, as shown by Example 12 of WO 94/16739, protein
microcapsules were dismissed as raising safety concerns (as well as efficacy
issues) when administered to mini-pigs. U.S. Patent Numbers 6,132,699 and
5,611,344 both describe methods of enhancing contrast using
perfluorocarbon gases in synthetic polymeric shells. U.S. Patent No.
5,837,221 describes a method of making a porous polymeric microparticle
having a hydrophobic agent incorporated into the polymer to increase
echogenicity.
Several ultrasound contrast agents have been approved in either the
United States or Europe for very limited cardiac applications. OPTISON
(Amersham, Mallinkrodt) consists of heat denatured human albumin
microcapsules containing the gas octafluoropropane. Each mL of
microsphere suspension contains 5-8 x108 microspheres with a mean
diameter in the 2-4.5 micron size range and 220 g octafluoropropane.
These microspheres have not been approved for myocardial blood flow
assessment and have only been approved for ventricular chamber
enhancement. At high bolus doses (5 mL suspension or 1100 g
octafluoropropane), ventricular chamber enhancement lasts up to 5 minutes.
DEFINITY (Bristol Myers Medical Imaging) consists of
octafluoropropane containing lipid microspheres where the lipid shell is
comprised of the phospholipids DPPA, DPPC, and mPEG-DPPE. Each mL
of suspension contains 1.2 x 1010 microparticles having a mean diameter in
the 1.1-3.3 micron size range and 1100 .tg of octafluoropropane. The agent
is only approved for ventricular chamber enhancement and not myocardial
blood flow assessment. At a bolus dose of 700 L (for a 70kg person) or
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5133 g of gas, the agent has an enhancement duration in the ventricular
chambers of approximately 3.4 minutes.
IMAGENT (Photogen Inc.) consists of lipid microspheres
containing pefluorohexane where the lipid shell is comprised of the
phospholipid DMPC. Each mL of suspension contains 1.4 x 109
microparticles having a mean diameter less than 3 microns and 92 g of
perfluorohexane. The agent is only approved for ventricular chamber
enhancement and not myocardial blood flow assessment. At a bolus dose of
0.43 mL (for a 70kg person) or 40 g of gas, the agent has a mean
enhancement duration in the ventricular chambers of approximately 2.6
minutes.
In all cases, these commercial agents have limited utility and are not
approved for applications other than ventricular chamber enhancement and
provide mean image enhancement durations in the ventricular chambers
lasting for periods of 5 minutes or less. There is a lack of commercial
ultrasound contrast agents which allow enhanced images of the
cardiovascular system, particularly of the myocardium and the ventricular
chambers, for long duration. The agents described in the prior art when
administered as a bolus or short infusion result in images of the myocardium
which last for significantly less time than the amount of time required to
conduct a complete examination of the heart. Typically, the prior art agents
provide images that last for well below one minute for the myocardium. An
agent which can provide enhanced image durations exceeding one minute in
the myocardium and/or greater than 5 minutes in the ventricular chambers is
desirable.
It is therefore an object of the invention to provide a dosage
formulation containing microparticles that provides enhanced images and
images of long duration, particularly for cardiac applications.
It is another object of the invention to provide a kit for administering
the dosage formulation containing microparticles for use in ultrasound
imaging techniques.
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Summary of the Invention
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 typically includes one, two or up to
five doses, most preferably one or two doses, of 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 administered to a patient in a dose effective to enhance
ultrasound imaging in the ventricular chambers for more than five minutes
and/or in the mycocardium for more than a minute, and a dose ranging from
0.025 to 8.0 mg microparticles/kg body weight. Preferably the dose
administered to a patient ranges from 0.05 to 4.0 mg microparticles/kg body
weight. In a preferred embodiment, the ultrasound imaging is enhanced in
the ventricular chambers for more than 9 minutes and/or in the myocardium
for more than 2 minutes.
The dosage formulation typically is provided in a vial or in a 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 by adding the
water to the vial or syringe of the dry powder and shaking to yield an
isosmotic or isotonic suspension of microparticles. In the preferred
embodiment of this dosage formulation, the suspension contains 1.0 - 3.5 x
109 microparticles /mL of suspension or 25-50 mg microparticles/mL of
suspension with the most preferred concentration yielding a suspension
containing 1.5-2.8 x 109 microparticles/mL of suspension or 30-45 mg
microparticles/mL of suspension. In a preferred embodiment, the
microparticles have a mean particle size less than 8 microns, most preferably
a mean particle size of 1.8-3.0 microns.
In the most preferred embodiment, the gas is CF4, C2F4, C2F6, C3F6,
C3F8, C4F8, C4Flo, or SF6. In preferred embodiments, the gas is n-
perfluorobutane (C4Flo) provided in an amount between 75-500 jig/mL of
administered volume of microparticle suspension; preferably the n-
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perfluorobutane is provided in an amount between 100-400 .xg/mL of
administered volume of microparticle suspension and most preferably
between 150-350 g/mL of administered volume of microparticle
suspension; or the gas is n-octafluoropropane provided in an amount between
75-375 g/mL of administered volume of microparticle suspension, most
preferably between 120-300 g/mL of administered volume of microparticle
suspension.
In the most preferred embodiment, the microparticle is formed of a
synthetic polymer such as poly(hydroxy acids) which include poly(lactic
acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid),
polyglycolides, polylactides, and poly(lactide-co-glycolide), polyanhydrides,
polyorthoesters, polyamides, polycarbonates, polyalkylenes such as
polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene
glycol), polyalkylene oxides such as poly(ethylene oxide) polyvinyl alcohols,
poly(valeric acid), and poly(lactide-co-caprolactone), derivatives,
copolymers and blends thereof and includes a hydrophobic compound
incorporated with the polymer at a ratio of between 0.01 and 30% by weight
of hydrophobic compound to weight of polymer, most preferably a lipid
incorporated with the polymer at a ratio of between 0.01 and 30% (weight
lipid/weight polymer). In a particularly preferred embodiment, the lipid is
dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine
(DMPC), dipentadecanoylphosphatidylcholine (DPDPC)
dilauroylphosphatidylcholine (DLPC), dipymitoylphosphatidylcholine
(DPPC), distearoylphosphatidylcholine (DSPC),
diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine
(DBPC), ditricosanoylphosphatidylcholine (DTPC),
dilignoceroylphatidylcholine (DLGPC); or a phosphatidylethanolamine.
Most preferably, the synthetic polymer in the microparticles is
poly(lactide-co-glycolide), with a lactide to glycolide ratio of 50:50 (i.e.
1:1)
and a weight average molecular weight in the range 20,000-40,000 Daltons,
and the hydrophobic compound in the microparticles is DAPC, in a ratio of 5
to 6.6% (weight DAPC/ weight polymer).
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The dosage formulation may be provided as a vial or a syringe of dry
powder containing microparticles or in a kit including a solution for
resuspending the microparticles. Typically the vial or syringe of dry powder
will also include excipients such as sugars or salts to make the solution
isosmotic or isotonic after reconstitution. This dosage formulation is then
administered to a 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 computerized tomography. The microparticles were
tested in clinical trials for cardiology applications such as myocardial blood
flow assessment and ventricular chamber enhancement.
Detailed Description of the Invention
Improved methods, microparticles, kits, and dosage formulations for
ultrasound imaging are described herein. The microparticles are useful in a
variety of diagnostic ultrasound imaging applications, particularly in
ultrasound procedures such as blood vessel imaging and echocardiography
such as myocardial blood flow assessment, myocardial blood volume
assessment and ventricular chamber enhancement.
I. Definitions
As generally used herein, the term "microparticle" includes
"microspheres" and "microcapsules", as well as other microparticles, unless
otherwise specified. Microparticles may or may not be spherical in shape.
"Microcapsules" are defined herein as microparticles having an outer
polymer shell surrounding a core of a gas. "Microspheres" as defined herein
can be solid polymeric spheres, or porous spheres with a honeycombed
structure or sponge like structure formed by pores throughout the polymer
that are filled with a gas. Some microspheres may contain an outer polymer
shell with a honeycombed structure or a sponge like structure formed by
pores throughout the polymer shell and the pores are filled with gas. For this
type of microsphere, this outer polymer shell surrounds an internal core of
gas.
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As generally used herein, the terms "dosage" and "dose" are used
synonymously to refer to the amount of a substance that is given at one time
or the amount of substance that is required to produce the desired diagnostic
or contrast effect.
As used herein, the term "dosage formulation' 'refers to a vial or
other container such as a syringe, containing one or more dosages of
substance required to produce the desired diagnostic or contrast effect.
As generally used herein "region of a patient" refers to a particular
area or portion of the patient. In some instances "region of patient' 'refers
to
regions throughout the entire patient. Examples of such regions are the
pulmonary region, the gastrointestinal region, the cardiovascular region
(including myocardial tissue or myocardium (i.e. heart muscle), ventricular
chambers, atrial chambers, valve function), the renal region as well as other
body regions, tissues, organs and the like, including the vasculature and
circulatory systems, and as well as diseased tissue, including cancerous
tissue. "Region of a patient" includes, for example, regions to be imaged
with diagnostic imaging. The "region of a patient" is preferably internal,
although it may be external.
As generally used herein "vasculature" denotes blood vessels
(including arteries, veins, capillaries and the like).
As generally used herein "gastrointestinal region" includes the region
defined by the esophagus, stomach, small and large intestines, and rectum.
As generally used herein "renal region" refers to the region defined
by the kidney and the vasculature that leads directly to and from the kidney,
and includes the abdominal aorta.
As generally used herein "region to be targeted" and "targeted
region" are used interchangeably to refer to a region of a patient where
delivery of an agent is desired.
As generally used herein "region to be imaged" and "imaging region"
are used interchangeably to refer to a region of a patient where imaging is
desired.
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As generally used herein "ventricular blood flow or ventricular
chamber enhancement" refers to the flow of blood through the ventricles of
the heart in one or more cardiac cycles.
As generally used herein "atrial blood flow" refers to the flow of
blood through the atria of the heart in one or more cardiac cycles.
As generally used herein "myocardial blood flow" refers to the flow
of blood in the vasculature of the heart muscle or myocardium, including the
blood vessels in the heart, in one or more cardiac cycles.
As generally used herein "myocardial blood volume" refers to the
volume of blood in the vasculature of the heart muscle or myocardium.
As generally used herein "cardiac cycle" refers to a complete
contractile period of the heart, and includes both the diastole and systole
periods.
As generally used herein "increased brightness" refers to an increase
in the brightness of an image compared to an image obtained without an
ultrasound contrast agent.
As generally used herein "enhanced image" refers to an image which
has increased brightness relative to an image obtained without an ultrasound
contrast agent.
As generally used herein "duration" refers to the total time over
which increased brightness of an image can be detected.
As generally used herein "coronary vasodilator" refers to a bioactive
agent such as dipyridamole or adenosine which, when administered to a
patient, causes dilation of the vasculature in the cardiovascular region.
II. Microparticles
In the preferred embodiment, the microparticles contain a polymer, a
lipid and a perfluorocarbon gas. Microparticles may consist of both
microspheres and microcapsules, or only microspheres or microcapsules.
Polymers
In the preferred embodiment, the microparticles are formed from
synthetic polymers. Synthetic polymers produce microparticles that are
biocompatible and are not contaminated by biological materials.
Additionally, synthetic polymers are preferred due to more reproducible
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synthesis and degradation both in vitro and in vivo. The polymer is selected
based on the time required for in vivo stability, i.e., that time required for
distribution to the site where imaging is desired, and the time required for
imaging. Synthetic polymers may be modified to produce microparticles
with different properties (e.g. changing molecular weight and/or functional
groups).
Representative synthetic polymers are: poly(hydroxy acids) such as
poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic
acid),
polyglycolides, polylactides, poly(lactide-co-glycolide) copolymers and
blends, polyanhydrides, polyorthoesters, polyamides, polycarbonates,
polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols
such as poly(ethylene glycol), polyalkylene oxides such as poly(ethylene
oxide) polyvinyl alcohols, poly(valeric acid), and poly(lactide-co-
caprolactone), derivatives, copolymers and blends thereof. As used herein,
"derivatives" include polymers having substitutions, additions of chemical
groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art.
Examples of preferred biodegradable polymers include polymers of
hydroxy acids such as lactic acid and glycolic acid, polylactide,
polyglycolide, poly(lactide-co-glycolide), and copolymers with PEG,
polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid),
poly(valeric acid), poly(lactide-co-caprolactone), blends and copolymers
thereof. The most preferred polymer is poly(lactide-co-glycolide) with a
lactide to glycolide ratio of 50:50 (i.e. 1:1) and the polymer having a weight
average molecular weight in the range 20,000-40,000 Daltons. The weight
average molecular weight (Mw,) of the polymer is the average molecular
weight calculated on the basis of the mass of molecules with a given
molecular weight within the distribution of individual polymer chains. MW
can be determined using gel permeation chromatography (GPC).
Hydrophobic Compounds
In the preferred embodiment, the polymer includes a hydrophobic
compound, as described in U.S. Patent No. 5,837,221. In general,
incorporation of compounds such as lipids which are hydrophobic and in an
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effective amount within the polymers, limits penetration and/or uptake of
water by the microparticles and thus limits gas loss from the microparticles.
This is effective in increasing the duration of enhanced imaging provided by
microparticles that contain a lipid, a synthetic polymer and a gas
encapsulated therein, especially fluorinated gases such as perfluorocarbons.
Lipids which may be used to stabilize gas inside the polymeric
microparticles include but are not limited to the following classes of lipids:
fatty acids and derivatives, mono-, di and triglycerides, phospholipids,
sphingolipids, cholesterol and steroid derivatives, terpenes and vitamins.
Fatty acids and derivatives thereof may include but are not limited to
saturated and unsaturated fatty acids, odd and even number fatty acids, cis
and 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,
molecules that have between 12 carbon atoms and 22 carbon atoms in either
linear or branched form. Examples of saturated fatty acids that may be used
include, but are not limited to, lauric, myristic, palmitic, and stearic
acids.
Examples of unsaturated fatty acids that may be used include, but are not
limited to lauric, physeteric, myristoleic, palmitoleic, petroselinic, and
oleic
acids. Examples of branched fatty acids that may be used include, but are
not limited to, isolauric, isomyristic, isopalmitic, and isostearic acids and
isoprenoids. 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-dioleoylphosphatidylethanol amine and
palmitoyl-homocysteine; and/or combinations thereof. Mono, di and
triglycerides or derivatives thereof that may be used include, but are not
limited to molecules that have fatty acids or mixtures of fatty acids between
6 and 24 carbon atoms, digalactosyldiglyceride, 1,2-dioleoyl-sn-glycerol;
1,2-dipalmitoyl-sn-3 succinylglycerol; and 1,3-dipalmitoyl-2-
succinylglycerol.
Phospholipids which may be used include but are not limited to
phosphatidic acids, phosphatidyl cholines with both saturated and
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unsaturated lipids, phosphatidyl ethanolamines, phosphatidylglycerols,
phosphatidylserines, phosphatidylinositols, lysophosphatidyl derivatives,
cardiolipin, and (3-acyl-alkyl phospholipids. Examples of phospholipids
include, but are not limited to, phosphatidylcholines such as
dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine
(DMPC), dipentadecanoylphosphatidylcholine (DPDPC),
dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine
(DPPC), distearoylphosphatidylcholine (DSPC),
diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine
(DBPC), ditricosanoylphosphatidylcholine (DTPC),
dilignoceroylphatidylcholine (DLPC); and phosphatidylethanolamines such
as dioleoylphosphatidylethanolamine or 1-hexadecyl-2-
palmitoylglycerophosphoethanolamine. Synthetic phospholipids with
asymmetric acyl chains (e.g., with one acyl chain of 6 carbons and another
acyl chain of 12 carbons) may also be used.
Sphingolipids which may be used include ceramides,
sphingomyelins, cerebrosides, gangliosides, sulfatides and lysosulfatides.
Examples of sphinglolipids include, but are not limited to, the gangliosides
GM 1 and GM2.
Steroids which may be used include but are not limited to cholesterol,
cholesterol sulfate, cholesterol hemisuccinate, 6-(5-cholesterol 3(3-yloxy)
hexyl-6-amino-6-deoxy-l-thio-a-D-galactopyranoside, 6-(5-cholesten-3 [3-
tloxy)hexyl-6-amino-6-deoxyl-l-thio-a-D mannopyranoside and cholesteryl)
4' -trimethyl 35 ammonio)butanoate.
Additional lipid compounds which may be used include tocopherol
and derivatives, and oils and derivatized oils such as stearlyamine.
A variety of cationic lipids such as DOTMA, N-[1-(2,3-dioleoyloxy)
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 may be used.
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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 0.01-30% (w lipid/w polymer);
preferably between 0.1-20% (w lipid/w polymer) and most preferably 1-12%
(w lipid/w polymer).
When formed by the methods described herein, the size of the
microparticles is consistently reproducible. As used herein, the terms "size"
or "diameter" in reference to particles refers to the number average particle
size, unless otherwise specified. An example of an equation that can be used
to define the number average particle size (Xõ) is shown below:
oo
nidi
X
n
ni
where n, = number of particles of a given diameter (d).
As used herein, the term "volume average diameter" refers to the
volume weighted diameter average. An example of equations that can be
used to define the volume average diameter (Xv) is shown below:
V3
nidi3
x
v
ni
i.1
where nz = number of particles of a given diameter (d;).
Particle size analysis can be performed on a Coulter counter, by light
microscopy, scanning electron microscopy, transmittance electron
microscopy, laser diffraction methods such as those using a Malvern
Mastersizer, light scattering methods or time of flight methods. As used
herein "Coulter method" refers to a method in which the powder is dispersed
in an electrolyte, and the resulting suspension analyzed using a Coulter
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Multisizer II fitted with a 50- m aperture tube. This method provides size
measurements and particle concentrations.
In the preferred embodiment for the preparation of injectable
microparticles capable of passing through the pulmonary capillary bed, the
microparticles have a diameter less than eight microns. Larger
microparticles may clog the pulmonary bed, and smaller microparticles may
not provide sufficient contrast effect. The preferred microparticle size for
an
intravenously administered ultrasound contrast agent is between 0.75
microns and 5 microns and is most preferably between 1.8 and 3.0 microns.
In the preferred embodiment, the microparticles have a honeycombed
structure or sponge like structure, formed by pores throughout the polymer or
the microparticles have a polymeric shell with a honeycombed or sponge
like, porous structure. In both cases the pores are filled with gas. These
microparticles are formed by spray drying a polymer solution containing a
pore forming agent such as a volatile salt as described below.
Ultrasound Contrast Imaging Agents
Examples of fluorinated gases include CF4, C2F4, C2F6, C3F6, C3F8,
C4F8, C4F10, and SF6. n-Perfluorobutane (C4F10) is particularly preferred
because it provides an insoluble gas that will not condense at the temperature
of use and is pharmacologically acceptable.
The amount of gas contained with the microparticles will depend on
the type of gas but is typically between 75-500 gg/mL of administered
volume of microparticle suspension. For n-perfluorobutane, the preferred
gas content is between 100-400 g/mL of administered volume of
microparticle suspension and most preferably is between 150-350 g/mL of
administered volume of microparticle suspension. For n-octafluoropropane,
the preferred gas content is between 75-375 gg/mL of administered volume
of microparticle suspension, and most preferably between 120-300 gg/mL of
administered volume of microparticle suspension.
III. Methods for making Microparticles
The microparticles may be produced by a variety of methods, and are
preferably produced by spray drying. A major criterion is that the polymer
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must be dissolved or melted with the hydrophobic compound or lipid, prior
to forming the microparticle.
Solvents
During formation, the polymer is generally dissolved in a solvent. As
defined herein, the polymer solvent is an organic solvent that is volatile or
has a relatively low boiling point or can be removed under vacuum and
which is acceptable for administration to humans in trace amounts, such as
methylene chloride. Other solvents, such as ethyl acetate, ethyl formate,
ethanol, methanol, dimethyl formamide (DMF), acetone, acetonitrile,
tetrahydrofuran (THF), formamide, acetic acid, dimethyl sulfoxide (DMSO)
and chloroform also may be utilized, or combinations thereof. In general,
the polymer is dissolved in the solvent to form a polymer solution having a
concentration of between 0.1 and 60% weight to volume (w/v), more
preferably between 0.25 and 30% (w/v) and most preferably between 0.5-
10% (w/v).
Spray Drying
Microparticles are preferably produced by spray drying by dissolving
a biocompatible polymer and lipid in an appropriate solvent, dispersing a
pore forming agent as a solid or as a solution into the polymer solution, and
then spray drying the polymer solution and the pore forming agent, to form
microparticles. As defined herein, the process of "spray drying" a solution
of a polymer and a pore forming agent refers to a process wherein the
polymer solution and pore forming agent are atomized to form a fine mist
and dried by direct contact with hot carrier gases. Using spray dryers
available in the art, the polymer solution and pore forming agent may be
atomized at the inlet port of the spray dryer, passed through at least one
drying chamber, and then collected as a powder. The temperature may be
varied depending on the gas or polymer used. The temperature of the inlet
and outlet ports can be controlled to produce the desired products.
The size and morphology of the microparticles formed during spray
drying is a function of the nozzle used to spray the polymer solution and the
pore forming agent, the nozzle pressure, the flow rate of the polymer solution
with the pore forming agent, the polymer used, the concentration of the
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polymer in solution, the type of polymer solvent, the type and the amount of
pore forming agent, the temperature of spraying (both inlet and outlet
temperature) and the polymer molecular weight. Generally, the higher the
polymer molecular weight, the larger the particle size, assuming the polymer
solution concentration is the same.
Typical process parameters for spray drying are as follows: inlet
temperature = 30-200 C, outlet temperature = 5-100 C, and polymer flow
rate = 10-5,000 ml/min.
A gaseous diagnostic agent may be encapsulated by emulsifying the
gas with the polymer solution and the pore forming agent prior to spray
drying. Alternatively, air filled microparticles can be produced during the
spray drying step and subsequently the air replaced with the perfluorocarbon
gas by applying a stream of the desired gas to the microparticles, or pulling
a
vacuum on the microparticles to remove the encapsulated air, then filling
with the desired perfluorocarbon gas. A lyophilizer or vacuum chamber may
be used if a vacuum step is used to exchange the gas.
Additives to Facilitate Microparticulate Formation
A variety of surfactants may be added during the formation of the
microparticles. Exemplary emulsifiers or surfactants which may be used
(0.1-15% w/ w polymer) include most physiologically acceptable
emulsifiers. Examples include natural and synthetic forms of bile salts or
bile acids, both conjugated with amino acids and unconjugated such as
taurodeoxycholate, and cholic acid.
Pore forming agents are included in the polymer solution in an
amount of between 0.01% and 90% weight to volume of polymer solution, to
increase pore formation. For example, in spray drying, a pore forming agent
such as a volatile salt, for example, ammonium bicarbonate, ammonium
acetate, ammonium carbonate, ammonium chloride or ammonium benzoate
or other volatile salt as either a solid or as a solution in a solvent such as
water can be used. The solid pore forming agent or the solution containing
the pore forming agent is then emulsified with the polymer solution to create
a dispersion or droplets of the pore forming agent in the polymer. This
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dispersion or emulsion is then spray dried to remove both the polymer
solvent and the pore forming agent. After the polymer is precipitated, the
hardened microparticles can be frozen and lyophilized to remove any pore
forming agent not removed during the polymer precipitation step.
The preferred microparticle is formed using the polymer,
poly(lactide-co-glycolide) with a lactide to glycolide ratio of 50:50 and
having a weight average molecular weight in the range 20,000-40,000
Daltons, and the phospholipid, diarachidoylphosphatidylcholine ((1,2-
diarachidoyl-sn-glycero-3-phosphocholine (DAPC) ) at a ratio of 5-6.6 %
(w DAPC/w polymer). The microparticles are further formulated in a
solution of mannitol and TWEEN 80 and processed to yield a dry powder
of microparticles which are backfilled on a lyophilizer with n-
perfluorobutane. The dry powder is reconstituted with 5 mL of sterile water
prior to use by adding the water to the vial of the dry powder and shaking to
yield a suspension of microparticles in isosmotic mannitol. The preferred
properties of the suspension are a gas content of 150-350 .tg/mL of n-
perfluorobutane per administered volume of microparticle suspension, 1.5-
2.8 x 109 microparticles/mL of administered volume of microparticle
suspension, 30-45mg microparticles/mL of administered volume of
microparticle suspension, and a mean particle size in the range 1.8-3.0
microns.
IV. Applications for the Microparticles
1. Formulations for Administration to a Patient
The microparticles may undergo further processing with excipients to
create a dry powder. The excipients provide tonicity or osmolarity or ease of
suspendability of the microparticles after reconstitution with a
pharmaceutically acceptable carrier prior to administration to a patient.
Excipients suitable for providing osmolarity or tonicity are sugars including
but not limited to mannitol, dextrose or glucose and salts including but not
limited to sodium chloride or sodium phosphate. Excipients suitable for
providing ease of suspendability of the microspheres include any
pharmaceutically acceptable wetting agent or surfactant including but not
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limited to polysorbate 80 (TWEEN 80), polysorbate 20 (TWEEN 20),
Pluronic or polyethylene glycol. Excipients suitable for providing
osmolarity or tonicity or that can be used as wetting agents are described in
references such as the Handbook of Pharmaceutical Excipients (Fourth
Edition, Royal Pharmaceutical Society of Great Britain, Science & Practice
Publishers) or Remingtons: The Science and Practice of Pharmacy
(Nineteenth Edition, Mack Publishing Company). The dry powder of
microparticles and excipients is created by suspending the microparticles in a
solution of excipients. Further size fractionation steps may be used if
needed. The microparticles in the solution of excipients are filled into vials
or syringes, frozen, and lyophilized to create the dry powder formulation. At
the conclusion of the lyophilization step, the microparticles are filled with
the perfluorocarbon gas by backfilling the lyophilizer with the
perfluorocarbon gas. The vials or syringes are then stoppered or capped and
in the case of vials, crimped. This results in a perfluorocarbon headspace in
the vial or syringe.
Alternatively, the microparticles can be dry blended with the
pharmaceutical excipients and then filled into vials or syringes. The
microparticles can be filled with the perfluorocarbon gas by applying a
vacuum after loading the vials or syringes on a lyophilizer or in a vacuum
chamber. The vials or syringes are then stoppered or capped and in the case
of vials, crimped. This results in a perfluorocarbon headspace in the vial or
syringe.
2. Dosage Units
Different size dosage units of microparticles may be used. For
example a small dosage unit may contain 25-75 mg of microparticles. An
intermediate dosage unit may contain 75-150 mg. A large dosage unit may
contain 150-250 mg of microparticles. An extra large dosage unit may
contain 250-1000 mg of microparticles.
When the suspension of microparticles is formed following
reconstitution, the mass concentration of microspheres in the suspension
typically ranges from 20 to 60 mg/mL. The preferred mass concentration of
microspheres in the suspension is 25-50 mg/mL; and the most preferred mass
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concentration of microspheres in the suspension is 30 to 45 mg/mL. The
preferred concentration of microparticles in the suspension is 1.0-3.5 x 109
microparticles/mL of suspension; and the most preferred concentration of
microparticles in the suspension is 1.5-2.8 x 109 microparticles/mL. The
microparticles have a preferred mean particle size of less than 8 microns,
most preferably in the range 1.8-3.0 microns.
Pharmaceutically acceptable carriers may include water for injection,
sterile water, saline, saline containing glycerol, saline containing TWEEN
20, saline containing TWEEN 80, isosmotic dextrose (5%), %2 isosmotic
dextrose (2.5%), isosmotic mannitol (5%), %2 isosmotic mannitol (2.5%),
isotonic mannitol containing TWEEN 20 and isotonic mannitol containing
TWEEN 80.
3. Kits
Kits for parenteral administration of the microparticles containing the
perfluorocarbon gas may be provided. The kit contains at least two
components. One component contains a dosage unit of the dry powder
contrast agent in a vial or syringe, and the other component contains a
pharmaceutically acceptable carrier in a vial or syringe. Prior to
administration to a patient, the pharmaceutically acceptable carrier is added
to the dosage unit of the dry powder contrast agent to form a suspension of
gas filled microparticles that are usable as an ultrasound imaging contrast
agent in diagnostic imaging by any route of administration.
4. Vials or Containers for microparticles
No specific vial or syringe or connection systems are required for the
kits; conventional vials, syringes and adapters may be used with the
microparticles. The only requirement for a vial is a good seal between the
stopper and the container. The quality of the seal, therefore, becomes a
matter of primary concern; any degradation of seal integrity could allow
undesirables substances to enter the vial or allow the gas to escape. In
addition to assuring sterility, vacuum retention is essential for products
stoppered at reduced pressures to assure safe and proper reconstitution. As
to the stopper, it may be a compound or multicomponent formulation based
on an elastomer, such as poly (isobutylene) or "butyl rubber" and must be
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impermeable to the gas used. The vial size is selected depending on the total
dosage of dry powder in the vial. Preferred vial sizes are 5mL, 10 mL, 20
mL and 30 mL. The syringe size is selected depending on the total dosage of
dry powder in the syringe. Preferred syringe sizes are 5mL, l OmL, 20mL,
and 50 mL syringes.
5. Diagnostic Applications
The microparticle compositions may be used in a many different
diagnostic applications including ultrasound imaging, magnetic resonance
imaging, fluoroscopy, x-ray, and computerized tomography.
In the preferred embodiment, the microparticles are used in
ultrasound procedures such as blood vessel imaging and echocardiography
including but not limited to ventricular chamber imaging, myocardial blood
flow assessment, myocardial blood volume assessment, diagnosis of
coronary artery disease, and ejection fraction assessment.
The microparticles may be used in vascular imaging, as well as in
applications to detect liver and renal diseases, in detecting and
characterizing
tumor masses and tissues, and in measuring peripheral blood velocity. The
microparticles also can be linked with ligands that minimize tissue adhesion
or that target the microparticles to specific regions of the body in vivo.
General Method of obtaining images
The microparticles in dry powder form are reconstituted with a
pharmaceutically acceptable carrier prior to administration, then an effective
amount for detection is administered to a patient using an appropriate route,
by injection into a blood vessel (such as intravenously (i.v.) or intra-
arterially
(i.a.)), or orally. The microparticle composition may be administered
intravenously to the patient as a bolus injection or short infusion (less than
minutes). Preferably the injection is administered over a time period
ranging from 15 seconds to 20 minutes, most preferably ranging from 30
seconds to 15 minutes. Typically, a dose ranging from 0.025 to 8 mg/kg
30 body weight per injection is administered intravenously to a patient,
preferably the dose ranges from 0.05 to 4 mg/kg.
For diagnostic ultrasound applications, energy is applied to at least a
portion of the patient to image the target tissue. A visible image of an
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internal region of the patient is then obtained, such that the presence or
absence of diseased tissue can be ascertained. Ultrasonic imaging
techniques, including second harmonic imaging and gated imaging, are well
know in the art and are described, for example, in Uhlendorf, IEEE
Transactions on Ultrasonics, Ferroelectrics, and Frequency Control,
14(1):70-79 (1994) and Sutherland, et al., Journal of the American Society of
Echocardiography, 7(5):441-458 (1994),
Ultrasound waves may be applied with a transducer. The ultrasound
can be pulsed or it may be continuous, if desired. Thus, diagnostic
ultrasound generally involves the application of echoes, after which, during a
listening period, the ultrasound transducer receives reflected signals.
Harmonics, ultraharmonics or subharmonics may be used. The second
harmonic mode may be beneficially employed, in which the 2x frequency is
received, where x is the incidental frequency. This may serve to decrease the
signal from the background material and enhance the signal from the
transducer using the imaging agents, which may be targeted to a desired site,
for example, blood clots. Other harmonic signals, such as odd harmonics
signals, for example, 3x or 5x, would be similarly received using this
method. Subharmonic signals, for example, x/2 and x/3, may also be
received and processed so as to form an image.
In addition, Power Doppler or Color Doppler may be applied. In the
case of Power Doppler, the relatively higher energy of the Power Doppler
may resonate the vesicles. This can create acoustic emissions which may be
in the subharmonic or ultraharmonic range or in some cases, in the same
frequency as the applied ultrasound.
Specific Imaging Applications
The microparticles described herein can be used in both cardiology
and radiology applications. For cardiology applications, the microparticle
compositions are administered to a patient and the patient is scanned using
an ultrasound machine to obtain visible images of the cardiovascular region.
Optionally the microparticle composition is administered in combination
with a pharmacological stressor or a physical stressor. Suitable
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pharmacological stressors include a coronary vasodilator such as
dipyridamole or adenosine, an inotropic agent (i.e. increases the strength of
heart contraction) such as dobutamine or a chronotropic agent (i.e. increases
the frequency of contraction) such as dobutamine. Suitable physical
stressors include physical exercise, such as by using a treadmill or a
stationary bicycle.
For radiology applications, the microparticle compositions are
administered to a patient and the patient is scanned using an ultrasound
machine to obtain visible images of the region of a patient to be examined.
The microparticles can be used to assess the function of the
cardiovascular system as well as to assess myocardial blood flow or
myocardial blood volume or to diagnose coronary heart disease (coronary
artery disease). For example the microparticles can enhance images of the
ventricular chambers and thus assist in regional cardiac function analysis
through wall motion analysis and assist in global cardiac function through
ejection fraction measurements. The microparticles can also be used to
assess myocardial blood flow to differentiate functioning cardiac tissue from
either ischemic (blood flow deficient) cardiac tissue or infarcted (dead)
cardiac tissue. The contrast signals detected in the myocardium can be used
as an estimate of myocardial blood volume since ultrasound contrasts agents
reside intravascularly following intravenous administration. The absence or
reduction in contrast intensity or image brightness in a particular myocardial
region over time is indicative of reduced blood flow (i.e. a defect).
Most often unless the patient has severe coronary heart disease, blood
flow to the various regions of the heart as assessed by techniques such as
ultrasound contrast will appear normal. In order to detect blood flow
abnormalities in patients without severe heart disease or to detect smaller
myocardial blood flow defects, it is necessary to increase the blood flow
requirements to the heart by inducing a state of stress. Stress can be induced
by having the patient exercise or by administering a pharmacological
compound such as a vasodilator, an inotropic agent or a chronotropic agent.
During exercise or pharmacological stress, blood flow defects can be more
easily detected because the ability to increase blood flow is reduced in
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regions supplied by coronary arteries with stenosis. A comparison of
ultrasound images of the myocardium following ultrasound contrast agent
administration can be made both in the pre-stress state (i.e. rest state) and
in
the stress state. A myocardial region without enhanced brightness found
during stress imaging but not during rest imaging is indicative of ischemia.
A myocardial region without enhanced brightness found during stress
imaging and during rest imaging is indicative of an infarct.
In one embodiment, the myocardial blood flow can be measured by
(1) administering a first injection of a microparticle composition to a
patient,
(2) scanning the patient using an ultrasound machine imaging to obtain a
visible image of the cardiovascular region, (3) inducing a state of stress in
the patient using a pharmacological stressor or exercise, (4) administering a
second injection of the microparticle composition and continuing the
scanning, and (5) assessing differences in the images obtained in steps (2)
and (4) either visually or using quantitative image analysis.
For radiology applications, the microparticles may be used to
improve the capabilities of ultrasound imaging for radiology indications,
including imaging of the kidney, liver and peripheral vascular disease,
increasing the visibility of blood flow and blood flow patterns and by
improving the detection of small lesions or structures deep within the body.
The microparticles can be used for both macrovascular and microvascular
indications. In macrovascular indications (the diagnosis of disease states and
conditions of major arteries and veins of the body), the microparticles may
aid in the detection of strokes and pre-stroke conditions through
visualization
of intracranial blood vessels, detecting atherosclerosis in large vessels such
as the carotid arteries by assessing the degree of carotid artery stenosis,
vascular graft patency and peripheral vascular thrombosis. For
microvascular indications (the diagnosis of disease states and through
analysis of patterns of small vessel blood flow), the microparticles may aid
in identifying lesions, tumors or other diseases in the liver (e.g. adenomas
or
hemangiomas), kidneys, spleen (e.g. splenic artery aneurysms), breasts and
ovaries and in other tissues and organs.
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Diseased tissues in a patient may be diagnosed by administering the
microparticle composition to the patient and scanning the patient using the
ultrasound imaging to obtain visible images of any diseased tissues in the
patient. Diseased tissues may manifest as a region of enhanced brightness or
a region that does not show enhanced brightness.
Enhanced Images obtained using Microparticle compositions
The microparticles produce an enhanced image following
administration. Enhanced images may be manifested by an increase in
brightness in the image compared to when no ultrasound contrast agent is
administered or by substantial elimination of artifacts in the image. Thus, in
connection with ultrasound imaging of the cardiovascular region, including
the heart tissue and the vasculature associated therewith, an enhanced image
may be manifested, for example, by increased brightness in the image of the
cardiovascular region and/or a substantial elimination in the occurrence of
artifacts in the image of the cardiovascular region. The images following a
single administration of the agent last for between 10 seconds and 60
minutes. The images preferably last for between 20 seconds and 30 minutes
and most preferably last for between 30 seconds and 20 minutes. In a
preferred embodiment, the ultrasound imaging is enhanced in the ventricular
chambers for more than five minutes or in the myocardium for more than
one minute.
The increase in brightness in the image may be assessed either
visually by the naked eye or using quantitative image analysis. With
particular reference to the gray scale (about 0 to about 255 VDUs or gray
levels) identified above, there is preferably an increase in the level of
brightness of at least about 10 VDUs (gray levels). More preferably, the
image has an increased brightness of greater than about 10 VDUs, 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 increased brightness is greater
than about 100 VDUs, for example, about 105, 110, 115, 120, 125, 130, 135,
140, 145, or 150 VDUs. In other embodiments, the increased brightness is
greater than about 150 VDUs, for example, about 155, 160, 165, 170, 175,
18-, 185, 190, 195, or 200 VDUs. Alternatively, the increased brightness is
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greater than about 200 VDUs, for example about 205, 210, 215, 220, 225,
230, 235, 240, 245, 250, or 255 VDUs.
The methods and compositions described above will be further
understood with reference to the following non-limiting examples.
Examples
Materials
Acetic acid, ammonium bicarbonate, mannitol USP, and polysorbate
80 (no animal-derived components) were purchased from Spectrum
Chemicals, Gardena, CA. Polymer (poly(lactide-co-glycolide) (PLGA)
(50:50)) and diarachidoylphosphatidylcholine (1,2-diarachidoyl-sn-glycero-
3-phosphocholine (DAPC)) were obtained from Boehringer Ingelheim
(Ingelheim, Germany) and Avanti (Alabaster, AL), respectively. Methylene
chloride was purchased from EM Science (EMD Chemicals, Gibbstown,
NJ). Vials (30 ml tubing vials) and stoppers (20 mm, gray, single-vent,
Fluro-Tec) were obtained from West Pharmaceutical Services (Lionville,
PA). n-Perfluorobutane (DFB) gas was purchased from F2 Chemicals Ltd,
Lancashire, UK.
Analytical Methods
Quantitation of Mass Concentration of Microparticles
The mass concentration of microparticles in vials was quantitated
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 microparticles was
determined based on a comparison of the amount of tin found in the
microparticles to the amount of tin found in the specific lot of polymer used
to make the microparticles. 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 phosphorous found in the microparticles in
comparison to the amount of phosphorus in the phospholipid itself. The
microparticle mass per mL of suspension was calculated by adding the
amount of polymer and phospholipid per vial and then dividing that sum by
the reconstitution volume (5 mL).
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Particle Size Analysis
A sample of reconstituted microparticles was added to an electrolyte
solution, and the resulting suspension analyzed for particle size and
microparticle concentration using a Coulter Multisizer II fitted with a 50 gm
aperture tube.
Gas Content of Microparticles
Vials of the dry powder were reconstituted with 5mL water and
shaken to create the microparticle suspension. The resulting suspension was
analyzed for DFB content by withdrawing a set of 0.3 mL aliquots through
the stopper using a needle and syringe. These aliquots were injected into
sealed headspace vials. The headspace vials equilibrated for at least 10
hours at room temperature. Samples were then heated 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 packed inlet
and a flame ionization detector. Quantitation 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
Sampling: Headspace, 1 mL sample loop
Detector: FID
Column: Supelco 60/80 Carbopack B 5%
Fluorocol
Inlet Temperature: 150 C
Detector Temperature: 325 C
Carrier Gas: Helium (25 mL/min)
FID Gases: Hydrogen (60 mL/min)
Air (350 mL/min)
Nitrogen (5 mL/min)
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Table 2: GC Temperature Program
Initial Rate Final Temp. Hold Time
Temp.
Initial Cond. 40 C N/A N/A 2.0 min
First Ramp 40 C 5 C/min 65 C 0.0 min
Second Ramp 65 C 10 C/min 130 C 0.0 min
Third Ramp 130 C 50 C/min 200 C 0.0 min
Final Cond. 200 C N/A N/A 3.1 min
Example 1: Production of Microparticles for Use as an Ultrasound
Contrast Agent
An organic solution was prepared by dissolving 176 g of PLGA, 10.6
g of diarachidoylphosphatidylcholine (1,2-diarachidoyl-sn-glycero-3-
phosphocholine (DAPC)), and 2.26 g of acetic acid in 5.88 L of methylene
chloride at 25 C. An aqueous solution composed of 68.5 g of ammonium
bicarbonate dissolved in 338 ml of water for injection was added to the
organic solution and homogenized for 10 minutes at 4310 RPM in a I OL
homogenization tank using a rotor-stator emulsifying mixer.
The resulting emulsion was spray dried using nitrogen as both the
atomizing and drying gas. Emulsions were spray dried on a bench top,
spray dryer using an air-atomizing nozzle from Spraying Systems (Wheaton,
IL) and a glass drying chamber/cyclone system from Buchi (Brinkmann,
Westbury, NY). Spray drying conditions were as follows: 40 ml/min
emulsion flow rate, 30 L/min atomization gas rate, 46 kg/hr drying gas rate,
and 12 C outlet temperature.
The spray dried product was further processed through dispersion,
freezing, and lyophilization steps. An aqueous vehicle was prepared by
dissolving 140 g of mannitol and 4.10 g of polysorbate 80 in 5.0 L of water.
The spray dried microparticles were dispersed in the vehicle at a
concentration of 25 mg/ml. The dispersion was deaggregated using a
stainless steel, 800 series, flow-cell sonicator from Misonix Incorporated
(Farmingdale, NY) and sieved through a 10" diameter vibratory sieve (RBF-
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10) from Vorti-Siv (Salem, OH). The sonicator was jacketed at 4 C to
prevent heating of the dispersion. The dispersion was sieved through 25 m
and 20 gm screens in series at 150 mL/min. The sieved dispersion was filled
into vials (10 ml fill in 30 ml vials), partially stoppered, and frozen by
immersion in liquid nitrogen.
Following freezing, the vials were lyophilized. At the conclusion of
lyophilization, the chamber was isolated, and n-perfluorobutane (DFB) was
backfilled into the vials to a pressure of -5 kilopascals prior to stoppering.
The dry powder was reconstituted with 5 mL of sterile water prior to
use by adding the water to the vial of the dry powder and shaking to yield a
suspension of microparticles in isosmotic mannitol. The suspension
contained 2.2 x 109 microparticles/mL of suspension, and 37 mg
microparticles/mL of suspension and the microparticles had a mean particle
size of 2.2 microns.
Example 2: Rate of Gas Leakage from the Microparticles
The rate of gas leakage from two separate batches (Batch 1 and Batch
2) of microparticles as produced by the methods of Example 1 was assessed
using gas chromatography (GC) as described in the analytical methods
sections. A third lot of microspheres (Batch 3) was produced similar to the
method of example 1, however, the phospholipid,
diarachidoylphosphatidylcholine (1,2-diarachidoyl-sn-glycero-3-
phosphocholine (DAPC)) was omitted during the production of the
microparticles.
Table 3: Gas Content and Rate of Gas Leakage for Microparticles
Gas Content Gas Content % Gas
( g/mL suspension) ( g/ml, suspension) Content Lost
Immediately 70 minutes Following over 70 minutes
Following Vial Vial Reconstitution
Reconstitution
Batch 1 341 312 9%
Batch 2 259 232 10%
Batch 3 139 18 87%
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The microparticles which contained DAPC lost approximately 10%
of the starting gas content after 70 minutes whereas the microparticles which
did not contain DAPC lost 87% of the starting gas content. Additionally, the
microparticles which contained DAPC had a higher starting initial gas
content relative to the microparticles without the DAPC. This indicates that
the inclusion of DAPC is important to the formation of the internal porous
structure of the microparticles during spray drying as well on retention of
gas
within the microparticles.
The total duration of intended use of an ultrasound contrast agent
following administration to a subject is generally on the order of 30 seconds
to 60 minutes depending on the type of cardiology or radiology ultrasound
examination conducted. Thus gas loss from the microparticles containing
the lipid DAPC is estimated to be insignificant over the period of the
ultrasound examination
Example 3: Cardiac Image Enhancement as a Function of Microparticle
Dose
Microparticles as produced by the method in Example 1 were studied
in healthy human adults. The dry powder was reconstituted prior to use by
adding 5 mL of sterile water to the vial and shaking the vial ten times. The
final concentration of microspheres in the resulting suspension was
approximately 37 mg/mL. Subjects received a single dose of either
0.5mg/kg, 2.0mg/kg or 4.0 mg/kg body weight. Subjects underwent
transthoracic ultrasound imaging using continuous harmonic imaging (frame
rate 15 Hz and transducer frequency 2.1/4.2 MHz). Images were visually
assessed for intensity and duration of enhancement.
The duration of enhancement in the ventricular chamber exceeded 9
minutes at both the 2 mg/kg and 4 mg/kg doses. The contrast effect was still
apparent in 13 out of 15 of the subjects at these two doses when the subjects
were re-imaged at 30 minutes, indicating the long duration of enhancement
provided by the microparticles.
The duration of ventricular chamber enhancement is summarized in
the Table 4.
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Table 4: Duration of Left Ventricular Image Enhancement
Dose Mean Duration of
(mg/kg body weight) Ventricular Chamber
Enhancement
(minutes)
0.5 2.6
2.0 >9.6
4.0 >9.6
Example 4: Comparison of Microparticles to Commercial Product for
Assessing Cardiac Images
A comparative cardiac ultrasound imaging study was conducted in
two adult men matched for body weight and cardiac function. The first
subject received a single administration of microparticles as produced by the
method of Example 1. The dry powder was reconstituted prior to use by
adding 5 mL of sterile water to the vial and shaking the vial ten times. The
final concentration of microspheres in the resulting suspension was
approximately 37 mg/mL and the gas content of the suspension was
approximately 250 g/mL suspension. The first subject received a dose of
4mg microparticle/kg which corresponds to a gas dose of 27 g/kg body
weight. The second subject received a single dose of the marketed
ultrasound contrast agent, OPTISON (Amersham Health) which contains
perfluoropropane containing albumin microspheres. The two subjects
received the same total amount of gas (27 g/kg body weight) which is the
acoustically active component. The two subjects underwent transthoracic
ultrasound imaging using continuous harmonic imaging (frame rate 15 Hz
and transducer frequency 2.1/4.2 MHz). Images were visually assessed for
intensity and duration of enhancement.
The duration of ventricular chamber enhancement and myocardial
enhancement is summarized in Table 5.
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Table 5: Duration of Image Enhancement with Different Ultrasound
Contrast Agents
Contrast Agent and Duration of Ventricular Duration of Myocardial
Dose of Gas Chamber Enhancement Enhancement (seconds)
Administered ( g/kg) (minutes)
Example # 1 > 9 160
Microparticles
(27gg/kg body weight)
OPTISON 1 10
(27 g/kg body weight)
The.microparticles produced using the method described in Example
1 provide enhanced images of both the ventricular chambers and the
myocardium which are significantly longer than OPTISON and which are
of appropriate duration to conduct a complete cardiac exam by ultrasound.
Example 5: Assessment of Myocardial Blood Flow to Assess Ischemia
Using Microparticle Formulations
Microparticles produced as per the method in Example 1 were
administered to a subject being evaluated for coronary heart disease. The
subject received two injections of the microparticles separated by 60
minutes. The first injection of the microparticles ("rest injection", 1.7
mg/kg) was used to assess the myocardium at rest. Prior to the second
injection of the microparticles, the subject was pharmacologically stressed
using the coronary vasodilator, dipyridamole (0.56 mg/kg). After the
induction of stress, the subject received a second injection of the
microparticles ("stress injection" 1.3 mg/kg) to assess the myocardium
under stress.
The comparison of the rest and stress images over time post
administration of the microparticles for the subject indicate a region of the
myocardium which has minimal increase in image enhancement and this
region becomes larger in size following the induction of the stress. This
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indicates the zone of myocardial tissue has both infarcted and ischemic
components. The detection of ischemia was confirmed using an alternate
diagnostic technique, nuclear imaging. Rest and stress nuclear perfusion
were conducted following the administration of 99Tc (MIBI) and the subject
was imaged using a commercial gamma counter. The defects noted on the
ultrasound rest and stress images were confirmed on the rest and stress
nuclear perfusion images.
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