U.S. patent application number 10/915921 was filed with the patent office on 2005-01-13 for paramagnetic polymerized protein microspheres and methods of preparation thereof.
This patent application is currently assigned to The Board of Trustees of the University of Illinois. Invention is credited to McDonald, Michael A., Watkin, Kenneth L..
Application Number | 20050008569 10/915921 |
Document ID | / |
Family ID | 26972050 |
Filed Date | 2005-01-13 |
United States Patent
Application |
20050008569 |
Kind Code |
A1 |
McDonald, Michael A. ; et
al. |
January 13, 2005 |
Paramagnetic polymerized protein microspheres and methods of
preparation thereof
Abstract
The present invention relates to a composition that includes
gadolinium particles encapsulated in microsphere shells. The
composition is suitable for use as a contrast agent with a
plurality of imaging modalities, including, for example,
ultrasound, magnetic resonance imaging, and computed temography.
The compositions also are useful for therapeutic applications,
including neutron capture therapy.
Inventors: |
McDonald, Michael A.;
(Champaign, IL) ; Watkin, Kenneth L.; (Champaign,
IL) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
TEN SOUTH WACKER DRIVE
SUITE 3000
CHICAGO
IL
60606
US
|
Assignee: |
The Board of Trustees of the
University of Illinois
Urbana
IL
|
Family ID: |
26972050 |
Appl. No.: |
10/915921 |
Filed: |
August 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10915921 |
Aug 11, 2004 |
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09976746 |
Oct 12, 2001 |
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6797257 |
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60300943 |
Jun 26, 2001 |
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Current U.S.
Class: |
424/1.11 |
Current CPC
Class: |
A61K 41/009 20130101;
A61K 9/5052 20130101; B82Y 5/00 20130101; A61K 49/225 20130101;
A61K 49/1821 20130101 |
Class at
Publication: |
424/001.11 |
International
Class: |
A61K 051/00 |
Claims
What is claimed:
1. A composition for use in vivo during neutron capture therapy
comprising a gadolinium particle or a gadolinium compound particle
encapsulated in a microsphere shell.
2. A composition in accordance with claim 1, wherein the
microsphere shell is spherical.
3. A composition in accordance with claim 2, wherein the gadolinium
compound particle is gadolinium oxide.
4. A composition in accordance with claim 1, wherein the
microsphere shell includes a protein substance.
5. A composition in accordance with claim 1, wherein the
microsphere shell includes a composition that is selected from the
group consisting of bovine serum albumin, human serum albumin,
lipids, liposomes, pepsin, gelatin, dextrose, dextrose-albumin,
conjugated antibodies, and combinations thereof.
6. A method of neutron capture therapy for treating cancerous cells
comprising administering to a patient a composition to a
predetermined area containing the cancerous cells, the composition
including a plurality of gadolinium particles or gadolinium
compound particles encapsulated in microsphere shells, and applying
a source of thermal neutron irradiation to the predetermined area
in a manner effective for causing the gadolinium particles or
gadolinium compound particles to release radiation for treating the
cancerous cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/976,746, filed on Oct. 12, 2001, which claims the
benefit of U.S. Provisional Application Ser. No. 60/300,943, filed
on Jun. 26, 2001. The parent applications are incorporated herein
by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to contrast agents and methods of
preparation thereof for use in various imaging modalities, and/or
for use in therapy.
[0004] 2. Description of Related Art
[0005] Introduction to Imaging Modalities,
[0006] Various in vivo imaging processes, including ultrasound,
magnetic resonance and computed tomography, are used as medical
diagnostic tools. The underlying principle of each imaging modality
is generally that the differences in a particular property or
properties (e.g., acoustic properties, proton density, etc.) of the
organs, tissue and other substances within the body at a location
to be examined are detected and then translated into an image. The
various modalities, however, rely on very different principles to
generate images. The effectiveness of any of these imaging
processes, and the resolution of the resulting images, in a great
part depends on the degree of contrast between the body parts that
the imaging equipment is able to detect so as to delineate the
features of the region of interest within the subject body area. As
a result, use of internally administered agents specifically
designed to enhance the degree of contrast detected with a
particular modality has become common. The differences in the
imaging techniques involved with various modalities, however, have
thus far generally restricted the use of any particular contrast
agent to one imaging modality.
[0007] Ultrasound
[0008] Ultrasound ("US") is an imaging process that relies on the
reflection of sound waves within the body to produce an image
thereof. High frequency sound (ultrasonic) waves, which are above
the range of sound audible to humans, are directed at the region of
interest within the body. The waves are reflected back wherever
there is a change in the physical parameters of the structures
within the body, e.g., a change in density between two adjacent
organs. The ultrasound equipment receives the reflected sound waves
and transmits them into an image based on the differing levels of
intensity of the reflected waves.
[0009] Use of a contrast agent enhances the differences in
intensities of the reflected waves. For example, intravenous
encapsulated microbubble contrast agents have become an established
clinical tool for enhancing medical diagnostic ultrasound and
Doppler sensitivity. Some current contrast agents function to
enhance the appearance of the blood pool and to define its
architecture and integrity. Other contrast agents provide passive,
targeted, organ-specific imaging based upon the biodistribution and
pharmacokinetics of the circulating contrast agent, localizing in,
for example, the liver, spleen, kidney and lung.
[0010] The interaction of encapsulated microbubble contrast agents
with ultrasound is complex. The microbubble response relative to a
driving acoustic pressure can be divided into three categories: (1)
linear scattering, (2) nonlinear scattering, and (3)
cavitation/destruction. Microbubbles produce linear scattering with
low acoustic driving pressures and produce non-linear scattering
with moderate acoustic driving pressures. At moderate acoustic
driving pressures, microbubbles exhibit pressure peaks at the
compressional phases of the source thereby providing both harmonic
and subharmonic energy greater than the surrounding medium. At very
high acoustic driving pressures microbubbles cavitate or destruct
as a result of fragmentation and deflation and thus create an
associated acoustic emission signal. The absolute values for low,
moderate and high acoustic driving pressures are not well defined
and depend upon not only the acoustic parameters of the ultrasonic
source but also the constituent physical properties of the
microbubbles themselves, as well as the fluid surrounding them.
[0011] A significant problem with the use of microbubble contrast
agents result from the machinery associated with the imaging
process. Typical medical diagnostic ultrasound imaging machinery
produces acoustic pressures that can range from 0.5 to 3 mega
pascals (MPa). This acoustic pressure range can destroy some
microbubble contrast agents during the imaging process, thus
reducing the efficacy of the contrast agent and also reducing the
effective imaging time (half-life) of the contrast agent.
[0012] Albunex.RTM. (from Molecular Biosystems, of San Diego,
Calif.), the first commercially available ultrasound contrast
agent, is a suspension of air-filled albumin microspheres produced
by sonication of a heated solution of 5% human albumin. The major
drawbacks associated with use of Albunex.RTM. as a contrast agent
for ultrasound are its short plasma half-life and its acoustic
instability relative to pressure changes. The plasma half-life of
radiolabeled Albunex.RTM. microbubbles after intravenous injection
is less than one minute. In addition, backscatter intensity falls
as pressure rises, an effect that has been demonstrated in vivo as
a systolic fall in videointensity following intravenous injection.
Moreover, albumin microbubbles cannot by used with other modalities
such as magnetic resonance imaging or computed tomography because
the microbubbles do not have the functional characteristics
required for such modalities.
[0013] With the development of medical ultrasonic contrast agents,
the theoretical behavior of encapsulated microbubbles has generated
substantial interest. Ye found that at frequencies below or
slightly higher than the resonance, acoustic scattering by
Albunex.RTM. bubbles is nearly omni-directional and bears
similarities to that by usual air bubbles. (Ye, "On Sound
Scattering and Attenuation of Albunex.RTM. Bubbles," J Acoust. Soc.
Am., 100(4) 2011-28, (1995)). The Ye reference also reveals that
the scattering by Albunex.RTM. bubbles can be highly anisotropic
when the frequency is above resonance. Work by de Jong showed large
differences in non-linear behavior between ideal and Albunex.RTM.
microspheres due to the additional restoring force and friction
inside the shell that surrounds the Albunex.RTM. microsphere. (de
Jong et al, "Higher Harmonics of Vibrating Gas-Filled Microspheres,
Part One: Simulations," Ultrasonics, 32(6) 447-453 (1994)).
[0014] Prior efforts to address the need for an increase in the
plasma half-life of medical ultrasonic contrast agents have focused
on: (1) strengthening the structure of the encapsulating shell, (2)
employing different substances for the encapsulating shell, or (3)
chemical modification of the microsphere surface, for example, by
pegylation. For example, the use of galactose with human serum
albumin microspheres appears to strengthen the shell, thereby
increasing the half-life to 3 to 6 minutes. (Goldberg, "Ultrasound
Contrast Agents," Clin. Diag. Ultrasound, 28:35-45 (1993)). Kimura
et al. utilized small unilamellar vesicle ("SUV"), large
unilamellar vesicle ("LUV") and multilamellar vesicle ("MLV") as
echogenic liposomes. (Kimura et al., "Preparation and
Characterization of Echogenic Liposome as an Ultrasound Contrast
Agent," Chem. Pharm. Bull., 46(10) 1493-96 (1998)). The acoustic
reflectivity obtained with the echogenic MLV was larger than that
of the gas bubbles enclosed within a surfactant mixture. A
half-lifetime of 39 minutes was observed for the MLV prepared from
egg-yolk phosphatidylcholine liposomes. The duration of
reflectivity was prolonged drastically to a half-lifetime of 866
minutes by incorporating cholesterol into the MLV, although,
significantly, the echogenicity was decreased by such
incorporation. Although there have been a number of important steps
at lengthening the effective imaging half-life of injectable
ultrasonic contrast agents using liposomes, there has been an
overall reduction in the echogenicity of these agents.
[0015] Thus, although there are a number of ultrasonic contrast
agents now available commercially, and despite significant research
directed to many of these agents, limitations still exist with
these agents. Furthermore, few ultrasonic contrast agents can be
used with other imaging modalities.
[0016] Magnetic Resonance
[0017] Another imaging technique is magnetic resonance ("MR")
imaging. This modality relies on detecting the emission of
electromagnetic radiation by certain atomic nuclei atomic nuclei in
the body upon application of pulsed radio frequency signals in the
presence of a magnetic field. The resulting magnetic echoes
produced when the signal is terminated ultimately are translated
into an image.
[0018] Use of certain contrast agents with MR is known in the art.
Contrast agents are commonly used intravenously to change the local
magnetic field in tissue. Generally, abnormal tissue will respond
differently in the presence of the contrast agent as compared to
normal tissue and will give off a different magnetic echo. Thus,
when the magnetic echoes are translated into an image, an image of
the tissue abnormalities is provided.
[0019] The use of gadolinium oxide (Gd.sub.2O.sub.3) particles
alone measuring less than 2 micrometers (.mu.m) in diameter as a
prototype MR contrast agent has been examined for imaging the liver
and spleen. (Burnett et al., "Gadolinium Oxide: A Prototype Agent
for Contrast Enhanced Imaging of the Liver and Spleen with Magnetic
Resonance," Magnetic Resonance Imaging, 3:65-71 (1985)).
[0020] Another study evaluated the effects of gadolinium
diethylenetriaminepentaacetic acid (Gd-DTPA), albumin Gd-DTPA, and
Gd.sub.2O.sub.3 on imaging of the spleen and renal cortex. (Daly et
al., "MR Image Time-Intensity Relations in Spleen and Kidney: A
Comparative Study Of GdDTPA, Albumin-(GdDTPA), And Gd.sub.2O.sub.3
Colloid," American Journal of Physiologic Imaging, 5:119-24
(1990)). The suspension of Gd.sub.2O.sub.3 used in the studies by
Burnett and Daly was synthesized by titrating a GdCl.sub.3 solution
with NaOH. With this method of preparation, residual GdCl.sub.3 is
likely to remain in the Gd.sub.2O.sub.3 preparation, such that
extreme toxicity from inadvertently incorporated free GdCl.sub.3 is
possible. With most chelated gadolinium contrast agents, only one
gadolinium atom per molecule is present in commercially-available
contrast media manufactured for use in MR imaging, so that the
enhancement capabilities of the contrast agent are limited. In
addition, synthesis of albumin particles and also albumin
microspheres tagged with gadolinium chelates on the surface would
also be expected to have decreased MR sensitivity due to the
limited number of sites for conjugation of the gadolinium chelate
to the microsphere surface.
[0021] Magnetite (Fe.sub.3O.sub.4) albumin microspheres ("MAM")
have been used as a superparamagnetic contrast agent for
reticuloendothelial MR imaging. (Widder et al., "Magnetite Albumin
Suspension: A Superparamagnetic Oral MR Contrast Agent," ARJ, 149:
839-43 (1987)). MAM was synthesized by combining 5% human serum
albumin ("HSA") and magnetite to create albumin microspheres using
a modified water-in-oil emulsion polymerization technique.
Nonlinear behavior of MAM with increased applied external magnetic
field over 0.3-0.9T was observed. The influence of magnetite on
T.sub.2 relaxation is believed to be due to local field
inhomogeneities generated by the large magnetic moment of
Fe.sub.3O.sub.4, which causes dephasing of proton spins and an
acceleration of T.sub.2 relaxation with negligible T.sub.1 effects.
Because iron oxide is predominately a T.sub.2 relaxation agent, MAM
has limited usefulness in conventional MR imaging. Additionally,
based on the lower density of iron oxide relative to other heavy
metals, iron oxide, and thus MAM, has a very limited utility for
other imaging modalities, such as computed tomography.
[0022] As with contrast agents for US, contrast agents for MR also
have limitations, both when used with MR and if used with other
imaging modalities. Few MR contrast agents have even been evaluated
for use with other imaging modalities.
[0023] Computer Tomography
[0024] Computed tomography ("CT"), also called computerized axial
tomography, is an imaging modality that utilizes a toroidal, or
donut-shaped x-ray camera to provide a cross-sectional image of the
body area of interest. Use of certain contrast agents to improve CT
images is known. Generally, the contrast agent localizes in a
particular body compartment and differentially opacifies normal or
abnormal tissue. The contrast agent causes the tissue to inhibit
passage of x-rays to produce a shadow of positive contrast in the
resulting image. Iodine-based contrast agents are considered to be
the industry standard with CT.
[0025] Gd-DTPA contrast agents have been used for certain limited
applications in conventional angiography and CT imaging. (Bloem and
Wondergem, "Gd-DTPA as a Contrast Agent in CT," Radiology,
171:578-79 (1989)). A major drawback associated with using Gd-DTPA
contrast agents for CT imaging is the fact that only one electron
dense (gadolinium) atom per molecule is present in
commercially-available contrast media. In comparison, two widely
used contrast agents, Optiray.RTM. (by Mallinckrodt, Inc., of St.
Louis, Mo.) and Ultravist 300.RTM. (by Berlex Laboratories, Inc.,
of Wayne and Montville, N.J. and Richmond, Calif.), contain three
electron dense (iodine) atoms per molecule. In addition, the molar
concentration of gadolinium in commercially-available
gadolinium-based contrast agents, such as Magnevist.RTM. (by Berlex
Laboratories, Inc., of Wayne and Montville, N.J. and Richmond,
Calif.), is 0.5 mol/L, which is one-fifth the molar concentration
of iodine in Optiray.RTM. (320 mg of iodine per mL, or 2.5 mol of
iodine per liter). Thus, presently available MR contrast agents
provide sub-optimal CT enhancement and/or are not well-suited for
use with other imaging modalities, such as CT and US.
[0026] Study of Contrast Agents in Different Imaging Modalities
[0027] To date, few contrast agents have been used for imaging
studies utilizing multiple imaging modalities. Correlative studies
using combinations of imaging methods, most notably CT and MR
imaging, are frequently performed in order to improve the accuracy
of diagnosis or assess the efficacy of treatment routines.
Magnevist.RTM. (Gd-DTPA) and a few other gadolinium-containing MR
contrast agents have been used for this purpose, but limitations
associated with the dosage and cost of commercially available MR
contrast agents have prevented widespread use. Further, these
agents would confer no obvious benefit to US imaging due to their
low compressibility and the high concentrations required in order
to provide effective US imaging.
[0028] Perfluorocarbon emulsions have been evaluated for contrast
image enhancement. Perflubron (perfluorooctyl bromide, "PFOB")
emulsified with egg yolk lecithin has been tested for use in US
(due to its high density), MR (fluorine nuclei imaging or as a
signal void for hydrogen nuclei imaging) and CT imaging (due to its
bromine atom). However, neither fluorine MR imaging nor signal void
imaging have found widespread use in hospital or clinical practice,
where T.sub.1 (and to a lesser extent, T.sub.2) imaging of protons
is typical. Also, PFOB is less dense radiographically, i.e. less
radio opaque than iodine-based CT contrast agents, making larger
doses necessary in order to achieve adequate x-ray attenuation.
[0029] Despite the significance of contrast agents in medical
diagnostics and the ever-present need for correlative studies, no
single commercially-available contrast agent provides effective,
cost-efficient image enhancement utilizing more than one imaging
modality.
BRIEF SUMMARY OF THE INVENTION
[0030] The invention relates to a new class of contrast agents,
namely paramagnetic protein microspheres, for use with multiple
imaging modalities. More particularly, this invention relates to
gadolinium oxide albumin microspheres ("GOAM"), in both unmodified
and surface-modified (including pegylation, antibody attachment,
etc.) forms, that are used as contrast agents with the more widely
used imaging modalities, including US, MR, and CT. In a preferred
embodiment, Gd.sub.2O.sub.3 molecules are encapsulated in albumin
microspheres. Unmodified and/or surface-modified GOAM of the
present invention can function as contrast imaging agents for
multiple imaging modalities, such as US, MR and CT.
[0031] With respect to US, these microspheres generally have the
potential to withstand withstand greater acoustic pressures than
prior contrast agents due to the synthesis method used in the
present invention. The presence of Gd.sub.2O.sub.3 sequestered
within albumin microspheres significantly enhances echogenicity of
the protein microspheres. The increased functionality of the GOAM
of the present invention as a US contrast agent derives from
increased echogenicity due to the effect of Gd.sub.2O.sub.3 on
density, compressibility, absorption cross-section, scattering
cross-section, and velocity of sound of the albumin microspheres.
Additionally, toxicity may be decreased because the overall
Gd.sub.2O.sub.3 concentration required for ultrasound image
enhancement is reduced due to gadolinium oxide being sequestered
within albumin micro spheres.
[0032] The GOAM of the present invention also can provide enhanced
CT imaging due to the high atomic weight and high k-edge of
gadolinium. Additionally, GOAM contains multiple Gd.sub.2O.sub.3
particles, each of which are made up of several gadolinium atoms,
improving the utility of GOAM as an x-ray attenuation agent for
CT.
[0033] T.sub.1 and T.sub.2 relaxation enhancement in MR imaging is
due to the paramagnetic properties of gadolinium, whose seven
unpaired electrons account for its high relaxivity, and the
super-paramagnetic and/or ferromagnetic properties of
Gd.sub.2O.sub.3, which will be non-specifically sequestered in
albumin microspheres, thereby allowing for increased interaction
with mobile protons, the potential for relaxation via physical
rotation of Gd.sub.2O.sub.3 and a decreased tumbling rate of
Gd.sub.2O.sub.3 when associated with albumin microspheres. In
addition, improved T.sub.1 and T.sub.2 relaxation at lower
concentrations of Gd.sub.2O.sub.3 is anticipated due to the
association of Gd.sub.2O.sub.3 with a macromolecule, i.e. an
albumin microsphere.
[0034] GOAM also may be used in therapeutic applications, such as
gadolinium neutron capture therapy, because of the high
cross-sectional density and high neutron capture rate of
gadolinium. Gadolinium has the highest thermal neutron capture
cross-section of any known element. GOAM also may be used to
encapsulate other therapeutic agents, such as antineoplastic
drugs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is a representative image at 40.times. magnification
of prior art unshelled air-filled microbubbles in oil;
[0036] FIG. 2 is a representative image at 40.times. magnification
of a prior art albumin microsphere;
[0037] FIG. 3 is a representative image at 40.times. magnification
of a population of Gd.sub.2O.sub.3 albumin microspheres in
accordance with the present invention, showing the gadolinium
particles inside of the microspheres;
[0038] FIG. 4a illustrates a cross-section of a plastic tube taken
at one end of the tube;
[0039] FIG. 4b is a representative image of the plastic tube of
FIG. 4a using B-mode ultrasound of oil;
[0040] FIG. 4c is a representative image of the plastic tube of
FIG. 4a using B-mode ultrasound of air-filled albumin microspheres
in oil;
[0041] FIG. 4d is a representative image of the plastic tube of
FIG. 4a using B-mode ultrasound of GOAM in oil;
[0042] FIG. 5a is a representative image of a simulation
illustrating total wave in the plane xz through the center of the
sphere, from left to right, at a time of 11.5 microseconds;
[0043] FIG. 5b is a representative image of the simulation of FIG.
5a at a time of 17.3 microseconds;
[0044] FIG. 5c is a representative image of the simulation of FIG.
5a at a time of 23.1 microseconds;
[0045] FIG. 5d is a representative image of the simulation of FIG.
5a at a time of 38.4 microseconds;
[0046] FIG. 6 illustrates RF acquisition data comparing GOAM of the
present invention, albumin microspheres, and free
Gd.sub.2O.sub.3;
[0047] FIG. 7 illustrates ultrasonic attenuation comparing GOAM of
the present invention, albumin microspheres, and free
Gd.sub.2O.sub.3 at three separate concentrations;
[0048] FIG. 8 illustrates integrated ultrasonic backscatter
coefficient comparing GOAM of the present invention, albumin
microspheres, and free Gd.sub.2O.sub.3 at three separate
concentrations;
[0049] FIG. 9 illustrates T.sub.1 magnetic resonance enhancement by
various contrast agents, including GOAM of the present invention;
and
[0050] FIG. 10 illustrates CT attenuation comparing GOAM of the
present invention, water, albumin microspheres, free
Gd.sub.2O.sub.3 at various concentrations, and stock solutions of
commercially available contrast agents.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The invention relates to paramagnetic compositions for use
with various imaging modalities. More particularly, the
paramagnetic compositions of the present invention comprise one or
more particles selected from the group consisting of gadolinium,
zinc, magnesium, manganese, calcium and compounds thereof; and one
or more microsphere shells encapsulating one or more particles,
wherein the composition is effective for enhancing images obtained
using more than one imaging modality as compared to images obtained
without the composition. The GOAM of the present invention can be
used as a contrast agent during medical diagnostic imaging
procedures. The composition is used with imaging techniques,
including ultrasound (US), magnetic resonance (MR), computed
tomography (CT) and the like, to obtain enhanced images of a
selected area of a patient's body. Use of the contrast agents of
the present invention allows for examination of a patient by
multiple imaging techniques, without the need for multiple contrast
agents or additional patient preparation between techniques, to
provide correlative studies for diagnostic purposes. A method of
synthesizing such compositions also is provided. Although it is
contemplated that contrast agents of the present invention may
include microspheres that include compounds comprising metals, such
as gadolinium, zinc, magnesium, manganese, calcium and the like, it
will be described by way of example principally in connection with
gadolinium oxide-containing protein microspheres.
[0052] As used herein, "contrast agent" and "imaging agent" relate
to any composition administered in vivo to obtain images of an area
of interest of a body. The images may be obtained using any imaging
technique known in the art. Preferably, use of such agent provides
an enhanced image of the body structures within the area of
interest as compared to an image obtained without use of any such
agent.
[0053] As used herein, "microsphere" means any microbubble within a
solution, the microbubble having an average diameter of no greater
than about 7 .mu.m, and more preferably between about 0.5 and about
4 .mu.m. Generally, a microsphere may be gas-filled, aqueous or
non-aqueous solution-filled and/or include particulate matter in
its outer shell. Preferably, in accordance with the invention, the
microsphere contains particulate matter.
[0054] As used herein, "paramagnetic compound" is intended to refer
to a compound that enhances the relaxation of hydrogen protons in
body tissue during MR imaging. Such a compound improves T.sub.1 and
T.sub.2 relaxation time and readily brightens tissues in which the
compound becomes localized.
[0055] FIG. 1 illustrates a typical air-in-oil (unshelled)
microbubble, as known in the art. The oil solution was first
sonicated and then air bubbles were created in the oil by blowing
in air. FIG. 2 shows a prior art albumin microsphere. Similar
microspheres and liposomes have been used as contrast agents with
US with limited benefits. The oil microsphere of FIG. 1 and the
albumin microsphere of FIG. 2 do not have the physical and
functional characteristics required to provide enhancement if used
with other imaging modalities, such at MR and CT.
[0056] As shown in FIG. 3, in accordance with the present
invention, a composition of gadolinium oxide-containing albumin
microspheres ("GOAM") is provided for use as a contrast agent. The
contrast agent includes a high-density paramagnetic particle
incorporated by polymerization in a protein shell. Preferably, the
gadolinium is provided as Gd.sub.2O.sub.3 particles, with at least
one encapsulated Gd.sub.2O.sub.3 particle per microsphere.
Preferably, each microsphere includes a plurality of particles.
Further, the gadolinium oxide preferably is present in the
microspheres in spherical form. The outer shell of the microsphere
may comprise proteins, such as bovine serum albumin ("BSA"), human
serum albumin ("HSA"), pepsin, conjugated antibodies or antibody
shells; lipids, such as phospholipids, glycolipids, and cholesterol
used in some liposome preparations; gelatin; and carbohydrates,
such as dextrose and dextrose-albumin, and combinations thereof, or
any other substance capable of imparting the characteristics of
elasticity, small size, spherical shape and having a metabolic
pathway, biodistribution, and subsequent elimination
pharmakokinetics. Preferably, a water-in-oil emulsion
polymerization method as known to those skilled in the art may be
modified to prepare the GOAM.
[0057] As an example, the GOAM may be prepared by first mixing
approximately 5 grams of BSA in 10 ml of distilled water and
passing the solution through a 0.2 .mu.m filter. One gram of
Gd.sub.2O.sub.3 is added to the aqueous solution. The colloid
solution includes Gd.sub.2O.sub.3 particles measuring between about
50 Angstroms (.ANG.) to about 2 .mu.M in diameter, preferably
between about 50 to about 750 .ANG., and more preferably between
about 200 to about 400 .ANG.. The BSA and Gd.sub.2O.sub.3 mixture
is first mixed in water and then added to approximately 40 ml of
oil, such as cottonseed, canola and the like, with stirring. The
mixture then is sonicated at an acoustic power of 70 watts/cm.sup.2
using a Misonix 2020XL sonicator fitted with a microprobe tip for
up to about 5 minutes. This solution is added dropwise to about 10
ml of oil preheated to between about 100.degree. C. and about
180.degree. C., and heated to between about 100.degree. C. and
about 180.degree. C. The solution is allowed to cool to room
temperature with stirring. The cooled GOAM solution is separated
from unused starting materials via filtered centrifugation. The
resulting solution is washed in either ether, ethanol, acetone, or
the like, and re-suspended in buffered saline solution or distilled
water. As an example, the resulting composition may have a bubble
concentration of between about 10.sup.6 to about 10.sup.9
bubbles/ml of solution and a gadolinium concentration of about 2 to
about 10 mg/l (as measured via ICP analysis).
[0058] FIG. 3 illustrates microspheres having an outer protein
shell surrounding a gadolinium compound. The albumin shell
encapsulates particles of Gd.sub.2O.sub.3. Preferably, the
Gd.sub.2O.sub.3 albumin microspheres measure between about 0.5 to
about 7 .mu.m in diameter and more preferably less than about 4
.mu.m in diameter.
[0059] The gadolinium oxide composition of the present invention is
particularly suitable for use as a contrast agent for a plurality
of imaging modalities. Use of contrast agents in accordance with
the present invention allows a reduced amount of gadolinium to be
administered while still maintaining the image-enhancing effects of
the contrast agent with MR and US imaging thereby reducing
potential toxic effects associated with gadolinium.
[0060] Pegylated gadolinium oxide albumin microspheres also can be
prepared from the synthesized GOAM. With pegylation, polyethylene
glycol ("PEG") chains can be added to the outer shells of the
microspheres. As an example, polyethylene glycol 2000 ("PEG 2000")
can be attached to the GOAM using various pegylation procedures.
The uptake of GOAM generally is altered, such that biodistribution
of the contrast agent in soft tissues, such as the liver and
spleen, changes. By surface modification of the GOAM, the half life
of the contrast agent in the blood pool is increased, allowing for
increased effectiveness of GOAM as a blood-pool enhancement
agent.
[0061] In another embodiment of the present invention, the
individual Gd.sub.2O.sub.3 particles may particles may be pegylated
and may then be encapsulated, if desired. The individual
Gd.sub.2O.sub.3 particles are stabilized with a carbohydrate
polyethylene glycol coat using a modified pegylation procedure. The
Gd.sub.2O.sub.3 particles preferably have diameters of between
about 200 to about 400 .ANG.. Using Gd.sub.2O.sub.3 particles in
this size range that have been pegylated will provide a relatively
high concentration of Gd.sub.2O.sub.3 and will modify the
biodistribution of the contrast agent in the body.
[0062] The contrast agents of the present invention can be used
with US, MR, and CT, which CT, which will allow correlative studies
to be performed. When used with US, both the microsphere shell and
the encapsulated particle interact with ultrasonic waves, altering
the scatter and absorption characteristics and thereby providing an
enhanced image. The encapsulated gadolinium compound reacts during
MR to alter the magnetic field of the tissue and acts as an
absorber of x-rays during CT, thereby providing enhanced images
with these modalities. The images obtained by the various
modalities with the contrast agents of the present invention have
increased clarity and contrast.
[0063] Use of the contrast agents provides a cost-effective means
of diagnosis. The contrast agents can be used with multiple
modalities, certain of which are less expensive to perform and may
be used as initial indicators for diagnosis. For example, imaging
with US is not as costly as with MR, and US may be conducted prior
to MR or other techniques to provide an initial diagnosis, such
that subsequent, more costly, tests may be more focused or possibly
avoided.
[0064] The contrast agents of the present invention also may be
used for certain therapeutic applications. More particularly,
gadolinium oxide-containing microspheres can be used with neutron
capture therapy in the treatment of cancer. Any procedure for
neutron capture therapy known to those of skill in the art may be
modified in accordance with the present invention. Generally, the
composition of gadolinium oxide-containing microspheres can be
prepared as described above. The gadolinium composition is
administered intravenously and/or otherwise localized to a tumor.
When the gadolinium nucleus is irradiated with neutrons, the
gadolinium produces several forms of radiation, including
.gamma.-rays, x-rays, internal conversion electrons and Auger
electrons, which help to kill the tumor. Because Gd.sub.2O.sub.3
has a very large thermal neutron capture cross-section (66 times
larger than that of boron-10), the range of radiation and the
corresponding killing efficacy are increased when compositions in
accordance with the present invention are used.
[0065] In accordance with the present invention there is also
provided a mathematical model that is free from certain limitations
of the models currently being used for contrast agents.
Additionally, the model is implemented into a simulation tool to
characterize newly created multimodal agents and thereby to evolve
improved designs with optimal characteristics.
[0066] A two-component simulation model is provided. The first part
uses Boundary Element Method ("BEM") to solve for the potential
flow. The second part uses a Finite Difference Time Domain
("3D-FDTD") model. This model uses the results of the BEM model to
simulate the bulk behavior of encapsulated microbubbles in solution
insonified by pulsed ultrasound waves. The 3D-FDTD method is used
for the simulation of acoustic wave propagation and scattering in
inhomogeneous media. This method exploits the true
three-dimensional aspect of the propagation problem by iteratively
solving in time steps the equation of motion and the equation of
continuity of the acoustic wave in the form of difference
equations, hence the name Finite Difference Time Domain ("FDTD").
The advantage of the FDTD method is the ability to simulate complex
structures in the time domain. This is especially important when
dealing with biological structures. In addition, transient behavior
as well as steady state behavior also can be studied with this
method.
[0067] Although not always explicitly stated, the near field has
always been modeled with incompressible potential flow assumption
(radial velocity approximately 1/r.sup.2 at a distance r from the
bubble center). The multi-scale rigorous mathematical model of the
present invention considers an inner potential region near the
bubble and an outer acoustic region far away. Rather than using a
radial equation, a boundary element method is applied to solve for
the potential flow in the near field, which furnishes nonlinear
shape oscillation and, therefore, the directional information of
the pressure and the velocity field around an agent. The velocity
potential .PHI.(x) is obtained by solving the discretized integral
equation: 1 ( x ) = S ( x 0 ) G n ( x - x 0 ) S ( x 0 ) - S G ( x -
x 0 ) n ( x 0 ) n ( x 0 ) S ( x 0 )
[0068] where (x,x.sub.0) is the Green's function of the Laplace
equation -[4.pi..vertline.x,x.sub.0.vertline.].sup.-1. The pressure
and the velocity fields obtained at the inscribing surface,
.differential..OMEGA..sub.s, are used to compute the scattered far
field in the acoustic region. In the far field the flow is
compressible yet linear:
(.gradient..sup.2+k.sub.m.sup.2).phi.(x)=0
[0069] where k.sub.m is the wave number based on the effective
sound speed in the medium containing agents. This equation is
solved for the velocity, .differential..PHI./.differential.n, given
the values of .PHI. at the surface. On the other hand, .PHI. at the
surface is obtained by the Bernoulli's equation, valid in a
potential flow: 2 t ( x ) + 1 2 ( x ) 2 = p .infin. - p L
[0070] The effects of internal pressure due to vapor (.nu.) and gas
(g) and surface tension (.sigma.) are represented in the liquid
pressure P.sub.L at the outer wall of the agent:
P.sub.L=P.sub..nu.+P.sub.g-C.sigma.
[0071] C being the curvature of the bubble surface.
[0072] Most contrast agents are made with an encapsulating shell,
however, very little is known about shell properties, which vary in
thickness, number of layers and other characteristics, depending on
the method used to create them. As mentioned before, various models
have been proposed with various degrees of detail for the elastic
shell. In Church's solution it is assumed that a continuous layer
of incompressible, solid elastic material separates the gas from
the bulk Newtonian liquid. A Rayleigh-Plesset-like equation
describing the dynamics of such surface-contaminated gas bubbles
was derived. Church found that the resonance frequencies of
individual bubbles tend to increase as the modulus of rigidity
increases. Encapsulated bubbles with shell rigidity greater than
approximately 85 mega pascals (MPa) provide a greater cross section
per unit attenuation in the lower biomedical frequency range than
do free bubbles of the equivalent size.
[0073] The need to simultaneously incorporate both non-linearity
and directionality is addressed by the present model. Non-linearity
is essential for harmonic and transient power scattering, both of
which promise better discrimination against background tissue
signals. On the other hand, directionality is an important observed
effect leading to significant modification of the contrast
response. This is especially important in the development of
medical imaging contrast agents in general and most specifically
with acoustic contrast agents.
[0074] The output from the boundary element model is used for the
input of the propagation model as described below.
[0075] Microbubbles in solution undergo nonlinear radial
oscillation when they are exposed to moderately strong (greater
than 100 KPa) ultrasound waves. These oscillations produce echoes
containing second and higher harmonics of the incident wave.
[0076] The pressure and the particle velocity in the coupled wave
equations are the instantaneous total pressure and the total
particle velocity at any point in the fluid medium. The equations
for a lossless medium with variable speed and density are the
following:
.gradient.p(x,y,z,t)=-.rho.(x,y,z,t).differential.u(x,y,z,t)/.differential-
.t, (1)
.gradient.u(x,y,z,t)=(-1/(.rho.(x,y,z,t)c.sup.2))
(.differential.p(x,y,z,t- )/.differential.t) (2)
[0077] The information needed to completely compute the different
fields over time is the initial fields' distribution and the
incident wave satisfying the two coupled equations. For the
computation, the fields in the medium are set to zero at the
initial time t=0. The two coupled equations are discretized to
obtain the FDTD equations. The practical implementation of the FDTD
method starts by partitioning the entire 3D space into small cubes
following the Yee cell method. (Yee, "Numerical Solution of Initial
Boundary Value Problems Involving Maxwell's Equations in Isotropic
Media," IEEE Trans. Antennas Prop., 14(8) 302-07 (1966)). Building
a medium consists of labeling each cube so that a given scattering
medium with specific material properties is obtained. The
computational complexity of the problem is O(n.sup.3) and the
storage requirement is also O(n.sup.3) where n is the number of
cells on each side of a cubic geometry. The transducer is modeled
as ideal point sources, generating a Gaussian spherical wave that
propagates through the 3D medium. The architecture of the code is
simple given the modular nature of each subroutine. Parallel
processing can be used for larger medium simulation. The procedure
for the simulations are: (1) generate the synthetic medium, (2)
compute analytically the propagation of the incident field in the
medium, and (3) compute the scattered pressure field and the
scattered velocity field at each point in the medium.
[0078] The FDTD method can predict field disturbances due to
short-range variations in medium density of the order of the
wavelength of the incident wave. Applications for this method
include the prediction of the acoustic field distribution in
inhomogeneous media such as biological tissues, prediction of
encapsulated microbubbles, insonification in different regimes,
tissue characterization and blood flow. Synthetic media can be
generated and used to compute the different scattered fields for
analysis. Additionally, this method also can be used by first
experimentally obtaining the medium parameters, i.e., by
reconstruction, or from knowledge of anatomy of the given tissue,
and then computing the various acoustic fields for specific
studies.
EXAMPLES
[0079] The following examples are intended to illustrate the
invention and not to limit or otherwise restrict the invention.
Example 1
Ultrasound Studies
[0080] Preliminary imaging studies were conducted to compare the
cross-sectional ultrasound images of oil (containing no contrast
agents), air-filled albumin microspheres, and unmodified and
surface-modified GOAM flowing through a tube. An Aloka SSD5500 PHD
ultrasound machine with a linear transducer (UST 5539 10 MHz) was
used to create traditional B-mode ultrasound images. In these
experiments, the above solutions were injected into clear plastic
Tygon tubing (OD=0.318 cm; ID=0.159 cm) (FIG. 4a) immersed in
degassed water at room temperature. Cross-sectional images of the
tube were captured using a personal computer, video frame grabber
and real time video capture software (Capture.COPYRGT., Watkin,
1997). These images are shown in FIGS. 4b-4d.
[0081] These images clearly demonstrate the full circumference
visualization capabilities of the various media--oil (no contrast
media), air-filled albumin microspheres, and GOAM. Differences are
clearly evident in the cross-sectional B-mode ultrasound images.
The inner circumference of the tube is not visible when imaging
with oil, which does not contain contrast media. Imaging with
flowing (and static) air filled albumin microspheres enhances the
tube image but the full circumference of the tube is not visible.
GOAM provides full circumferential tube delineation and
enhancement. This demonstrates the potential utility of modified
GOAM for mapping of blood vessels, especially the delineation of
small blood vessels. Modified GOAM has the potential to enhance
visualization of flow in small vessels of the heart and perhaps
enhance low velocity ultrasound spectral Doppler signals. Moreover,
full vessel circumference imaging is an essential prerequisite to
3D imaging studies.
Example 2
Physical Characterization of Unmodified and Surface-Modified
GOAM
[0082] The determination of the size distribution, concentration,
and size fractionation of the synthesized GOAM is accomplished via
Coulter counter analysis. In addition, optical microscopic images
(Bausch & Lomb) are used to verify the size and conformation of
GOAM.
Example 3
Acoustic Simulations
[0083] Two different simulation approaches are required to describe
the characteristics of the acoustic driving forces on the developed
microsphere as well as the acoustic propagation of the scattered
ultrasonic energy. One approach uses boundary element method (BEM)
modeling to describe the acoustic behavior of the microsphere.
Finite difference time domain modeling (FDTD) is used to examine
the backscattering properties of the reflected acoustic pressure
waves.
Example 4
Ultrasound Characterization of Unmodified and Surface-Modified
GOAM
[0084] General Procedures
[0085] The in vitro ultrasonic characteristics of synthesized
unmodified or surface-modified GOAM are determined at different
concentrations of Gd.sub.2O.sub.3 at constant temperatures. The
following characteristics are determined: bubble size-istribution,
life time, effect of ultrasound machine power, effect of suspension
condition--dilution and carrier medium, attenuation (as a function
of frequency), sound velocity, normalized backscatter coefficient,
and scattering.
[0086] The acquisition of all ultrasound signals is accomplished
using an Aloka 5500 PHD RF machine using two different ultrasound
transducers (Aloka UST 5539 10 MHz linear small parts transducer
and Aloka UST 9119 2-5 MHz curvilinear abdominal transducer). The
Aloka 5500 PHD RF stores simultaneously, in real time, multiple
frames of RF data from all the transducer elements as well as the
corresponding B-mode images. These data are then ported to a PC
(Intel 800 MHz PIII) for off-line analyses of the independent RF
element data as well as the associated B-mode images.
[0087] Forward scattering data acquisition is accomplished using a
high performance needle hydrophone data acquisition system
(Precision Acoustics, Ltd. Digital Acquisition System) with a 0.04
mm 9 micron PVDF probe). These data are stored on a personal
computer.
[0088] A Bausch and Lomb optical microscope is used to acquire
digitized optical images of the microbubbles. Digital image
acquisition is accomplished using a Sony CCD camera (Model 1250)
linked to a Pinnacle Systems video frame grabber board (Miro DC30
plus) and a PC (Intel 800 MHz PIII) and stored on the hard drive.
All images are acquired in RGB mode with an image size of
608.times.456 pixels. Specially developed image capture software
(Capture .COPYRGT., Watkin, 1998-2001) permits real time image
capture at 30 fps.
[0089] Both in vitro and in vivo B-mode imaging data are
simultaneously recorded using a separate computer acquisition
system. Digital image acquisition is accomplished using a Pinnacle
Systems video frame grabber board (Miro DC30 plus) and a PC (Intel
800 MHz PIII) and stored on the hard drive. All images are acquired
in RGB mode with an image size of 608.times.456 pixels. Specially
developed image capture software (Capture C), Watkin, 1998-2001)
permits real time image capture at 30 fps. This acquisition system
is connected directly to the color video output of the Aloka 5500
PHD RF machine.
[0090] An acrylic imaging tank, (25 cm.times.15 cm.times.15 cm)
with a 10 cm.times.10 cm thin membrane window at one end for
acoustic monitoring is filled with freshly degassed, de-ionized
water at constant temperature (22.degree. C.). Ready-to-use
cellulose dialysis tubes (240 .mu.m) are fixed across the width of
the imaging tank at 1 cm, 2 cm and 3 cm depths from the imaging
window. These tubes are filled with the contrast media selected for
each experiment. Rinsing protocols are used following the injection
of each contrast media.
[0091] Bubble Size-Distribution
[0092] Two different methods are used to determine the size and
distribution of the unmodified and surface-modified GOAM. An
optical microscope is used for optical verification of the sizes
and distributions of the microbubbles at 10.times. and 40.times.
power. Calibration is provided by precision graticule slides.
[0093] More precise bubble sizing and distribution data is
determined using a Beckman-Coulter Multisizer Z2.
[0094] GOAM Life Time
[0095] The echogenicity of unmodified and surface-modified GOAM is
tested over an extended period of time to assess the time period
during which GOAM remain stable within a specially constructed
imaging vial. A small imaging vial with a thin acoustic membrane is
used for this purpose. Ultrasound imaging acoustic power is fixed
at a mechanical index of 0.7. A fixed concentration of microbubbles
is used. The concentration is in the linear range of the
backscatter/concentration plot.
[0096] Effects of Acoustic Power
[0097] Ultrasound pressure waves of current B-mode imaging machines
typically destroy microbubbles. Fixed concentrations of the
contrast agent are stabilized in cellulose dialysis tubes to
determine the characteristics of unmodified and surface-modified
GOAM in a commercial diagnostic ultrasonic field. The two different
ultrasound transducers described above are used to acquire the RF
data and B-mode images. The acoustic power of the ultrasound
machine as reflected by the mechanical index ("MI") provided on the
machine is set in 0.1 MI steps, from 0.2 to 0.8 MI. The tube
contains fresh contrast agents for each MI level test for each
transducer. The backscatter coefficient for each step for each
contrast agent is determined.
[0098] Effects of Suspension Condition
[0099] Different suspension conditions affect the properties of
ultrasound contrast agents. The effects are tested by changing
different air concentrations, diluting the contrast agent, and
utilizing different carrier media.
[0100] The effects of air concentration are assessed for both
unmodified and surface-modified GOAM at fixed concentrations using
the methods described in Sboros. (Sboros et al., "An In Vitro
Comparison of Ultrasonic Contrast Agents in Solutions with Varying
Air Levels," Ultrasound in Med. & Biol., 26:807-18 (2000) which
is incorporated by reference herein). Sterile water is used as the
suspension medium. A sterile bag filled with sterile water is
infused with helium or air to achieve partial oxygen pressures
(pO.sub.2) of 1.5 or 24.7 kPa, respectively. These suspensions are
injected slowly in the cellulose dialysis tubing. The imaging data
is gathered under these conditions using the Aloka 5500 PHD RF
machine to acquire the RF data and B-mode images. Microbubble
concentration and size are determined for the suspensions.
Normalized ultrasonic backscatter vs. concentration is
examined.
[0101] In vitro characterization of ultrasonic contrast media
conducted in aqueous solutions do not necessarily adequately
simulate the behavior of contrast agent in the circulatory system.
Therefore, different concentration levels of GOAM are suspended
with sterilized water, saline, plasma and whole blood at 37.degree.
C. The contrast agent is suspended in an imaging cell similar to
that described by Lazewatsky and colleagues. (Lazewatsky et al.,
"The Effect of Dilution on the Measurement of In-vitro Properties
of Ultrasound Contrast Agents," Proceeding of 1999 IEEE Ultrasonics
Symposium, 1737-42 (1999) which is incorporated by reference
herein). The two different ultrasound transducers described above
are used to acquire the RF data and B-mode images. Time-video
intensity along with backscatter data are acquired using the video
and RF data acquisitions systems described above.
[0102] Attenuation as a Function of Frequency
[0103] The in vitro enhancement and attenuation properties of
unmodified and surface-modified GOAM are examined using the methods
described by de Jong (de Jong and Hoff, "Ultrasound Scattering
Properties of Albunex.RTM. Microspheres," Ultrasonics, 31(3) 175-81
(1993) which is incorporated by reference herein) and by Forsberg
et al. (Forsberg et al., "In Vio Evaluation of a New Contrast
Agent," Proceeding of 1994 IEEE Ultrasonics Symposium, 1555-58
(1994); "Quantitative Acoustic Characterization of a New
Surfactant-Based Ultrasound Contrast Agent," Ultrasound in Med.
& Biol., 23:1201-08 (1997) which both are incorporated by
reference herein) using a flow pump to provide flow through the
dialysis tubing in the imaging tank described above. Frequency
dependent dose attenuation is determined, as well as the
time-attenuation dose dependence curves.
[0104] Sound Velocity
[0105] Because unmodified and surface-modified GOAM have
particulate gadolinium encased within the microbubble, it is
important to determine the effects of the embedded gadolinium on
microbubble sound velocity. Different concentrations of GOAM are
utilized for this test. A modified version of the displacement
method described by Hall et al. (Hall et al., "Experimental
Determination of Phase Velocity of Perfluorocarbons: Application to
Targeted Contrast Agents," IEEE Transactions on Ultrasonics,
Ferroelectrics and Frequency Control, 47:75-84 (2000) which is
incorporated by reference herein) is employed. The imaging tank
described above is employed along with the cellulose tubing. A
polished stainless steel plate is placed 0.5 cm behind the
cellulose tube. The two different ultrasound transducers described
above are used to acquire the RF data and B-mode images. The tube
is filled with sterilized water. Then the tube is filled with
different concentrations of the contrast agents. Full rinsing is
conducted between each injection of different contrast media. Sound
velocity for each concentration is determined.
[0106] Scattering
[0107] Because unmodified and surface-modified GOAM have
particulate gadolinium encased within the microbubble, it is also
important to determine the effects of the embedded gadolinium on
microbubble directional scattering. Different concentrations of
GOAM are utilized for this test. The two different ultrasound
transducers described above are used to acquire the RF data and
B-mode images The cellulose tube is filled with sterilized water
first. Then the tube is filled with different concentrations of the
contrast agents. Full rinsing is conducted between each injection
of different contrast media. A needle hydrophone (Precison
Acoustics Ltd.) is utilized to record the acoustic wave at
45.degree., 90.degree., 135.degree. and 180.degree. relative to the
circumference of cellulose tubing.
[0108] Blood Flow Imaging
[0109] The dynamic blood flow imaging characteristics of different
concentrations of unmodified and surface-modified GOAM are examined
using sterile water, plasma, and whole blood. Two different flow
systems are used. The first consists of a tissue mimicking flow
phantom (ATS Laboratories, Bridgeport, Conn.) with a 6 mm diameter
flow channel for simulating large vessels. The second system
utilizes the tank and cellulose tubing apparatus described above
which simulate smaller capillaries. (This system lacks a tissue
mimicking interface.) A precision flow pump provides both constant
and pulsatile flows. These imaging data are recorded directly on to
the hard disk of a PC using the color video output of the Aloka
5500 PHD connected to the image acquisition system described above.
The effects of different concentrations on the spectral Doppler
signals from the Aloka 5500 are examined along with the effects on
both color Doppler and "Power Flow" imaging in B-mode.
Example 5
MR Characterization
[0110] T.sub.1 and T.sub.2 relaxation are determined for unmodified
and surface-modified GOAM, GOAM made from pegylated Gd.sub.2O.sub.3
colloid solution, Gd.sub.2O.sub.3 colloid solution and GOAM having
encapsulated pegylated Gd.sub.2O.sub.3 at various concentrations
using different pH and temperatures. Imaging of the above reagents
is conducted in sterile water, plasma, and whole blood in order to
better approximate physiological conditions.
[0111] Images are obtained on a 3.0T imaging spectrometer with
image processing and display system. A partial saturation pulsing
sequence is utilized for T.sub.1 weighted 2D acquisitions. T.sub.1
values are derived by an inversion recovery method and curve fitted
by the least squares techniques (i.e. Niesman et al., "Liposome
Encapsulated MgCl as Liver Specific Contrast Agent for Magnetic
Resonance Imaging," Investigative Radiology, 25:545-51 (1990) which
is incorporated by reference herein). T.sub.2 values are derived by
the Carr-Purcell-Meiboom-Gill pulse sequence with curve fitting by
the least squares method.
[0112] Additional studies involve measurement of T.sub.1 and
T.sub.2 relaxation at different magnetic field strengths using a 3T
spectrometer.
Example 6
Simulation of Ultrasound Wave Propagation
[0113] Ultrasound wave propagation in a synthetically generated
medium was simulated. The simulation consisted of a bubble with an
outer diameter of 250 .mu.m and inner diameter of 225 .mu.m. The
bubble was surrounded by an albumin shell of thickness 12.5 .mu.m.
FIGS. 5a-5d illustrate a sequence of snapshots of the absolute
value of the scattered wave. The simulation shows the progress of
an ultrasound wave toward a single spherical target. FIGS. 5a-5d
illustrate that the ultrasonic wave hitting the sphere, as well as
the wave being reflected (the backscattered wave), is accurately
simulated.
Example 7
CT Characterization
[0114] CT attenuation of unmodified and surface-modified GOAM, GOAM
made from pegylated Gd.sub.2O.sub.3 colloid solution,
Gd.sub.2O.sub.3 colloid solution and GOAM having encapsulated
pegylated Gd.sub.2O.sub.3 at various concentrations, as well as the
lowest dose providing acceptable enhancement are determined. Serial
dilutions of the above reagents in normal saline or distilled water
are placed in a tissue equivalent phantom and positioned in the
center of the gantry of the CT scanner. The phantom containing the
above reagents is imaged under the following constant scanning
parameters: slice thickness of 10 mm, 120 keV, 250 mA, scan time of
1 second and a 25 cm field of view. Attenuation measurements
obtained from region of interest circles of approximately 80 mm3
(10 mm thickness) are plotted against the concentrations of the
reagents, and a linear regression analysis is performed. The
attenuation of each dilution is recorded as mean Hounsfield units
using region of interest analysis.
Example 8
RF Data Acquisition
[0115] FIG. 6 illustrates RF acquisition data for B-mode ultrasound
imaging comparing GOAM of the present invention, air-filled albumin
microspheres, and free Gd.sub.2O.sub.3. The photographs in the left
column show an ultrasound image of a cross-sectional view of a 1.5
ml eppendorf tube on its side and having either GOAM, albumin
microspheres or free Gd.sub.2O.sub.3 contained within. The GOAM
solution has a bubble concentration of 10.sup.6 bubbles/ml and a
Gd.sub.2O.sub.3 concentration of 0.02 mmol. The albumin
microspheres solution has a bubble concentration of 10.sup.6
bubbles/ml. The free Gd.sub.2O.sub.3 has a concentration of 200
mmol. To the right of each image is a plot of RF data illustrating
amplitude over time as the ultrasound wave passed through the
eppendorf tube from the top, through the contrast agent and then
through the bottom. This test demonstrates that GOAM provides much
greater RF attenuation than the other contrast agents.
Example 9
Ultrasonic Attenuation
[0116] As shown in FIG. 7, ultrasonic attenuation of GOAM of the
present invention is compared to that of air-filled albumin
microspheres, and free Gd.sub.2O.sub.3. Ultrasonic attenuation
(dB/cm/MHz) is plotted for albumin microspheres (bubble
concentration of 10.sup.6 bubbles/ml), GOAM (bubble concentration
of 10.sup.6 bubbles/ml and Gd.sub.2O.sub.3 concentration of 0.02
mmol), and free Gd.sub.2O.sub.3 at concentrations of 200 mmol, 4
mmol and 2 mmol, respectively. This test demonstrates that GOAM has
greater ultrasonic attenuation than the other contrast agents.
Example 10
Integrated Ultrasonic Backscatter Coefficient
[0117] As shown in FIG. 8, the ultrasonic backscatter coefficient
of GOAM of the present invention, air-filled albumin microspheres,
and free Gd.sub.2O.sub.3 at three separate concentrations is
compared. The integrated ultrasonic backscatter coefficient (dB) is
plotted for albumin microspheres (bubble concentration of 10.sup.6
bubbles/ml), GOAM (bubble concentration of 10.sup.6 bubbles/ml and
Gd.sub.2O.sub.3 concentration of 0.02 mmol), and free
Gd.sub.2O.sub.3 at concentrations of 200 mmol, 4 mmol and 2 mmol,
respectively. This test demonstrates that GOAM has a greater
integrated ultrasonic backscatter coefficient than the other
media.
Example 11
Second MR Characterization
[0118] FIG. 9 illustrates magnetic resonance enhancement of various
contrast agents. Vials containing the various contrast agents (or
water) were inserted into a portion of beef. The contrast agents
included, starting from the top row, from right to left, moving
down:
[0119] First (top) row: Isovue.RTM. 300 (by Bracco Spa of Italy)
(788 mmol), ProHance.RTM. (by Bracco Spa of Italy) (500 mmol);
[0120] Second row: Free Gd.sub.2O.sub.3 (20 mmol, 100 mmol and 200
mmol, respectively);
[0121] Third row: Free Gd.sub.2O.sub.3 (0.02 mmol, 0.4 mmol and 1.0
mmol, respectively); and
[0122] Fourth (bottom) row: Water, Air-filled albumin microspheres
and GOAM of the present invention (bubble concentration of 10.sup.6
bubbles/ml and Gd.sub.2O.sub.3 concentration of 0.02 mmol).
[0123] This test demonstrates that GOAM provides enhanced MR
imaging.
Example 12
CT Characterization
[0124] FIG. 10 illustrates CT attenuation comparing GOAM of the
present invention, water, albumin microspheres, free
Gd.sub.2O.sub.3 at various concentrations, and commercially
available contrast agents. CT attenuation (Hounsfield units) is
plotted for water, albumin microspheres (bubble concentration of
10.sup.6 bubbles/ml), GOAM (bubble concentration of 10.sup.6
bubbles/ml and Gd.sub.2O.sub.3 concentration of 0.02 mmol), free
Gd.sub.2O.sub.3 at concentrations of 0.4 mmol, 1.0 mmol, 10 mmol,
20 mmol, and 100 mmol, respectively, Isovue.RTM. 300 (by Bracco Spa
of Italy) (788 mmol) and ProHance.RTM. (by Bracco Spa of Italy)
(500 mmol). This test demonstrates that GOAM has greater CT
attenuation as compared to albumin microspheres. Additionally, this
test suggests that attenuation will increase as greater
concentrations of Gd.sub.2O.sub.3 are incorporated into the
GOAM.
[0125] Many modifications and variations may be made in the
techniques and compositions described and illustrated herein
without departing from the spirit and scope of the present
invention. Accordingly, the techniques and compositions described
and illustrated herein should be understood to be illustrative only
and not limiting upon the scope of the present invention.
* * * * *