Note: Descriptions are shown in the official language in which they were submitted.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
SITE SPECIFIC BINDING SYSTEM,
NUCLEAR IMAGING COMPOSITIONS AND METHODS
Background of the Invention
This invention relates to a novel site specific
binding system and novel compositions, and more
particularly, to such a system and compositions which are
useful in improved methods for ultrasonic imaging, drug
or chemotherapeutic agent delivery, and diagnostic assays
and detection systems.
Heretofore, with respect to ultrasonic imaging,
although ultrasonic contrast agents based upon "bubble"
technology have been demonstrated to develop an acoustic
impedance mismatch by virtue of gas encapsulated either
in protein (Feinstein et al., J. Am. Coll. Cardiol. 1990;
16:316-324 and Keller et al., J. Am. Soc. Echo. 1989;
2:48-52), polysaccharide (Corday et al., J. Am. Coll.
Cardiol. 1984; 3:978-85) biodegradable polymers
(Schneider et al., Invest. Radiol., 1993; 27:134-139 and
Bichon et al., European Patent Application No.
890810367.4: 1990) or lipids (D'Arrigo et al., J.
Neurormag., 1991; 1:134-139; Simon et al., Invest.
Radiol., 1992; 27:29-34; and Unger et al., Radiology
1992; 195:453-456), no experimental evidence of site-
specific targeting of an acoustic contrast or imaging
agent with resultant changes in the acoustic properties
of the targeted tissue, surface or support are known.
This lack of results has occurred despite numerous
methods described in the literature for modifying such
agents for targeting purposes, and the failure of past
targeting approaches may be due to the chemical nature of
the agents, production process limitations or particle
instabilities.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
2
Nongaseous acoustic contrast agents have been
described including lipid emulsions (Fink etp al.,
Ultrason. Imaging, 1985 7:191-197) liposomes (Lanza et
al., J. Am. Coll. Cardiol., 1992 (abstract); 19 (3 Suppl
A) 114A), and perfluorocarbon emulsions (Mattrey et al.,
Radiology 1982; 145: 759-762 and Mattrey et al.,
Ultrasound Med. 1983; 2:173-176). As with the contrast
agents discussed above, no demonstration of site targeted
emulsion or liposome has been reported. Again, such
failure may reflect instability of the particles, process
incompatibilities or the chemical nature of the contrast
agent. Lipid emulsions were evaluated by Fink et al.
supia and did not exhibit adequate echogenicity in
studies examining hepatic imaging. A unique chemical
formulation of liposomes described by Lanza et al. supra
was suggested to have the potential to be a targetable
ultrasonic contrast but such has not been demonstrated to
date. Perfluorocarbon emulsions, Perflubron
(perfluorooctylbromide, P100) and Flusol
(perfluorodecalin and perfluorotripropylamine, F20) have
been used as ultrasonic contrast agents and have been
reported to accumulate in liver, spleen and tumors
secondary to phagocytic uptake of emulsion particles at
these sites (Mattrey et al. 1983, supra). These
perfluorocarbon emulsions have also been noted to enhance
Doppler signals and opacify lumens. Fluorocarbons and
fluorocarbon emulsions for use as contrast agents are
disclosed in U.S. Patent Nos. 4,927,623, 5,077,036,
4,838,274, 5,068,098, 5,114,703, 5,362,477, 5,362,478,
5,171,755, 5,304,325, 5,350,571 and 5,403,575. However,
no demonstration of perfluorocarbon emulsions as a ligand
targeted acoustic contrast system has been reported.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
3
Previous descriptions of tissue or organ targeting
in biomedical ultrasonics has referred to the collection
of acoustically reflective particles within or around
structural tissue abnormalities. Localized acoustic
enhancement of tissue pathologies (e.g. malignancies) has
not been ligand-directed but rather has depended upon
differential dynamic rates of particle uptake and/or
clearance between normal and malignant tissues. Such
contrast agents have included aqueous solutions (Ophir et
al., Ultrason. Imaging 1979, 1:265-279; Ophir et al.,
Ultrasound Med. Biol. 1989, 15:319-333; and Tyler et al.,
Ultrason. Imaging, 3:323-329), emulsions (Fink et al.
Ultrason. Imaging, 1985, 7:191-197), and suspensions
(Mattrey et al. 1982 supra and Mattrey et al., Radiology,
1987, 163:339-343). Although the possibility of ligand-
directed ultrasonic contrast targeting with acoustically
reflective liposomes has been suggested, no successful
applications of this concept have been reported (Lanza et
al. 1992, supra and Valentini et al., J. Am. Coll.
Cardiol., 1995, 25:16A). Previous approaches to
targeting in vivo of particles have involved direct
conjugation of a ligand (e.g. monoclonal antibody) to a
vesicle by a variety of methods (see, for example,
Torchlin et al., Biochem. Biophys. Res. Commun. 1978,
85:983-990; Endoh et al., J. Immunol. Methods, 1981,
44:79-85; Hashimoto et al., J. Immunol. Methods, 1983,
62:155-162 and Martin et al., Biochemistry, 1981,
20:4229-4238).
There remains a need for new and improved
methodologies for ligand-based binding systems which can
be adapted as an ultrasonic contrast system permitting
detection of molecular moieties such as peptides,
carbohydrates or nucleic acids and whose uses can range
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
4
from ultrasound-based ELISA-like laboratory diagnostic
assays in liquid and solid phase systems and in cell
cultures; electrophoretic, chromatographic and
hybridization detection systems to the detection of
thrombi, infections, cancers and infarctions in patients
with the use of conventional ultrasonic imaging methods.
Summary of the Invention
Among the several objects of the invention may be
noted the provision of a novel method for ligand-based
binding of lipid encapsulated particles to molecular
epitopes on a surface in vivo or in vitro, the provision
of such a method in which the ligand is conjugated to the
lipid encapsulated particles through an avidin-biotin
interaction and the resulting conjugate is bound to
molecular epitopes on a surface; the provision of such a
method which is useful for enhancing the acoustic
reflectivity of a biological surface for ultrasonic
imaging; the provision of a method of this type wherein
the conjugate formed is effective for imaging by x-ray,
ultrasound, magnetic resonance or positron emission
tomography; the provision of compositions for use in
ultrasonic imaging of a biological surface and for
enhancing the acoustic reflectivity of such a surface;
the provision of ultrasonic contrast agents which become
highly reflective when bound to the desired site or
biological surface through the ligand-based binding
system of the invention; and the provision of such
methods and compositions which are capable of targeting
and altering the echogenic properties of a tissue surface
for improved and specific identification of pathological
processes. Other objects will be in part apparent and in
part pointed out hereinafter.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
Briefly, in its broadest embodiment, the present
invention is directed to a method for ligand-based
binding of lipid encapsulated particles to molecular
epitopes on a surface in vivo or in vitro which comprises
5 sequentially administering (a) a site-specific ligand
activated with a biotin activating agent; (b) an avidin
activating agent; and (c) lipid encapsulated particles
activated with a biotin activating agent, whereby the
ligand is conjugated to the particles through an avidin-
biotin interaction and the resulting conjugate is bound
to the molecular epitopes on such surface. The conjugate
is effective for imaging by x-ray, ultrasound, magnetic
resonance or positron emission tomography. In a more
specific embodiment, the invention is directed to a
method for enhancing the acoustic reflectivity of a
biological surface through the sequential administration
of the above-noted components whereby the resulting
conjugate is bound to a natural or synthetic surface to
enhance the acoustic reflectivity thereof for ultrasonic
imaging. The invention is also directed to compositions
for use in ultrasonic imaging of such surfaces and for
enhancing the acoustic reflectivity thereof.
Brief Description of the Drawings
Figure 1 is a graph showing changes in aggregate
particle size of biotinylated and control perfluorocarbon
emulsions with increasing avidin concentration;
Figure 2 shows ultrasonic images of control and
biotinylated perfluorocarbon emulsion before and after
the addition of avidin;
Figure 3 is a graphic illustration of dialysis
tubing images and region of interest placement for gray
scale analysis;
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
6
Figure 4 is a graph showing changes in average pixel
gray scale associated with the addition of avidin to
control or biotinylated perfluorocarbon emulsion;
Figure 5 is a graph showing the effect of control
and biotinylated perfluorocarbon emulsion on apparent
backscatter transfer function and integrated backscatter
of avidinized nitrocellulose membranes;
Figure 6 is a graph showing the apparent backscatter
transfer function of biotinylated and control
perfluorocarbon emulsions targeted to D-dimer covalently
conjugated to nitrocellulose membranes;
Figure 7 is a graph showing the apparent backscatter
transfer function (dB) of biotinylated and control
perfluorocarbon emulsions at low ultrasonic frequencies;
Figure 8 is a graph showing the apparent backscatter
transfer function of biotinylated and control
perfluorocarbon large particle size emulsions targeted to
avidinized nitrocellulose membranes;
Figure 9 shows ultrasonic images of plasma thrombi
before and after exposure to control or biotinylated
emulsions;
Figure 10 is a graph showing the average pixel
grayscale level of plasma thrombi pre-targeted with
antifibrin monoclonal antibody and exposed to control or
biotinylated perfluorocarbon emulsion;
Figure 11 shows ultrasonic images of femoral artery
thrombus acoustically enhanced with biotinylated
perfluorocarbon emulsion in vivo;
Figure 12 is a graph showing the net change in
apparent backscatter transfer function of biotinylated
and control perfluorocarbon emulsions targeted to
prostate specific antigen in prostatic carcinoma relative
to normal regions;
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
7
Figure 13 is a graph showing the net change in
integrated backscatter between normal prostatic stroma
and cancer regions for control versus biotinylated
perfluorocarbon emulsions;
Figure 14 is a graph showing the net change in
apparent backscatter transfer function of biotinylated
and control perfluorocarbon emulsions targeted to OC-125
antigen in ovarian carcinoma relative to normal regions;
Figure 15 is a graph showing the net change in
integrated backscatter between normal ovarian tissue and
carcinoma regions for control versus biotinylated
perfluorocarbon emulsions;
Figure 16 shows ultrasonic and optical images of
tonsil using perfluorocarbon contrast and horseradish
peroxidase targeted to epithelium with anticytokeratin
antibodies;
Figure 17 shows peak detected ultrasonic images of
tonsil epithelium acoustically enhanced with
anticytokeratin antibody targeted perfluorocarbon
emulsion.
Fig. 18 is a graph showing avidin titration curves
for three biotinylated perfluorocarbon emulsions
incorporating varying concentrations of gadolinium-DTPA-
BOA;
Fig. 19 shows the enhancement of the acoustic
reflectivity of plasma clots treated with targeted
perfluorocarbon dual ultrasonic and MRI contrast; and
Fig. 20 shows the femoral artery thrombus detected
by both magnetic resonance and ultrasound.
Description of the Preferred Embodiments
In accordance with the present invention, it has now
been found that a ligand-based binding system having
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
8
broad application may be achieved through ligand-based
binding of lipid encapsulated particles to molecular
epitopes on a surface in vivo or in vitro by sequentially
administering (a) a site-specific ligand activated with a
biotin activating agent; (b) an avidin activating agent;
and (c) lipid encapsulated particles activated with a
biotin activating agent, whereby the ligand is conjugated
to the lipid encapsulated particles through an avidin-
biotin interaction or complexing and the resulting
conjugate is bound to the molecular epitopes on the
surface. The ligand-based binding system of the present
invention thus permits detection of molecular moieties
such as peptides, carbohydrates or nucleic acids with a
specific ligand probe (e.g. an antibody or antibody
fragment) complexed or conjugated with avidin and biotin,
the latter being carried by lipid encapsulated particles
(e.g. biotinylated lipid encapsulated emulsion or
liposome). The ligand-based binding system of the
invention may be employed in an ultrasonic contrast agent
system, ultrasound-based ELISA-like laboratory diagnostic
assays in liquid and solid phase systems and in cell
cultures, electrophoretic, chromatographic and
hybridization detection systems, and for the detection of
thrombi, infections, cancers and infarctions in patients
with the use of conventional ultrasonic imaging methods.
The invention may also be applied for therapeutic
purposes by delivery of chemotherapeutic agents or drugs
to desired sites due to the specificity of the binding
system coupled with the ability to monitor the progress
of the therapeutic treatment through repeated imaging at
such sites. In this regard, the above-referred to
conjugate of the ligand to the lipid encapsulated
particles through an avidin-biotin interaction or
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
9
complexing is effective for imaging by x-ray, ultrasound,
magnetic resonance or positron emission tomography.
In one embodiment of the invention, there is
provided a method for enhancing the reflectivity of a
biological surface by sequentially administering to the
surface (a) a site-specific ligand activated with a
biotin activating agent; (b) an avidin activating agent;
and (c) lipid encapsulated particles activated with a
biotin activating agent; whereby the ligand is conjugated
to the lipid encapsulated particles through an avidin-
biotin interaction and the resulting conjugate is bound
to the biological surface to enhance the acoustic
reflectivity thereof for ultrasonic imaging. This novel
triphasic approach utilizes an avidin-biotin interaction
to permit administration of the targeting ligand separate
from the acoustic lipid encapsulated particles. In a
specific application of the method in accordance with the
invention, a biotinylated ligand is first systemically
administered to a patient to pretarget the tissue or
biological surface of interest and to circulate for a
period of time necessary or sufficient to optimize the
percentage bound. In the second phase, avidin is
administered, circulates and binds to the biotinylated
ligand attached to the target tissue or surface and to
any residual, free circulating ligand. Avidin cross-
linking increases the avidity and stability of the ligand
on the target tissue or surface while promoting the rapid
clearance of circulating avidin-ligand complexes via the
reticuloendothelial system. In the third phase, the
biotinylated lipid encapsulated particles are
administered, binding to avidin through unoccupied biotin
binding sites, and imparting increased acoustic contrast
to the targeted tissue surface. Repeated sequential
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
administration of avidin and the biotinylated lipid
encapsulated particles may be carried out to amplify the
acoustic contrast effect of the lipid encapsulated
particles bound to the targeted surface.
5 In the practice of the invention, the ligand
employed may be, for example, constituted by monoclonal
or polyclonal antibodies, viruses, chemotherapeutic
agents, receptor agonists and antagonists, antibody
fragments, lectin, albumin, peptides, hormones, amino
10 sugars, lipids, fatty acids, nucleic acids and cells
prepared or isolated from natural or synthetic sources.
In short, any site-specific ligand for any molecular
epitope or receptor to be detected through the practice
of the invention may be utilized.
The ligand is activated with a biotin activating
agent. As employed herein, the term "biotin activating
agent" or "biotinylated" encompasses biotin, biocytin and
other biotin analogs such as biotin amido caproate N-
hydroxysuccinimide ester, biotin 4-amidobenzoic acid,
biotinamide caproyl hydrazide and other biotin
derivatives and conjugates. Other derivatives include
biotin-dextran, biotin-disulfide-N-hydroxysuccinimide
ester, biotin-6 amido quinoline, biotin hydrazide, d-
biotin-N hydroxysuccinimide ester, biotin maleimide, d-
biotin p-nitrophenyl ester, biotinylated nucleotides and
biotinylated amino acids such as Ne-biotinyl-l-lysine.
In the second phase, as previously mentioned, an
avidin activating agent is administered. As employed
herein, the term "avidin activating agent" or
"avidinized" encompasses avidin, streptavidin and other
avidin analogs such as streptavidin or avidin conjugates,
highly purified and fractionated species of avidin or
streptavidin, and non or partial amino acid variants,
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
11
recombinant or chemically synthesized avidin analogs with
amino acid or chemical substitutions which still
accommodate biotin binding.
The lipid encapsulated particles or contrast agent
employed in the third phase may be constituted, for
example, by a biotinylated emulsion or liposome which may
contain a gas, liquid or solid. In a specific example,
the lipid encapsulated particles may be constituted by a
perfluorocarbon emulsion, the emulsion particles having
incorporated into their outer coating a biotinylated
lipid compatible moiety such as a derivatized natural or
synthetic phospholipid, a fatty acid, cholesterol,
lipolipid, sphingomyelin, tocopherol, glucolipid,
stearylamine, cardiolipin, a lipid with ether or ester
linked fatty acids or a polymerized lipid. Thus, the
biotinylated contrast agent constituting the lipid
encapsulated particles may be produced by incorporating
biotinylated phosphatidylethanolamine into the outer
lipid monolayer of a perfluorocarbon emulsion.
Perfluorocarbon emulsions are particularly well
suited for biomedical applications and for use in the
practice of the present invention. They are known to be
stable, biologically inert and readily metabolized,
primarily by trans-pulmonic alveolae evaporation.
Further, their small particle size easily accommodate
transpulmonic passage and their circulatory half-life (4-
8 hours) advantageously exceeds that of other agents.
Also, perfluorocarbons have been used to date in a wide
variety of biomedical applications, including use as
artificial blood substitutes. For use in the present
invention, various fluorocarbon emulsions may be employed
including those in which the fluorocarbon is a
fluorocarbon-hydrocarbon, a perfluoroalkylated ether,
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
12
polyether or crown ether. Useful perfluorocarbon
emulsions are disclosed in U.S. Patent Nos. 4,927,623,
5,077,036, 5,114,703, 5,171,755, 5,304,325, 5,350,571,
5,393,524, and 5,403,575 and include those in which the
perfluorocarbon compound is perfluorotributylamine,
perfluorodecalin, perfluorooctylbromide,
perfluorodichlorooctane, perfluorodecane,
perfluorotripropylamine, perfluorotrimethylcyclohexane or
other perfluorocarbon compounds. Further, mixtures of
such perfluorocarbon compounds may be incorporated in the
emulsions utilized in the practice of the invention. As
a specific example of a perfluorocarbon emulsion useful
in the invention may be mentioned a
perfluorodichlorooctane emulsion wherein the lipid
coating thereof contains between approximately 50 to 99.5
mole percent lecithin, preferably approximately 55 to 70
to mole percent lecithin, 0 to 50 mole percent
cholesterol, preferably approximately 25 to 45 mole
percent cholesterol and approximately 0.5 to 10 mole
percent biotinylated phosphatidylethanolamine, preferably
approximately 1 to 5 mole percent biotinylated
phosphatidylethanolamine. Other phospholipids such as
phosphatidylserine may be biotinylated, fatty acyl groups
such as stearylamine may be conjugated to biotin, or
cholesterol or other fat soluble chemicals may be
biotinylated and incorporated in the lipid coating for
the lipid encapsulated particles. The preparation of an
exemplary biotinylated perfluorocarbon for use in the
practice of the invention is described hereinafter in
accordance with known procedures.
When the lipid encapsulated particles are
constituted by a liposome rather than an emulsion, such a
liposome may be prepared as generally described in the
CA 02373993 2001-11-23
WO 00/71172 PCTIUS99/11491
13
literature (see, for example, Kimelberg et al., CRC Crit.
Rev. Toxicol. 6,25 (1978) and Yatvin et al., Medical
Physics, Vol. 9, No. 2, 149 (1982)). Liposomes are known
to the art and generally comprise lipid materials
including lecithin and sterols, egg phosphatidyl choline,
egg phosphatidic acid, cholesterol and aipha-tocopherol.
With respect to the particle size of the lipid
encapsulated particles constituted by a perfluorocarbon
emulsion or liposome, the particle size may range between
approximately 0.05 to 5 microns and preferably between
approximately 0.05 and 0.5 micron. Small size particles
are thus preferred because they circulate longer and tend
to be more stable than larger particles.
As indicated, the ligand is conjugated to the lipid
encapsulated particles or perfluorocarbon emulsion
through an avidin-biotin interaction. The ligand may
also be conjugated to the emulsion directly or indirectly
through intervening chemical groups or conjugated
directly or indirectly to biotin or a biotin analog
through intervening chemical groups such as an alkane
spacer molecule or other hydrocarbon spacer. The use of
spacer molecules between the ligand and biotin or between
biotin and the emulsion is not required but aids in
rendering the biotin more available for binding to
avidin.
As previously mentioned, the emulsion or liposome
constituting the lipid encapsulated particles or vesicles
may contain a gas, liquid or solid. The gas may be
nitrogen, oxygen, carbon dioxide or helium and may, for
example, be evolved from the fluorocarbon component of
the emulsions described above.
Alternatively, but less preferably, the ligand-based
binding method of the invention may be carried out by
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
14
sequentially administering a site-specific ligand
activated with a biotin or avidin activating agent and
lipid encapsulated particles activated with a biotin or
avidin activating agent, a biotin activating agent being
used where an avidin activating agent was employed in the
first step and an avidin activating agent being used
where a biotin activating agent was employed in the first
step. The direct conjugation of the ligand to a
perfluorocarbon emulsion, for example, is less preferable
since it may accelerate in vivo clearance of the emulsion
contrast agent.
In the practice of the invention, it has been
unexpectedly found that the individual components of the
ultrasonic contrast agents as described above are poorly
reflective or have low echogenicity in the bloodstream
but become highly reflective when the ligand-avidin-
emulsion complex is formed in vivo at the desired site or
biological surface and thereby substantially enhances the
acoustic reflectivity thereof for ultrasonic imaging.
This is in sharp contrast to previously known sonographic
contrast agents which are inherently bright or of high
reflectivity in the bloodstream. The improved acoustic
reflectivity achieved through the present invention
provides the advantage of enhancing the signal-to-noise
ratio because the background contrast from lipid
encapsulated particles in the blood is minimal. Thus,
the present invention offers an improved noninvasive
method for forming an acoustic contrast agent which can
be targeted in vitro or in vivo and which when bound to a
specific desired site alters the acoustic reflectivity of
a tissue surface or support media in a manner detectable
with ultrasonic transducers suitable for biomedical and
diagnostic applications within a frequency range of at
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
least 5 to 50 MHz (nominal center frequencies may be
wider ranging based on the knowledge that these are broad
band transducers). The method of the invention
advantageously provides a practical means for detecting
5 any molecular epitope or receptor for which a
biotinylated monoclonal antibody or other ligand is
available without the need for use of ionizing radiation
with or without associated invasive procedures in various
clinical applications and while employing standard,
10 commercially available ultrasonic technology. The
present invention does not employ ultrasonic contrast
systems or agents to delineate blood flow as in the prior
art but rather to detect physiologic and pathologic
events by sensing the accumulation of the contrast agent
15 at specific binding sites.
In the application of the invention to diagnostic
assays such as ultrasound-based ELISA-type laboratory
diagnostic assays in liquid and solid phase systems, the
surface on which ligand-based binding of lipid
encapsulated particles to molecular epitopes occurs may
be, for example, nylon, nitrocellulose membranes or a gel
as well as a biological surface.
The ligand-based binding system of the invention may
also be applied to provide a chemotherapeutic agent or
gene therapy delivery system combined with ultrasonic
imaging. For example, chemotherapeutic agents or immune
activating drugs such as tissue plasminogen activator,
adriamycin, vincristine, urokinase, streptokinase,
methotrexate, cytarabine, thioguanine, doxorubicin, 5-
fluorouracil, cisplatin, etoposide, ifosfamide,
asparginase, deoxycoformycin, hexamethyl melamine and
including radioactive agents may be incorporated in the
lipid encapsulated particles and become part of the
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
16
conjugate bound to a specific biological surface site for
therapeutic action. The present invention would also
advantageously permit the site to be ultrasonically
imaged in order to monitor the progress of the therapy on
the site and to make desired adjustments in the dosage of
therapeutic agent subsequently directed to the site. The
invention thus provides a noninvasive means for the
detection and therapeutic treatment of thrombi,
infections, cancers and infarctions in patients while
employing conventional ultrasonic imaging systems.
Further, as indicated, the invention may be applied
to nuclear medicine imaging by adding a radioactive or
chemical moiety to the surface and/or formulating said
moiety inside the emulsion. These agents can be
transported and concentrated at sites specifically
targeted by the ligand where the added moiety or moieties
provide additional imaging and/or therapeutic character
or capability. Radionuclides such as technetium-99m
(99mTc) and other radioactive moieties known to the art
such as indium, gallium, thallium and iodine may be
chelated and applied to a lipid encapsulated emulsion for
nuclear imaging in accordance with the invention. Thus,
for example, 99mTc-pertechnetate has been successfully
incorporated directly into an emulsion and found to have
10 fold improved 99mTC retention over simple aqueous
injections.
Radionuclides, such as technetium-99m, are routinely
attached to lipid surfaces, including red cells (Vorne et
al., Clin Nucl Med 1992; 17:14-17; Suzman et al. Ann
Surg 1996; 224:29-36; and Ng et al., Dis Colon Rectum,
1997; 40:471-7) and leukocytes (Mortelmans et al., J Nucl
Med., 1989; 30:2022-2028; and Vinjamuri et al., Lancet.,
1996; 347:233-235) for human diagnostic imaging and
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
17
liposomes (Umbrain et al., Br J Anaesth., 1995; 75:311-
318; and Umbrain et al., Acta Anaesthesiol Scand., 1997;
41:25-34) on a more experimental basis. The most common
approaches have involved the use of stannous (II)
chloride or stannous oxinate to anchor 99mTc to the lipid
membrane (Umbrain et al., Br J Anaesth., 1995; 75:311-
318). More recently, 99mTc-hexamethyl-propyleneamineoxime
(99mTc-HM-PAO) has gained favor as a lipophilic chelator,
able to diffuse through cell membranes, and is now
approved and routinely used for human cerebral tumors
imaging and leukocyte/red cell tagging applications
(Mortelmans et al., J Nucl Med., 1989; 30:2022-2028; and
Umbrain et al., Acta Anaesthesiol Scand., 1997; 41:25-
34). No investigator has to date reported conjugation or
incorporation of a radionuclide such as 99mTc to an
emulsion for ligand-targeted diagnostic imaging and/or
therapeutic applications.
The following examples illustrate the practice of
the invention.
Example 1
The procedure for preparing a biotinylated lipid
encapsulated perfluorodichlorooctane emulsion for use in
ultrasound imaging is as follows.
The biotinylated lipid perfluorodichlorooctane
(PFDCO) emulsion is comprised of the following
components: PFDCO (40% v/v), safflower oil (2.0%w/v), a
surfactant co-mixture (2.0% w/v) and glycerin (1.7% w/v).
The surfactant co-mixture is composed of approximately 64
mole% lecithin, 35 mole % cholesterol and 1 mole % N-(6-
biotinoyl)amino)
hexanoyl)dipalmitoyl-L-alpha-phosatidylethanolamine.
These components are weighed together into a test tube
and dissolved in chloroform. The chloroform is stripped
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
18
from the material and the resulting surfactant mixture is
dried in a 50 C vacuum oven overnight. The co-mixture is
dispersed into water by sonication resulting in a
liposome suspension. The suspension is transferred into
a 30 mL capacity blender cup (Dynamics Corporation of
America, New Hartford, CT) along with the PFDCO and oil.
The mixture is blended for 30-60 seconds to a pre-
emulsion. The preemulsified sample is transferred to the
reservoir of a microfluidizer, model S110 (Microfluidics,
Newton, MA), and emulsified for three minutes at 10,000
psi. To prevent the emulsion from heating excessively
during homogenization, the shear valve and mixing coil of
the microfluidizer are immersed in a room temperature
water bath during processing. The final temperature of
the emulsion is approximately 35 C. The finished
emulsion is bottled in 10 mL serum vials, blanketed with
nitrogen gas and sealed with stopper/crimp seal. The
average particle size of the finished product, measured
by a laser light scatter particle sizer (Brookhaven
Instruments Corporation, Holtsville, NY), is 250 nm.
Example 2
The incorporation of biotinylated
phosphatidylethanolamine into the encapsulating lipid
monolayer of perfluorocarbon emulsion is prepared as
described in Example one and demonstrated to increase
aggregate particle size in the presence of titrated
concentrations of avidin (Pierce, Rockford, IL 61105).
An identically prepared control emulsion is prepared
which incorporates nonbiotinylated
phosphatidylethanolamine into the outer lipid monolayer
of the perfluorocarbon emulsion. Avidin is resuspended
in isotonic phosphate buffered saline (PBS, Fisher Inc.,
Fair Lawn, NJ). Within a polystyrene cuvette, a 3.0 ml
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
19
reaction mixture is prepared containing PBS, biotinylated
or control perfluorocarbon emulsion (20 l) and avidin,
at 0.0, 0.5, 1.0, 1.5 or 2.0 g/ml. Contents are mixed
by gentle inversion and react for thirty minutes at room
temperature. Emulsion particle sizes are determined in
triplicate with a Brookhaven BI-90 particle size analyzer
(Holtsville, NY) at 37 C. Aggregate particle size of the
biotinylated emulsion increased progressively from a
baseline of 263 2.54 nm to greater than 2000 nm with
increasing concentration of avidin (Figure 1). Marked
flocculation and sedimentation are noted when avidin
concentrations exceed 2.0 g/ml. The particle size of
the control emulsion is
234 3.81 nm in diameter and addition of 2.0 g of avidin
to the reaction mixture does not affect particle size.
These results clearly demonstrate that the biotinylated
phosphatidylethanolamine is incorporated and oriented
appropriately into the outer lipid monolayer of the
perfluorocarbon emulsion and that surface biotins are
adequately available to avidin in the media. Multiple
biotin binding sites on the avidin molecule as well as
multiple biotin residues on the surface of the emulsion
progresses towards a rapid complexing of particles in
vitro.
Example 3
Biotinylated perfluorocarbon emulsion particles,
approximately 250 nm in diameter, with low independent
acoustic reflectivity, are complexed with avidin in
solution which eventuates aggregation and enhances
echogenicity. Biotinylated and control perfluorocarbon
emulsion (200 l) prepared as described previously are
diluted in PBS (15 ml) and placed within dialysis tubing
(Spectra/Por 4, 25 mm, MWCO 12,000-14,000, Spectrum
Medical Industries, Inc., Los Angeles, CA),
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
ultrasonically imaged within a PBS water bath at room
temperature using a 7.5 MHz focused transducer and a
Hewlett Packard (HP) Sonos 2500 Phased Array Imaging
System (Andover, MA). Real-time images are recorded to
5 SVHS video tape for subsequent image analysis. Pixel
grayscale and homogeneity are assessed on selected
freeze-frame images using NIH Image 1.47 (National
Institutes of Health). Avidin (30 g/ml) is added to
each emulsion suspension, mixed by gentle inversion and
10 allowed to complex for 30 minutes. The emulsion
suspensions are optically opaque but ultrasonically
undetected prior to the addition of avidin. Complexing
of the biotinylated perfluorocarbon emulsion ensues
rapidly with the addition of avidin and a white,
15 flocculant precipitate soon appears. Avidin induces no
changes in control emulsion suspension. Insonification
of the suspensions reveals that the biotinylated
perfluorocarbon emulsion particles opacify the dialysis
tubing; whereas, the control particles are not
20 appreciated acoustically (Figure 2). Gray scale echo
intensity analysis of freeze-frame images of the control
and biotinylated emulsion suspensions before and after
avidin are summarized in Figures 3 and 4. The increased
average grayscale level of the biotinylated emulsion
(71.3 22.1) suspension relative to its pre-avidin pixel
gray scale level (2.2 4.4) demonstrates the acoustic
enhancement achieved. Average pixel gray scale levels of
the control emulsion before (3 7.33) and after (1.0 1.3)
avidin addition are similar. These results demonstrate
the low acoustic reflectivity of the perfluorocarbon
emulsion when imaged as independent particles in
comparison with the enhanced echogenic nature of the
aggregated biotinylated particles. The lack of acoustic
CA 02373993 2007-08-30
21
change in the control emulsion suspension in the presence
of avidin confirms the ligand specificity of the
biotinylated emulsion.
Example 4
Biotinylated perfluorocarbon emulsion, approximately
250 nm diameter, are specifically targeted to avidin,
covalently bound to a modified nitrocellulose membrane
and increases the acoustic reflectivity of the membrane
surface at high ultrasonic frequencies (30 to 60 MHz).
Briefly, nitrocellulose membranes (S+S NCw, Schleicher &
Schuell, Keane, NH) were conjugated to avidin using a
diaminohexane (Sigma Chemical Co., St. Louis, MO) spacer
and glutaraldehyde-(Sigma Chemical Co., St. Louis, MO)
activation as described by Masson et al. (Electrophoresis
1993, 14, 860-865) . Nitrocellulose discs (2 cm diameter)
are soaked in 2.5% diaminohexane dissolved in deionized
water for 60 minutes with constant slow rotary agitation.
Membranes are washed with 1M acetic acid for 6-7 hours
followed by an 18+ hour deionized water wash with
constant agitation. The membranes are placed in 1%
glutaraldehyde in 0.1M sodium bicarbonate buffer, pH 10.0
for 15 minutes then washed for three hours with deionized
water. Nitrocellulose membranes are stored and dried at
4 C until use; storage does not exceed three days.
Fifty (50) l of avidin (250 g) are spotted
dropwise upon the center of six membranes with a
microliter syringe and allowed to dry. Each membrane is
extensively washed with a 0.1% TweenTM-20 (Sigma Chemical
Co., St. Louis, MO) in PBS then placed in 3% bovine serum
albumin (BSA, crystallized, Sigma Chemical Company, St.
Louis, MO) dissolved in PBS-0.1% Tween-20 for 20 minutes
to blockade nonspecific protein binding sites around the
periphery of the disc. After the BSA blockade, each disc
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
22
is extensively washed with PBS and placed in 300 l of
either biotinylated or control perfluorocarbon emulsion
suspended in 4 ml PBS for 20 minutes. Unbound emulsion
is removed in serial PBS washes. Each disc is reexposed
to avidin and control or biotinylated emulsion to ensure
saturated coverage of the nitrocellulose surface. The
nitrocellulose discs are washed and stored in PBS at 4 C
until imaged with acoustic microscopy.
For acoustic microscopic imaging, each
nitrocellulose disc is placed flat above a polished
stainless steel plate in a polystyrene holder with a 2 X
2 cm central window removed. The mounted specimen is
immersed into PBS at ambient temperature for ultrasonic
insonification. A custom designed acoustic microscope,
utilizing a 50MHz (nominal frequency) broadband, focused,
piezoelectric delay-line transducer (1/4 inch diameter,
1/2 inch focal length, Model V390, Panametrics Co.,
Waltham, MA) operated in the pulse-echo mode is utilized
for insonification. Backscattered radio frequency (RF)
data is collected and digitized at 500 megasamples per
second utilizing a Tektronix DSA 601 digitizing
oscilloscope (Beaverton, OR) with 8-bit resolution. A
variable gain system is used to increase the effective
dynamic range of this digitizer. Radio frequency data
are acquired from approximately 100 independent sites
from each region of interest with 100 micron lateral step
resolution.
A radio frequency peak-detected scan of the data is
converted into a gray scale (0=lowest soattering,
255=highest scattering) map to allow selection of regions
of interest for integrated backscatter analysis. Radio
frequency (RF) ultrasonic data are stored in a raster
scan format and analyzed with custom software. Segments
CA 02373993 2001-11-23
WO 00/71172 PCTIUS99/11491
23
of the RF lines are gated for integrated backscatter
analysis to encompass the front and back surfaces of the
nitrocellulose disc. The data are multiplied by a
rectangular window and their power spectra are determined
by fast-Fourier transformation. The power spectra from
the specimens referenced to the power spectrum returned
from a near-perfect steel planar reflector and the
frequency-dependent backscatter transfer function across
the useful bandwidth of the transducer (30 to 60 MHz) are
computed and expressed in decibels relative to acoustic
scattering from the near perfect steel plate reflector
(Wong et al., Ultrasound in Med & Biol. 1993; 19: 365-
374). Integrated backscatter (IB) is computed as the
average of the frequency-dependent backscatter transfer
function across the useful bandwidth of the transducer.
Discs incubated with biotinylated perfluorocarbon
emulsion have central regions with high acoustic
scattering in comparison with the peripheral (i.e.
background) regions of the same disc. Nitrocellulose
discs incubated with the control emulsion have no central
high scattering regions and no differences in acoustic
character is detected by changes in the RF signature
between the central and peripheral regions of the disc.
IB from the centrally-located, biotinylated emulsion
region (-17.8 0.2 db) is 6.3 0.1 dB(4-fold) greater
(p<0.05) than IB from the analogous region on the control
disc (-24.1 0.2 dB). The frequency-dependent variation
in apparent backscatter transfer function (mean SEM)
from the avidin spotted regions of the biotinylated and
control emulsion discs are presented in Figure 5. A
smooth and consistently greater acoustic response is
noted across the frequency spectrum due to the bound
biotinylated emulsion. These results demonstrate the
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
24
effectiveness of the biotinylated perfluorocarbon
emulsion to specifically target a surface bound antigen
and dramatically alter the acoustic reflectivity of the
surface with the bathing medium, increasing the
ultrasonic backscattered power at high frequencies.
Example 5
Biotinylated perfluorocarbon emulsion (250 nm
diameter) is specifically targeted to D-dimer covalently
attached to a modified nitrocellulose membrane utilizing
a biotinylated anti-D-dimer F(ab) fragment-avidin complex
and results in a marked increase in the acoustic power
reflected from the surface. D-dimer is covalently linked
to nitrocellulose discs modified with a diaminohexane
spacer arm and activated with glutaraldehyde as
previously described in Example 4. Fifty (50) g of D-
dimer is spotted with a microliter syringe upon the
center of three of six membranes and allowed to air dry.
Unbound D-dimer is exhaustively washed from the membranes
with phosphate buffered saline (PBS)-0.1% Tween-20.
Nonspecific protein binding sites of all membranes are
blocked with 3% bovine serum albumin (BSA) in PBS-0.1%
Tween-20 for 20 minutes followed by serial PBS washes.
D-dimer spotted membranes are incubated with 12.5 g
biotinylated anti-D-dimer F(ab) antibody in 4.0 ml 3% BSA
for 2 hours, washed with PBS buffer and then incubated
with 250 g avidin in 4 ml PBS for 30 min. After
removing unbound avidin with PBS washes, the discs are
exposed to either biotinylated or control perfluorocarbon
emulsion (300 l) in 4.0 ml PBS for 20 minutes. Excess
emulsion is removed with PBS buffer washes. Discs are
reexposed to avidin and perfluorocarbon emulsion as
described above and the membranes are stored in PBS at
4 C until imaging.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
For acoustic microscopic imaging, each
nitrocellulose disc is placed flat above a polished
stainless steel plate in a polystyrene holder, immersed
in PBS at ambient temperature, and insonified with a
5 custom designed acoustic microscope, utilizing a 50MHz
(nominal frequency) broadband, focused, piezoelectric
delay-line transducer (1/4 inch diameter, 1/2 inch focal
length, Model V390, Panametrics Co., Waltham, MA)
operated in the pulse-echo mode. Backscattered radio
10 frequency (RF) data is collected and digitized at 500
megasamples per second utilizing a Tektronix DSA 601
digitizing oscilloscope (Beaverton, OR) with 8-bit
resolution. A variable gain system is used to increase
the effective dynamic range of this digitizer. Radio
15 frequency data are acquired from approximately 100
independent sites from each region of interest with 100
micron lateral step resolution.
A radio frequency peak-detected scan of the data is
converted into a gray scale (0=lowest scattering,
20 255=highest scattering) map of the disc to allow visual
inspection and selection of regions of interest for
integrated backscatter (IB) analysis. Radio frequency
(RF) ultrasonic data are stored in a raster scan format
and analyzed with custom software. Segments of the RF
25 lines are gated for integrated backscatter analysis to
encompass the front and back surfaces of the
nitrocellulose disc. The data are multiplied by a
rectangular window and their power spectra are determined
by fast-Fourier transformation. The power spectra from
the specimens referenced to the power spectrum returned
from a near-perfect steel planar reflector and the
frequency-dependent backscatter transfer function across
the useful bandwidth of the transducer (30 to 60 MHz) are
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
26
computed and expressed in decibels relative to acoustic
scattering from the near perfect steel plate reflector
(Wong et al., Ultrasound in Med & Biol. 1993; 19: 365-
374). Integrated backscatter is computed as the average
of the frequency-dependent backscatter transfer function
across the useful bandwidth of the transducer.
Biotinylated, anti-D-dimer F(ab) fragment is
specifically bound to the central region of the D-dimer
spotted discs and crosslinked by avidin through its
biotin moiety. As in previous examples, biotinylated
perfluorocarbon emulsion specifically binds to the
antibody bound avidin; whereas, the nonspecific binding
of the control emulsion have no binding and are not
detected acoustically. IB of the biotinylated emulsion
coated nitrocellulose (-18.0 0.2 dB) was greater by
4.6 0.1 dB (p<0.05) than that from the control disc (-
22.6 0.1 dB) over the 30 to 60 MHz frequency range. The
frequency-dependent variation in apparent backscatter
transfer function (mean SEM) of the biotinylated and
control emulsion discs are presented in Figure 6. A
smooth and consistently greater acoustic response is
noted across the frequency spectrum due to the bound
biotinylated emulsion. These data confirm and extend the
findings of Example 4 with avidin alone, demonstrating
that biotinylated perfluorocarbon emulsion bound through
a specific, targeting ligand system can significantly
enhance the acoustic backscatter of a solid support
surf ace .
Example 6
Biotinylated perfluorocarbon emulsion (250 nm
diameter) is specifically targeted to avidin conjugated
to nitrocellulose discs and insonified at clinically
relevant frequencies (5 to 15 MHz) and significantly
CA 02373993 2001-11-23
WO 00/71172 PCTIUS99/11491
27
increases the acoustic backscatter of the membrane.
Briefly, nitrocellulose membranes (S+S NCTM, Schleicher &
Schuell, Keane, NH) are conjugated to avidin using a
diaminohexane (Sigma Chemical Co., St. Louis, MO) spacer
and glutaraldehyde (Sigma Chemical Co., St. Louis, MO)
activation as described by Masson et al. (Electrophoresis
1993, 14, 860-865). Nitrocellulose discs (2 cm diameter)
are soaked in 2.5% diaminohexane dissolved in deionized
water for 60 minutes with constant, slow rotary
agitation. Membranes are transferred to and washed with
1M acetic acid for 6-7 hours then transferred for
continued washing in deionized water for at least 18
additional hours with constant agitation. The membranes
are placed in 1% glutaraldehyde in 0.1M sodium
bicarbonate buffer, pH 10.0 for 15 minutes. After
glutaraldehyde activation is complete, the membranes are
washed with continued agitation for three hours. The
nitrocellulose membranes stored and dried at 4 C until
use; storage does not exceed three days.
Fifty (50) l of avidin (250 g) are spotted
dropwise upon the center of a nitrocellulose membrane
with a microliter syringe and allowed to dry. Each
membrane is washed with 0.1% Tween-20 (Sigma Chemical
Co., St. Louis, MO) in phosphate buffered saline (PBS)
then placed in 3% bovine serum albumin (BSA,
crystallized, Sigma Chemical Company, St. Louis, MO)
dissolved PBS-0.1% Tween-20 for 20 minutes to blockade
nonspecific protein binding sites around the periphery of
the disc. After the BSA blockade, each disc is washed
with PBS and placed in 300 l of either biotinylated or
control perfluorocarbon emulsions suspended in 4 ml PBS
for 20 minutes with mild, rotary agitation. The unbound
emulsion is removed with washes of PBS. Each disc is
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
28
reexposed to avidin, washed with PBS, reexposed to
control or biotinylated perfluorocarbon emulsion and
rewashed with PBS as previously described. The
nitrocellulose discs are stored in PBS at 4 C until
imaged with the acoustic microscope.
For acoustic microscopic imaging, each
nitrocellulose disc is placed flat above a polished
stainless steel plate in a polystyrene holder with a 2 cm
x 2 cm central window removed. The mounted specimen is
immersed into PBS at ambient temperature for ultrasonic
insonification. A custom designed acoustic microscope,
utilizing a 10 MHz (nominal frequency) broadband,
focused, piezoelectric delay-line transducer (1/2 inch
diameter, 2 inch focal length, Model V311, Panametrics
Co., Waltham, MA) operated in the pulse-echo mode is
utilized for insonification. Backscattered radio
frequency (RF) data is collected and digitized at 500
megasamples per second utilizing a Tektronix DSA 601
digitizing oscilloscope (Beaverton, OR) with 8-bit
resolution. A variable gain system is used to increase
the effective dynamic range of this digitizer. Radio
frequency data are acquired from approximately 100
independent sites from each region of interest with 250
micron lateral step resolution.
A radio frequency peak-detected scan of the data is
converted into a gray scale (0=lowest scattering,
255=highest scattering) map of the disc to allow visual
inspection and selection of regions of interest for
integrated backscatter analysis. Radio frequency
ultrasonic data are stored in a raster scan format and
analyzed with custom software. Segments of the RF lines
are gated for integrated backscatter analysis to
encompass the front and back surfaces of the
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
29
nitrocellulose disc. The data are multiplied by a
rectangular window and their power spectra are determined
by fast-Fourier transformation. The power spectra from
the specimens referenced to the power spectrum returned
from a near-perfect steel planar reflector and the
frequency-dependent backscatter transfer function across
the useful bandwidth of the transducer (5 to 15 MHz) are
computed and expressed in decibels relative to acoustic
scattering from the near perfect steel plate reflector
(Wong et al., Ultrasound in Med & Biol. 1993; 19: 365-
374). Integrated backscatter (IB) is computed as the
average of the frequency-dependent backscatter transfer
function across the useful bandwidth of the transducer.
Discs incubated with biotinylated perfluorocarbon
emulsion have central regions with high acoustic
scattering relative to the peripheral regions of the same
disc or the central regions of the control emulsion disc.
Nitrocellulose discs incubated with the control emulsion
have no high scattering regions. ID of the biotinylated
emulsion coated nitrocellulose (0.5 0.5 dB) was greater
by 9.6 0.1 dB (8-fold) (p<0.05) than that from the
control disc (-9.2 0.5 dB) over the 5 to 15 MHz frequency
range. The frequency-dependent variation in apparent
backscatter transfer function (mean SEM) of the
biotinylated and control emulsion discs are presented in
Figure 7. A smooth and consistently greater acoustic
response is noted across the frequency spectrum due to
the bound biotinylated emulsion. These data confirm and
extend the findings of Examples 4 and 5 with avidin and
D-dimer, demonstrating that biotinylated perfluorocarbon
emulsion bound through a specific, targeting ligand
system can significantly enhance the acoustic backscatter
of a solid support surface and that this improved
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
acoustic backscatter is detected at low, clinically
useful ultrasonic frequencies (5 to 15 MHz) as well as
high frequencies (30 to 60 MHz).
Example 7
5 Biotinylated perfluorocarbon contrast, approximately
3000 nm diameter, is specifically targeted to avidin
conjugated to nitrocellulose discs and insonified at
clinically relevant frequencies (at least 5 to 15 MHz
,
bandinidth). Briefly, nitrocellulose membranes (S+S NCTM
10 Schleicher & Schuell, Keane, NH) are conjugated to avidin
using a diaminohexane (Sigma Chemical Co., St. Louis, MO)
spacer and glutaraldehyde (Sigma Chemical Co., St. Louis,
MO) activation as described by Masson et al.
(Electrophoresis 1993, 14, 860-865). Nitrocellulose
15 discs (2 cm diameter) are soaked in 2.5% diaminohexane
dissolved in deionized water for 60 minutes with
constant, slow rotary agitation. Membranes are
transferred to and washed with 1M acetic acid for 6-7
hours then transferred for continued washing in deionized
20 water for at least.18 additional hours with constant
agitation. The membranes are placed in 1% glutaraldehyde
in 0.1M sodium bicarbonate buffer, pH 10.0 for 15
minutes. After glutaraldehyde activation is complete,
the membranes are washed with continued agitation for
25 three hours. The nitrocellulose membranes stored and
dried at 4 C until use; storage does not exceed three
days.
Fifty (50) l of avidin (250 g) are spotted
dropwise upon the center of two of four membranes with a
30 microliter syringe and allowed to dry. Each membrane is
washed with 0.1% Tween-20 (Sigma Chemical Co., St. Louis,
MO) in phosphate buffered saline (PBS) then placed in 3%
bovine serum albumin (BSA, crystallized, Sigma Chemical
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
31
Company, St. Louis, MO) dissolved PBS-0.1% Tween-20 for
20 minutes to blockade nonspecific protein binding sites
around the periphery of the disc. After the BSA
blockade, each disc is washed with PBS and placed in 300
l of either biotinylated or control perfluorocarbon
emulsions, approximately 3000 nm particle size, suspended
in 4 ml PBS for 20 minutes with mild, rotary agitation.
The unbound emulsion is removed with washes of PBS. Each
disc is reexposed to avidin, washed with PBS, exposed to
perfluorocarbon emulsion and rewashed with PBS as
previously described. The nitrocellulose discs are
stored in PBS at 4 C until imaged with the acoustic
microscope.
For acoustic microscope imaging, each nitrocellulose
disc is placed flat above a polished stainless steel
plate in a polystyrene holder with a 2 cm x 2 cm central
window removed. The mounted specimen is immersed into
PBS at ambient temperature for ultrasonic insonification.
A custom designed acoustic microscope, utilizing a
broadband 10 MHz (nominal frequency) focused,
piezoelectric delay-line transducer (1/2 inch diameter, 2
inch focal length, Model V311, Panametrics Co., Waltham,
AM) operated in the pulse-echo mode is utilized for
insonification. Backscattered radio frequency (RF) data
is collected and digitized at 500 megasamples per second
utilizing a Tektronic DSA 601 digitizing oscilloscope
(Beaverton, OR) with 8-bit resolution. A variable gain
system is used to increase the effective dynamic range of
this digitizer. Radio frequency data are acquired from
approximately 100 independent sites from each region of
interest with 250 micron lateral step resolution.
A radio frequency peak-detected scan of the data is
converted into a gray scale (0=lowest scattering,
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
32
255=highest scattering) map of the disc to allow visual
inspection and selection of regions of interest for
integrated backscatter analysis. Radio frequency
ultrasonic data are stored in a raster scan format and
analyzed with custom software. Segments of the RF lines
are gated for integrated backscatter (IB) analysis to
encompass the front and back surfaces of the
nitrocellulose disc. The data are multiplied by a
rectangular window and their power spectra are determined
by fast-Fourier transformation. The power spectra from
the specimens referenced to_the power spectrum returned
from a near-perfect steel planar reflector and the
frequency-dependent backscatter transfer function across
the useful bandwidth of the transducer (5 to 15 MHz) are
computed and expressed in decibels relative to acoustic
scattering from the near perfect steel plate reflector
(Wong et al., Ultrasound in Med & Biol. 1993; 19: 365-
374). Integrated backscatter is computed as the average
of the frequency-dependent backscatter transfer function
across the useful bandwidth of the transducer.
Discs incubated with biotinylated perfluorocarbon
emulsion have central regions with high acoustic
scattering relative to the peripheral regions of the same
disc and central regions of the control disc.
Nitrocellulose discs incubated with the control emulsion
have no central high scattering regions and no
differences in acoustic character are detected between
the central and peripheral regions of the disc. IB of
the biotinylated emulsion coated nitrocellulose (-2.4 0.7
dB) was greater by 8.8 0.3 dB (approximately 8-fold
(p<0.05)) than that from the control disc (-11.2 0.4 dB)
over the 5 to 15 MHz frequency range. The frequency-
dependent variation in apparent backscatter transfer
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
33
function (mean SEM) of the biotinylated and control
emulsion discs are presented in Figure 8. A smooth and
consistently greater acoustic response is noted across
the frequency spectrum due to the bound biotinylated
emulsion. These data confirm and extend the findings of
Examples 4, 5 and 6 with avidin and D-dimer,
demonstrating that biotinylated perfluorocarbon emulsions
with large particle sizes can be bound through a
specific, targeting ligand system and significantly
enhance the acoustic backscatter of a solid support
surface. This improved acoustic backscatter is detected
at clinically relevant ultrasonic frequencies, 5 to 15
MHz.
Example 8
Biotinylated perfluorocarbon emulsion is targeted to
a plasma thrombi using biotinylated antifibrin monoclonal
antibodies (NIB1H10; Tymkewycz et al. 1993. Blood
Coagulation and Fibrinolysis 4:211-221) and avidin. In a
representative study (1 of 5), whole porcine blood is
obtained and anticoagulated (9:1, v/v) with sterile
sodium citrate. Blood is centrifuged at 1500 RPM at room
temperature and the plasma fraction is obtained and
stored at 4 C. Two porcine plasma thrombi are produced
by combining plasma, 100 mM calcium chloride (3:1 v/v)
and 2-5 U thrombin in a plastic tube through which 5-0
Vicryl suture is passed. Thrombi are allowed to
coagulate at room temperature.
One thrombus is incubated with 150 g antifibrin
monoclonal antibody in 10 ml PBS with 1% bovine serum
albumin (BSA) for two hours and a second control thrombus
is incubated in PBS with 1% BSA. The antibody treated
thrombus is then incubated with 0.5 mg avidin in 10 ml
PBS with 1% BSA for 30 minutes. The control thrombus
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
34
remains in PBS with 1% BSA. Both thrombi are washed
extensively with PBS. Each thrombus is incubated with
300 1/10 ml PBS of either biotinylated or control
emulsion for 30 minutes. All thrombi are reexposed to
emulsion twice to ensure uniform coverage and
ultrasonically insonified (Figure 9). Ultrasonic imaging
is performed using a 7.5 MHz focused, linear phased array
transducer and a Hewlett Packard Sonos 2500 Imaging
System (Hewlett Packard, Inc., Andover, MA). All
ultrasonic recordings are produced with fixed gain,
compensation and time-gain compensation levels and are
recorded on to SVHS videotape for subsequent image
analysis. Average pixel grayscale over an extensive
region of interest was sampled for 21 independent freeze-
frame images for each thrombus using NIH Image 1.47
(National Institutes of Health; Figure 10). The
biotinylated perfluorocarbon emulsion is found to provide
a marked acoustic enhancement of the surface. Average
pixel grayscale levels of the biotinylated emulsion
thrombus are 79.5 2.5 whereas the brightness of the
control was markedly less (34.8 2.2, p<0.05). These
results demonstrate the ability of biotinylated
perfluorocarbon emulsion to target and acoustically
enhance a biological tissue (i.e. thrombus) in vitro.
Example 9
Biotinylated perfluorocarbon emulsion is targeted
via biotinylated antifibrin antibodies (NIB5F3 and
NIB1H10 Tymkewycz et al. 1993. Blood Coagulation and
Fibrinolysis 4:211-221) to an isolated femoral artery
thrombus in six mongrel dogs. A mongrel dog is
anesthetized with sodium pentobarbital induction and
halothane anesthesia. The right femoral artery and all
branches are isolated at the level of the saphenous
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
branch. A silver plated copper wire attached to a 22 ga.
right angled needle point, insulated with plastic tubing
(polyethylene P-240), is inserted into the femoral artery
and secured with 4-0 Prolene suture. A current of 200-
5 400 A is applied for up to two hours. Thrombus
formation is monitored with continuous wave doppler and
discontinued after an approximately 50% increase in
circulation velocity is noted distal to the electrical
injury. Adventitial discoloration secondary to the
10 current is appreciated proximal to the entry point of the
wire. A 20 ga. catheter is inserted into a proximal
branch of the femoral artery and secured with 4-0 silk
suture. A pressurized 0.9% NaCl drip is attached through
a three-way stopcock to the catheter. Blood flow into
15 the isolated segment is disrupted by proximal snare
ligature. Excess blood is flushed from the arterial
segment to inhibit further thrombus formation by infusion
of saline for 15 minutes. The distal draining branches
of the femoral artery are ligated or snared with suture.
20 Biotinylated antifibrin monoclonal antibody (50 g/1.0 ml
PBS) is injected via the catheter and flushed with a few
drops of saline. The antibody is allowed to incubate for
one hour then the snare ligature distal to the wire
insertion is released and excess antibody is flushed
25 through with saline for five minutes. The distal femoral
artery is reoccluded and avidin (250 g/1.0 ml PBS) is
infused and incubates for 30 minutes. The distal
ligature is again released and excess avidin is flushed
through with saline for five minutes. The distal
30 ligature is reestablished and biotinylated
perfluorocarbon emulsion is infused and incubates for 30
minutes. After the initial exposure of the thrombus to
the emulsion, the unbound emulsion is washed through with
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
36
saline. Thrombi are each exposed to avidin and
biotinylated perfluorocarbon emulsion as described above.
In three animals, the contra lateral artery is also
isolated, partially occluded with electrically induced
thrombi and exposed to a control perfluorocarbon emulsion
analagous to the administration of biotinylated emulsion
described above. Femoral arteries exposed to either
control or biotinylated perfluorocarbon emulsion are
ultrasonically imaged at 7.5 MHz with a focused, linear
phased array transducer and a clinical Hewlett-Packard
Sonos 2500 Ultrasonic Imaging System before and after
contrast administration. Acutely formed thrombi, both
control and contrast targeted, are not ultrasonically
appreciated. For 6 of 6 femoral arteries, partially
occlusive thrombi are markedly enhanced using the
antifibrin targeted biotinylated perfluorocarbon
contrast. In 3 of 3 femoral arteries thrombi, exposure
to the control perfluorocarbon emulsion does not
accentuate their acoustic reflectivity and these thrombi
remain ultrasonically undetectable. Figure 11 reveals a
representative example of a femoral artery site of
thrombus formation after electrical induction before and
after exposure to antifibrin antibody and biotinylated
contrast. In the pre-contrast image, the femoral artery
is observed with a bright echogenic wire point anode
protruding into the lumen but no thrombus is appreciated.
After treatment with the biotinylated contrast emulsion,
a large partially occluded thrombus is clearly noted by
the enhanced acoustic reflectivity (Figure 11). Again,
no thrombus is appreciated in the control artery before
or after exposure to control emulsion. These results
demonstrate the concept of using bound perfluorocarbon
emulsion to acoustically enhance biological surfaces,
CA 02373993 2007-08-30
37
such as thrombotic tissue, in vivo to enable detection
with a commercially available ultrasound imaging system.
Example 10
Biotinylated perfluorocarbon emulsion, approximately
250 nm diameter, is targeted to prostatic carcinoma using
monoclonal antibodies specific for prostate specific
antigen (PSA) and are acoustically detected using polar,
high frequency, high resolution acoustic microscopy.
Representative examples of human prostatic carcinoma
tissues are routinely processed by immersion fixation in
10% neutral buffered formalin and embedded in paraffin.
Twenty micron sections are prepared for acoustic
microscopy; 5 micron sections are used for optical
studies. All histologic sections are mounted on acid
cleaned glass slides that have been coated with poly-L-
lysine. All mounted sections are heated at 55 C for 1
hour in an oven.
Prior to immunostaining, all sections are dewaxed in
three changes of Americlear', and dehydrated in successive
changes of 95% and 100% ethanol. Endogenous peroxidase
activity is blocked only in sections prepared for optical
studies by immersion in absolute methanol containing 0.6%
(v/v) hydrogen peroxide for 30 minutes. These and all
sections for acoustic microscopy are then rehydrated
through graded ethanols and distilled water and placed in
isotonic PBS (pH 7.4). All sections are incubated with
target specific monoclonal antibodies.
Prostate sections are incubated with anti-PSA
primary monoclonal antibodies per the recommendations of
the vendor for 18 hours at 4 C in moisture chambers.
After primary incubation, sections are rinsed in isotonic
PBS, then overlain with a polyclonal biotinyl-horse anti-
mouse immunoglobin (VectaStain Elite Kits, Vector
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
38
Laboratories, Burlingame, CA) for 1 hour at room
temperature. After rinsing in PBS, a 30 micron section
is prepared for acoustic microscopy. A section for light
microscopy (5 micron) is incubated with avidin-biotin-
peroxidase complex (VectaStain Elite Kit, Vector Lab) for
1 hour at room temperature. This section is rinsed in
phosphate buffer (pH 7.6) and immersed in a solution of
3,31-diaminobenzidine tetrahydrochloride (Sigma
Chemicals, St. Louis, MO; 0.5 mg/ml in phosphate buffer,
pH 7.6, containing 0.003% [v/v] hydrogen peroxide) for
approximately ten minutes. The chromogenic precipitate
is optically enhanced by brief immersion of stained
sections in 0.125% (w/v) osmium tetroxide. The section
is then rinsed in tap water, counterstained in Harris'
hematoxylin, dehydrated in graded ethanols and
Americlear, and mounted in a synthetic mounting medium.
After the second biotinylated antibody is incubated
and washed, slides for acoustic microscopy are incubated
in avidin (1.0 mg/-20cc PBS) using a bath on a rotating
table for 30 min. Excess avidin is washed away with
isotonic PBS buffer, pH 7.4-7.5 in three minute washes.
Slides are incubated with biotinylated or control
perfluorocarbon emulsion for twenty minutes (0.5 cc/-20.0
ml PBS), washed briefly with isotonic PBS 3X for 5
minutes each and reincubated with avidin (1.0 mg/-20cc)
for 15 minutes. Excess avidin is rinsed off with three,
5 min. washes in PBS. The slide is then reincubated at
above concentrations with biotinylated or control
perfluorocarbon emulsion for 20 minutes. Unbound
emulsion is washed away in three changes of PBS (5
minutes each) and the slides are transferred to the
acoustic microscope for analysis.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
39
The mounted specimens are each immersed into
isotonic, phosphate buffered saline at room temperature
for ultrasonic insonification. A custom designed
acoustic microscope is used to collect ultrasonic data.
The microscope consists of 50 MHz broadband, focused,
piezoelectric delay-line transducer (1/4 inch diameter,
1/2 inch focal length, 62 micron beam diameter, Model
V390, Panametrics Co., Waltham, MA) operated in the
pulse-echo mode. A Tektronix DSA 601 digitizing
oscilloscope (Beaverton, OR) is used to digitize 35
degree polar backscattered radio frequency (rf) data at
500 megasamples per second with 8-bit resolution. A
variable gain system is used to increase the effective
dynamic range of this digitizer. Radio frequency data is
acquired from approximately 100 independent sites from
each specimen with 50 micron lateral step resolution.
The rf data is stored in a low resolution raster
scan format and analyzed with custom software. Segments
of the rf lines are gated for integrated backscatter
analysis to encompass the front surface (i.e. excluding
the back wall). The gated data are multiplied by a
Hamming window and their power spectra are determined by
fast-Fourier transformation. Power spectra within a
tissue section are compared directly without reference to
a steel plate. Integrated backscatter (IB) is computed
from the average of the frequency-dependent backscatter
transfer function across the useful bandwidth of the
transducer (30 to 55 MHz). Immunostained tissues are
reviewed using a Nikon Optiphot-2 microscope for regions
of PSA positive staining and the acoustic characteristics
are compared.
The net change in the apparent backscatter transfer
function between the normal prostatic stroma and
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
carcinomatous regions are clearly increased in sections
treated with PSA targeted biotinylated versus the control
perfluorocarbon emulsion across the frequency spectrum
(30 to 55 MHz; Figure 12). Biotinylated perfluorocarbon
5 emulsion increases (p<0.05) the integrated backscatter
from regions of prostatic cancer (47.17 dB) versus normal
stromal (40.79 1.18 dB) by 6.38 dB (approximately 4-
fold). In the control tissue sections, the integrated
backscatter from the region of prostatic carcinoma
10 (39.63 1.63 dB) was greater (p<0.05) than that from the
normal stromal areas (36.13 2.17 dB) by approximately 3.5
dB (2-fold), reflecting inherent differences in acoustic
character between normal and cancerous prostatic tissue.
However, the targeted biotinylated perfluorocarbon
15 emulsion amplified (p<0.05) these inherent differences by
approximately 2-fold (2.87 dB; Figure 13). These results
clearly demonstrate the ability of site-targeted
biotinylated perfluorocarbon emulsion to specifically
enhance acoustic detection of prostate cancer in vitro.
20 Example 11
Biotinylated perfluorocarbon emulsion, approximately
250 nm diameter, is targeted to ovarian carcinoma using
monoclonal antibodies specific for OC-125 antigen and are
acoustically detected using polar, high frequency, high
25 resolution acoustic microscopy. Representative examples
of human ovarian, carcinoma tissues are routinely
processed by immersion fixation in 10% neutral buffered
formalin and embedded in paraffin. Twenty micron
sections are prepared for acoustic microscopy; 5 micron
30 sections are used for optical studies. All histologic
sections are mounted on acid cleaned glass slides that
have been coated with poly-L-lysine. All mounted
sections are heated to 55 C for 1 hour in an oven.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
41
Prior to immunostaining, all sections are dewaxed in
three changes of Americlear, and dehydrated in successive
changes of 95% and 100% ethanol. Endogenous peroxidase
activity is blocked only in sections prepared for optical
studies by immersion in absolute methanol containing 0.6%
(v/v) hydrogen peroxide for 30 minutes. These and all
sections for acoustic microscopy are then rehydrated
through graded ethanols and distilled water and placed in
isotonic PBS (pH 7.4). All sections are incubated with
target specific monoclonal antibodies.
Ovarian sections are incubated with anti-OC-125
primary monoclonal antibodies per the recommendations of
the vendor for 18 hours at 4 C in moisture chambers.
After primary incubation, sections are rinsed in isotonic
PBS, then overlain with a polyclonal biotinyl-horse anti-
mouse immunoglobin (VectaStain Elite Kits, Vector
Laboratories, Burlingame, CA) for 1 hour at room
temperature. After rinsing in PBS, duplicate 30 micron
sections are prepared for acoustic microscopy. Sections
for light microscopy (5 micron) are incubated with
avidin-biotin-peroxidase complex (VectaStain Elite Kit,
Vector Lab) for 1 hour at room temperature. Sections are
rinsed in phosphate buffer (pH 7.6) and immersed in a
solution at 3,3'-diaminobenzidine tetrahydrochloride
(Sigma Chemicals, St. Louis, MO; 0.5 mg/ml in phosphate
buffer, pH 7.6, containing 0.0003% [v/v] hydrogen
peroxide) for approximately ten minutes. The chromogenic
precipitate is optically enhanced by brief immersion of
stained sections in 0.125% (w/v) osmium tetroxide.
Sections are then rinsed in tap water, counterstained in
Harris' hematoxylin, dehydrated in graded ethanols and
Americlear, and mounted in a synthetic mounting medium.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
42
After the second biotinylated antibody is incubated
and washed, slides are incubated in avidin (1.0 mg/-20 cc
PBS) using a bath on a rotating table for 30 min. Excess
avidin is washed away with isotonic PBS buffer, pH 7.4-
7.5 in three 5 minute washes. The prepared slides are
incubated with biotinylated or control perfluorocarbon
emulsion for twenty minutes (0.5 cc/-20.0 ml PBS), washed
briefly with isotonic PBS 3X for 5 minutes each and
rewashed in avidin (1.0 mg/-20 cc) for 15 minutes.
Excess avidin is rinsed off with three, 5 min. washes in
PBS. Slides are then reincubated at above concentrations
with biotinylated or control perfluorocarbon emulsion for
minutes. Unbound emulsion is washed away in three
changes of PBS (5 minutes each) and the slides are
15 transferred to the acoustic microscope for analysis.
The mounted specimens are each immersed into
isotonic, phosphate buffered saline at room temperature
for ultrasonic insonification. A custom designed
acoustic microscope is used to collect ultrasonic data.
20 The microscope consists of 50MHz broadband, focused,
piezoelectric delay-line transducer (1/4 inch diameter,
1/2 inch focal length, 62 micron beam diameter, Model
V390, Panametrics Co., Waltham, MA) operated in the
pulse-echo mode. A Tektronix DSA 601 digitizing
oscilloscope (Beaverton, OR) is used to digitize 35
degree polar backscattered radio frequency (rf) data at
500 megasamples per second with 8-bit resolution. A
variable gain system is used to increase the effective
dynamic range of this digitizer. Radio frequency data is
acquired from approximately 100 independent sites from
each specimen with 50 micron lateral step resolution.
The rf data are stored in a low resolution raster
scan format and analyzed with custom software. Segments
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
43
of the rf lines are gated for integrated backscatter
analysis to encompass the front surface (i.e. excluding
the back wall). The gated data are multiplied by a
Hamming window and their power spectra are determined by
fast-Fourier transformation. Integrated backscatter is
computed from the average of the frequency-dependent
backscatter transfer function across the useful bandwidth
of the transducer (30 to 55 MHz). The power spectra from
the specimens are referenced to the power spectrum
returned from a glass microscope slide. IB is expressed
in decibels relative to the scattering from the glass
slide. Immunostained tissues are reviewed using a Nikon
Optiphot-2 microscope for regions of PSA positive
staining and the acoustic characteristics are compared.
The net change in the apparent backscatter transfer
function between the normal ovarian stroma and
carcinomatous regions are clearly increased in sections
treated with OC-125 targeted biotinylated versus the
control perfluorocarbon emulsion across the frequency
spectrum (30 to 55 MHz; Figure 14). Biotinylated
perfluorocarbon emulsion increases (p<0.05) the
integrated backscatter from regions of ovarian cancer (-
28.19 1.39 dB) versus normal stromal (-38.75 0.84 dB) by
10.57 dB (greater than 8-fold). In the control tissue
sections, the integrated backscatter from the region of
ovarian carcinoma (-33.49 0.86 dB) was greater (p <0.05)
than the normal stromal areas (-40.21 0.61 dB),
approximately 6.72 dB (4-fold), reflecting inherent
differences in acoustic character between normal and
cancerous ovarian tissue. However, the targeted
biotinylated perfluorocarbon emulsion amplified (p<0.05)
these inherent differences by approximately 2-fold (3.84
dB; Figure 15). These results clearly demonstrate the
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
44
ability of site-targeted biotinylated perfluorocarbon
emulsion to specifically enhance acoustic detection of
ovarian cancer in vitro.
Example 12
Biotinylated perfluorocarbon emulsion, approximately
250 nm diameter, is targeted to the epithelial capsule of
tonsil using monoclonal antibodies specific for
cytokeratin, CD-20, and BCL-2 antigens and are
acoustically detected using polar, high frequency, high
resolution acoustic microscopy. Representative examples
of human tonsil are routinely processed by immersion
fixation in 10% neutral buffered formalin and embedded in
paraffin. Twenty micron sections are prepared for
acoustic microscopy; a 5 micron section is used for
optical studies. All histologic sections are mounted on
acid cleaned glass slides that have been coated with
poly-L-lysine. All mounted sections are heated at 55 C
for 1 hour in an oven.
Prior to immunostaining, all sections are dewaxed in
three changes of Americlear, and dehydrated in successive
changes of 95% and 100% ethanol. Endogenous peroxidase
activity is blocked only in sections prepared for optical
studies by immersion in absolute methanol containing 0.6%
(v/v) hydrogen peroxide for 30 minutes. These and all
sections for acoustic microscopy are then rehydrated
through graded ethanols and distilled water and placed in
isotonic PBS (pH 7.4). All sections are incubated with
target specific monoclonal antibodies.
Tonsil sections are incubated with a mixture of
anti-CD-20, BCL-2, and cytokeratin primary monoclonal
antibodies per the recommendations of the vendor for 18
hours at 4 C in moisture chambers. After primary
incubation, sections are rinsed in isotonic PBS, then
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
overlain with a polyclonal biotinyl-horse anti-mouse
immunoglobin (VectaStain Elite Kits, Vector Laboratories,
Burlingame, CA) for 1 hour at room temperature. After
rinsing in PBS, duplicate 30 micron sections are prepared
5 for acoustic microscopy. Sections for light microscopy
(5 micron) are incubated with avidin-biotin-peroxidase
complex (VectaStain Elite Kit, Vector Lab) for 1 hour at
room temperature. Sections are rinsed in phosphate
buffer (pH 7.6) and immersed in a solution of 3,3'-
10 diaminobenzidine tetrahydrochloride (Sigma Chemicals, St.
Louis, MO; 0.5 mg/ml in phosphate buffer, pH 7.6,
containing 0.003% [v/v] hydrogen peroxide) for
approximately ten minutes. The chromogenic precipitate
are optically enhanced by brief immersion of stained
15 sections in 0.125% (w/v) osmium tetroxide. Sections are
then rinsed in tap water, counterstained in Harris'
hematoxylin, dehydrated in graded ethanols and
Americlear, and mounted in a synthetic mounting medium.
After the second biotinylated antibody is incubated
20 and washed, one slide is incubated in avidin (1.0 mg/-20
cc PBS) using a bath on a rotating table for 30 min.
Excess avidin is washed away with isotonic PBS buffer, pH
7.4-7.5 in three 5 minute washes. The prepared slide is
incubated with biotinylated perfluorocarbon emulsion for
25 twenty minutes (0.5 cc/-20.0 ml PBS), washed briefly with
isotonic PBS 3X for 5 minutes each and rewashed in avidin
(1.0 mg/-20 cc) for 15 minutes. Excess avidin is rinsed
off with three, 5 minute washes in PBS. The slides are
reincubated at above concentrations with biotinylated
30 perfluorocarbon emulsion for 20 minutes. Unbound
emulsion is washed away in three changes of PBS (5
minutes each) and the slide is transferred to the
acoustic microscope for analysis.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
46
The mounted specimen is immersed into isotonic,
phosphate buffered saline at room temperature for
ultrasonic insonification. A custom designed acoustic
microscope is used to collect ultrasonic data. The
microscope consists of a 50 MHz broadband, focused,
piezoelectric delay-line transducer (1/4 inch diameter,
1/2 inch focal length, 62 micron beam diameter, Model
V390, Panametrics Co., Waltham, MA) operated in the
pulse-echo mode. A Tektronix DSA 601 digitizing
oscilloscope (Beaverton, OR) is used to digitize 35
degree polar backscattered radio frequency (rf) data at
500 megasamples per second with 8 bit resolution. A
variable gain system is used to increase the effective
dynamic range of this digitizer. Radio frequency data
are collected from the entire specimen and a peak
detected image is created of the section and compared
with the immunostained tissue image.
Immunostained tissue is examined and imaged using a
Nikon Optiphot-2 microscope with a Javlin Chromachip II
camera attachment. Images are routed through a Panasonic
digital mixer model WJ-AVE5 to Panasonic SVHS video
recorders, models AG-1960 or AG-1970 and displayed upon
an Sony Trinitron monitor. Images are captured using
NuVista software (Truevision, Inc., Indianapolis, IN
46256) executing on a Macintosh LCIII microcomputer.
Figure 16 compares tonsil acoustically imaged as a
radio frequency peak detected scan at 100 micron lateral
step resolution (a) with an optically imaged section
immunostained with horseradish peroxidase (b). The
epithelial capsule targeted by a mixture of anti-
cytokeratin antibodies is distinctly stained with
horseradish peroxidase and homologous regions in the
acoustic image are "brightened" by the targeted
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
47
biotinylated acoustic contrast. In Figure 17 the radio
frequency peak detected acoustic image at 100 micron step
resolution (a) is enhanced to 50 micron lateral step
resolution. The targeted biotinylated perfluorocarbon
contrast is clearly seen acoustically enhancing the
epithelial rim of the tonsil, analogous to the optical
immunostained image. This example clearly demonstrates
the fidelity of biotinylated perfluorocarbon contrast
targeting for enhanced acoustic contrast of tissues, such
as lymph nodes.
Example 13
Method to Prepare control and biotinylated
perfluorocarbon microemulsions incorporating gadolinium
DTPA into the outer lipid membrane.
The biotinylated perfluorocarbon contrast agent was
produced by incorporating biotinylated
phosphatidylethanolamine into the outer lipid monolayer
of a perfluorocarbon microemulsion. Briefly, the
emulsion was comprised of perfluorodichlorooctane (40%,
v/v, PFDCO, Minnesota Manufacturing and Mining, St. Paul,
MN), safflower oil (2.0%, w/v), a surfactant co-mixture
(2.0%, w/v) and glycerin (1.7%, w/v). The surfactant co-
mixture include (50 to 70 mole % lecithin (Pharmacia
Inc., Clayton, NC), 0 to 35 mole % cholesterol (Sigma
Chemical Co. St. Louis, MO) and 0.5 to 1 mole % N-(6-
(biotinoyl)amino)hexanoyl)-dipalmitoyl-L-alpha-
phosphatidylethanolamine Pierce, Rockford, IL) and 0 to
30% gadolinium (diethylenetriaminepentaacetic acid
bis(oleylamide) (Gd-DTPA-BOA), (Gateway Chemical
Technology, St. Louis, MO) which were dissolved in
chloroform. The chloroform-lipid mixture was evaporated
under reduced pressure, dried in a 50 C vacuum oven
overnight and dispersed into water by sonication,
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
48
resulting in a liposome suspension. The liposome
suspension was transferred into a blender cup (dynamics
Corporation of America, New Hartford, CT) with
perfluorodichlorooctane, safflower oil and distilled,
deionized water and emulsified for 30 to 60 seconds. The
emulsified mixture was transferred to an S100
Microfluidics emulsifier (Microfluidics, Newton, MA) and
continuously processed at 10,000 PSI for three minutes.
The completed emulsion was vialed, blanketed with
nitrogen and sealed with stopper crimp seal until use. A
control emulsion was prepared identically except a
nonbiotinylated phosphatidylethanolamine was substituted
into the surfactant co-mixture. Biotinylated and control
perfluorocarbon emulsion particle sizes were determined
in triplicate at 37 C with a Brookhaven BI-90 laser light
scatter submicron particle size analyzer (Brookhaven
Instruments Corporation, Holtsville, NY).
Examp l e 14
Demonstration of the effect of gadolinium incorporation
on increases in particle size associated with avidin
addition.
Biotinylated, gadolinium DTPA perfluorocarbon
emulsions (30 ,c.cl) were added to 2.97 ml of isotonic
phosphate buffered saline (PBS), pH 7.4 and avidin in a
polystyrene cuvette. Avidin (Pierce, Inc., Rockford, IL)
was dissolved in PBS and was present in the cuvette to
final concentrations of 0 to 10 ,ug/ml. All samples were
prepared in duplicate, were mixed by gentle inversion,
and continuously agitated at low speed on a rotary table
for 30 minutes at room temperature. Emulsion particles
sizes were determined in triplicate at 37 C with a
Brookhaven BI-90 laser light scatter submicron particle
size analyzer (Brookhaven Instruments Corporation,
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
49
Holtsville, NY). Figure 18 reveals that the baseline
particle size for three emulsions incorporating
gadolinium remained around 250 nm. Addition of avidin
increased particle size in a dose related manner.
Incremental increases in emulsion particle size were
slightly but not detrimentally smaller for the higher
concentrations of gadolinium incorporation.
Example 15
Demonstrates the ability of biotinylated perfluorocarbon
emulsions incorporating gadolinium DTPA into their outer
membrane to target and acoustically enhance human plasma
clots.
Whole human blood was obtained fresh and
anticoagulated (9:1, v/v) with sterile sodium citrate.
In a series of trials, plasma clots (6) were produced by
combining plasma and 100 mM calcium chloride (3:1 v/v)
with 5 units of thrombin (Sigma Chemical Company, St.
Louis, MO) in a plastic mold on a nitrocellulose surface.
The plasma was allowed to coagulate slowly at room
temperature. Half of the clots (3) were incubated
individually with 150 ug biotinylated antifibrin
monoclonal antibody (NIB 5F3; NIBSC, Herts, United
Kingdom) in 10 ml PBS for two hours; the remaining clots
(3) were maintained in PBS. The antibody treated clots
were then incubated with avidin (50,ug/ml PBS) for 30
minutes followed by 10% gadolinium, biotinylated
perfluorocarbon emulsion (30 1/ml PBS for 30 minutes.
The control clots were treated similarly with control
perfluorocarbon emulsion (30,ul/ml PBS). Targeted and
control clots were retreated with avidin and targeted or
control perfluorocarbon emulsion, respectively, to
optimize surface saturation prior to ultrasonic
interrogation. Clots on nitrocellulose disks were placed
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
in a waterbath and imaged with 30MHz intravascular
catheters (Boston Scientific Corporation, Maple Grove,
MN) and a conventional ultrasonic scanner with a 7.5 MHz
linear array transducer (Hewlett Packard Inc.)
5 Ultrasonic images were recorded on to Super-VHS videotape
for subsequent image analysis. Figure 19 clearly
demonstrates the enhanced acoustic reflectivity of the
plasma clot surface with the target acoustic contrast
which is not appreciated in the control. These results
10 demonstrate that the targeting capability and the
enhanced acoustic reflectivity effects of the
biotinylated perfluorocarbon emulsion was retained.
Example 16
The Efficacy of the Dual Ultrasound/MRI Contrast Agent in
15 solution to provide T1 shortening in a concentration
dependent manner.
Biotinylated emulsions incorporating gadolinium DTPA
at overall concentrations of 0.2, 0.4 and 0.6 mole-% into
the outer lipid membrane were prepared as described in
20 Example 13. The dual contrast particles were serially
diluted with PBS into 3cc plastic tubes and magnetic
resonance imaging was performed using a Philips Gyroscan
S15 ACS-NT (1.5T). A Look-Locker MR pulse sequence was
used to map the longitudinal relaxation curve. Briefly,
25 an inversion pulse was applied, followed by acquisition
of a series of images with small flip angles and short
inter-image spacing. The signal intensity changes
between images were directly related to the actual
relaxation curve, and Tl (the spin-lattice relaxation
30 time) was determined from this relationship. The pulse
sequence parameters used in this experiment were TR 50
ms, TE 10 ms, flip angle 5 degrees, matrix 64 x 64, field
of view 160 x 104 mm, 20 images, delay after inversion
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
51
pulse 16 ms. The experiment was repeated with a Tr of 25
ms in order to measure very short T1's at high GD3+
concentration. T1 parametric maps were generated where
pixel intensity is the Tl value in milliseconds. Table 1
reveals the direct dependence of T1 shortening on
gadolinium concentration. T1 shortening was greater for
particles containing higher concentrations of gadolinium
whether achieved by formulation or dilution.
Table 1. T1 dependence on Gd concentration
0.2% Gd 0.4% Gd 0.6% Gd
Formulation [Gd] mM T1 [Gd] mM T1 [Gd] mM T1
Dilution (ms) (ms) (ms)
1:16 (2.5% 0.000125 972 2 0.000252 492 1 0.000314 519 1
PFC)
1:8 (5% 0.000249 878 7 0.000505 339 2 0.000628 414 4
PFC)
1:4 (10% 0.000498 430 7 0.00101 189 2 0.001256 156 2
PFC)
1:2 (20% 0.000996 169 1 0.00202 92 1 0.002511 90 1
PFC)
1:1 (40% 0.001992 65 1 0.00404 <50 0.005022 <50
PFC)
The control emulsion (i.e. no gadolinium) had a mean %1 of
1788 9 ms. T1's shorter 50 ms than could not be resolved
25with the present technique. The relaxivity for each
preparation (0.2%, 0.4%, and 0.6% [Gd]) was determined by
computing the slope of the line relating [Gd] in mM to
1/%1 (Table 2). The relaxivity of the 0.2 gadolinium
emulsions was the greatest; whereas, the relaxivity of the
300.4% and 0.6 contrast formulations were shorter and
similar.
CA 02373993 2001-11-23
WO 00/71172 PCTIUS99/11491
52
Table 2. Relaxivity of preparations
slope (ms-mM) intercept r
0.2% 7.89 -0.00093 0.99
0.4o 5.06 0.00049 0.99
0.6% 4.36 0.00034 0.99
Example 17
The Efficacy of the Dual Ultrasound/MRI Contrast Agent
targeted in vitro to human plasma clots to provide T1
shortening
Biotinylated emulsions incorporating gadolinium
at overall concentrations of 10, 20 and 30 mole % into the
outer lipid membrane were prepared as described in Example
13. The actual weight percentage of gadolinium in the
applied emulsions are 0.25% (10%), 0.43% (20%), and 0.54%
15(30%). Whole human blood was obtained fresh and
anticoagulated (9:1, v/v) with sterile sodium citrate. In
a series of trials, plasma clots (6) were produced by
combining plasma and 1000 mM calcium chloride (3:1, v/v)
with 5 units of thrombin (Sigma Chemical Company, St.
20Louis, MO) in a plastic mold on a nitrocellulose surface.
The clot dimensions when formed on the nitrocellulose
support membrane were: thickness<0.5 mm; diameter - 1 cm.
The plasma was allowed to coagulate slowly at room
temperature. Half of the clots (3) were incubated
25individually with 150 ,ug biotinylated antifibrin
monoclonal antibody (NIB 5F3; NIBSC, Herts, United
Kingdom) in 10 ml PBS for two hours; the remaining clots
(3) were maintained in PBS. The antibody treated clots
were then incubated with avidin (50,ug/ml PBS) for 30
30minutes followed by 10% gadolinium, biotinylated
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
53
perfluorocarbon emulsion (3041/ml PBS) for 30 minutes.
The control clots were treated similarly with control
perfluorocarbon emulsion (30,ul/ml PBS). Half of the
targeted and control clots were retreated with avidin and
5targeted or control perfluorocarbon emulsion,
respectively, to optimize surface saturation prior to
imaging.
Clots exposed to control and targeted gadolinium
contrast were encased with 10% gelatin in P-30, plastic
lOpetri dishes and magnetic resonance imaging was performed
using a Philips Gyroscan S15 ACS-NT (1.5T). A Look-Locker
pulse sequence was used to map the longitudinal relaxation
curve. Briefly, an inversion pulse was applied, followed
by acquisition of a series of images with small flip
15angles and short inter-image spacing. The pulse sequence
parameters were TR 50 ms, TE 10 ms, flip angle 5 degrees,
matrix 64 x 64, field of view 160 x 104 mm, 20 images,
delay after inversion pulse 16 ms. T1 was determined from
the resulting parametric Tl map in each clot and in the
20surrounding gel. T2 (spin-spin relaxation time) was
determined from an 8 echo spin-echo sequence with TE 30
ms, and TR 8000 ms. The image voxel dimension for this
experiment was - 2.5x2.0x2.0 mm. The mean Ti value for
the gel was 582 8 ms. T2 values for all samples fell in a
25narrow range between 80 and 92 ms. T2 of the gel was 91
ms. Adding Gd to the preparation resulted in a measurable
and significant drop in T1 which plateaued at the lowest
paramagnetic concentration (Table 3). Because of the
partial volume effect involved in this measurement (i.e.,
30only a thin layer of gadolinium emulsion on a clot surface
relative to the voxel dimension, or approximately 11:1 gel
substrate to gadolinium-emulsion), the contrast
enhancement effect is actually remarkably sensitive.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
54
Table 3. Ti dependence on [Gd] targeted to fibrin clots
imbedded in gelatin.
[Gd] 1 exposure to targeting 2 exposures to targeting
system system
Ti (ms) Tl (ms)
0.0% 725+16 824+41
0.25% 662 23 696 15
0.43% 642+21 660+11
0.54% 666+17 667+13
Example 18
In situ targeting of canine thrombi in vivo for magnetic
10resonance imaging using dual contrast agent.
In accordance with approved animal protocols,
dogs weighing 20-30Kg were anesthetized with sodium
pentobarbital (30mg/kg, i.iv.) followed by 1% halothane in
oxygen. The right femoral artery and all branches between
15the inguinal ligament and the saphenous artery were
exposed. One proximal arterial branch, slightly distal to
the inguinal ligament, was selected for cannulation. All
other branches were ligated. The tip of a 23ga needle
crimped on silver plated copper wire was inserted
20obliquely into the femoral artery 2-3 cm proximal to the
saphenous branch and secured with 4-0 Prolene suture
through connective tissue on either side. Anodal current
(200-400 uA) was applied for 90 to 120 minutes to induce a
partially occlusive thrombus. A Doppler flow probed
25placed proximally was used to monitor the development of
thrombus. Partial distal constriction of the femoral
artery was used to facilitate thrombus formation.
After a thrombus had been formed, a 20 ga.
catheter was inserted into the preserved proximal branch
30of the artery and a pressurized 0.9% NaCl drip was
CA 02373993 2001-11-23
WO 00/71172 PCTIUS99/11491
attached through a three-way stopcock to the catheter.
Saline was allowed to flush the artery and antegrade blood
flow through the femoral artery was stopped by placement
of a snare 1 - 2 cm proximal to the catheter. Continued
5blood flow through the distal femoral arterial containing
the thrombus was prevented for the duration of the study.
After blood was flushed from the isolated
arterial segment with continuous saline infusion, the
distal femoral artery was occluded transiently with a
10snare. For contrast targeted thrombi, biotinylated
antifibrin monoclonal antibody (150,ug NIB 5F3 or NIB 1H10
in 0.5 ml of PBS, pH 7.2-7.4) was injected through the
three-way stopcock and incubated in the vessel for one
hour. The distal snare on the femoral artery was then
15released and unbound antibody was flushed away with 0.9%
saline. After re-establishing the distal arterial
occlusion, 0.5 mg of avidin (Pierce, Rockford, IL) in 0.5
ml of PBS was injected into the segment and incubated
within artery for 30 minutes. Again, the distal occlusion
20was released and unbound avidin was flushed from the lumen
with 0.9% NaCl. The distal arterial occlusion was re-
established and 0.2 ml of biotinylated emulsion was
injected into the vessel lumen and incubated for 30
minutes.
25 Arteries were ultrasonically imaged after
thrombus formation (baseline and after each administration
of antibody, avidin and perfluorocarbon emulsion with a
7.5 M.Hz linear array transducer using a commercially
available imaging system. The acoustically reflective
30needle electrode was used to localize regions of
thrombosis for insonification. After all data were
collected, the presence of thrombus was confirmed in each
animal by incision of the artery at the end of-study.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
56
After ultrasonic imaging, the femoral arterial
segment was perfused with formalin in situ for 30 minutes
and then excised with a rigid support to preserve
conformation. The arterial segment was placed in a
5formalin container and transferred to the MRI scanner for
imaging. Magnetic resonance imaging was performed using a
Philips Gyroscan S15 ACS-NOT (1.5T) using the Look-Locker
technique with a TR of 100 ms, TE 10 ms, flip angle 5
degrees, matrix 64 x 64, field of view 160 x 104 mm, 20
10images, delay after inversion pulse 16 ms. Slice
thickness was 4 mm.
The measured T1 of the formalin background was
2319 12 ms, and the measured Ti of the thrombus was
1717 + 173 ms. This difference in Tl between the clot and
15background resulted in high contrast as shown in Figure
20. The location and dimensions of the enhanced Tl signal
were analogous to the result obtained by ultrasound Figure
20 and confirmed dissection of the artery. A second
arterial thrombus preparation imaged with the same
20magnetic resonance techniques yielded analogous results.
In this experiment, the Ti of the clot was 1572 173 ms,
and the background Tl was 2319 12 ms.
In view of the above, it will be seen that the
several objects of the invention are achieved and other
25advantageous results attained.
Example 19
Stannous (II) chloride method to prepare control and
biotinylated perfluorocarbon microemulsions incorporating
99Tc into the lipid membrane.
30 The biotinylated perfluorocarbon contrast agent
was produced by incorporating biotinylated
phosphatidylethanolamine into the outer lipid monolayer of
a perfluorocarbon microemulsion as previously
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
57
demonstrated. Briefly, the emulsion is comprised of
perfluorodichlorooctane (40% v/v, PFDCO, Minnesota
Manufacturing and Mining, St. Paul, MN), safflower oil
(2.0%, w/v), a surfactant co-mixture (2.0%, w/v) and
5glycerin (1.7% w/v). The surfactant co-mixture included
50-70 mole % lecithin (Pharmacia Inc., Clayton, NC), 0 to
35 mole % cholesterol (Sigma Chemical Co., St. Louis, MO)
and 0.5 to 1 mole % N-(6-biotinoyl)amino)hexanoyl)-
dipalmitoyl-L-alpha-phosphatidylethanolamine Pierce,
10Rockford, IL) which is dissolved in chloroform. The
chloroform-lipid mixture is evaporated under reduced
pressure, dried in a 50 C vacuum oven overnight and
dispersed into water by sonication, resulting in a
liposome suspension. The liposome suspension is
15transferred into a blender cup (Dynamics Corporation of
America, New Hartford, CT) with perfluorodichlorooctane,
safflower oil, glycerin, and distilled, deionized water
and emulsified for 30 to 60 seconds. The emulsified
mixture is transferred to an S110 Microfluidics emulsifier
20(Microfluidics, Newton, MA) and continuously processed at
10,000 PSI for three minutes. The completed emulsion is
vialed, blanketed with nitrogen and sealed with stopper
crimp seal until use. A control emulsion is prepared
identically except a nonbiotinylated
25phosphatidylethanolamine is substituted into the
surfactant co-mixture. Biotinylated and control
perfluorocarbon emulsion particle sizes are determined in
triplicate at 37 C with a laser light scatter submicron
particle size analyzer.
30 Stannous (II) chloride (SnC12=2Hz0) 2 to 10
mmol/l in NaCl 150 mmol/l and 1 to 10 mCi of 99t Tc-
pertechnetate may be combined with 1 to 3 g of a 40%
perfluorocarbon emulsion with 2% lipid surfactant (as
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
58
described above) for 30 minutes at room temperature.
Unbound 99m Tc-pertechnetate may be removed by repeated
centrifugation and washing of the emulsion with NaCl 150
mmol/l or by routine column chromatography (Sepharose 4B
5or equivalent). Labelling efficiencies (%) are obtained
by the ratio of counts per minute in the emulsion
pellet/total counts per minute added x 100 or by counts
per minute eluting with the particles from the
column/total counts per minute engaged on the
10chromatography column.
Example 20
Stannous oxinate method to prepare control and
biotinylated perfluorocarbon microemulsions incorporating
99aI'c into the lipid membrane.
15 The biotinylated perfluorocarbon contrast agent
was produced by incorporating biotinylated
phosphatidylethanolamine into the outer lipid monolayer of
a perfluorocarbon microemulsion as demonstrated
previously. Stannous oxinate is added to the lipid
20surfactant for direct incorporation into the lipid
membrane. Brief, the emulsion was comprised of
perfluorodichlorooctane (40% v/v, PFDCO, Minnesota Mining
and Manufacturing, St. Paul, MN), safflower oil (2.0%,
w/v, optional), a surfactant co-mixture (2.0%, w/v) and
25glycerin (1.7%, w/v). The surfactant co-mixture included
(50 to 70 mole % lecithin (Pharmacia Inc., Clayton, NC), 0
to 35 mole % cholesterol (Sigma Chemical Co., St. Louis,
MO) and 0.5 to 1 mole % N-(6-(biotinoyl)amino)hexanoyl)-
dipalmitoyl-L-alpha-phosphatidylethanolamine Pierce,
30Rockford, IL), and stannous oxinate (500 ,ug) which were
dissolved in chloroform or chloroform/methanol. The
chloroform-lipid mixture was evaporated under reduced
pressure, dried in a 50 C vacuum oven overnight and
CA 02373993 2007-08-30
59
dispersed into water by sonication, resulting in a
liposome suspension. The liposome suspension was
transferred into a blender cup (Dynamics Corporation of
America, New Hartford, CT) with perfluorodichiorooctane,
5safflower oil (optional), glycerin, and distilled,
deionized water and emulsified for 30 to 60 seconds. The
emulsified mixture was transferred to an S110
Microfluidics emulsifier (Microfluidics, Newton, MA) and
continuously processed at 10,000 PSI for three minutes.
lOThe completed emulsion was vialed, blanketed with nitrogen
and sealed with stopper crimpseal until use. A control
emulsion was prepared identically except a nonbiotinylated
phosphatidylethanolamine was substituted into the
surfactant co-mixture. Biotinylated and control
15 perfluorocarbon emulsion particle sizes were determined in
triplicate at 37 C with a laser light scatter submicron
particle size analyzer.
Emulsion particles (2.5g of 40% emulsion -
approximately 20mg lipid surfactant), washed by
20centrifugation of free stannous oxinate, is combined with
99niTc technetate (3 to 10 mCi) for 30 minutes in room
temperature. Unbound 99oiTc-pertechnetate is removed by
repeated centrifugation and washing of the emulsion with
NaCl 150 mmol/l or by routine column chromatography
25(Sepharose' 4B or equivalent). Labelling efficiencies W
are obtained by the ratio of counts per minute in the
emulsion pellet/total counts per minute added x 100 or by
counts per minute eluting with the particles from the
column/total counts per minute engaged on the
30 chromatography column.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
Example 21
Method to prepare control and biotinylated perfluorocarbon
microemulsions incorporating 99mTc into the lipid membrane
using exametazine.
5 The biotinylated perfluorocarbon contrast agent
was produced by incorporating biotinylated
phosphatidylethanolamine into the outer lipid monolayer of
a perfluorocarbon microemulsion. Briefly, the emulsion
was comprised of perfluorodichlorooctane (40% v/v, PFDCO,
10Minnesota Mining and Manufacturing, St. Paul, MN),
safflower oil (2.0%, w/v), a surfactant co-mixture (2.0%
w/v) and glycerin (1.7%, w/v). The surfactant co-mixture
included 50 to 70 mole % lecithin (Pharmacia In., Clayton,
NC), 0 to 35 mole % cholesterol (Sigma Chemical Co., St.
15Louis, MO) and 0.5 to 1 mole % N-(6-
biotinyoyl)amino)hexanoyl)-dipalmitoyl-L-alpha-
phosphatidylethanolamine Pierce, Rockford, IL) which were
dissolved in chloroform. The chloroform-lipid mixture was
evaporated under reduced pressure, dried in a 50 C vacuum
20oven overnight and dispersed into water by sonication,
resulting in a liposome suspension. The liposome
suspension was transferred into a blender cup (Dynamics
Corporation of America, New Hartford, CT) with
perfluorodichlorooctane, safflower oil, glycerin, and
25distilled, deionized water and emulsified for 30 to 60
seconds. The emulsified mixture was transferred to an
S110 Microfluidics emulsifier (Microfluidics, Newton, MA)
and continuously processed at 10,000 PSI for three
minutes. The completed emulsion was vialed, blanketed
30with nitrogen and sealed with stopper crimp seal until
use. A control emulsion was prepared identically except a
nonbiotinylated phosphatidylethanolamine was substituted
into the surfactant co-mixture. Biotinylated and control
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
61
perfluorocarbon emulsion particle sizes were determined in
triplicate at 37 C with a laser light scatter submicron
particle size analyzer.
An HM-PAO (exametazine) kit (Ceretec , Nycomed
5Amersham Inc., Arlington Heights, IL) containing 0.5 mg
HMPAO and 4.0 g SnC12 is reconstituted with 5-10 mCi of
99mTcO4 in 5 ml of 0.9% NaCl and incubated for 5 minutes.
One ml aliquot of 99mTc-HMPAO were incubated with 5 ml of
perfluorocarbon emulsion for 15 minutes. The mixture was
10centrifuged and washed with phosphate buffered saline.
Labelling efficiencies (%) are obtained by the ratio of
counts per minute in the emulsion pellet/total counts per
minute added x 100.
Example 22
15Demonstration of the effect of "m'I'c-pertechnetate
incorporation on increases in particle size associated
with avidin addition.
Biotinylated, 99mTc-perfluorocarbon emulsions
(30 l) are added to 2.97 ml of isotonic phosphate
20buffered saline (PBS), pH 7.4 and avidin in a polystyrene
cuvette. Avidin (Pierce, In., Rockford, IL) was dissolved
in PBS and was present in the cuvette to final
concentrations of 0 to 10 g/ml. All samples were
prepared in duplicate, were mixed by gentle inversion, and
25continuously agitated at low speed on a rotary table for
30 minutes at room temperature. Emulsion particle sizes
were determined in triplicate at 37 C with a laser light
scatter submicron particle size analyzer. Addition of
avidin increases particle size in a dose related manner as
30previously demonstrated for the ultrasonic and MRI
emulsion preparations.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
62
Example 23
Demonstrates the ability of biotinylated perfluorocarbon
emulsions incorporating 99mTc-pertechnetate into their
membrane to target and acoustically enhance human plasma
5clots.
Whole human blood is obtained fresh and
anticoagulated (9:1, v/v) with sterile sodium citrate. In
a series of trials, plasma clots (6) are produced by
combining plasma and 100 mM calcium chloride (3:1 v/v)
lOwith 5 units of thrombin (Sigma Chemical Co., St. Louis,
MO) in a plastic mold on a nitrocellulose surface. The
plasma is allowed to coagulate slowly at room temperature.
Half of the clots (3) are incubated individually with 150
gg biotinylated antifibrin monoclonal antibody (NIB 5F3;
15NIBSC, Herts, United Kingdom) in 10 ml PBS for two hours;
the remaining clots (3) were maintained in PBS. All clots
are incubated with avidin (50 g/ml PBS) for 30 minutes
followed by 99tnTc-pertechnetate, biotinylated
perfluorocarbon emulsion (30 g/ml PBS) for 30 minutes.
20Antibody targeted and control clots are retreated with
avidin and targeted or control perfluorocarbon emulsion,
respectively, to optimize surface saturation prior to
ultrasonic interrogation and gamma counting. Clots on
nitrocellulose disks are placed in a waterbath and imaged
25with 30MHz intravascular catheters (Boston Scientific
Corporation, Maple Grove, MN) and a conventional
ultrasonic scanner (Hewlett Packard Inc.). Ultrasonic
images are recorded onto Super-VHS videotape for
subsequent image analysis. Enhanced acoustic reflectivity
30of the plasma clot surface noted with the targeted
acoustic contrast is not appreciated in the control clots,
as previously demonstrated.
CA 02373993 2001-11-23
WO 00/71172 PCT/US99/11491
63
The control and targeted clots are placed in a
gamma counter and differences in radioactivity determined.
Targeted clots bear significantly more radioactivity than
control clots, demonstrating the specific targeting of
599mTc with emulsion particle. The formulation provides for
both acoustic and radioactive diagnostic imaging.
These results show that the addition of a
radionuclide such as technetium-99m does not impair the
binding of the emulsion particles to avidin or the ability
l0of the particles to bind targeted pathologic tissue and
does not interfere with the acoustic reflectivity
enhancement imparted by the bound emulsion particles.
Thus, these results are essentially analogous to the
results previously demonstrated for gadolinium-DTPA-BOA
15except that 99mTc has been substituted for the paramagnetic
complex.
In view of the above, it will be seen that the
several objects of the invention are achieved and other
advantageous results attained.
20 As various changes could be made in the above
methods and compositions without departing from the scope
of the invention, it is intended that all matter contained
in the above description and shown in the accompanying
drawings shall be interpreted as illustrative and not in a
25limiting sense.
