Note: Descriptions are shown in the official language in which they were submitted.
WO 92/22247 I'CI'/US92/02615
2110491
GA6! FILLED LIPOSOMES
AND THEIR USE AS ULTRASONIC CONTRAST AGENTS
BACRGROtTND OF THE INVENTION
Field of the Invention
This invention relates to the field of ultrasonic
imaging and, more specifically, to gas filled liposomes
prepared using vacuum drying gas instillation methods, and
to gas filled liposomes substantially devoid of liquid in
the interior thereof. The invention also relates to
methods of and apparatus for preparing such liposomes and
to methods for employing such liposomes in ultrasonic
imaging applications.
Background of the Invention
There are a variety of imaging techniques which
have been used to detect and diagnose disease in animals
and humans. One of the first techniques used for
diagnostic imaging was X-rays. The images obtained
through this technique reflect the electron density of the
object being imaged. Contrast agents such as barium or
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iodine have ~tdnu qArt over the years to attenuate or block
X-rays such thait the contrast between various structures
is increased. For example, barium is used for gastro-
intestinal studlies to define the bowel lumen and visualize
the mucosal surfaces of the bowel. Iodinated contrast
media is used i.ntravascularly to visualize the arteries in
an X-ray process called angiography. X-rays, however, are
known to be som,ewhat dangerous, since the radiation
employed in X-rays is ionizing, and the various
deleterious effects of ionizing radiation are cumulative.
Magnetic resonance imaging (MRI) is another
important imaging technique, however this technique has
various drawbacks such as expense and the fact that it
cannot be conducted as a portable examination. In
addition, MRI is not available at many medical centers.
Radionuclide:s, employed in nuclear medicine,
provide a further imaging technique. In employing this
technique, radionuclides such as technetium labelled
compounds are injected into the patient, and images are
obtained from gamma cameras. Nuclear medicine techniques,
however, suffer from poor spatial resolution and expose
the animal or p-atient to the deleterious effects of
radiation. Furthermore, there is a problem with the
handling and disposal of radionuclides.
Ultrasound, a still further diagnostic imaging
technique, is uinlike nuclear medicine and X-rays in that
it does not expose the patient to the harmful effects of
ionizing radiation. Moreover, unlike magnetic resonance
imaging, ultrasound is relatively inexpensive and can be
conducted as a portable examination. In using the
ultrasound techiiique, sound is transmitted into a patient
or animal via a transducer. When the sound waves
propagate throucjh the body, they encounter interfaces from
tissues and flu:ids. :Depending on the acoustic properties
of the tissues and fliuids in the body, the ultrasound
sound waves are partially or wholly reflected or absorbed.
When sound waves are reflected by an interface they are
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detected by the receiver in the transducer and processed
to form an image. The acoustic properties of the tissues
and fluids witlhin the body determine the contrast which
appears in the resultant image.
Advances have been made in recent years in
ultrasound technology. However, despite these various
technological improvements, ultrasound is still an
imperfect tool in a number of respects, particularly with
regard to the imaging and detection of disease in the
liver and splecsn, kidneys, heart and vasculature,
including measiiring blood flow. The ability to detect and
measure these things depends on the difference in acoustic
properties between tissues or fluids and the surrounding
tissues or f1u:Lds. As a result, contrast agents have been
sought which w:Lll increase the acoustic difference between
tissues or f1uids and the surrounding tissues or fluids in
order to improve ultrasonic imaging and disease detection.
The principles underlying image formation in
ultrasound have directed researchers to the pursuit of
gaseous contrast agents. Changes in acoustic properties
or acoustic impedance are most pronounced at interfaces of
different substances with greatly differing density or
acoustic impedaLnce, particularly at the interface between
solids, liquids: and qases. When ultrasound sound waves
encounter such interjEaces, the changes in acoustic
impedance result in a more intense reflection of sound
waves and a mor=e intense signal in the ultrasound image.
An additional f'actor affecting the efficiency or
reflection of sound iLs the elasticity of the reflecting
interface. The greater the elasticity of this interface,
the more efficient the reflection of sound. Substances
such as gas bubbles present highly elastic interfaces.
Thus, as a result of the foregoing principles, researchers
have focused on the development of ultrasound contrast
agents based on gas bubbles or gas containing bodies.
However, despite the theoretical reasons why such contrast
WO 92/22247 2 110 4 91 PCT/US92/026"
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agents should be effective, overall the diagnostic results
to date have been somewhat disappointing.
New and/or better contrast agents for ultrasound
imaging are needed. The present invention is directed to
addressing these and/or other important needs.
BUMMARY OF THE INVENTION
The present invention provides contrast agents
for ultrasonic imaging.
Specifically, in one embodiment, the present
invention provides ultrasound contrast agents comprising
gas filled liposomes prepared by vacuum drying gas
instillation methods, such liposomes sometimes being
referred to herein as vacuum dried gas instilled
liposomes. In another embodiment, the invention is
directed to contrast agents comprising gas filled
liposomes substantially devoid of liquid in the interior
thereof.
In a further embodiment, the subject invention
provides methods for preparing the liposomes of the
subject invention, said methods comprising: (i) placing
liposomes urider negative pressure; (ii) incubating the
liposomes under the negative pressure for a time
sufficient to remove substantially all liquid from the
liposomes; and (iii) instilling selected gas into the
liposomes until ambient pressures are achieved. Methods
employing the foregoing steps are referred to herein as
the vacuum drying gas instillation methods.
In a still further embodiment, the invention
provides apparatus for preparing the liposomes of the
invention using the vacuum drying gas instillation
methods, said apparatus comprising: (i) a vessel
containing liposomes; (ii) means for applying negative
pressure to the vessel to draw liquid from the liposomes
contained therein; (iii) a conduit connecting the negative
pressurizing means to the vessel, the conduit directing
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the flow of said liquid; and (iv) means for introducing a
gas into the liposomes in the vessel.
In additional embodiments, the invention
contemplates methods for providing an image of an internal
region of a patient, and/or for diagnosing the presence of
diseased tissue in a patient, comprising: (i) administering
to the patient the liposomes of the present invention; and
(ii) scanning the patient using ultrasonic imaging to obtain
visible images of the region of the patient, and/or of any
diseased tissue in the patient.
Finally, the present invention provides diagnostic
kits for ultrasonic imaging which include the contrast
agents of the invention.
According to one aspect of the present invention,
there is provided a contrast agent for ultrasonic imaging
comprising gas filled liposomes in an aqueous carrier that
are at least 90% devoid of liquid in the interior thereof.
According to another aspect of the present
invention, there is provided a method of preparing a
composition comprising gas filled liposomes in an aqueous
carrier for use in contrast agents for ultrasonic imaging,
comprising the following steps: (i) placing liposomes under
negative pressure; (ii) incubating said liposomes under said
negative pressure for a time sufficient to remove at least
90% of the liquid from the interior of said liposomes; and
(iii) instilling gas into said liposomes until ambient
pressures are achieved; wherein said liposomes are suspended
in aqueous carrier.
According to still another aspect of the present
invention, there is provided a kit for ultrasonic imaging
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comprising gas filled liposomes which, when suspended in an
aqueous carrier, are at least 90% devoid of liquid in the
interior thereof and printed material comprising
instructions for use of the kit in ultrasonic imaging.
According to yet another aspect of the present
invention, there is provided a composition comprising, a
gas-filled liposome at least 90% devoid of liquid in the
interior thereof, and an aqueous carrier therefor.
According to a further aspect of the present
invention, there is provided use of a contrast agent as
described herein for ultrasonic imaging of an internal
bodily region of a patient.
According to yet a further aspect of the present
invention, there is provided a method for providing an image
of an internal bodily region of a patient comprising:
(a) administering to the patient a contrast agent comprising
gas filled liposomes in an aqueous carrier that are at least
90% devoid of liquid in the interior thereof; and (b)
scanning the patient using ultrasonic imaging to obtain
visible images of the region.
According to still a further aspect of the present
invention, there is provided a method for diagnosing the
presence of diseased tissue in a patient comprising:
(a) administering to the patient a contrast agent comprising
gas filled liposomes in an aqueous carrier that are at least
90% devoid of liquid in the interior thereof; and
(b) scanning the patient using ultrasonic imaging to obtain
visible images of the region.
According to another aspect of the present
invention, there is provided a stabilized gas bubble
comprising gas encapsulated by one or more lipid materials,
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said stabilized gas bubble being at least 90% devoid of
liquid in the interior thereof and suspended in an aqueous
medium.
According to yet another aspect of the present
invention, there is provided a contrast agent adapted for
injection into the body of a patient and comprising a
stabilized gas bubble as described herein.
Surprisingly, the gas filled liposomes prepared by
the vacuum drying gas instillation method, and the gas
filled liposomes substantially devoid of liquid in the
interior thereof which may be prepared in accordance with
the vacuum drying gas instillation method, possess a number
of unexpected, but highly beneficial, characteristics. The
liposomes of the invention exhibit intense echogenicity on
ultrasound, are highly stable to pressure and/or possess a
long storage life, either when stored dry or suspended in a
liquid medium. Also surprising is the ability of the
liposomes during the vacuum drying gas instillation process
to fill with gas and resume their original circular shape,
rather than irreversibly collapse into a cup-like shape.
Indeed, despite the theoretical reasons why the
prior art ultrasound contrast agents based on gas bubbles or
gas containing bodies should be effective, the diagnostic
results had remained largely disappointing. A number of the
gaseous ultrasound contrast agents developed by prior
researchers involved unstabilized bubbles, and it has been
found that the instability of these contrast agents severely
diminishes the diagnostic usefulness of such agents. Other
gaseous ultrasound contrast agents developed by prior
researchers have involved gas bubbles
WO 92/22247 21 10 4 91 P('T/US92/026"
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stabilized in constructs which also contain a substantial
amount of liquid, and it has been found that the presence
of a substantial amount of liquid in the construct leads
to less satisfactory diagnostic results. Indeed, the
presence of liquid in the construct has been found to
disadvantageously alter the resonant characteristics of
the gas in the construct, and has been found to hasten the
diffusion of further liquid into (and concomitantly gas
out of) the construct. The present invention provides new
and/or better contrast agents for ultrasound imaging in an
effort to address these and/or other important needs.
These and other features of the invention and the
advantages thereof will be set forth in greater detail in
the figures and the description below.
BRIEF DESCRIPTION OF THE FIGURES
FIGURE 1 shows an apparatus according to the
present invention for preparing the vacuum dried gas
instilled liposomes and the gas filled liposomes
substantially devoid of liquid in the interior thereof
prepared by the vacuum drying gas instillation method.
FIGURE 2 is a graphical representation of the dB
reflectivity of gas filled liposomes substantially devoid
of liquid in the interior thereof prepared by the vacuum
drying gas instillation method. The data was obtained by
scanning with a 7.5 megahertz transducer using an Acoustic
ImagingTT' Model 5200 scanner (Acoustic Imaging, Phoenix,
Arizona), and was generated by using the system test
software to measure reflectivity. The system was
standardized prior to each experiment with a phantom of
known acoustic impedance.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to ultrasound
contrast agents comprising gas filled liposomes prepared
by vacuum drying gas instillation methods, such liposomes
sometimes being referred to herein as vacuum dried gas
,WO 92/22247 2 110 4 9 t PC,T/US92/02615
- 7 -
instilled liposomes. The present invention is further
directed to coiitrast agents comprising gas filled
liposomes substantially devoid of liquid in the interior
thereof.
The vacuum drying gas instillation method which
may be employeci to p:repare both the gas filled liposomes
prepared by the vacuum drying gas instillation method, and
the gas filled liposomes substantially devoid of liquid in
the interior thereof, contemplates the following process.
First, in accordance with the process, the liposomes are
placed under neagativia pressure (that is, reduced pressure
or vacuum conditions). Next, the liposomes are incubated
under that negative pressure for a time sufficient to
remove substant:ially all liquid from the liposomes,
thereby resulti.ng in substantially dried liposomes. By
removal of substantially all liquid, and by substantially
dried liposomes, as those phrases are used herein, it is
meant that the liposomes are at least about 90% devoid of
liquid, preferably at least about 95% devoid of liquid,
most preferably about 100% devoid of liquid. Finally, the
liposomes are instilled with selected gas by applying the
gas to the liposomes until ambient pressures are achieved,
thus resulting in the subject vacuum dried gas instilled
liposomes of the present invention, and the gas filled
liposomes of the inveantion substantially devoid of liquid
in the interior thereof. By substantially devoid of
liquid in the interior thereof, as used herein, it is
meant liposomes havirig an interior that is at least about
90% devoid of liquid, preferably at least about 95% devoid
of liquid, most prefe:rably about 100% devoid of liquid.
Unexpectedly, the liposomes prepared in
accordance with the vacuum dried gas instillation method,
and the gas filled li.posomes, substantially devoid of
liquid in the interior thereof, possess a number of
surprising yet highly beneficial characteristics. The
liposomes of the invention exhibit intense echogenicity on
ultrasound, are highly stable to pressure, and/or
WO 92/22247 2110 4. 01 PCT/US92/02F'-'
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generally possess a long storage life, either when stored
dry or suspended in a liquid medium. The ecogenicity of
the liposomes is of obvious importance to the diagnostic
applications of the invention, and is illustrated in
Figure 2. Preferably, the liposomes of the invention
possess a reflectivity of greater than 2 dB, preferably
between about 4 dB and about 20 dB. Within these ranges,
the highest reflectivity for the liposomes of the
invention is exhibited by the larger liposomes, by higher
concentrations of liposomes, and/or where higher
ultrasound frequencies are employed. The stability of the
liposomes is also of great practical importance. The
subject liposomes tend to have greater stability during
storage than other gas filled liposomes produced via known
procedures such as pressurization or other techniques. At
72 hours after formation, for example, conventionally
prepared liposomes often are essentially devoid of gas,
the gas having diffused out of the liposomes and/or the
liposomes having ruptured and/or fused, resulting in a
concomitant loss in reflectivity. In comparison, gas
filled liposomes of the present invention generally have a
shelf life stability of greater than about three weeks,
preferably a shelf life stability of greater than about
four weeks, more preferably a shelf life stability of
greater than about five weeks, even more preferably a
shelf life stability of greater than about three months,
and often a shelf life stability that is even much longer,
such as over six months, twelve months, or even two years.
Also unexpected is the ability of the liposomes
during the vacuum drying gas instillation process to fill
with gas and resume their original circular shape, rather
than collapse into a cup-shaped structure, as the prior
art would cause one to expect. See, e.g., Crowe et al.,
Archives of Biochemistry and Biophysics, Vol. 242, pp.
240-247 (1985); Crowe et al., Archives of Biochemistry and
Biophysics, Vol. 220, pp. 477-484 (1983); Fukuda et al.,
J. Am. Chem. Soc., Vol. 108, pp. 2321-2327 (1986); Regen
WO 92/2224" 2110491 PCT/US92/02615
- 9 -
et al., J. Am. Chem. Soc., Vol. 102, pp. 6638-6640 (1980).
The liposomes subjected to the vacuum drying gas
instillation method of the invention may be prepared using
any one of a variety of conventional liposome preparatory
techniques which will be apparent to those skilled in the
art. These technique:; include freeze-thaw, as well as
techniques such as sonication, chelate dialysis,
homogenization, solvent infusion, microemulsification,
spontaneous formation, solvent vaporization, French
pressure cell technique, controlled detergent dialysis,
and others. The: size of the liposomes can be adjusted, if
desired, prior t.o vacuum drying and gas instillation, by a
variety of procedures including extrusion, filtration,
sonication, homogenization, employing a laminar stream of
a core of liquicl introduced into an immiscible sheath of
liquid, and similar methods, in order to modulate
resultant liposc-mal biodistribution and clearance.
Extrusion under pressure through pores of defined size is,
however, the preferred means of adjusting the size of the
liposomes. The foregoing techniques, as well as others,
are discussed, for example, in U.S. Patent No. 4,728,578;
U.K. Patent Application GB 2193095 A; U.S. Patent No.
4,728,575; U.S. Paten't No. 4,737,323; International
Application PCT/US85/101161; Mayer et al., Biochimica et
Biophysica Acta,_ Vol. 858, pp. 161-168 (1986); Hope et
al., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65
(1985); U.S. Patent No. 4,533,254; Mayhew et al., Methods
in Enzymology, Vol. 149, pp. 64-77 (1987); Mayhew et al.,
Biochimica et B:iophysica Acta, Vol 755, pp. 169-74 (1984);
Cheng et al, Investictative Radiology, Vol. 22, pp. 47-55
(1987); PCT/US89/05040; U.S. Patent No. 4,162,282; U.S.
Patent No. 4,310,505; U.S. Patent No. 4,921,706; and
Liposomes Technol oqY, Gregoriadis, G., ed., Vol. I, pp.
29-37, 51-67 and 79-108 (CRC Press Inc, Boca Raton, FL,
1984).
63189-331
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-
Although any of a
number of varying techniques can be employed, preferably
the liposomes are prepared via microemulsification
techniques. The liposomes produced by the various
5 conventional procedures can then be employed in the vacuum
drying gas instillation method of the present invention,
to produce the liposomes of the present invention.
The materials which may be ut~ilized in preparing
liposomes to be employed in the vacuum drying gas
10 instillation method of the present invention include any
of the materials or combinations thereof known to those
skilled in the art as suitable for liposome construction.
The lipids used may be of either natural or synthetic
origin. Such materials include, but are not limited to,
lipids such as fatty acids, lysolipids,,
dipalmitoylphosphatidylcholine, phosphatidylchol.ine,
phosphatidic acid, sphingomyelin, cholesterol, cholesterol
hemisuccinate, tocopherol hemisuccinate,
phosphatidylethanolamine, phosphatidyl-inositol,
lysolipids, sphingomyelin, glycosphingolipids,
glucolipids, glycolipids, suiphatides, lipids with ether
and ester-linked fatty acids, polymerized lipids, diacetyl
phosphate, stearylamine, distearoylphosphatidylcholine,
phosphatidylserine, sphingomyelin, cardiolipin,
phospholipids with short chain fatty acids of 6-8 carbons
in length, synthetic phospholipids with asymmetric acyl
chains (e.g., with one acyl chain of 6 carbons and another
acyl chain of 12 carbons), 6-(5-cholesten-30-yloxy)-l-
thio-p-D-galactopyranoside, digalactosyldiglyceride, 6-(5-
cholesten-3p-yloxy)hexyl-6-amino-6-deoxy-l-thio-p-D-
galactopyranoside, 6-(5-cholesten-3p-yloxy)hexyl-=6-amino-
6-deoxyl-l-thio-a-D-mannopyranoside, dibehenoyl-
phosphatidylcholine, dimyristoylphosphatidylcholine,
dilauroylphosphatidylcholine, and dioleoyl-
phosphatidylcholine, and/or combinations thereof. Other
useful lipids or combinations thereof apparent to those
skilled in the art which are in keeping with the spirit of
~
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63189-331
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the present invention are also encompassed by the present
invention. For example, carbohydrates bearing lipids may
be employed for in vivo targeting as described in U.S.
Patent No. 4,310,505. Of particular interest for use in
the present invention are lipids which are in the gel
state (as compared with the liquid crystalline state) at
the temperature at which the vacuum drying gas
instillation is performed. The phase transition
temperatures of various lipids will be readily apparent to
those skilled in the art and are described, for example,
in Liposome Technologv, Gregoriadis, G., ed., Vol. I, pp.
1-18 (CRC Press, Inc. Boca Raton, FL 1984).
In addition, it has been found that
the incorporation of at least a small amount of negatively
charged lipid into any liposome membrane, although not
required, is beneficial to providing highly stable
liposomes. By at least a small amount, it is meaint about
1 mole percent of the total lipid. Suitable negatively
charged lipids will be readily apparent to those skilled
in the art, and include, for example phosphatidylserine
and fatty acids. Most preferred for reasons of the
combined ultimate ecogenicity and stability following the
vacuum drying gas instillation process are liposoines
prepared from dipalmitoyl- phosphatidy3choline.
By way of general guidance, dipalmitoyl-
phosphatidylcholine liposomes may be prepared by
suspending dipalmitoylphosphatidylcholine lipids in
phosphate buffered saline or water, and heating the lipids
to about 50 C, a temperature which is slightly above the
45 C temperature required for transition of the
dipalmitoyl-
phosphatidylcholine lipids from a gel state to a liquid
crystalline state, to form liposomes. To prepare
multilamellar vesicles of.a rather heterogeneous size
distribution of around 2 microns, the liposomes may then
be mixed gently by hand while keeping the liposome
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solution at a temperature of about 50 C. The temperature
is then lowered to room temperature, and the liposomes
remain intact. Extrusion of
dipalmitoylphosphatidylcholine liposomes through
polycarbonate filters of defined size may, if desired, be
employed to make liposomes of a more homogeneous size
distribution. A device useful for this technique is an
extruder device (Extruder DeviceT~', Lipex Biomembr.anes,
Vancouver, Canada) equipped with a thermal barrel so that
extrusion may be conveniently accomplished above the gel
state-liquid crystalline transition temperature for
lipids.
Alternatively, and again by way of general
guidance, conventional freeze-thaw procedures may be used
to produce either oligolamellar or unilamellar
dipalmitoylphosphatidylcholine liposomes. After the
freeze-thaw procedures, extrusion procedures as described
above may then be performed on the liposomes.
The liposomes thus prepared may then be subjected
to the vacuum drying gas instillation process of the
present invention, to produce the vacuum dried gas
instilled liposomes, and the gas filled liposomes
substantially devoid of liquid in the interior thereof, of
the invention. In accordance with the process of the
invention, the liposomes are placed into a vessel suitable
for subjecting to the liposomes to negative pressure (that
is, reduced pressure or vacuum conditions). Negative
pressure is then applied for a time sufficient to remove
substantially all liquid from the liposomes, thereby
resulting in substantially dried iiposomes. As those
skilled in the art would recognize, once armed with the
present disclosure, various negative pressures can be
employed, the important parameter being that substantially
all of the liquid has been removed from the liposomes.
Generally, a negative pressure of at least about 700 mm Hg
and preferably in the range of between about 700 mm Hg and
about 760 mm Hg (gauge pressure), applied for about 24 to
twO 92/22247 PCT/US92/02615
13 -
about 72 hours, is sufficient to remove substantially all
of the liquid i:rom tlhe liposomes. Other suitable
pressures and time periods will be apparent to those
skilled in the art, in view of the disclosures herein.
Finally, a selected gas is applied to the
liposomes to iristill the liposomes with gas until ambient
pressures are achieved, thereby resulting in the vacuum
dried gas instilled :liposomes of the invention, and in the
gas filled liposomes substantially devoid of liquid in the
interior thereof. Preferably, gas instillation occurs
slowly, that is, over a time period of at least about 4
hours, most pre:ferab:ly over a time period of between about
4 and about 8 hours. Various biocompatible gases may be
employed. Such gases include air, nitrogen, carbon
dioxide, oxygen, argon, xenon, neon, helium, or any and
all combinatioris theireof. Other suitable gases will be
apparent to thc-se skilled in the art, the gas chosen being
only limited by the proposed application of the liposomes.
The above described method for production of
liposomes is referred to hereinafter as the vacuum drying
gas instillatic-n process.
If desired, the liposomes may be cooled, prior to
subjecting the liposomes to negative pressure, and such
cooling is preferred. Preferably, the liposomes are
cooled to below 0 C, more preferably to between about -
10 C and about -20 C,, and most preferably to -10 C, prior
to subjecting the liposomes to negative pressure. Upon
reaching the desired negative pressure, the liposomes
temperature is then preferably increased to above 0 C,
more preferably to between about 10 C and about 20 C, and
most preferably to 10 C, until substantially all of the
liquid has beert removed from the liposomes and the
negative pressure is discontinued, at which time the
temperature is then permitted to return to room
temperature.
If the liposomes are cooled to a temperature
below 0 C, it is preferable that the vacuum drying gas
WO 92/22247 2110 4 91 PCr/US92/0261
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instillation process be carried out with liposomes either
initially prepared in the presence of cryoprotectants, or
liposomes to which cryoprotectants have been added prior
to carrying out the vacuum drying gas instillation process
of the invention. Such cryoprotectants, while not
mandatorily added, assist in maintaining the integrity of
liposome membranes at low temperatures, and also add to
the ultimate stability of the membranes. Preferred
cryoprotectants are trehalose, glycerol,
polyethyleneglycol (especially polyethyleneglycol of
molecular weight 400), raffinose, sucrose and sorbitol,
with trehalose being particularly preferred.
It has also been surprisingly discovered that the
liposomes of the invention are highly stable to changes in
pressure. Because of this characteristic, extrusion of
the liposomes through filters of defined pore size
following vacuum drying and gas instillation can be
carried out, if desired, to create liposomes of relatively
homogeneous and defined pore size.
For storage prior to use, the liposomes of the
present invention may be suspended in an aqueous solution,
such as a saline solution (for example, a phosphate
buffered saline solution), or simply water, and stored
preferably at a temperature of between about 2 C and about
10 C, preferably at about 4 C. Preferably, the water is
sterile. Most preferably, the liposomes are stored in a
hypertonic saline solution (e.g., about 0.3 to about 0.5%
NaCl), although, if desired, the saline solution may be
isotonic. The solution also may be buffered, if desired,
to provide a pH range of pH 6.8 to pH 7.4. Suitable
buffers include, but are not limited to, acetate, citrate,
phosphate and bicarbonate. Dextrose may also be included
in the suspending media. Preferably, the aqueous solution
is degassed (that is, degassed under vacuum pressure)
prior to suspending the liposomes therein. Bacteriostatic
agents may also be included with the liposomes to prevent
bacterial degradation on storage. Suitable bacteriostatic
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agents include but are not limited to benzalkonium
chloride, benzethonium chloride, benzoic acid, benzyl
alcohol, butylparaben, cetylpyridinium chloride,
chlorobutanol, chlorocresol, methylparaben, phenol,
potassium benzioate, potassium sorbate, sodium benzoate and
sorbic acid. i3ne or more antioxidants may further be
included with 'the gas filled liposomes to prevent
oxidation of the lipid. Suitable antioxidants include
tocopherol, ascorbic acid and ascorbyl palmitate.
Liposomes prepared in the various foregoing manners may be
stored for at :least several weeks or months. Liposomes of
the present invention may alternatively, if desired, be
stored in their dried, unsuspended form, and such
liposomes also have a shelf life of greater than several
weeks or months. Specifically, the liposomes of the
present invent:Lon, stored either way, generally have a
shelf life stability of greater than about three weeks,
preferably a shelf life stability of greater than about
four weeks, more preferably a shelf life stability of
greater than about five weeks, even more preferably a
shelf life stability of greater than about three months,
and often a she:lf life stability that is even much longer,
such as over si.x months, twelve months or even two years.
As another aspect of the invention, useful
apparatus for preparing the vacuum dried gas instilled
liposomes, and the gas filled liposomes substantially
devoid of liqui.d in the interior thereof, of the invention
is also presented. Specifically, there is shown in Figure
1 a preferred apparatus for vacuum drying liposomes and
instilling a gas into the dried liposomes. The apparatus
is comprised of a vessel 8 for containing liposomes 19.
If desired, the apparatus may include an ice bath 5
containing dry ice 17 surrounding the vessel 8. The ice
bath 5 and dry ice 17 allow the liposomes to be cooled to
below 0 C. A vacuum pump 1 is connected to the vessel 8
via a conduit 15 for applying a sustained negative
pressure to the vessel. In the preferred embodiment, the
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pump 1 is capable of applying a negative pressure of at
least about 700 mm Hg, and preferably a negative pressure
in the range of about 700 mm Hg to about 760 mm Hg (gauge
pressure). A manometer 6 is connected to the conduit 15
to allow monitoring of the negative pressure applied to
the vessel 8.
In order to prevent liquid removed from the
liposomes from entering the pump 1, a series of traps are
connected to the conduit 15 to assist in collecting the
liquid (and liquid vapor, all collectively referred to
herein as liquid) drawn from the liposomes. In a
preferred embodiment, two traps are utilized. The first
trap is preferably comprised of a flask 7 disposed in an
ice bath 4 with dry ice 17. The second trap is preferably
comprised of a column 3 around which tubing 16 is
helically arranged. The column 3 is connected to the
conduit 15 at its top end and to one end of the tubing 16
at its bottom end. The other end of the tubing 16 is
connected to the conduit 15. As shown in Figure 1, an ice
bath 2 with dry ice 17 surrounds the column 3 and tubing
16. If desired, dry ice 17 can be replaced with liquid
nitrogen, liquid air or other cryogenic material. The ice
baths 2 and 4 assist in collecting any liquid and
condensing any liquid vapor drawn from the liposomes for
collection in the traps. In preferred embodiments of the
present invention the ice traps 2 and 4 are each
maintained at a temperature of least about -70 C.
A stopcock 14 is disposed in the conduit 15
upstream of the vessel 8 to allow a selected gas to be
introduced into the vessel 8 and into the liposomes 19
from gas bottle 18.
Apparatus of the present invention are utilized
by placing the liposomes 19 into vessel 8. In a
preferable embodiment, ice bath 5 with dry ice 17 is used
to lower the temperature of the liposomes to below 0 C,
more preferably to between about -10 C and about -20 C,
and most preferably to -10 C. With stopcocks 14 and 9
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closed, va,.;uum pump 1's turned on. Stopcocks 10, 11 12
and 13 are then carefully opened to create a vacuum in
vessel 8 by mei3ns of vacuum pump 1. The pressure is
gauged by means of manometer 6 until negative pressure of
at least about 700 mm Hg and preferably in the range of
between about '700 mm Hg and about 760 mm Hg (gauge
pressure) is achieved. In preferred embodiments of the
present invention vessel 7, cooled by ice bath 4 with dry
ice 17, and co:Lumn 3 and coil 16, cooled by ice bath 2
with dry ice 17, together or individually condense liquid
vapor and trap liquid drawn from the liposomes so as to
prevent such l:iquids and liquid vapor from entering the
vacuum pump 1. In preferred embodiments of the present
invention, the tempe:rature of ice traps 2 and 4 are each
maintained at a temperature of at least about -70 C. The
desired negative pressure is generally maintained for at
least 24 hours as liquid and liquid vapor is removed from
the liposomes 3_9 in vessel 8 and frozen in vessels 3 and
7. Pressure within ithe system is monitored using
manometer 6 and is gcanerally maintained for about 24 to
about 72 hours, at which time substantially all of the
liquid has beeri removed from the liposomes. At this
point, stopcock 10 is slowly closed and vacuum pump 1 is
turned off. Stopcock 14 is then opened gradually and gas
is slowly intrciduced into the system from gas bottle 18
through stopcock 14 via conduit 15 to instill gas into the
liposomes 19 in vessel 8. Preferably the gas instillation
occurs slowly aver a time period of at least about 4
hours, most preferably over a time period of between about
4 and about 8 hours, until the system reaches ambient
pressure.
The vacuum dried gas instilled liposomes and the
gas filled liposomes substantially devoid of liquid in the
interior thereof of the present invention have superior
characteristics for ultrasound contrast imaging.
Specifically, the present invention is useful in imaging a
patient generally, arid/or in diagnosing the presence of
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diseased tissue in a patient. The patient may be any type
of mammal, but is most preferably human. Thus, in further
embodiments of the present invention, a method of
providing an image of an internal bodily region of a
patient is provided. This method comprises administering
the liposomes of'the invention to the patient and scanning
the patient using ultrasonic imaging to obtain visible
images of the region.I A method is also provided for
diagnosing the presence of diseased tissue in a patient,
said method comprising administering to a patient
liposomes of the present invention, and then scanning the
patient using ultrasonic imaging to obtain visible images
of any diseased tissue in the patient. By region of-a
patient, it is meant the whole patient, or a particular
area or portion of the patient. For example, by using the
method of the invention, a patient's heart, and a
patient's vasculature (that is,:venous or arterial
systems), may be visualized and/or diseased tissue may be
diagnosed. In visualizing a patient's vasculature, blood
flow may be measured, as will be well understood by those
skilled in the art in view of the present disclosure. The
invention is also particularly useful in visualizing
and/or diagnosing disease in a patient's left heart, a
region not easily imaged heretofore by ultrasound.. Liver,
spleen and kidney regions of a patient may also be readily
visualized and/or disease detected therein using the
present methods.
Liposomes of the present invention may be of
varying sizes, but preferably are of a size'range wherein
they have a mean outside diameter between about 30
nanometers and about 10 microns, with the preferable mean
outside diameter being about 2 microns. As is known to
those skilled in the art, liposome size influences
biodistribution and, therefore, different size liposomes
may be.selected for various purposes. For intravascular
use, for example, liposome size is generally no larger
than about 5 microns, and generally no smaller than about
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30 nanometers, in mean outside diameter. To provide
ultrasound enhancement of organs such as the liver and to
allow differentiation of tumor from normal tissue, smaller
liposomes, between about 30 nanometers and about 100
nanometers in mean outside diameter, are useful.
Any of the various types of ultrasound imaging
devices can be employed in the practice of the invention,
the particular type or model of the device not being
critical to the method of the invention. Generally, for
the diagnostic uses of the present invention, ultrasound
frequencies between about 3.0 to about 7.5 megahertz are
employed.
As one skilled in the art would recognize,
administration of contrast imaging agents of the present
invention may be carried out in various fashions, such as
intravascularly, intralymphatically, parenterally,
subcutaneously, intramuscularly, intraperitoneally,
interstitially, hyperbarically, orally, or intratumorly
using a variety of dosage forms. One preferred route of
administration is intravascularly. For intravascular use
the contrast algent is generally injected intravenously,
but may be injected intraarterially as well. The useful
dosage to be administered and the mode of administration
will vary depe:nding upon the age, weight, and mammal to be
diagnosed, and the particular diagnostic application
intended. Typically dosage is initiated at lower levels
and increased iuntil the desired contrast enhancement is
achieved. Genierally, the contrast agents of the invention
are administered in the form of an aqueous suspension such
as in water or a saline solution (e.g., phosphate buffered
saline). Prefierably, the water is sterile. Also
preferably the saline solution is a hypertonic saline
solution (e.g., about 0.3 to about 0.5% NaCl), although,
if desired, the saline solution may be isotonic. The
solution also inay be buffered, if desired, to provide a pH
range of pH 6.13 to pH 7.4. In addition, dextrose may be
preferably inc:Luded in the media. Preferably, the aqueous
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solution is degassed (that is, degassed under vacuum
pressure) prior to suspending the liposomes therein.
Kits useful for ultrasonic imaging in accordance
with the present invention comprise gas filled liposomes
prepared by a vacuum drying gas instillation methods, and gas filled liposomes
substantially devoid of liquid in the
interior thereof, in addition to conventional ultrasonic
imaging kit component.s. Such conventional ultrasonic
imaging kit component.s are well known, and include, for
example, filters to remove bacterial contaminants or to
break up liposomal ac[gregates prior to administration.
The liposomes of the present invention are
believed to differ from the liposomes of the prior art in
a number of respects, both in physical and in functional
characteristics. For example, the liposomes of the
invention are substaritially devoid of liquid in the
interior thereof. By definition, liposomes in the prior
art have been characterized by the presence of an aqueous
medium. See, e.g., I)orland's Illustrated Medical
Dictionary, p. 946, 27th ed. (W.B. Saunders Company,
Philadelphia 1988). Moreover, the present liposomes
surprisingly exhibit intense ecogenicity on ultrasound,
and possess a long storage life, characteristics of great
benefit to the use of the liposomes as ultrasound contrast
agents.
There are various other applications for
liposomes of the inviantion, beyond those described in
detail herein. Such additional uses, for example, include
such applications as hyperthermia potentiators for
ultrasound and as driug delivery vehicles. Such additional
uses and other relatied subject matter are described and
claimed in applicant's patent applications filed
concurrently herewith entitled "Novel Liposomal Drug
Delivery Systerns" and "Method For Providing Localized
Therapeutic Heat to Biological Tissues and Fluids Using
Gas Filled Liposomes",=
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The p:resent invention is further described in the
following examples. Examples 1-8 are actual examples that
describe the p:reparation and testing of the vacuum dried
gas instilled :Liposomes, the gas filled liposomes being
substantially devoid of any liquid in the interior
thereof. Examp:Les 9-11 are prophetic examples that
describe the use of the liposomes of the invention. The
following examples should not be construed as limiting the
scope of the appended claims.
EXAMPLES
Examnle 1
Dipalmitoylphosphatidylcholine (1 gram) was
suspended in 10 ml phosphate buffered saline, the
suspension was heated to about 50 C, and then swirled by
hand in a rounci bottom flask for about 30 minutes. The
heat source was removed, and the suspension was swirled
for two additional hours, while allowing the suspension to
cool to room temperature, to form liposomes.
The liposomes thus prepared were placed in a
vessel in an apparatus similar to that shown in Figure 1,
cooled to about: -10'C, and then subjected to high negative
vacuum pressure. The temperature of the liposomes was
then raised to about 10"C. High negative vacuum pressure
was maintained for about 48 hours. After about 48 hours,
nitrogen gas wais graciually instilled into the chamber over
a period of about 4 hours, after which time the pressure
returned to ambient pressure. The resulting vacuum dried
gas instilled l.iposomes, the gas filled liposomes being
substantially dlevoid of any liquid in the interior
thereof, were t:hen suspended in 10 cc of phosphate
buffered saline: and stored at about 4 C for about three
months.
Example 2
To test the liposomes of Example 1
ultrasonographically, a 250 mg sample of these liposomes
was suspended in 300 cc of degassed phosphate buffered
saline (that is, degassed under vacuum pressure). The
- - ----------
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liposomes were then scanned in vitro at varying time
intervals with a 7.5 mHz transducer using an Acoustic
TM
Imaging Model 5200 scanner (Acoustic Imaging, Phoenix, AZ)
and employing the system test software to measure dB
reflectivity. The system was standardized prior to
testing the liposomes with a phantom of known acoustic
impedance. A graph showing dB reflectivity is provided in
Figure 2.
Example 3
Dipalmitoylphosphatidylcholine (1 gram) and the
cryoprotectant trehalose (1 gram) were suspended in 10 ml
phosphate buffered saline, the suspension was heated to
about 500C, and then swirled by hand in a round bottom
flask for about 30 minutes. The heat source was removed,
and the suspension was swirled for about two additional
hours, while allowing the suspension to cool to room
temperature, to form liposomes.
The liposomes thus prepared were then vacuum
dried and gas instilled, substantially following the
procedures shown in Example 1, resulting in vacuum dried
gas instilled liposomes, the gas filled liposomes being
substantially devoid of any liquid in the interior
thereof. The liposomes were then suspended in 10 cc of
phosphate buffered saline, and then stored at about 4'C
for several weeks.
Example 4
To test the liposomes of Example 3
ultrasonographically, the procedures of Example 2 were
substantially followed. The dB reflectivity of the
liposomes were similar to the dB reflectivity reported in
Example 2.
Example 5
Dipalmitoylphosphatidylcholine (1 gram) was
suspended in 10 ml phosphate buffered saline, the
suspension was heated to about 50'C, and then swirled by
hand in a round bottom flask for about 30 minutes. The
suspension was then subjected to 5 cycles of extrusion
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through an extruder device jacketed with a thermal barrel
(Extruder DeviceT"', Lipex Biomembranes, Vancouver, Canada),
both with and without conventional freeze-thaw treatment
prior to extrusion, while maintaining the temperature at
about 50 C. The heat source was removed, and the
suspension was swirled for about two additional hours,
while allowing the suspension to cool to room temperature,
to form liposo:mes.
The liposomes thus prepared were then vacuum
dried and gas instilled, substantially following the
procedures shown in Example 1, resulting in vacuum dried
gas instilled liposomes, the gas filled liposomes being
substantially i3evoid of any liquid in the interior
thereof. The :Liposomes were then suspended in 10 cc of
phosphate buffered saline, and then stored at about 4 C
for several weiaks.
Example 6
To test the liposomes of Example 5
ultrasonograph:ically, the procedures of Example 2 were
substantially followed. The dB reflectivity of the
liposomes were similar to the dB reflectivity reported in
Example 2.
Example 7
In orcier to test the stability of the liposomes
of the invention, the liposomes suspension of Example 1
was passed through 2 micron polycarbonate filters in an
extruder device (Extruder DeviceT"', Lipex Biomembranes,
Vancouver, Canada) five times at a pressure of about 600
psi. After extrusion treatment, the liposomes were
studied ultrasonographically, as described in Example 2.
Surprisingly, even after extrusion under high pressure,
the liposomes of the invention substantially retained
their echogenicity.
Example 8
The li.posomes of Example 1 were scanned by
ultrasound using transducer frequencies varying from 3 to
7.5 mHz. The results indicated that at a higher frequency
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of ultrasound, the echogenicity decays more rapidly,
reflecting a relatively high resonant frequency and higher
energy associated with the higher frequencies.
The following examples, Examples 9, 10, and 11,
are prophetic examples.
Example 9
A patient with suspected myocardial ischemia is
administered an intravenous dose of 500 mg of vacuum dried
gas instilled liposomes encapsulating nitrogen gas, the
gas filled liposomes being substantially devoid of liquid
in the interior thereof, with a mean diameter of 2
microns, and the left ventricular myocardium is studied
ultrasonographically.
Example 10
A patient with suspected myocardial ischemia is
administered an intravenous dose of 500 mg of vacuum dried
gas instilled liposomes encapsulating nitrogen gas, the
gas filled liposomes being substantially devoid of liquid
in the interior thereof, with a mean diameter of 2
microns, and the left ventricular myocardium is studied
ultrasonographically.
Example 11
A patient with suspected hepatic metastases is
administered an intravenous dose of 500 mg of vacuum dried
gas instilled liposomes encapsulating nitrogen gas, the
gas filled liposomes being substantially devoid of liquid
in the interior thereof, and the liver is examined
ultrasonographically.
Various modifications of the invention in
addition to those shown and described herein will be
apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall
within the scope of the appended claims.
