U.S. patent application number 09/291277 was filed with the patent office on 2001-06-07 for contrast agents.
Invention is credited to ERIKSEN, MORTEN, FRIGSTAD, SIGMUND, OSTENSEN, JONNY, RONGVED, PAL.
Application Number | 20010002993 09/291277 |
Document ID | / |
Family ID | 26310262 |
Filed Date | 2001-06-07 |
United States Patent
Application |
20010002993 |
Kind Code |
A1 |
OSTENSEN, JONNY ; et
al. |
June 7, 2001 |
CONTRAST AGENTS
Abstract
Ultrasonic visualisation of a subject, particularly of perfusion
in the myocardium and other tissues, is performed using novel
gas-containing contrast agent preparations which promote
controllable and temporary growth of the gas phase in vivo
following administration and can therefore act as deposited
perfusion tracers. The preparations include a coadministerable
composition comprising a diffusible component capable of inward
diffusion into the dispersed gas phase to promote temporary growth
thereof. In cardiac perfusion imaging the preparations may
advantageously be coadministered with vasodilator drugs such as
adenosine in order to enhance the differences in return signal
intensity from normal and hypoperfused myocardial tissue
respectively.
Inventors: |
OSTENSEN, JONNY; (OSLO,
NO) ; ERIKSEN, MORTEN; (OSLO, NO) ; FRIGSTAD,
SIGMUND; (TRONDHEIM, NO) ; RONGVED, PAL;
(OSLO, NO) |
Correspondence
Address: |
ROYAL N. RONNING, JR., VICE PRESIDENT
AMERSHAM PHARMACIA BIOTECH INC.
800 CENTENNIAL AVENUE
P.O. BOX 1327
PISCATAWAY
NJ
08855-1327
US
|
Family ID: |
26310262 |
Appl. No.: |
09/291277 |
Filed: |
April 14, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09291277 |
Apr 14, 1999 |
|
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PCT/GB97/02898 |
Oct 21, 1997 |
|
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60044452 |
Apr 29, 1997 |
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Current U.S.
Class: |
424/9.52 |
Current CPC
Class: |
A61K 49/223 20130101;
Y10S 977/929 20130101; Y10S 977/928 20130101 |
Class at
Publication: |
424/9.52 |
International
Class: |
A61B 008/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 21, 1996 |
DE |
9621884.7 |
Apr 23, 1997 |
DE |
9708239.0 |
Claims
What is claimed is:
1. A combined preparation for simultaneous, separate or sequential
use as a contrast agent in ultrasound imaging, said preparation
comprising: i) an injectable aqueous medium having gas dispersed
therein; and ii) a composition comprising a diffusible component
capable of diffusion in vivo into said dispersed gas so as at least
transiently to increase the size thereof.
2. A combined preparation as claimed in claim 1 wherein the
dispersed gas comprises air, nitrogen, oxygen, carbon dioxide,
hydrogen, an inert gas, a sulphur fluoride, selenium hexafluoride,
an optionally halogenated silane, a low molecular weight
hydrocarbon, a ketone, an ester, a halogenated low molecular weight
hydrocarbon or a mixture of any of the foregoing.
3. A combined preparation as claimed in claim 2 wherein the gas
comprises a perfluorinated ketone, perfluorinated ether or
perfluorocarbon.
4. A combined preparation as claimed in claim 3 wherein the
perfluorocarbon comprises a perfluoroalkane, perfluoroalkene or
perfluorocycloalkane.
5. A combined preparation as claimed in claim 2 wherein the gas
comprises sulphur hexafluoride or a perfluoropropane,
perfluorobutane or perfluoropentane.
6. A combined preparation as claimed in any of the preceding claims
wherein the dispersed gas is stabilised by a coalescence-resistant
surface membrane, a filmogenic protein, a polymer material, a
non-polymeric and non-polymerisable wall-forming material or a
surfactant.
7. A combined preparation as claimed in claim 6 wherein said
surfactant comprises at least one phospholipid.
8. A combined preparation as claimed in claim 7 wherein at least
75% of the said surfactant material comprises phospholipid
molecules individually bearing net overall charge.
9. A combined preparation as claimed in claim 8 wherein at least
75% of the film-forming surfactant material comprises one or more
phospholipids selected from phosphatidylserines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids
and cardiolipins.
10. A combined preparation as claimed in claim 9 wherein at least
80% of said phospholipids comprise phosphatidylserines.
11. A combined preparation as claimed in any of the preceding
claims wherein the composition comprising the diffusible component
is formulated for administration cutaneously, subcutaneously,
intramuscularly, intravenously or by inhalation.
12. A combined preparation as claimed in any of claims 1 to 10
wherein the composition comprising the diffusible component further
comprises a carrier liquid.
13. A combined preparation as claimed in claim 12 wherein the
diffusible component is dispersed in an aqueous carrier liquid in
the form of an oil-in-water emulsion or microemulsion.
14. A combined preparation as claimed in claim 13 wherein the
diffusible component comprises an aliphatic ether, polycyclic oil,
polycyclic alcohol, heterocyclic compound, aliphatic hydrocarbon,
cycloaliphatic hydrocarbon or halogenated low molecular weight
hydrocarbon.
15. A combined preparation as claimed in claim 14 wherein the
diffusible component comprises a perfluorocarbon.
16. A combined preparation as claimed in claim 15 wherein the
perfluorocarbon comprises a perfluoroalkane, perfluoroalkene,
perfluorocycloalkane, perfluorocycloalkene or perfluorinated
alcohol.
17. A combined preparation as claimed in claim 16 wherein the
diffusible component comprises perfluoropentane, perfluorohexane or
perfluorodimethylcyclobutane.
18. A combined preparation as claimed in any of claims 13 to 17
wherein the emulsion is stabilised by a phospholipid
surfactant.
19. A combined preparation as claimed in claim 18 wherein at least
75% of the said phospholipid surfactant comprises molecules
individually bearing net overall charge.
20. A combined preparation as claimed in claim 19 wherein at least
75% of the phospholipid surfactant is selected from
phosphatidylserines, phosphatidylglycerols, phosphatidylinositols,
phosphatidic acids and cardiolipins.
21. A combined preparation as claimed in claim 20 wherein at least
80% of said phospholipid surfactant comprises
phosphatidylserines.
22. A combined preparation as claimed in any of the preceding
claims which further includes a vasodilator drug.
23. A combined preparation as claimed in claim 22 wherein said
vasodilator drug is adenosine.
24. A combined preparation as claimed in any of claims 1 to 21
which further includes a therapeutic agent.
25. A combined preparation as claimed in any of claims 1 to 21
which further includes contrast-enhancing moieties for an imaging
modality other than ultrasound.
26. A method of generating enhanced images of a human or non-human
animal subject which comprises the steps of: i) injecting a
physiologically acceptable aqueous medium having gas dispersed
therein into the vascular system of said subject; ii) before,
during or after injection of said aqueous medium administering to
said subject a composition comprising a diffusible component
capable of diffusion in vivo into said dispersed gas so as at least
transiently to increase the size thereof; and iii) generating an
ultrasound image of at least a part of said subject.
27. A method as claimed in claim 26 wherein the composition
comprising the diffusible component is administered cutaneously,
subcutaneously, intramuscularly, intravenously or by
inhalation.
28. A method as claimed in claim 26 or claim 27 wherein a
vasodilator drug is coadministered to the subject.
29. A method as claimed in claim 28 wherein said vasodilator drug
is adenosine.
Description
[0001] This invention relates to ultrasound imaging, more
particularly to novel contrast agent preparations and their use in
ultrasound imaging, for example in visualising tissue
perfusion.
[0002] It is well known that contrast agents comprising dispersions
of microbubbles of gases are particularly efficient backscatterers
of ultrasound by virtue of the low density and ease of
compressibility of the microbubbles. Such microbubble dispersions,
if appropriately stabilised, may permit highly effective ultrasound
visualisation of, for example, the vascular system and tissue
microvasculature, often at advantageously low doses.
[0003] The use of ultrasonography to measure blood perfusion (i.e.
blood flow per unit of tissue mass) is of potential value in, for
example, tumour detection, tumour tissue typically having different
vascularity from healthy tissue, and studies of the myocardium,
e.g. to detect myocardial infarctions. A problem with the
application of existing ultrasound contrast agents to cardiac
perfusion studies is that the information content of images
obtained is degraded by attenuation caused by contrast agent
present in the ventricles of the heart.
[0004] The present invention is based on the finding that
ultrasonic visualisation of a subject, in particular of perfusion
in the myocardium and other tissues, may be achieved and/or
enhanced by means of gas-containing contrast agent preparations
which promote controllable and temporary growth of the gas phase in
vivo following administration. Thus, for example, such contrast
agent preparations may be used to promote controllable and
temporary retention of the gas phase, for example in the form of
microbubbles, in tissue microvasculature, thereby enhancing the
concentration of gas in such tissue and accordingly enhancing its
echogenicity, e.g. relative to the blood pool.
[0005] It will be appreciated that such use of gas as a deposited
perfusion tracer differs markedly from existing proposals regarding
intravenously administrable microbubble ultrasound contrast agents.
Thus it is generally thought necessary to avoid microbubble growth
since, if uncontrolled, this may lead to potentially hazardous
tissue embolisation. Accordingly it may be necessary to limit the
dose administered and/or to use gas mixtures with compositions
selected so as to minimise bubble growth in vivo by inhibiting
inward diffusion of blood gases into the microbubbles (see e.g.
WO-A-9503835 and WO-A-9516467).
[0006] In accordance with the present invention, on the other hand,
a composition comprising a dispersed gas phase is coadministered
with a composition comprising at least one substance which has or
is capable of generating a gas or vapour pressure in vivo
sufficient to promote controllable growth of the said dispersed gas
phase through inward diffusion thereto of molecules of gas or
vapour derived from said substance, which for brevity is
hereinafter referred to as a "diffusible component", although it
will be appreciated that transport mechanisms other than diffusion
may additionally or alternatively be involved in operation of the
invention, as discussed in greater detail hereinafter.
[0007] This coadministration of a dispersed gas phase-containing
composition and a composition comprising a diffusible component
having an appropriate degree of volatility may be contrasted with
previous proposals regarding administration of volatile substances
alone, e.g. in the form of phase shift colloids as described in
WO-A-9416739. Thus the contrast agent preparations of the present
invention permit control of factors such as the probability and/or
rate of growth of the dispersed gas by selection of appropriate
constituents of the coadministered compositions, as described in
greater detail hereinafter, whereas administration of the
aforementioned phase shift colloids alone may lead to generation of
microbubbles which grow uncontrollably and unevenly, possibly to
the extent where at least a proportion of the microbubbles may
cause potentially dangerous embolisation of, for example, the
myocardial vasculature and brain (see e.g. Schwarz, Advances in
Echo-Contrast [1994(3)], pp. 48-49).
[0008] It has also been found that administration of phase shift
colloids alone may not lead to reliable or consistent in vivo
volatilisation of the dispersed phase to generate gas or vapour
microbubbles. Grayburn et al. in J. Am. Coll. Cardiol. 26(5)
[1995], pp. 1340-1347 suggest that preactivation of
perfluoropentane emulsions may be required to achieve myocardial
opacification in dogs at effective imaging doses low enough to
avoid haemodynamic side effects. An activation technique for such
colloidal dispersions, involving application of hypobaric forces
thereto, is described in WO-A-9640282; typically this involves
partially filling a syringe with the emulsion and subsequently
forcibly withdrawing and then releasing the plunger of the syringe
to generate a transient pressure change which causes formation of
gas microbubbles within the emulsion. This is an inherently
somewhat cumbersome technique which may fail to give consistent
levels of activation.
[0009] It is stated in U.S. Pat. No. 5,536,489 that emulsions of
water-insoluble gas-forming chemicals such as perfluoropentane may
be used as contrast agents for site-specific imaging, the emulsions
only generating a significant number of image-enhancing gas
microbubbles upon application of ultrasonic energy to a specific
location in the body which it is desired to image. Our own research
has shown that emulsions of volatile compounds such as
2-methylbutane or perfluoropentane give no detectable echo
enhancement either in vitro or in vivo when ultrasonicated at
energy levels which are sufficient to give pronounced contrast
effects using two component contrast agents in accordance with the
present invention.
[0010] According to one aspect of the invention there is provided a
combined preparation for simultaneous, separate or sequential use
as a contrast agent in ultrasound imaging, said preparation
comprising:
[0011] i) an injectable aqueous medium having gas dispersed
therein; and
[0012] ii) a composition comprising a diffusible component capable
of diffusion in vivo into said dispersed gas so as at least
transiently to increase the size thereof.
[0013] According to a further aspect of the invention there is
provided a method of generating enhanced images of a human or
non-human animal subject which comprises the steps of:
[0014] i) injecting a physiologically acceptable aqueous medium
having gas dispersed therein into the vascular system of said
subject;
[0015] ii) before, during or after injection of said aqueous medium
administering to said subject a composition comprising a diffusible
component capable of diffusion in vivo into said dispersed gas so
as at least transiently to increase the size thereof; and
[0016] iii) generating an ultrasound image of at least a part of
said subject.
[0017] This method according to the invention may advantageously be
employed in visualising tissue perfusion in a subject, the increase
in size of the dispersed gas being utilised to effect enrichment or
temporary retention of gas in the microvasculature of such tissue,
thereby enhancing its echogenicity.
[0018] Any biocompatible gas may be present in the gas dispersion,
the term "gas" as used herein including any substances (including
mixtures) at least partially, e.g. substantially or completely in
gaseous (including vapour) form at the normal human body
temperature of 37.degree. C. The gas may thus, for example,
comprise air; nitrogen; oxygen; carbon dioxide; hydrogen; an inert
gas such as helium, argon, xenon or krypton; a sulphur fluoride
such as sulphur hexafluoride, disulphur decafluoride or
trifluoromethylsulphur pentafluoride; selenium hexafluoride; an
optionally halogenated silane such as methylsilane or
dimethylsilane; a low molecular weight hydrocarbon (e.g. containing
up to 7 carbon atoms), for example an alkane such as methane,
ethane, a propane, a butane or a pentane, a cycloalkane such as
cyclopropane, cyclobutane or cyclopentane, an alkene such as
ethylene, propene, propadiene or a butene, or an alkyne such as
acetylene or propyne; an ether such as dimethyl ether; a ketone; an
ester; a halogenated low molecular weight hydrocarbon (e.g.
containing up to 7 carbon atoms); or a mixture of any of the
foregoing. Advantageously at least some of the halogen atoms in
halogenated gases are fluorine atoms; thus biocompatible
halogenated hydrocarbon gases may, for example, be selected from
bromochlorodifluoromethane, chlorodifluoromethane,
dichlorodifluoromethane, bromotrifluoromethane,
chlorotrifluoromethane, chloropentafluoroethane,
dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene,
ethylfluoride, 1, 1-difluoroethane and perfluorocarbons.
Representative perfluorocarbons include perfluoroalkanes such as
perfluoromethane, perfluoroethane, perfluoropropanes,
perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture
with other isomers such as perfluoro-isobutane), perfluoropentanes,
perfluorohexanes or perfluoroheptanes; perfluoroalkenes such as
perfluoropropene, perfluorobutenes (e.g. perfluorobut-2-ene),
perfluorobutadiene, perfluoropentenes (e.g. perfluoropent-1-ene) or
perfluoro-4-methylpent-2-ene; perfluoroalkynes such as
perfluorobut-2-yne; and perfluorocycloalkanes such as
perfluorocyclobutane, perfluoromethylcyclobutane,
perfluorodimethylcyclob- utanes, perfluorotrimethylcyclobutanes,
perfluorocyclopentane, perfluoromethylcyclopentane,
perfluorodimethylcyclopentanes, perfluorocyclohexane,
perfluoromethylcyclohexane or perfluorocycloheptane. Other
halogenated gases include methyl chloride, fluorinated (e.g.
perfluorinated) ketones such as perfluoroacetone and fluorinated
(e.g. perfluorinated) ethers such as perfluorodiethyl ether. The
use of perfluorinated gases, for example sulphur hexafluoride and
perfluorocarbons such as perfluoropropane, perfluorobutanes,
perfluoropentanes and perfluorohexanes, may be particularly
advantageous in view of the recognised high stability in the
bloodstream of microbubbles containing such gases. Other gases with
physicochemical characteristics which cause them to form highly
stable microbubbles in the bloodstream may likewise be useful.
[0019] The dispersed gas may be administered in any convenient
form, for example using any appropriate gas-containing ultrasound
contrast agent formulation as the gas-containing composition.
Representative examples of such formulations include microbubbles
of gas stabilised (e.g. at least partially encapsulated) by a
coalescence-resistant surface membrane (for example gelatin, e.g.
as described in WO-A-8002365), a filmogenic protein (for example an
albumin such as human serum albumin, e.g. as described in U.S. Pat.
Nos. 4,718,433, 4,774,958, 4,844,882, EP-A-0359246, WO-A-9112823,
WO-A-9205806, WO-A-9217213, WO-A-9406477 or WO-A-9501187), a
polymer material (for example a synthetic biodegradable polymer as
described in EP-A-0398935, an elastic interfacial synthetic polymer
membrane as described in EP-A-0458745, a microparticulate
biodegradable polyaldehyde as described in EP-A-0441468, a
microparticulate N-dicarboxylic acid derivative of a polyamino
acid--polycyclic imide as described in EP-A-0458079, or a
biodegradable polymer as described in WO-A-9317718 or
WO-A-9607434), a nonpolymeric and non-polymerisable wall-forming
material (for example as described in WO-A-9521631), or a
surfactant (for example a polyoxyethylene-polyoxypropylene block
copolymer surfactant such as a Pluronic, a polymer surfactant as
described in WO-A-9506518, or a film-forming surfactant such as a
phospholipid, e.g. as described in WO-A-9211873, WO-A-9217212,
WO-A-9222247, WO-A-9428780, WO-A-9503835 or WO-A-9729783).
[0020] Other useful gas-containing contrast agent formulations
include gas-containing solid systems, for example microparticles
(especially aggregates of microparticles) having gas contained
therewithin or otherwise associated therewith (for example being
adsorbed on the surface thereof and/or contained within voids,
cavities or pores therein, e.g. as described in EP-A-0122624,
EP-A-0123235, EP-A-0365467, WO-A-9221382, WO-A-9300930,
WO-A-9313802, WO-A-9313808 or WO-A-9313809). It will be appreciated
that the echogenicity of such microparticulate contrast agents may
derive directly from the contained/associated gas and/or from gas
(e.g. microbubbles) liberated from the solid material (e.g. upon
dissolution of the microparticulate structure).
[0021] The disclosures of all of the above-described documents
relating to gas-containing contrast agent formulations are
incorporated herein by reference.
[0022] Gas microbubbles and other gas-containing materials such as
microparticles preferably have an initial average size not
exceeding 10 .mu.m (e.g. of 7 .mu.m or less) in order to permit
their free passage through the pulmonary system following
administration, e.g. by intravenous injection. However, larger
microbubbles may be employed where, for example, these contain a
mixture of one or more relatively blood-soluble or otherwise
diffusible gases such as air, oxygen, nitrogen or carbon dioxide
with one or more substantially insoluble and non-diffusible gases
such as perfluorocarbons. Outward diffusion of the
soluble/diffusible gas content following administration will cause
such microbubbles rapidly to shrink to a size which will be
determined by the amount of insoluble/non-diffusible gas present
and which may be selected to permit passage of the resulting
microbubbles through the lung capillaries of the pulmonary
system.
[0023] Since dispersed gas administered in accordance with the
invention is caused to grow in vivo through interaction with
diffusible component, the minimum size of the microbubbles,
solid-associated gas etc. as administered may be substantially
lower than the size normally thought necessary to provide
significant interaction with ultrasound (typically ca. 1-5 .mu.m at
conventionally-employed imaging frequencies); the dispersed gas
moieties may therefore have sizes as low as, for example, 1 nm or
below. The invention may accordingly permit use of gas-containing
compositions which have not hitherto been proposed for use as
ultrasound contrast agents, e.g. because of the low size of the
dispersed gas moieties.
[0024] Where phospholipid-containing compositions are employed in
accordance with the invention, e.g. in the form of
phospholipid-stabilised gas microbubbles, representative examples
of useful phospholipids include lecithins (i.e.
phosphatidylcholines), for example natural lecithins such as egg
yolk lecithin or soya bean lecithin, semisynthetic (e.g. partially
or fully hydrogenated) lecithins and synthetic lecithins such as
dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or
distearoylphosphatidylcholine; phosphatidic acids;
phosphatidylethanolamines; phosphatidylserines;
phosphatidylglycerols; phosphatidylinositols; cardiolipins;
sphingomyelins; fluorinated analogues of any of the foregoing;
mixtures of any of the foregoing and mixtures with other lipids
such as cholesterol. The use of phospholipids predominantly (e.g.
at least 75%) comprising molecules individually bearing net overall
charge, e.g. negative charge, for example as in naturally occurring
(e.g. soya bean or egg yolk derived), semisynthetic (e.g. partially
or fully hydrogenated) and synthetic phosphatidylserines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids
and/or cardiolipins, for example as described in WO-A-9729783, may
be particularly advantageous.
[0025] Representative examples of gas-containing microparticulate
materials which may be useful in accordance with the invention
include carbohydrates (for example hexoses such as glucose,
fructose or galactose; disaccharides such as sucrose, lactose or
maltose; pentoses such as arabinose, xylose or ribose; .alpha.-,
.beta.- and .gamma.-cyclodextrins; polysaccharides such as starch,
hydroxyethyl starch, amylose, amylopectin, glycogen, inulin,
pulullan, dextran, carboxymethyl dextran, dextran phosphate,
ketodextran, aminoethyldextran, alginates, chitin, chitosan,
hyaluronic acid or heparin; and sugar alcohols, including alditols
such as mannitol or sorbitol), inorganic salts (e.g. sodium
chloride), organic salts (e.g. sodium citrate, sodium acetate or
sodium tartrate), X-ray contrast agents (e.g. any of the
commercially available carboxylic acid and non-ionic amide contrast
agents typically containing at least one 2, 4, 6-triiodophenyl
group having substituents such as carboxyl, carbamoyl,
N-alkylcarbamoyl, N-hydroxyalkylcarbamoyl, acylamino,
N-alkylacylamino or acylaminomethyl at the 3- and/or 5-positions,
as in metrizoic acid, diatrizoic acid, iothalamic acid, ioxaglic
acid, iohexol, iopentol, iopamidol, iodixanol, iopromide,
metrizamide, iodipamide, meglumine iodipamide, meglumine
acetrizoate and meglumine diatrizoate), and polypeptides and
proteins (e.g. gelatin or albumin such as human serum albumin).
[0026] Other gas-containing materials which may be useful in
accordance with the invention include gas-containing material
stabilised by metals (e.g. as described in U.S. Pat. Nos. 3,674,461
or 3,528,809), gas-containing material stabilised by synthetic
polymers (e.g. as described in U.S. Pat. No. 3,975,194 or by
Farnand in Powder Technology 22 [1979], pp. 11-16), commercially
available microspheres of the Expancel.RTM. type, e.g. Expancel 551
DE (see e.g. Eur. Plast. News 9(5) [1982], p. 39, Nonwovens
Industry [1981], p. 21 and Mat. Plast. Blast. 10 [1980], p. 468),
commercially available microspheres of the Ropaque.RTM. type (see
e.g. J. Coatings Technol. 55(707) [1983], p. 79), micro- and
nano-sized gas-containing structures such as zeolites, inorganic or
organic aerogels, nanosized open void-containing chemical
structures such as fullerenes, clathrates or nanotubes (e.g. as
described by G. E. Gadd in Science 277 (5328) (1997), pp. 933-936),
and natural surfactant-stabilised microbubble dispersions (e.g. as
described by d'Arrigo in "Stable Gas-in-Liquid Emulsions, Studies
in physical and theoretical chemistry" 40--Elsevier, Amsterdam
[1986]).
[0027] A wide range of diffusible components may be used in
accordance with the invention, including gases/vapours, volatile
liquids, volatile solids and precursors capable of gas generation,
e.g. upon administration, the principal requirement being that the
component should either have or be capable of generating a
sufficient gas or vapour pressure in vivo (e.g. at least 10 torr)
so as to be capable of promoting inward diffusion of gas or vapour
molecules into the dispersed gas. It will be appreciated that
mixtures of two or more diffusible components may if desired be
employed in accordance with the invention; references herein to
"the diffusible component" are to be interpreted as including such
mixtures. Similarly, references to administration of a diffusible
component are intended also to embrace administration of two or
more such components, either as mixtures or as plural
administrations.
[0028] The composition comprising the diffusible component may take
any appropriate form and may be administered by any appropriate
method, the route of administration depending in part on the area
of the subject which is to be investigated. Thus, for example, oral
administration of an appropriate composition comprising a
diffusible component may be particularly useful where it is desired
to promote temporary retention of gas in the tissue of the
gastrointestinal wall. In representative embodiments of such
applications the gas dispersion may be injected intravenously in
doses similar to those used in echocardiography and the diffusible
component may be formulated as an orally administrable emulsion,
e.g. a perfluorocarbon-in-water emulsion as described in further
detail hereinafter, for example being used at a dose of 0.2-1.0
.mu.l perfluorocarbon/kg. Following administration and distribution
of the two compositions, growth of the gas dispersion in the
capillary blood pool in the gastric or intestinal wall may enhance
contour contrast from these regions. It will be appreciated that
the reverse combination of an orally administrable gas dispersion
and intravenously injectable diffusible component may be useful in
providing contour contrast from the inner wall or mucosa of the
gastrointestinal system.
[0029] It may be advantageous when using such orally administrable
gas dispersion or diffusible component compositions to incorporate
chemical groups or substances which promote adhesion to the wall of
the gastrointestinal tract, for example by admixture with the
composition or by attachment to a component thereof, e.g. a
surfactant or other stsbilising moiety, since this may stimulate
growth of the dispersed gas phase by enhancing its contact with the
diffusible component. Examples of such adhesion-promoting
groups/substances have previously been described in relation to,
for example, gastrointestinal X-ray contrast agents, and include
acrylic esters as described in WO-A-9722365, iodophenol sulphonate
esters as described in U.S. Pat. No. 5,468,466 and iodinated phenyl
esters as described in U.S. Pat. No. 5,260,049.
[0030] Inhalation of a suitably volatile diffusible component may,
for example, be used to promote growth of the administered gas
dispersion immediately following its passage through the lung
capillaries, e.g. so that the gas then becomes temporarily retained
in the capillaries of the myocardium. In such embodiments growth of
the dispersed gas may be further increased by raising the lung
pressure of the diffusible component, e.g. by an excess of up to
0.5 bar, for example by using a respirator or by having the subject
exhale against a resistance.
[0031] Intramuscular or subcutaneous injection of appropriately
formulated diffusible component compositions, e.g. incorporating a
physiologically acceptable carrier liquid, may, for example,
advantageously be employed where it is desired specifically to
limit the effect of the component to a particular target area of
the subject. One example of a composition for subcutaneous
injection comprises nanoparticles such as are used for lymph
angiography. Subcutaneously injected diffusible component may be
taken up by the lymph system, where it may cause growth of an
intravenously injected gas dispersion, thereby facilitating imaging
of lymph nodes. The reverse combination of a subcutaneously
injected gas diapersion and intravenously injected diffusible
component may similarly be employed.
[0032] Intravenous injection of appropriately formulated diffusible
component compositions, e.g. incorporating a physiologically
acceptable carrier liquid, permits considerable versatility in
operation of the invention since, as discussed in greater detail
hereinafter, the constituents of the gas dispersion and diffusible
component compositions may be selected to control parameters such
as the onset and rate of growth of the dispersed gas and thus the
parts of the body in which tissue echogenicity may be enhanced by
temporary retention of gas, for example in the microvasculature
thereof.
[0033] Appropriate topical formulations may be applied cutaneously
so as to promote transcutaneous absorption of the diffusible
component. Such administration may have applications in imaging
and/or therapy of the skin, subcutis and adjacent regions and
organs, for example in targeting the peripheral circulation of body
extremities such as legs.
[0034] Diffusible components for administration orally or by
injection may, for example, be formulated as solutions in or
mixtures with water and/or one or more water-miscible and
physiologically acceptable organic solvents, such as ethanol,
glycerol or polyethylene glycol; dispersions in an aqueous medium,
for example as the oil phase or a constituent of the oil phase of
an oil-in-water emulsion; microemulsions, i.e. systems in which the
substance is effectively dissolved in the hydrophobic interiors of
surfactant micelles present in an aqueous medium; or in association
with microparticles or nanoparticles dispersed in an appropriate
carrier liquid, for example being adsorbed on microparticle or
nanoparticle surfaces and/or contained within voids, cavities or
pores of microparticles or nanoparticles, or encapsulated within
microcapsules.
[0035] Where a diffusible component is to be administered as a
solution, the partial pressure derived therefrom in vivo will
depend on the concentration of the component, e.g. in the blood
stream, and the corresponding pressure of pure component material,
for example in accordance with Raoult's law in a system approaching
ideality. Thus if the component has low water solubility it is
desirable that it should have a sufficient vapour pressure in pure
form at normal body temperature, e.g. at least 50 torr, preferably
at least 100 torr. Examples of relatively water-insoluble
components with high vapour pressures include gases such as those
listed hereinbefore as possible microbubble gases.
[0036] Representative examples of more highly
water-soluble/water-miscible diffusible components, which may
therefore exhibit lower vapour pressures at body temperature,
include aliphatic ethers such as ethyl methyl ether or methyl
propyl ether; aliphatic esters such as methyl acetate, methyl
formate or ethyl formate; aliphatic ketones such as acetone;
aliphatic amides such as N,N-dimethylformamide or
N,N-dimethylacetamide; and aliphatic nitrites such as
acetonitrile.
[0037] It may, however, be preferred to employ a substantially
water-immiscible diffusible component formulated as an emulsion
(i.e. a stabilised suspension) in an appropriate aqueous medium,
since in such systems the vapour pressure in the aqueous phase of
the diffusible component will be substantially equal to that of
pure component material, even in very dilute emulsions. In such
embodiments the diffusible component may, for example, be
formulated as part of a proprietary registered pharmaceutical
emulsion, such as Intralipid.RTM. (Pharmacia).
[0038] The diffusible component in such emulsions is advantageously
a liquid at processing and storage temperature, which may for
example be as low as -10.degree. C. if the aqueous phase contains
appropriate antifreeze material, while being a gas or exhibiting a
substantial vapour pressure at body temperature. Appropriate
compounds may, for example, be selected from the various lists of
emulsifiable low boiling liquids given in the aforementioned
WO-A-9416379, the contents of which are incorporated herein by
reference. Specific examples of emulsifiable diffusible components
include aliphatic ethers such as diethyl ether; polycyclic oils or
alcohols such as menthol, camphor or eucalyptol; heterocyclic
compounds such as furan or dioxane; aliphatic hydrocarbons, which
may be saturated or unsaturated and straight chained or branched,
e.g. as in n-butane, n-pentane, 2-methylpropane, 2-methylbutane, 2,
2-dimethylpropane, 2, 2-dimethylbutane, 2, 3-dimethylbutane,
1-butene, 2-butene, 2-methylpropene, 1, 2-butadiene, 1,
3-butadiene, 2-methyl-1-butene, 2-methyl-2-butene, isoprene,
1-pentene, 1, 3-pentadiene, 1, 4-pentadiene, butenyne, 1-butyne,
2-butyne or 1, 3-butadiyne; cycloaliphatic hydrocarbons such as
cyclobutane, cyclobutene, methylcyclopropane or cyclopentane; and
halogenated low molecular weight hydrocarbons (e.g. containing up
to 7 carbon atoms). Representative halogenated hydrocarbons include
dichloromethane, methyl bromide, 1, 2-dichloroethylene, 1,
1-dichloroethane, 1-bromoethylene, 1-chloroethylene, ethyl bromide,
ethyl chloride, 1-chloropropene, 3-chloropropene, 1-chloropropane,
2-chloropropane and t-butyl chloride. Advantageously at least some
of the halogen atoms are fluorine atoms, for example as in
dichlorofluoromethane, trichlorofluoromethane, 1, 2-dichloro-1,
2-difluoroethane, 1, 2-dichloro-1, 1, 2, 2-tetrafluoroethane, 1, 1,
2-trichloro-1, 2, 2-trifluoroethane, 2-bromo-2-chloro-1, 1,
1-trifluoroethane, 2-chloro-1, 1, 2-trifluoroethyl difluoromethyl
ether, 1-chloro-2, 2, 2-trifluoroethyl difluoromethyl ether,
partially fluorinated alkanes (e.g. pentafluoropropanes such as 1
H, 1 H, 3 H-pentafluoropropane, hexafluorobutanes,
nonafluorobutanes such as 2 H-nonafluoro-t-butane, and
decafluoropentanes such as 2 H, 3 H-decafluoropentane), partially
fluorinated alkenes (e.g. heptafluoropentenes such as 1 H, 1 H, 2
H-heptafluoropent-1-ene, and nonafluorohexenes such as 1 H, 1 H, 2
H-nonafluorohex-1-ene), fluorinated ethers (e.g. 2, 2, 3, 3,
3-pentafluoropropyl methyl ether or 2, 2, 3, 3, 3-pentafluoropropyl
difluoromethyl ether) and, more preferably, perfluorocarbons.
Examples of perfluorocarbons include perfluoroalkanes such as
perfluorobutanes, perfluoropentanes, perfluorohexanes (e.g.
perfluoro-2-methylpentane), perfluoroheptanes, perfluorooctanes,
perfluorononanes and perfluorodecanes; perfluorocycloalkanes such
as perfluorocyclobutane, perfluorodimethyl-cyclobutanes,
perfluorocyclopentane and perfluoromethylcyclopentane;
perfluoroalkenes such as perfluorobutenes (e.g. perfluorobut-2-ene
or perfluorobuta-1, 3-diene), perfluoropentenes (e.g.
perfluoropent-1-ene) and perfluorohexenes (e.g.
perfluoro-2-methylpent-2-ene or perfluoro-4-methylpent-2-ene);
perfluorocycloalkenes such as perfluorocyclopentene or
perfluorocyclopentadiene; and perfluorinated alcohols such as
perfluoro-t-butanol.
[0039] Such emulsions may also contain at least one surfactant in
order to stabilise the dispersion; this may be the same as or
different from any surfactant(s) used to stabilise the gas
dispersion. The nature of any such surfactant may significantly
affect factors such as the rate of growth of the dispersed gas
phase. In general a wide range of surfactants may be useful, for
example selected from the extensive lists given in EP-A-0727225,
the contents of which are incorporated herein by reference.
Representative examples of useful surfactants include fatty acids
(e.g. straight chain saturated or unsaturated fatty acids, for
example containing 10-20 carbon atoms) and carbohydrate and
triglyceride esters thereof, phospholipids (e.g. lecithin),
fluorine-containing phospholipids, proteins (e.g. albumins such as
human serum albumin), polyethylene glycols, and block copolymer
surfactants (e.g. polyoxyethylene-polyoxypropylene block copolymers
such as Pluronics, extended polymers such as acyloxyacyl
polyethylene glycols, for example polyethyleneglycol methyl ether
16-hexadecanoyloxy-hexadecanoate, e.g. wherein the polyethylene
glycol moiety has a molecular weight of 2300, 5000 or 10000), and
fluorine-containing surfactants (e.g. as marketed under the trade
names Zonyl and Fluorad, or as described in WO-A-9639197, the
contents of which are incorporated herein by reference).
Particularly useful surfactants include phospholipids comprising
molecules with net overall negative charge, such as naturally
occurring (e.g. soya bean or egg yolk derived), semisynthetic (e.g.
partially or fully hydrogenated) and synthetic phosphatidylserines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids
and/or cardiolipins.
[0040] The droplet size of the dispersed diffusible component in
emulsions intended for intravenous injection should preferably be
less than 10 .mu.m, e.g. less than 7 .mu.m, and greater than 0.1
.mu.m in order to facilitate unimpeded passage through the
pulmonary system.
[0041] As noted above, water-immiscible diffusible components may
also be formulated as microemulsions. Such systems are advantageous
by virtue of their thermodynamic stability and the fact that the
diffusible component is in practice uniformly distributed
throughout the aqueous phase; microemulsions therefore have the
appearance of solutions but may exhibit the properties of emulsions
as regards the partial pressure of the dispersed phase.
[0042] Gas precursors which may be used include any biocompatible
components capable of gas generation in vivo, i.e. at body
temperature and physiological pH. Representative examples include
inorganic and organic carbonates and bicarbonates, and
nitrogen-generating substances such as pyrazolines, pyrazoles,
triazolines, diazoketones, diazonium salts, tetrazoles and azides.
It will be appreciated that in such systems it is the ultimately
generated gas which is the actual diffusible component.
[0043] In order to ensure maximum volatilisation of the diffusible
component following administration and to enhance growth of the
dispersed gas, both of which are endothermic processes, it may be
advantageous to manipulate the temperature of the solution or
suspension of the diffusible component and/or the gas dispersion
prior to administration and/or to incorporate exothermically
reactive constituents therein; the use of such constituents which
react exothermically under the influence of ultrasound radiation
may be particularly advantageous.
[0044] Growth of the dispersed gas phase in vivo may, for example,
be accompanied by expansion of any encapsulating material (where
this has sufficient flexibility) and/or by abstraction of excess
surfactant from the administered material to the growing gas-liquid
interfaces. It is also possible, however, that stretching of the
encapsulating material and/or interaction of the material with
ultrasound may substantially increase its porosity. Whereas such
disruption of encapsulating material has hitherto in many cases
been found to lead to rapid loss of echogenicity through outward
diffusion and dissolution of the gas thereby exposed, we have found
that when using contrast agent preparations in accordance with the
present invention, the exposed gas exhibits substantially
stability. Whilst not wishing to be bound by theoretical
calculations, we believe that the exposed gas, e.g. in the form of
liberated microbubbles, may be stabilised, e.g. against collapse of
the microbubbles, by the supersaturated environment generated by
the diffusible component, which provides an inward pressure
gradient to counteract the outward diffusive tendency of the
microbubble gas. The exposed gas surface, by virtue of the
substantial absence of encapsulating material, may cause the
contrast agent preparation to exhibit exceptionally favourable
acoustic properties as evidenced by high backscatter and low energy
absorption (e.g. as expressed by high backscatter: attenuation
ratios); this echogenic effect may continue for a significant
period, even during continuing ultrasound irradiation.
[0045] The stabilising effect of coadministered diffusible
component may therefore be used to great advantage to enhance both
the duration and magnitude of the echogenicity of existing
gas-containing contrast agent formulations in cases where these
parameters may be insufficient when the contrast agent composition
is administered alone. Thus, for example, the duration of effect of
albumin-based contrast agents is often severely limited by collapse
of the encapsulating albumin material, either as a result of
systolic pressure changes in the heart or venous system or as a
consequence of ultrasound irradiation, but may be substantially
enhanced by coadministration with a diffusible component in
accordance with the present invention.
[0046] In a representative embodiment of the method of the
invention a composition comprising a gas dispersion and a
composition comprising a diffusible component suspension are
selected such that at least a proportion of the dispersed gas
passes through the lungs and then undergoes rapid growth following
passage from the lungs through inward diffusion of the diffusible
component, so as temporarily to be retained in the myocardium and
thereby permit ultrasonic visualisation of myocardial perfusion. As
the concentration of volatile diffusible component in the
bloodstream falls away, e.g. as the component is cleared from the
blood, for example by removal through the lungs and exhalation by
the subject, by metabolism or by redistribution to other tissues,
the diffusible component will typically diffuse out of the
dispersed gas, which will therefore shrink towards its initial
smaller size, and ultimately once more becoming free flowing in the
bloodstream, typically being removed therefrom by the
reticuloendothelial system. This pattern of a substantial transient
increase in echogenicity followed by disappearance of contrast
effect is markedly different from any echogenic properties
exhibited by either of the two compositions when administered
alone. It will be appreciated from the foregoing that control of
the duration of retention of the dispersed gas may therefore be
achieved by appropriate adjustment of the dose and/or formulation
of the diffusible component.
[0047] Other capillary systems, such as but not limited to those of
the kidney, liver, spleen, thyroid, skeletal muscle, breast and
penis, may similarly be imaged.
[0048] It will be appreciated that factors such as the rate and/or
extent of growth of the dispersed gas may in general be controlled
by appropriate selection of the gas and any encapsulating
stabilising material and, more particularly, the nature of the
diffusible component and the manner in which it is formulated,
including the nature of any surfactant employed and the size of the
dispersed phase droplets where the component is formulated as an
emulsion; in this last context, for a given amount of emulsified
diffusible component, a reduction in droplet size may enhance the
rate of transfer of diffusible component relative to that from
larger droplets since more rapid release may occur from smaller
droplets having higher surface area:volume ratios. Other parameters
permitting control include the relative amounts in which the two
compositions are administered and, where these are administered
separately, the order of administration, the time interval between
the two administrations, and possible spatial separation of the two
administrations. In this last respect it will be appreciated that
the inherent diffusivity of the diffusible component may permit its
application to different parts of the body in a wide variety of
ways, for example by inhalation, cutaneously, subcutaneously,
intravenously, intramuscularly or orally, whereas the available
forms of administration for the dispersed gas may be somewhat more
limited.
[0049] Particularly important parameters with regard to the
diffusible component are its solubility in water/blood and its
diffusibility (e.g. as expressed by its diffusion constants), which
will determine its rate of transport through the carrier liquid or
blood, and its permeability through any membrane encapsulating the
dispersed gas. The pressure generated by the diffusible component
in vivo will also affects its rate of diffusion into the dispersed
gas, as will its concentration. Thus, in accordance with Fick's
law, the concentration gradient of diffusible component relative to
the distance between, for example, individual gas microbubbles and
emulsion droplets, together with the diffusion coefficient of the
diffusible substance in the surrounding liquid medium, will
determine the rate of transfer by simple diffusion; the
concentration gradient is determined by the solubility of the
diffusible component in the surrounding medium and the distance
between individual gas microbubbles and emulsion droplets.
[0050] The effective rate of transport of the diffusible component
may, if desired, be controlled by adjusting the viscosity of the
dispersed gas phase composition and/or the diffusible component
composition, for example by incorporating one or more biocompatible
viscosity enhancers such as X-ray contrast agents, polyethylene
glycols, carbohydrates, proteins, polymers or alcohols into the
formulation. It may, for example, be advantageous to coinject the
two compositions as a relatively high volume bolus (e.g. having a
volume of at least 20 ml in the case of a 70 kg human subject),
since this will delay complete mixing of the constituents with
blood (and thus the onset of growth of the dispersed gas) until
after entry into the right ventricle of the heart and the lung
capillaries. The delay in growth of the dispersed gas may be
maximised by employing carrier liquid which is undersaturated with
respect to gases and any other diffusible components as
hereinbefore defined, e.g. as a result of being cooled.
[0051] As noted above, transport mechanisms other than diffusion
may be involved in operation of the invention. Thus, for example,
transport may also occur through hydrodynamic flow within the
surrounding liquid medium; this may be important in vessels and
capillaries where high shear rate flow may occur. Transport of
diffusible component to the dispersed gas may also occur as a
result of collision or near-collision processes, e.g. between gas
microbubbles and emulsion droplets, for example leading to
adsorption of diffusible component at the microbubble surface
and/or penetration of diffusible component into the microbubble,
i.e. a form of coalescence. In such cases the diffusion coefficient
and solubility of the diffusible component have a minimal effect on
the rate of transfer, the particle size of the diffusible component
(e.g. the droplet size where this is formulated as an emulsion) and
the collision frequency between microbubbles and droplets being the
principal factors controlling the rate and extent of microbubble
growth. Thus, for example, for a given amount of emulsified
diffusible component, a reduction in droplet size will lead to an
increased overall number of droplets and so may enhance the rate of
transfer by reducing the mean interparticle distance between the
gas microbubbles and emulsion droplets and thus increasing the
probability of collision and/or coalescence. It will be appreciated
that the rate of transfers proceeding through collision processes
may be markedly enhanced if additional oscillatory movement is
imparted to the gas microbubbles and emulsion droplets of the
diffusible component through application of ultrasonic energy. The
kinetics of collision processes induced by such ultrasonic energy
may differ from the kinetics for transport of diffusible component
in carrier liquid and/or blood, for example in that specific energy
levels may be necessary to initiate coalescence of colliding gas
microbubbles and emulsion droplets. Accordingly it may be
advantageous to select the size and therefore the mass of the
emulsion droplets so that they generate sufficient collisional
force with the oscillating microbubbles to induce coalescence.
[0052] As also noted above, the permeability of any material
encapsulating the dispersed gas phase is a parameter which may
affect the rate of growth of the gas phase, and it may therefore be
desirable to select a diffusible component which readily permeates
any such encapsulating material (which may, for example, be a
polymer or surfactant membrane, e.g. a monolayer or one or more
bilayers of a membrane-forming surfactant such as a phospholipid).
We have found, however, that substantially impermeable
encapsulating material may also be used, since it appears that
sonication, including sonication at lower and higher frequencies
than normally used in medical ultrasound imaging (e.g. in the range
10 Hz to 1 GHz, preferably between 1 kHz and 10 MHz) and with
either continuous radiation or simple or complex pulse patterns, of
combined contrast agent preparations administered according to the
invention may itself promote or enhance growth of the dispersed
gas. Such growth may, for example, be induced by the ultrasound
irradiation used to effect an investigation or by preliminary
localised irradiation, e.g. serving to effect temporary retention
of gas in the microvasculature of a particular target organ.
Alternatively, activation of growth of the dispersed gas may be
induced by aplication of sufficient amounts of other forms of
energy, for exaple shaking, vibration, an electric field, radiation
or particle bombardment, e.g. with neutral particles, ions or
electrons.
[0053] Whilst we do not wish to be bound by theoretical
considerations it may be that ultrasonication at least transiently
modifies the permeability of the encapsulating material, the
diffusibility of the diffusible component in the surrounding liquid
phase and/or the frequency of collisions between emulsion droplets
and the encapsulated microbubbles. Since the effect may be observed
using extremely short ultrasound pulses (e.g. with durations of ca.
0.3 .mu.s in B-mode imaging or ca. 2 .mu.s in Doppler or second
harmonic imaging) it seems unlikely to be an example of rectified
diffusion, in which ongoing ultrasound irradiation produces a
steady increase in the equilibrium radii of gas bubbles (see
Leighton, E. G. --"The Acoustic Bubble", Academic Press [1994], p.
379), and it may be that the ultrasound pulses disrupt the
encapsulating membrane and so enhance growth of the dispersed gas
through inward diffusion of diffusible component into the
thus-exposed gas phase.
[0054] If desired, either the dispersed gas or the diffusible
component may comprise an azeotropic mixture or may be selected so
that an azeotropic mixture is formed in vivo as the diffusible
component mixes with the dispersed gas. Such azeotrope formation
may, for example, be used effectively to enhance the volatility of
relatively high molecular weight compounds, e.g. halogenated
hydrocarbons such as fluorocarbons (including perfluorocarbons)
which under standard conditions are liquid at the normal human body
temperature of 37.degree. C., such that they may be administered in
gaseous form at this temperature. This has substantial benefits as
regards the effective echogenic lifetime in vivo of contrast agents
containing such azeotropic mixtures since it is known that
parameters such as the water solubility, fat solubility,
diffusibility and pressure resistivity of compounds such as
fluorocarbons decrease with increasing molecular weight.
[0055] In general, the recognised natural resistance of azeotropic
mixtures to separation of their constituents will enhance the
stability of contrast agent components containing the same, both
during preparation, storage and handling and following
administration.
[0056] Azeotropic mixtures useful in accordance with the invention
may, for example, be selected by reference to literature relating
to azeotropes, by experimental investigation and/or by theoretical
predictions, e.g. as described by Tanaka in Fluid Phase Equilibria
24 (1985), pp. 187-203, by Kittel, C. and Kroemer, H. in Chapter 10
of Thermal Physics (W. H. Freeman & Co., New York, USA, 1980)
or by Hemmer, P. C. in Chapters 16-22 of Statistisk Mekanikk
(Tapir, Trondheim, Norway, 1970), the contents of which are
incorporated herein by reference.
[0057] One literature example of an azeotrope which effectively
reduces the boiling point of the higher molecular weight component
to below normal body temperature is the 57:43 w/w mixture of 1, 1,
2-trichloro-1, 2, 2-trifluoromethane (b.p. 47.6.degree. C.) and 1,
2-difluoromethane (b.p. 29.6.degree. C.) described in U.S. Pat. No.
4,055,049 as having an azeotropic boiling point of 24.9.degree. C.
Other examples of halocarbon-containing azeotropic mixtures are
disclosed in EP-A-0783017, U.S. Pat. Nos. 5,599,783, 5,605,647,
5,605,882, 5,607,616, 5,607,912, 5,611,210, 5,614,565 and
5,616,821, the contents of which are incorporated herein by
reference.
[0058] Simons et al. in J. Chem. Phys. 18(3) (1950), pp. 335-346
report that mixtures of perfluoro-n-pentane (b.p. 29.degree. C.)
and n-pentane (b.p. 36.degree. C.) exhibit a large positive
deviation from Raoult's law; the effect is most pronounced for
approximately equimolar mixtures. In practice the boiling point of
the azeotropic mixture has been found to be about 22.degree. C. or
less. Mixtures of perfluorocarbons and unsubstituted hydrocarbons
may in general exhibit useful azeotropic properties; strong
azeotropic effects have been observed for mixtures of such
components having substantially similar boiling points. Examples of
other perfluorocarbon:hydrocarbon azeotropes include mixtures of
perfluoro-n-hexane (b.p. 59.degree. C.) and n-pentane, where the
azeotrope has a boiling point between room temperature and
35.degree. C., and of perfluoro-4-methylpent-2-ene (b.p. 49.degree.
C.) and n-pentane, where the azeotrope has a boiling point of
approximately 25.degree. C.
[0059] Other potentially useful azeotropic mixtures include
mixtures of halothane and diethyl ether and mixtures of two or more
fluorinated gases, for example perfluoropropane and fluoroethane,
perfluoropropane and 1, 1, 1-trifluoroethane, or perfluoroethane
and difluoromethane.
[0060] It is known that fluorinated gases such as perfluoroethane
may form azeotropes with carbon dioxide (see e.g. WO-A-9502652).
Accordingly, administration of contrast agents containing such
gases may lead to in vivo formation of ternary or higher azeotropes
with blood gases such as carbon dioxide, thereby further enhancing
the stability of the dispersed gas.
[0061] Where the two compositions of combined contrast agent
preparations according to the invention are to be administered
simultaneously they may, for example, be injected from separate
syringes via suitable coupling means or may be premixed, preferably
under controlled conditions such that premature microbubble growth
is avoided.
[0062] Compositions intended for mixing prior to simultaneous
administration may advantageously be stored in appropriate dual or
multi-chamber devices. Thus, for example, the gas dispersion
composition or a dried precursor therefor [e.g. comprising a
lyophilised residue of a suspension of gas microbubbles in an
amphiphilic material-containing aqueous medium, particularly
wherein the amphiphilic material consists essentially of
phospholipid predominantly (e.g. at least 75%, preferably
substantially completely) comprising molecules which individually
have an overall net (e.g. negative) charge] may be contained in a
first chamber such as a vial, to which a syringe containing the
diffusible component composition is sealing connected; the syringe
outlet is closed, e.g. with a membrane or plug, to avoid premature
mixing. Operation of the syringe plunger ruptures the membrane and
causes the diffusible component composition to mix with the gas
dispersion component or to mix with and reconstitute a precursor
therefor; following any necessary or desired shaking and/or
dilution, the mixture may be withdrawn (e.g. by syringe) and
administered.
[0063] Alternatively the two compositions may be stored within a
single sealed vial or syringe, being separated by, for example, a
membrane or plug; an overpressure of gas or vapour may be applied
to either or both compositions. Rupture of the membrane or plug,
e.g. by insertion of a hypodermic needle into the vial, leads to
mixing of the compositions; this may if desired be enhanced by
hand-shaking, whereafter the mixture may be withdrawn and
administered. Other embodiments, for example in which a vial
containing a dried precursor for the gas dispersion composition is
fitted with a first syringe containing a redispersion fluid for
said precursor and a second syringe containing the diffusible
component composition, or in which a vial containing
membrane-separated diffusible component composition and dried
precursor for the gas dispersion composition is fitted with a
syringe containing redispersion fluid for the latter, may similarly
be used.
[0064] In embodiments of the invention in which the gas dispersion
composition and diffusible component composition are mixed prior to
administration, either at the manufacturing stage or subsequently,
the mixture will typically be stored at elevated pressure or
reduced temperature such that the pressure of the diffusible
component is insufficient to provide growth of the dispersed gas.
Activation of growth of the dispersed gas may be induced simply by
release of excess pressure or by the heating to body temperature
which will follow administration of the mixture, or it may if
desired be brought about by preheating the mixture immediately
before administration.
[0065] In embodiments of the invention in which the gas dispersion
composition and diffusible component composition are administered
separately, the timing between the two administrations may be used
to influence the area of the body in which growth of the dispersed
gas phase predominantly occurs. Thus, for example, the diffusible
component may be injected first and allowed to concentrate in the
liver, thereby enhancing imaging of that organ upon subsequent
injection of the gas dispersion. Where the stability of the gas
dispersion permits, this may likewise be injected first and allowed
to concentrate in the liver, with the diffusible component then
being administered to enhance the echogenicity thereof.
[0066] Imaging modalities which may be used in accordance with the
invention include two- and three-dimensional imaging techniques
such as B-mode imaging (for example using the time-varying
amplitude of the signal envelope generated from the fundamental
frequency of the emitted ultrasound pulse, from sub-harmonics or
higher harmonics thereof or from sum or difference frequencies
derived from the emitted pulse and such harmonics, images generated
from the fundamental frequency or the second harmonic thereof being
preferred), colour Doppler imaging, Doppler amplitude imaging and
combinations of these last two techniques with any of the other
modalities described above. For a given dose of the gas dispersion
and diffusible component compositions, the use of colour Doppler
imaging ultrasound to induce growth of the dispersed gas has been
found to give stronger contrast effects during subsequent B-mode
imaging, possibly as a result of the higher ultrasound intensities
employed. To reduce the effects of movement, successive images of
tissues such as the heart or kidney may be collected with the aid
of suitable synchronisation techniques (e.g. gating to the ECG or
respiratory movement of the subject). Measurement of changes in
resonance frequency or frequency absorption which accompany growth
of the dispersed gas may also usefully be made to detect the
contrast agent.
[0067] It will be appreciated that the dispersed gas content of
combined contrast agent preparations according to the invention
will tend to be temporarily retained in tissue in concentrations
proportional to the regional rate of tissue perfusion. Accordingly,
when using ultrasound imaging modalities such as conventional or
harmonic B-mode imaging where the display is derived directly from
return signal intensities, images of such tissue may be interpreted
as perfusion maps in which the displayed signal intensity is a
function of local perfusion. This is in contrast to images obtained
using free-flowing contrast agents, where the regional
concentration of contrast agent and corresponding return signal
intensity depend on the actual blood content rather than the rate
of perfusion of local tissue.
[0068] In cardiac studies, where perfusion maps are derived from
return signal intensities in accordance with this embodiment of the
invention, it may be advantageous to subject a patient to physical
or pharmacological stress in order to enhance the distinction, and
thus the difference in image intensities, between normally perfused
myocardium and any myocardial regions supplied by stenotic
arteries. As is known from radionucleide cardiac imaging, such
stress induces vasodilatation and increased blood flow in healthy
myocardial tissue, whereas blood flow in underperfused tissue
supplied by a stenotic artery is substantially unchanged since the
capacity for arteriolar vasodilatation is already exhausted by
inherent autoregulation seeking to increase the restricted blood
flow.
[0069] The application of stress as physical exercise or
pharmacologically by administration of adrenergic agonists may
cause discomfort such as chest pains in patient groups potentially
suffering from heart disease, and it is therefore preferable to
enhance the perfusion of healthy tissue by administration of a
vasodilating drug, for example selected from adenosine,
dipyridamole, nitroglycerine, isosorbide mononitrate, prazosin,
doxazosin, dihydralazine, hydralazine, sodium nitroprusside,
pentoxyphylline, amelodipine, felodipine, isradipine, nifedipine,
nimodipine, verapamil, diltiazem and nitrous oxide. In the case of
adenosine this may lead to in excess of fourfold increases in
coronary blood flow in healthy myocardial tissue, greatly
increasing the uptake and temporary retention of contrast agents in
accordance with the invention and thus significantly increasing the
difference in return signal intensities between normal and
hypoperfused myocardial tissue. Because an essentially physical
entrapment process is involved, retention of contrast agents
according to the invention is highly efficient; this may be
compared to the uptake of radionucleide tracers such as thallium
201 and technetium sestamibi, which is limited by low contact time
between tracer and tissue and so may require maintenance of
vasodilatation for the whole period of blood pool distribution for
the tracer (e.g. 4-6 minutes for thallium scintigraphy) to ensure
optimum effect. The contrast agents of the invention, on the other
hand, do not suffer such diffusion or transport limitations, and
since their retention in myocardial tissue may also rapidly be
terminated, for example by cessation of growth-generating
ultrasound irradiation, the period of vasodilatation needed to
achieve cardiac perfusion imaging in accordance with this
embodiment of the invention may be very short, for example less
than one minute. This will reduce the duration of any possible
discomfort caused to patients by administration of vasodilator
drugs.
[0070] In view of the fact that the required vasodilatation need
only be short lasting, adenosine is a particularly useful
vasodilating drug, being both an endogenous substance and having a
very short-lasting action as evidenced by a blood pool half-life of
only 2 seconds. Vasodilatation will accordingly be most intense in
the heart, since the drug will tend to reach more distal tissues in
less than pharmacologically active concentrations. It will be
appreciated that because of this short half-life, repeated
injection or infusion of adenosine may be necessary during cardiac
imaging in accordance with this embodiment of the invention; by way
of example, an initial administration of 150 .mu.g/kg of adenosine
may be made substantially simultaneously with administration of the
contrast agent composition, followed 10 seconds later by slow
injection of a further 150 .mu.g/kg of adenosine, e.g. over a
period of 20 seconds.
[0071] Contrast agent preparations in accordance with the invention
may advantageously be employed as delivery agents for bioactive
moieties such as therapeutic drugs (i.e. agents having a beneficial
effect on a specific disease in a living human or non-human
animal), particularly to targeted sites. Thus, for example,
therapeutic compounds may be present in the dispersed gas, may be
linked to part of an encapsulating wall or matrix, e.g. through
covalent or ionic bonds, if desired through a spacer arm, or may be
physically mixed into such encapsulating or matrix material; this
last option is particularly applicable where the therapeutic
compound and encapsulating or matrix material have similar
polarities or solubilities.
[0072] The controllable growth properties of the dispersed gas may
be utilised to bring about its temporary retention in the
microvasculature of a target region of interest; use of ultrasonic
irradiation to induce growth and thus retention of the gas and
associated therapeutic compound in a target structure is
particularly advantageous. Localised injection of the gas
dispersion composition or, more preferably, the diffusible
component composition, e.g. as hereinbefore described, may also be
used to concentrate growth of the dispersed gas in a target
area.
[0073] The therapeutic compound, which may if desired be coupled to
a site-specific vector having affinity for specific cells,
structures or pathological sites, may be released as a result of,
for example, stretching or fracture of the encapsulating or matrix
material caused by growth of the dispersed gas, solubilisation of
the encapsulating or matrix material, or disintegration of
microbubbles or microparticles (e.g. induced by ultrasonication or
by a reversal of the concentration gradient of the diffusible
component in the target area). Where a therapeutic agent is
chemically linked to an encapsulating wall or matrix, the linkage
or any spacer arm associated therewith may advantageously contain
one or more labile groups which are cleavable to release the agent.
Representative cleavable groups include amide, imide, imine, ester,
anhydride, acetal, carbamate, carbonate, carbonate ester and
disulphide groups which are biodegradable in vivo, e.g. as a result
or hydrolytic and/or enzymatic action.
[0074] Representative and non-limiting examples of drugs useful in
accordance with this embodiment of the invention include
antineoplastic agents such as vincristine, vinblastine, vindesine,
busulfan, chlorambucil, spiroplatin, cisplatin, carboplatin,
methotrexate, adriamycin, mitomycin, bleomycin, cytosine
arabinoside, arabinosyl adenine, mercaptopurine, mitotane,
procarbazine, dactinomycin (antinomycin D), daunorubicin,
doxorubicin hydrochloride, taxol, plicamycin, aminoglutethimide,
estramustine, flutamide, leuprolide, megestrol acetate, tamoxifen,
testolactone, trilostane, amsacrine (m-AMSA), asparaginase
(L-asparaginase), etoposide, interferon a-2a and 2b, blood products
such as hematoporphyrins or derivatives of the foregoing;
biological response modifiers such as muramylpeptides; antifungal
agents such as ketoconazole, nystatin, griseofulvin, flucytosine,
miconazole or amphotericin B; hormones or hormone analogues such as
growth hormone, melanocyte stimulating hormone, estradiol,
beclomethasone dipropionate, betamethasone, cortisone acetate,
dexamethasone, flunisolide, hydrocortisone, methylprednisolone,
paramethasone acetate, prednisolone, prednisone, triamcinolone or
fludrocortisone acetate; vitamins such as cyanocobalamin or
retinoids; enzymes such as alkaline phosphatase or manganese
superoxide dismutase; antiallergic agents such as amelexanox;
anticoagulation agents such as warfarin, phenprocoumon or heparin;
antithrombotic agents; circulatory drugs such as propranolol;
metabolic potentiators such as glutathione; antituberculars such as
p-aminosalicylic acid, isoniazid, capreomycin sulfate, cyclosexine,
ethambutol, ethionamide, pyrazinamide, rifampin or streptomycin
sulphate; antivirals such as acyclovir, amantadine, azidothymidine,
ribavirin or vidarabine; blood vessel dilating agents such as
diltiazem, nifedipine, verapamil, erythritol tetranitrate,
isosorbide dinitrate, nitroglycerin or pentaerythritol
tetranitrate; antibiotics such as dapsone, chloramphenicol,
neomycin, cefaclor, cefadroxil, cephalexin, cephradine,
erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin,
bacampicillin, carbenicillin, dicloxacillin, cyclacillin,
picloxacillin, hetacillin, methicillin, nafcillin, penicillin or
tetracycline; antiinflammatories such as diflunisal, ibuprofen,
indomethacin, meclefenamate, mefenamic acid, naproxen,
phenylbutazone, piroxicam, tolmetin, aspirin or salicylates;
antiprotozoans such as chloroquine, metronidazole, quinine or
meglumine antimonate; antirheumatics such as penicillamine;
narcotics such as paregoric; opiates such as codeine, morphine or
opium; cardiac glycosides such as deslaneside, digitoxin, digoxin,
digitalin or digitalis; neuromuscular blockers such as atracurium
mesylate, gallamine triethiodide, hexafluorenium bromide,
metocurine iodide, pancuronium bromide, succinylcholine chloride,
tubocurarine chloride or vecuronium bromide; sedatives such as
amobarbital, amobarbital sodium, apropbarbital, butabarbital
sodium, chloral hydrate, ethchlorvynol, ethinamate, flurazepam
hydrochloride, glutethimide, methotrimeprazine hydrochloride,
methyprylon, midazolam hydrochloride, paraldehyde, pentobarbital,
secobarbital sodium, talbutal, temazepam or triazolam; local
anaesthetics such as bupivacaine, chloroprocaine, etidocaine,
lidocaine, mepivacaine, procaine or tetracaine; general
anaesthetics such as droperidol, etomidate, fentanyl citrate with
droperidol, ketamine hydrochloride, methohexital sodium or
thiopental and pharmaceutically acceptable salts (e.g. acid
addition salts such as the hydrochloride or hydrobromide or base
salts such as sodium, calcium or magnesium salts) or derivatives
(e.g. acetates) thereof; and radiochemicals, e.g. comprising
beta-emitters. Of particular importance are antithrombotic agents
such as vitamin K antagonists, heparin and agents with heparin-like
activity such as antithrombin III, dalteparin and enoxaparin; blood
platelet aggregation inhibitors such as ticlopidine, aspirin,
dipyridamole, iloprost and abciximab; and thrombolytic enzymes such
as streptokinase and plasminogen activator. Other examples of
therapeutics include genetic material such as nucleic acids, RNA,
and DNA of natural or synthetic origin, including recombinant RNA
and DNA. DNA encoding certain proteins may be used in the treatment
of many different types of diseases. For example, tumour necrosis
factor or interleukin-2 may be provided to treat advanced cancers;
thymidine kinase may be provided to treat ovarian cancer or brain
tumors; interleukin-2 may be provided to treat neuroblastoma,
malignant melanoma or kidney cancer; and interleukin-4 may be
provided to treat cancer.
[0075] Contrast agent preparations in accordance with the invention
may be used as vehicles for contrast-enhancing moieties for imaging
modalities other than ultrasound, for example X-ray, light imaging,
magnetic resonance and, more preferably, scintigraphic imaging
agents. Controlled growth of the dispersed gas phase may be used to
position such agents in areas of interest within the bodies of
subjects, for example using ultrasound irradiation of a target
organ or tissue to induce the desired controlled growth and
temporary retention of the agent, which may then be imaged using
the appropriate non-ultrasound imaging modality.
[0076] Contrast agent preparations in accordance with the invention
may also be used as vehicles for therapeutically active substances
which do not necessarily require release from the preparation in
order to exhibit their therapeutic effect. Such preparations may,
for example, incorporate radioactive atoms or ions such as
beta-emitters which exhibit a localised radiation-emitting effect
following growth of the dispersed gas phase and temporary retention
of the agent at a terget site. It will be appreciated that such
agents should preferably be designed so that subsequent shrinkage
and cessation of retention of the dispersed gas does not occur
until the desired therapeutic radiation dosage has been
administered.
[0077] Contrast agent preparations in accordance with the invention
may additionally exhibit therapeutic properties in their own right.
Thus, for example, the dispersed gas may be targeted to capillaries
leading to tumours and may act as cell toxic agents by blocking
such capillaries. Thus it is possible by applying localised
ultrasonic energy to obtain a controlled and localised embolism;
this may be of importance as such or in combination with other
therapeutic measures. Concentrations of dispersed gas in
capillaries may also enhance absorption of ultrasonic energy in
hyperthermic therapy; this may be used in,for example, treatment of
liver tumours. Irradiation with a relatively high energy (e.g. 5 W)
focused ultrasound beam, e.g. at 1.5 MHz, may be appropriate in
such applications.
[0078] It will be appreciated that the present invention extends to
preparations comprising an aqueous medium having gas dispersed
therein and a composition comprising a diffusible component as
general compositions of matter and to their use for non-imaging
agent purposes.
[0079] The following non-limitative Examples serve to illustrate
the invention.
[0080] Example 1--Preparations
[0081] a) Perfluorobutane gas dispersion
[0082] Hydrogenated phosphatidylserine (100 mg) in a 2% solution of
propylene glycol in purified water (20 ml) was heated to 80.degree.
C. for 5 minutes and the resulting dispersion was allowed to cool
to room temperature overnight. 1 ml portions were transferred to 2
ml vials, the headspace above each portion was flushed with
perfluorobutane gas, and the vials were shaken for 45 seconds using
an Espe CapMix.RTM. mixer for dental materials, yielding milky
white microbubble dispersions with a volume median diameter of 5.0
.mu.m, measured using a Coulter Counter (all Coulter Counter
measurements were made at room temperature using an instrument
fitted with a 50 .mu.m aperture and having a measuring range 1-30
.mu.m; Isoton II was used as electrolyte).
[0083] b) Dispersion of lyophilised perfluorobutane gas
dispersion
[0084] A sample of the milky white dispersion from Example 1(a) was
washed three times by centrifugation and removal of the
infranatant, whereafter an equal volume of 10% sucrose solution was
added. The resulting dispersion was lyophilised and then
redispersed in distilled water, yielding a milky white microbubble
dispersion with a volume median diameter of 3.5 .mu.m, measured
using a Coulter Counter.
[0085] c) 2-Methylbutane emulsion
[0086] Hydrogenated phosphatidylserine (100 mg) in purified water
(20 ml) was heated to 80.degree. C. for 5 minutes and the resulting
dispersion was cooled to 0.degree. C. overnight. 1 ml of the
dispersion was transferred to a 2 ml vial, to which was added 200
.mu.l of 2-methylbutane (b.p. 28.degree. C.). The vial was then
shaken for 45 seconds using a CapMix.RTM. to yield an emulsion of
diffusible component which was stored at 0.degree. C. when not in
use. The volume median diameter of the emulsion droplets was 1.9
.mu.m, measured using a Coulter Counter.
[0087] d) Perfluoropentane emulsion
[0088] The procedure of Example 1(c) was repeated except that the
2-methylbutane was replaced by perfluoropentane (b.p. 29.degree.
C.). The thus-obtained emulsion of diffusible component was stored
at 0.degree. C. when not in use.
[0089] e) 2-Chloro-1-1, 2-trifluoroethyl difluoromethyl ether
emulsion
[0090] The procedure of Example 1(c) was repeated except that the
2-methylbutane was replaced by 2-chloro-1, 1, 2-trifluoroethyl
difluoromethyl ether (b.p. 55-57.degree. C.). The thus-obtained
emulsion of diffusible component was stored at 0.degree. C. when
not in use.
[0091] f) 2-Bromo-2-chloro-1, 1, 1-trifluoroethane emulsion
[0092] The procedure of Example 1(c) was repeated except that the
2-methylbutane was replaced by 2-bromo-2-chloro-1, 1,
1-trifluoroethane (b.p. 49.degree. C.). The thus-obtained emulsion
of diffusible component was stored at 0.degree. C. when not in
use.
[0093] g) 1-Chloro-2, 2, 2-trifluoroethyl difluoromethyl ether
emulsion
[0094] The procedure of Example 1(c) was repeated except that the
2-methylbutane was replaced by 1-chloro-2, 2, 2-trifluoroethyl
difluoromethyl ether (b.p. 49.degree. C.). The thus-obtained
emulsion of diffusible component was stored at 0.degree. C. when
not in use.
[0095] h) Dispersion of gas-containing polymer/human serum albumin
particles
[0096] Human serum albumin-coated gas-containing particles of
polymer made from ethylidene bis(16-hydroxyhexadecanoate) and
adipoyl chloride, prepared according to Example 3(a) of
WO-A-9607434, (100 mg) were crushed in a mortar and dispersed in
0.9% aqueous sodium chloride (10 ml) by shaking on a laboratory
shaker for 24 hours.
[0097] i) Dispersion of gas-containing polymer/gelatin
particles
[0098] Gelatin-coated gas-containing particles of polymer made from
ethylidene bis(16-hydroxyhexadecanoate) and adipoyl chloride,
prepared according to Example 3(e) of WO-A-9607434, (100 mg) were
crushed in a mortar and dispersed in 0.9% aqueous sodium chloride
(10 ml) by shaking on a laboratory shaker for 24 hours.
[0099] j) 2-Methylbutane emulsion
[0100] The procedure of Example 1(c) was repeated except that the
emulsion was diluted 10 times prior to use and was stored in an ice
bath when not in use.
[0101] k) Perfluoropentane emulsion
[0102] The procedure of Example 1(d) was repeated except that the
emulsion was diluted 10 times prior to use and was stored in an ice
bath when not in use.
[0103] l) Perfluoropentane emulsion
[0104] Hydrogenated phosphatidylserine (100 mg) in purified water
(20 ml) was heated to 80.degree. C. for 5 minutes and the resulting
dispersion was cooled to 0.degree. C. overnight. 1 ml of the
dispersion was transferred to a 2 ml vial, to which was added 100
.mu.l of perfluoro-n-pentane (b.p. 29.degree. C.). The vial was
then shaken for 75 seconds using a CapMix.RTM. to yield an emulsion
of diffusible component which was stored at 0.degree. C. when not
in use. The volume median diameter of the emulsion droplets was 2.9
.mu.m, measured using a Coulter Counter.
[0105] m) Perfluorobutane emulsion
[0106] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by perfluorobutane (b.p. -2.degree.
C.). The thus-obtained emulsion of diffusible component was stored
at 0.degree. C. when not in use.
[0107] n) Perfluoropentane emulsion prepared by sonication
[0108] Hydrogenated phosphatidylserine (500 mg) in purified water
(100 ml) was heated to 80.degree. C. for 5 minutes and the
resulting dispersion was allowed to cool to room temperature
overnight. 10 ml of the dispersion were transferred to a 30 ml
vial, to which perfluoropentane (1 ml) was then added. Sonication
of the resulting mixture for two minutes gave a dispersion of
diffusible component wherein the drops had a mean diameter <1
.mu.m.
[0109] o) Perfluoropentane emulsion
[0110] The procedure of Example 1(1) was repeated except that the
volume of perfluoropentane employed was reduced to 60 .mu.l. The
thus-obtained emulsion of diffusible component was stored at
0.degree. C. when not in use.
[0111] p) Perfluoropentane emulsion
[0112] The procedure of Example 1(1) was repeated except that the
volume of perfluoropentane employed was reduced to 20 .mu.l. The
thus-obtained emulsion of diffusible component was stored at
0.degree. C. when not in use.
[0113] q) PerfluoroDentane:uerfluoro-4-methylpent-2-ene (1:1)
emulsion
[0114] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by a mixture of 50 .mu.l of
perfluoropentane (b.p. 29.degree. C.) and 50 .mu.l of
perfluoro-4-methylpent-2-ene (b.p. 49.degree. C.). The
thus-obtained emulsion of diffusible components was stored at
0.degree. C. when not in use. The volume median diameter of the
emulsion droplets was 2.8 .mu.m, measured using a Coulter
Counter.
[0115] r) Perfluoropentane:1 H, 1 H, 2 H-heptafluoropent-1-ene
(1:1) emulsion
[0116] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by a mixture of 50 .mu.l of
perfluoropentane (b.p. 29.degree. C.) and 50 .mu.l of 1 H, 1 H, 2
H-heptafluoropent-1-ene (b.p. 30-31.degree. C.). The thus-obtained
emulsion of diffusible components was stored at 0.degree. C. when
not in use.
[0117] s) Perfluoropentane emulsion stabilised by
distearoylphosphatidylch- oline:distearoylphosphatidylserine
(1:1)
[0118] The procedure of Example 1(1) was repeated except that the
hydrogenated phosphatidylserine was replaced by a mixture of
distearoylphosphatidylcholine (50 mg) and
distearoylphosphatidylserine, sodium salt (50 mg). The
thus-obtained emulsion of diffusible component was stored at
0.degree. C. when not in use.
[0119] t) Perfluoropentane emulsion stabilised by
distearoylphosphatidylch- oline:distearoylphosphatidylserine
(3:1)
[0120] The procedure of Example 1(1) was repeated except that the
hydrogenated phosphatidylserine was replaced by a mixture of
distearoylphosphatidylcholine (75 mg) and
distearoylphosphatidylserine, sodium salt (25 mg). The
thus-obtained emulsion of diffusible component was stored at
0.degree. C. when not in use.
[0121] u) Perfluoropentane emulsion stabilised by
distearoylphosphatidylch- oline:distearoylphosphatidylglycerol
(3:1)
[0122] The procedure of Example 1(1) was repeated except that the
hydrogenated phosphatidylserine was replaced by a mixture of
distearoylphosphatidylcholine (75 mg) and
distearoylphosphatidylglycerol, sodium salt (25 mg). The
thus-obtained emulsion of diffusible component was stored at
0.degree. C. when not in use.
[0123] v) Perfluoropentane emulsion stabilised by hydrogenated
phosphatidylcholine:hydrogenated phosphatidylserine (11:1)
[0124] The procedure of Example 1(1) was repeated except that the
hydrogenated phosphatidylserine was replaced by 100 mg of a mixture
of hydrogenated phosphatidylcholine and hydrogenated
phosphatidylserine (11:1). The thus-obtained emulsion of diffusible
component was stored at 0.degree. C. when not in use.
[0125] w) Perfluoro-4-methylpent-2-ene emulsion stabilised by
distearoylphosphatidylcholine:distearoylphosphatidylserine
(3:1)
[0126] The procedure of Example 1(1) was repeated except that the
hydrogenated phosphatidylserine was replaced by a mixture of
distearoylphosphatidylcholine (75 mg) and
distearoylphosphatidylserine, sodium salt (25 mg) and the
perfluoropentane was replaced by perfluoro-4-methylpent-2-ene. The
thus-obtained emulsion of diffusible component was stored at
0.degree. C. when not in use.
[0127] x) Perfluoropentane:perfluoro-4-methylpent-2-ene (1:1)
emulsion stabilised by
distearoylphosphatidylcholine:distearoylphosphatidylserine
(3:1)
[0128] The procedure of Example 1(w) was repeated except that the
perfluoro-4-methylpent-2-ene was replaced by a mixture of 50 .mu.l
of perfluoropentane and 50 .mu.l of perfluoro-4-methylpent-2-ene.
The thus-obtained emulsion of diffusible components was stored at
0.degree. C. when not in use.
[0129] y) Perfluoropentane:perfluoro-4-methylpent-2-ene (1:1)
emulsion stabilised by
distearoylphosphatidvlcholine:distearoylphosphatidylglycero- l
(3:1)
[0130] The procedure of Example 1(x) was repeated except that the
distearoylphosphatidylserine, sodium salt was replaced by
distearoylphosphatidylglycerol, sodium salt. The thus-obtained
emulsion of diffusible components was stored at 0.degree. C. when
not in use.
[0131] z) Perfluorodecalin:perfluorobutane emulsion
[0132] Hydrogenated phosphatidylserine (100 mg) in aqueous glycerol
(5.11%)/propylene glycol (1.5%) (20 ml) was heated to 80.degree. C.
for 5 minutes and the resulting dispersion was cooled to 0.degree.
C. overnight. 1 ml of the dispersion was transferred to a 2 ml
vial, to which was added 100 .mu.l of perfluorodecalin (b.p.
141-143.degree. C.) saturated with perfluorobutane (b.p. -2.degree.
C.). The vial was then shaken for 60 seconds using a CapMix.RTM. to
yield an emulsion of diffusible component which was stored at
0.degree. C. when not in use.
[0133] aa) Perfluorodecalin:perfluoropropane emulsion
[0134] The procedure of Example 1(z) was repeated except that the
perfluorodecalin saturated with perfluorobutane was replaced by
perfluorodecalin saturated with perfluoropropane (b.p. -39.degree.
C.). The thus-obtained emulsion of diffusible component was stored
at 0.degree. C. when not in use.
[0135] ab) Perfluorodecalin:sulphur hexafluoride emulsion
[0136] The procedure of Example 1(z) was repeated except that the
perfluorodecalin saturated with perfluorobutane was replaced by
perfluorodecalin saturated with sulphur hexafluoride (b.p.
-64.degree. C.). The thus-obtained emulsion of diffusible component
was stored at 0.degree. C. when not in use.
[0137] ac) Perfluoropentane emulsion stabilised with Fluorad
FC-170C
[0138] 1 ml of a dispersion of Fluorad FC-170C (200 mg) in purified
water (20 ml) was transferred to a 2 ml vial, to which was added
100 .mu.l of perfluoro-n-pentane. The vial was then shaken for 75
seconds using a CapMix.RTM. to yield an emulsion of diffusible
component which was stored at 0.degree. C. when not in use.
[0139] ad) Perfluoropentane emulsion stabilised with Pluronic
F68:Fluorad FC-170C
[0140] 100 .mu.l of a 10% Pluronic F68 solution was added to 200
.mu.l of 1% Fluorad FC170C and 700 .mu.l purified water. The
resulting mixture was transferred to a 2 ml vial, to which was
added 100 .mu.l of perfluoro-n-pentane. The vial was then shaken
for 75 seconds using a CapMix.RTM. to yield an emulsion of
diffusible component which was stored at 0.degree. C. when not in
use. A sample of this emulsion was transferred to a screw-topped
plastic vial (2.8 ml) which was then sonicated in a water bath for
2 minutes (pulse sonication: 1 per second). The volume median
diameter of the sonicated emulsion droplets was 0.99 .mu.m,
measured using a Coulter Counter.
[0141] ae) Perfluoropentane emulsion stabilised with Pluronic
F68:Fluorad FC-170C and prenared by homogenisation
[0142] 1 ml of a 10% Pluronic F68 solution was added to 2 ml of 1%
Fluorad FC170C and 7 ml purified water, whereafter 1 ml of
perfluoro-n-pentane was added to the resulting mixture. The
thus-obtained dispersion was then homogenised by rotor/stator
homogenisation for 2 minutes at 23000 rpm. The resulting emulsion
was transferred to a screw-topped plastic vial (10 ml) and
sonicated in a water bath for 2 minutes (pulse sonication: 1 per
second).
[0143] af) Perfluoropentane emulsion
[0144] Hydrogenated phosphatidylserine (250 mg) in purified water
(100 ml) was heated to 80.degree. C. for 5 minutes and the
resulting dispersion was cooled to 0.degree. C. overnight. 1 ml of
the dispersion was transferred to a 2 ml vial, to which was added
100 .mu.l of perfluoropentane. The vial was shaken for 75 seconds
using a CapMix.RTM. to yield an emulsion of diffusible component
which was stored at 0.degree. C. when not in use.
[0145] ag) Dispersion of lyophilised Derfluorobutane gas
dispersion
[0146] A sample of the milky white dispersion from Example 1(a) was
washed three times by centrifugation and removal of the
infranatant, whereafter an equal volume of 10% sucrose solution was
added. The resulting dispersion was lyophilised and then
redispersed in distilled water, yielding a milky white microbubble
dispersion with a volume mean diameter of 2.6 .mu.m, measured using
a Coulter Counter.
[0147] ah) Perfluoropropane gas dispersion
[0148] The procedure of Example 1(a) was repeated except that the
perfluorobutane gas was replaced by perfluoropropane gas. The
resulting milky white microbubble dispersion had a volume median
diameter of 2.6 .mu.m, measured using a Coulter Counter.
[0149] ai) Perfluoropentane emulsion
[0150] Hydrogenated phosphatidylserine (100 mg) in purified water
(100 ml) was heated to 80.degree. C. for 5 minutes and the
resulting dispersion was cooled to 0.degree. C. overnight. 1 ml of
the dispersion was transferred to a 2 ml vial, to which was added
100 .mu.l of perfluoropentane. The vial was shaken for 75 seconds
using a CapMix.RTM. to yield an emulsion of diffusible component
which was stored at 0.degree. C. when not in use.
[0151] aj) Perfluoropentane emulsion stabilised with Brij 58:
Fluorad FC-170C, prepared by shaking
[0152] Brij 58 (400 mg) was added to a solution of 0.1% Fluorad
FC-170C (10 ml) and stirred at room temperature for one hour. 1 ml
of the resulting solution was transferred to a 2 ml vial, to which
was added perfluoropentane (100 .mu.l). The vial was then shaken
for 75 seconds using a CapMix.RTM. to yield an emulsion of
diffusible component which was stored at 0.degree. C. when not in
use.
[0153] ak) Perfluoropentane emulsion stabilised with Brij58:
Fluorad FC-170C, prepared by sonication
[0154] Brij58 (400 mg) was added to a solution of 0.1% Fluorad
FC-170C (10 ml) and stirred at room temperature for one hour.
Perfluoropentane (1 ml) was then added and the resulting mixture
was sonicated for 2 minutes to yield an emulsion of small drops of
the diffusible component. This emulsion was stored at 0.degree. C.
when not in use.
[0155] al) Perfluoro-4-methylpent-2-ene emulsion
[0156] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by perfluoro-4-methylpent-2-ene (b.p.
49.degree. C.). The thus-obtained emulsion of diffusible component
was stored at 0.degree. C. when not in use.
[0157] am) 1 H, 1 H, 2 H-Heptafluoropent-1-ene emulsion
[0158] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by 1 H, 1H, 2 H-heptafluoropent-1-ene
(b.p. 30-31.degree. C.). The thus-obtained emulsion of diffusible
component was stored at 0.degree. C. when not in use.
[0159] an) Perfluorocyclopentene emulsion
[0160] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by perfluorocyclopentene (b.p.
27.degree. C.). The thus-obtained emulsion of diffusible component
was stored at 0.degree. C. when not in use.
[0161] ao) Perfluorodimethylcyclobutane emulsion
[0162] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by perfluorodimethylcyclobutane
(mixture of 1, 2- and 1, 3-isomers, b.p. 45.degree. C.). The
thus-obtained emulsion of diffusible component was stored at
0.degree. C. when not in use.
[0163] ap) Emulsion of an azeotropic perfluorohexane : n-pentane
mixture
[0164] 4.71 g (0.014 mol) perfluoro-n-hexane (boiling point
59.degree. C.) (Fluorochem Ltd.) and 0.89 g (0.012 mol) n-pentane
(boiling point 36.degree. C.) (Fluka AG) were mixed in a vial to
give an azeotropic mixture shown to boil readily at 35.degree. C.
In another vial, hydrogenated phosphatidylserine (100 mg) in
purified water (20 ml) was heated to 80.degree. C. for 5 minutes
and the resulting dispersion was cooled to room temperature. 1 ml
of the phospholipid dispersion was transferred to a 2 ml vial to
which was added 100 .mu.l of the azeotropic mixture. The vial was
then shaken for 45 seconds using a CapMix.RTM. to yield an emulsion
of diffusible component which was stored at room temperature when
not in use.
[0165] aq) Perfluorodimethylcyclobutane emulsion
[0166] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by perfluorodimethylcyclobutane
(>97% 1, 1-isomer, balance being 1, 2- and 1, 3-isomers). The
thus-obtained emulsion of diffusible component was stored at
0.degree. C. when not in use.
[0167] ar) Perfluorohexane emulsion
[0168] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by perfluorohexane (b.p. 57.degree.
C.). The thus-obtained emulsion of diffusible component was stored
at 0.degree. C. when not in use.
[0169] as) Perfluorodimethylcyclobutane emulsion stabilised with
fluorinated surfactant
[0170] The procedure of Example 1(aq) is repeated except that the
hydrogenated phosphatidylserine is replaced by either
perfluorinated distearoylphosphatidylcholine (5 mg/ml) or a mixture
of perfluorinated distearoylphosphatidylcholine and hydrogenated
phosphatidylserine (3:1, total lipid concentration 5 mg/ml). The
thus-obtained emulsions of diffusible component was stored at
0.degree. C. when not in use.
[0171] at) 2, 2, 3, 3, 3-Pentafluoropropyl methyl ether
emulsion
[0172] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by 2, 2, 3, 3, 3-pentafluoropropyl
methyl ether (b.p. 46.degree. C.). The thus-obtained emulsion of
diffusible component was stored at 0.degree. C. when not in
use.
[0173] au) 2 H, 3 H-Decafluoropentane emulsion
[0174] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by 2 H, 3 H-decafluoropentane (b.p.
54.degree. C.). The thus-obtained emulsion of diffusible component
was stored at 0.degree. C. when not in use.
[0175] av) Perfluorodimethylcyclobutane emulsion stabilised by
lysophosphatidylcholine
[0176] The procedure of Example 1(aq) was repeated except that the
hydrogenated phosphatidylserine was replaced by
lysophosphatidylcholine. The thus-obtained emulsion of diffusible
component was stored at 0.degree. C. when not in use.
[0177] aw) Perfluorodimethylcyclobutane emulsion estabilised by
hydrogenated phosphatidylserine:lysotphosphatidylcholine (1:1)
[0178] The procedure of Example 1(aq) was repeated except that the
hydrogenated phosphatidylserine was replaced by a mixture of
hydrogenated phosphatidylserine and lysophosphatidyicholine (1:1).
The thus-obtained emulsion of diffusible component was stored at
0.degree. C. when not in use.
[0179] ax) Perfluorodimethylcyclobutane emulsion stabilised by a
polyethylene glycol 10,000-based surfactant
[0180] The procedure of Example 1(aq) was repeated except that the
hydrogenated phosphatidylserine dispersion was replaced by a
solution of
.alpha.-(16-hexadecanoyloxyhexadecanoyl)-.omega.-methoxypolyethylene
glycol 10,000 in water (10 mg/ml). The thus-obtained emulsion of
diffusible component was stored at 0.degree. C. when not in
use.
[0181] ay) Perfluorodimethylcyclobutane emulsion stabilised by a
polyethylene glycol 10,000-based surfactant
[0182] The procedure of Example 1(aq) was repeated except that the
hydrogenated phosphatidylserine dispersion was replaced by a
solution of
.alpha.-(16-hexadecanoyloxy-hexadecanoyl)-.omega.-methoxypolyethylene
glycol 10,000 in water (20 mg/ml). The thus-obtained emulsion of
diffusible component was stored at 0.degree. C. when not in
use.
[0183] az) Perfluorobutane-filled microbubbles encapsulated by
phosphatidylserine and RGDC-Mal-polyethylene-glycol
2000-distearoylphosphatidylethanolamine
[0184] To a mixture of phosphatidylserine (4.5 mg) and
Mal-polyethylene glycol 2000-distearoylphosphatidylethanolamine
(0.5 mg) in a vial was added a solution of 1.4% propylene
glycol/2.4% glycerol in water (1 ml). The dispersion was heated to
80.degree. C. for 5 minutes, cooled to room temperature and then
flushed with perfluorobutane gas. The vial was shaken for 45
seconds using a CapMix.RTM. and then placed on a roller table.
After centrifugation the infranatant was exchanged with a solution
of RGDC in sodium phosphate buffer having a pH of 7.5, after which
the vial was placed on the roller table for several hours.
[0185] ba) Perfluorobutane-filled microbubbles encapsulated by
phosphatidylserine and
dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000
[0186] To a vial containing phosphatidylserine and
dipalmitoylphosphatidyl- ethanolamine-polyethylene glycol 2000
(ratio 10:1) is added a solution of 2% propylene glycol in water to
give a lipid concentration of 5 mg/ml. The dispersion is heated to
80.degree. C. for 5 minutes and then cooled to room temperature,
whereafter the headspace is flushed with perfluorobutane gas. The
vial is shaken for 45 seconds using a CapMix.RTM. and is then
placed on a roller table. After washing by centrifugation and
removal of infranatant, an equal volume of water containing 10%
sucrose is added. The resulting dispersion is lyophilised and then
redispersed by adding water, yielding a milky white microbubble
dispersion.
[0187] bb) Perfluorobutane-filled microbubbles encapsulated by
phosphatidylserine and
distearoylphosphatidylethanolamine-polyethylene glycol 5000
[0188] To a vial containing phosphatidylserine and
distearoylphosphatidyle- thanolamine-polyethylene glycol 5000
(ratio 10:1) is added a solution of 2% propylene glycol in water to
give a lipid concentration of 5 mg/ml. The dispersion is heated to
80.degree. C. for 5 minutes and then cooled to room temperature,
whereafter the headspace is flushed with perfluorobutane gas. The
vial is shaken for 45 seconds using a CapMix.RTM. and is then
placed on a roller table. After washing by centrifugation and
removal of infranatant, an equal volume of water containing 10%
polyethylene glycol is added. The resulting dispersion is
lyophilised and then redispersed, yielding a milky white
microbubble dispersion.
[0189] bc) Perfluorobutane-filled microbubbles encapsulated by
nhosphatidylserine and
dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000
[0190] To a vial containing phosphatidylserine and
dipalmitoylphosphatidyl- ethanolamine-polyethylene glycol 2000
(ratio 10:1) is added a solution of 2% propylene glycol in water to
give a lipid concentration of 5 mg/ml. The dispersion is heated to
80.degree. C. for 5 minutes and then cooled to room temperature,
whereafter the headspace is flushed with perfluorobutane gas. The
vial is shaken for 45 seconds using a CapMix.RTM. and is then
placed on a roller table. After washing by centrifugation and
removal of infranatant, an equal volume of water is added, yielding
a milky white microbubble dispersion.
[0191] bd) Perfluorobutane-filled microbubbles encapsulated by
piodyhati ylserine and
distearoylphosphatidylethanolamine-polyethylene glycol 5000
[0192] To a vial containing phosphatidylserine and
distearoylphosphatidyle- thanolamine-polyethylene glycol 5000
(ratio 10:1) is added a solution of 2% propylene glycol in water to
give a lipid concentration of 5 mg/ml. The dispersion is heated to
80.degree. C. for 5 minutes and then cooled to room temperature,
whereafter the headspace is flushed with perfluorobutane gas. The
vial is shaken for 45 seconds using a CapMix.RTM. and is then is
placed on a roller table. After washing by centrifugation and
removal of infranatant, an equal volume of water is added, yielding
a milky white microbubble dispersion.
[0193] be) perfluorodimethylcyclobutane emulsion stabilised by
phosphatidylserine and
dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000
[0194] The procedure of Example 1(aq) is repeated except that the
hydrogenated phosphatidylserine is replaced by a mixture of
hydrogenated phosphatidylserine and
dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (ratio
10:1). The thus-obtained emulsion of diffusible component is stored
at 0.degree. C. when not in use.
[0195] bf) Perfluorodimethylcyclobutane emulsion stabilised by
phosphatidylserine and
distearoylphosphatidylethanolamine-polyethylene glycol 5000
[0196] The procedure of Example 1(aq) is repeated except that the
hydrogenated phosphatidylserine is replaced by a mixture of
hydrogenated phosphatidylserine and
distearoylphosphatidylethanolamine-polyethylene glycol 5000 (ratio
10:1). The thus-obtained emulsion of diffusible component is stored
at 0.degree. C. when not in use.
[0197] bg) Lyophilised perfluorobutane-filled microbubbles
redispersed in an emulsion
[0198] A sample of the milky white dispersion prepared as described
in Example 1(bp) was washed three times by centrifugation and
removal of the infranatant, whereafter an equal volume of 10%
sucrose solution was added. The resulting dispersion was
lyophilised and then redispersed in an emulsion prepared as
described in Example 1(aq) just prior to use.
[0199] bh) Avidinylated perfluorodimethylcyclobutane emulsion
droplets
[0200] Distearoylphosphatidylserine (4.5 mg) and
biotin-dipalmitoylphospha- tidylethanolamine (0.5 mg) were weighed
into a clean vial and 1.0 ml of a solution of 2% propylene glycol
was added. Following heating to 80.degree. C. the mixture was
cooled to room temperature. 100 .mu.l of
perfluorodimethylcyclobutane were added and the vial was shaken for
75 seconds using a CapMix.RTM. to yield an emulsion of diffusible
component. A diluted sample of the emulsion (100 .mu.l emulsion in
1 ml water) was incubated with excess avidin and placed on a roller
table. The diluted emulsion was then washed extensively with water
and concentrated by centrifuging.
[0201] bi) 1 H-Tridecafluorohexane emulsion
[0202] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by 1 H-tridecafluorohexane (b.p.
71.degree. C.). The thus-obtained emulsion of diffusible component
was stored at 0.degree. C. when not in use.
[0203] bj) Perfluoroheptane emulsion
[0204] The procedure of Example 1(1) was repeated except that the
perfluoropentane was replaced by perfluoroheptane (b.p.
80-85.degree. C.). The thus-obtained emulsion of diffusible
component was stored at 0.degree. C. when not in use.
[0205] bk) Perfluorodimethylcyclobutane emulsion with
phosphatidylserine and fluorescent streptavidin
[0206] Distearoylphosphatidylserine (4.5 mg) and
biotin-dipalmitoylphospha- tidylethanolamine (0.5 mg) are weighed
into a clean vial and 1.0 ml of a solution of 2% propylene glycol
is added. Following heating to 80.degree. C. the mixture is cooled
to room temperature. 100 .mu.l of perfluorodimethylcyclobutane are
added and the vial is shaken for 75 seconds using a CapMix.RTM.
mixer to yield an emulsion of diffusible component. A diluted
sample of the emulsion (100 .mu.l emulsion in 1 ml water) is
incubated with excess fluorescent streptavidin in phosphate buffer
and placed on a roller table. The diluted emulsion is then washed
extensively with water and concentrated by centrifuging.
[0207] bl) Dispersion of lyophilised perfluorobutane gas
dispersion
[0208] A sample of the milky white dispersion prepared as described
in Example 1(a) was washed three times by centrifugation and
removal of the infranatant, whereafter an equal volume of 10%
sucrose solution was added. The resulting dispersion was
lyophilised and then redispersed in distilled water just prior to
use.
[0209] bm) Perfluorodimethvlcyclobutane emulsion stabilised by
sterilised phosphatidylserine
[0210] The procedure of Example 1(aq) was repeated except that the
hydrogenated phosphatidylserine was replaced by a sterilised
solution of hydrogenated phosphatidylserine. The thus-obtained
emulsion of diffusible component was stored at 0.degree. C. when
not in use.
[0211] bn) Perfluoropronane gas dispersion
[0212] The procedure of Example 1(a) was repeated except that the
perfluorobutane gas was replaced by perfluoropropane gas.
[0213] bo) Dispersed Echovist
[0214] Echovist granulate (Schering AG) (0.25 g) was dispersed in
an emulsion (1.15 ml) prepared as described in Example 1(aq).
[0215] bp) Perfluorobutane gas dispersion
[0216] Hydrogenated phosphatidylserine (500 mg) was added to a
solution of 1.5% propylene glycol/5.11% glycerol in water (100 ml)
and heated to 80.degree. C. for 5 minutes, whereafter the resulting
dispersion was allowed to cool to ambient temperature. 1 ml
portions were transferred to 2 ml vials, the headspace above each
portion was flushed with perfluorobutane gas, and the vials were
shaken for 45 seconds using a CapMix.RTM., whereafter the vials
were placed on a roller table.
[0217] bg) Preparation of biotinylated perfluorobutane
microbubbles
[0218] Distearoylphosphatidylserine (4.5 mg) and
biotin-dipalmitoylphospha- tidylethanolamine (0.5 mg) were weighed
into a clean vial and 1.0 ml of a solution of 1.4% propylene
glycol/2.4% glycerol was added. Following heating to 78.degree. C.
the mixture was cooled to room temperature and the head space was
flushed with perfluorobutane gas. The vial was shaken for 45
seconds using a CapMix.RTM. mixer and was then placed on a roller
table for 16 hours. The resulting microbubbles were washed
extensively with deionised water.
[0219] br) Aerogels
[0220] To "spatula edge" pyrolysed resorcinol-formaldehyde aerogel
particles (provided by Dr. Pekala, Lawrence Livermore National
Laboratory) were added 300 .mu.l water, a droplet of pH9 buffer and
5-10 droplets of 1% Pluronic F68. The aerogel particles sedimented
quickly, but did not aggregate.
[0221] ba) Small bubbles
[0222] A rubber tube, 8 mm inner diameter and approximately 20 cm
long, was placed vertically in a stand, capped at the bottom end
and filled with a microbubble dispersion made according to Example
1(a) (except that an Ystral.RTM. rotor stator homogeniser was used
to make the microbubble dispersion). After two hours, a syringe
connected to a canula was inserted into the rubber tube close to
the bottom, and a 1 ml fraction of the size-fractionated
microbubble dispersion was collected. Coulter counter analysis
revealed the thus-obtained microbubble dispersion to have a median
diameter of 1.2 .mu.m.
[0223] bt) Perfluorobutane gas dispersion stabilised by 5%
albumin:5% dextrose (1:3)
[0224] 20% human serum was diluted to 5% with purified water. A 5
ml sample of the diluted albumin was further diluted with 5%
glucose (15 ml) and the resulting mixture was transferred to a
vial. The head space was flushed with perfluorobutane gas and the
vial was sonicated for 80 seconds, giving a milky white microbubble
dispersion.
[0225] bu) Dispersion of Buckminsterfullerene C.sub.60
[0226] Buckminsterfullerene C.sub.60 was added to 2.5 k human serum
albumin (1 ml) in a 2 ml vial which was shaken for 75 seconds using
a CapMix.RTM..
[0227] bv) Sulphur hexafluoride gas dispersion
[0228]
Distearoylphosphatidylcholine:dipalmitoylphosphatidylglycerol
(10:1) stabilised microbubbles were made as described in Example 5
of WO-A-9409829. Thus 50 mg distearoylphosphatidylcholine, 5 mg
dipalmitoylphosphatidylglycerol and 2.2 g polyethylene glycol 4000
were dissolved in 22 ml t-butanol at 60.degree. C., and the
solution was rapidly cooled to -77.degree. C. and lyophilised
overnight. 100 mg of the resulting powder were placed in a vial,
and the head space was evacuated and then filled with sulphur
hexafluoride. 1 ml purified water was added just before use, giving
a microbubble dispersion.
[0229] bw) 2-Methylbutane emulsion
[0230] Hydrogenated phosphatidylserine (100 mg) in purified water
(20 ml) was heated to 80.degree. C. for 5 minutes and the resulting
dispersion was cooled in refrigerator overnight. 1 ml of the
dispersion was transferred to a 2 ml vial, to which was added 100
.mu.l of 2-methylbutane. The vial was shaken for 75 seconds using a
CapMix.RTM. to yield an emulsion of diffusible component which was
stored at 0.degree. C. when not in use.
[0231] bx) Lyophilised perfluorobutane gas dispersion in aqueous
sodium bicarbonate
[0232] A sample of the milky white dispersion from Example 1(a) was
washed three times by centrifugation and removal of the
infranatant, whereafter an equal volume of 10% sucrose solution was
added. The resulting dispersion was lyophilised and then
redispersed in 0.1 M sodium bicarbonate solution.
[0233] by) Perfluorobutane gas dispersion
[0234] A perfluorobutane gas dispersion was prepared as Example
1(a). The dispersion was washed three times with purified water by
centrifugation and removal of the infranant, yielding a milky white
microbubble dispersion.
[0235] bz) Perfluorobutane gas dispersion with iron oxide
particles
[0236] To 1 ml of a perfluorobutane gas dispersion prepared as in
Example 1(by) was added 1 ml purified water. The pH was raised to
11.2 with ammonium hydroxide and the dispersion was heated for 5
minutes at 60.degree. C. Uncoated iron oxide particles (0.3 ml, 4.8
mg Fe/ml) were added and the dispersion was allowed to stand for 5
minutes. The pH was lowered to 5.9 with hydrochloric acid, yielding
a brown dispersion which after a while gave a top layer with brown
particles, a clear non-coloured infranant and no precipitate.
[0237] ca) Perfluorobutane gas dispersion with iron oxide
particles
[0238] To 1 ml of a perfluorobutane gas dispersion prepared as in
Example 1(by) was added 0.3 ml uncoated iron oxide particles (4.8
mg Fe/ml) at pH 7, yielding a brown dispersion which on standing
gave a top layer with brown microbubbles, a clear infranant and no
precipitate.
[0239] cb) [comparative]
[0240] To 1 ml of a solution of hydrogenated phosphatidylserine in
purified water (5 mg/ml) was added 0.3 ml uncoated iron oxide
particles (4.8 mg Fe/ml) yielding a brown dispersion which after
standing gave a brown precipitate.
[0241] cc) Perfluorobutane gas dispersion with iron oxide particles
coated with oleic acid
[0242] 1.3 mmol FeCl.sub.2 4 H.sub.2O (0.259 g) and 2.6 mmol FeCl
.sub.36 H.sub.2O (0.703 g) were dissolved in 10 ml purified water
and 1.5 ml ammonium hydroxide were added. The resulting iron oxide
particles were washed five times with purified water (25 ml).
Diluted ammonium hydroxide was added to the particles and the
suspension was heated to 80.degree. C. Oleic acid (0.15 g) was
added, and the dispersion was allowed to stand for 5 minutes at
ambient temprature. Purified water (10 ml) was added and the pH was
lowered to 5.4 with hydrochloric acid. The dispersion was sonicated
for 15 minutes, whereafter the infranant was removed and the
particles were suspended in 2-methylbutane (5 ml), yielding a fine
black dispersion.
[0243] 25 mg distearoylphosphatidylcholine and 2.5 mg
dimyristoylphosphatidylglycerol were dissolved in 11 ml t-butanol
at 60.degree. C. and 0.1 ml iron oxide particles from above was
added, together with 1.1 g polyethylene glycol 4000. The dispersion
was heated for 10 minutes at 60.degree. C., rapidly cooled to
-77.degree. C. and lyophilised. 100 mg of the lyophilisate were
introduced into a 2 ml vial, which was then evacuated and flushed
twice with perfluorobutane gas. The lyophilisate was then dispersed
in 1 ml purified water and washed twice with purified water by
centrifugation with removal of the infranant and the precipitate.
After standing the resulting dispersion had a light grey and
floating top layer.
[0244] Example 2--In vitro characterisation of microbubble growth
by microscopy/visual observation
[0245] a) One drop of the perfluorobutane gas dispersion from
Example 1(a) at ca. 4.degree. C. was diluted with one drop of
air-supersaturated purified water at ca. 4.degree. C. on a
microscope object glass cooled to ca. 4.degree. C. and investigated
at 400 X magnification. The microbubbles were observed to vary in
size from 2 to 5 .mu.m. The temperature was then raised to ca.
40.degree. C., whereupon a significant increase in the size of the
microbubbles was observed, the larger microbubbles growing most in
size. The number of microbubbles was significantly reduced after
about 5 minutes.
[0246] b) [comparative] One drop of the 2-methylbutane emulsion
from Example 1(c), cooled in an ice bath to ca. 0.degree. C., was
placed on a microscope object glass cooled to ca. 0.degree. C. and
investigated at 400 X magnification. The oil phase droplets of the
emulsion were observed to vary in size from 2 to 6 82 m. The
temperature was then raised to ca. 40.degree. C. No microbubble
formation was observed.
[0247] c) A sample of the perfluorobutane gas dispersion from
Example 1(a) (0.5 ml) was diluted with purified water (50 ml) and
cooled to 0.degree. C. A portion of this diluted disperion (1 ml)
was mixed with a portion of the 2-methylbutane emulsion from
Example 1(c) (100 .mu.l). One drop of the resulting mixture was
placed on a microscope object glass maintained at 0.degree. C. by
means of a heating/cooling stage and covered with a cover glass,
also at 0.degree. C. The temperature of the object glass was
gradually increased to 40.degree. C. using the heating/cooling
stage. Rapid and substantial microbubble growth was observed by
microscopy and was confirmed by size and distribution measurements
made using a Malvern Mastersizer.
[0248] d) [comparative] A sample of the perfluorobutane gas
dispersion from Example 1(a) (0.5 ml) was diluted with purified
water (50 ml) and cooled in an ice bath to 0.degree. C. A portion
of this diluted disperion (1 ml) was mixed with 100 .mu.l of a 5
mg/ml dispersion of hydrogenated phosphatidylserine in purified
water, also at 0.degree. C. One drop of the resulting mixture was
placed on a microscope object glass cooled to 0.degree. C. and
investigated at 400 X magnification. The microbubbles were observed
to vary in size from 2 to 5 82 m. The temperature was then raised
to ca. 40.degree. C., whereupon a significant increase in the size
of the microbubbles was observed, although the increase was less
heavy and less rapid then that observed in Example 2(c).
[0249] e) A sample of the perfluorobutane gas dispersion from
Example 1(a) was diluted with purified water (1:1) and cooled to
0.degree. C. A drop of the 2-chloro-1, 1, 2-trifluoroethyl
difluoromethyl ether emulsion from Example 1(e) was added to the
diluted microbubble dispersion on a microscope object glass
maintained at 0.degree. C. by means of a heating/cooling stage and
covered with a cover glass, also at 0.degree. C. The temperature of
the object glass was gradually increased to 40.degree. C. using the
heating/cooling stage. Rapid and substantial microbubble growth was
observed by microscopy.
[0250] f) A sample of the perfluorobutane gas dispersion from
Example 1(a) was diluted with purified water (1:1) and cooled to
0.degree. C. A drop of the 2-bromo-2-chloro-1, 1, 1-trifluoroethane
emulsion from Example 1(f) was added to the diluted microbubble
dispersion on a microscope object glass maintained at 0.degree. C.
by means of a heating/cooling stage and covered with a cover glass,
also at 0.degree. C. The temperature of the object glass was
gradually increased to 40.degree. C. using the heating/cooling
stage.
[0251] Rapid and substantial microbubble growth was observed by
microscopy.
[0252] g) A sample of the perfluorobutane gas dispersion from
Example 1(a) was diluted with purified water (1:1) and cooled to
0.degree. C. A drop of the 1-chloro-2, 2, 2-trifluoroethyl
difluoromethyl ether emulsion from Example 1(g) was added to the
diluted microbubble dispersion on a microscope object glass
maintained at 0.degree. C. by means of a heating/cooling stage and
covered with a cover glass, also at 0.degree. C. The temperature of
the object glass was gradually increased to 40.degree. C. using the
heating/cooling stage. Rapid and substantial microbubble growth was
observed by microscopy.
[0253] h) One drop of the dispersion of polymer/human serum albumin
microparticles from Example 1(h) and one drop of the
perfluoropentane emulsion from Example 1(k) were placed on a
microscope object glass warmed to 50.degree. C. and investigated at
400 X magnification. Significant growth of microbubbles was
observed as the drops mixed.
[0254] i) One drop of the dispersion of polymer/human serum albumin
microparticles from Example 1(h) and one drop of the 2-methylbutane
emulsion from Example 1(j) were placed on a microscope object glass
warmed to 40.degree. C. and investigated at 400 X magnification.
Significant, rapid and heavy growth of microbubbles was observed as
the drops mixed.
[0255] j) One drop of the dispersion of polymer/gelatin
microparticles from Example 1(i) and one drop of the
perfluoropentane emulsion from Example 1(k) were placed on a
microscope object glass warmed to 50.degree. C. and investigated at
400 X magnification. Significant growth of microbubbles was
observed as the drops mixed.
[0256] k) One drop of the dispersion of polymer/gelatin
microparticles from Example 1(i) and one drop of the 2-methylbutane
emulsion from Example 1(j) were placed on a microscope object glass
warmed to 40.degree. C. and investigated at 400 X magnification.
Significant, rapid and heavy growth of microbubbles was observed as
the drops mixed.
[0257] l) [comparative] One drop of the perfluoropentane emulsion
from Example 1(k) was placed on a microscope object glass warmed to
5.degree. C. and investigated at 400 X magnification. No
microbubble formation was observed.
[0258] m) [comparative] One drop of the 2-methylbutane emulsion
from Example 1(j) was placed on a microscope object glass warmed to
40.degree. C. and investigated at 400 X magnification. No
microbubble formation was observed.
[0259] n) [comparative] One drop of the dispersion of polymer/human
serum albumin microparticles from Example 1(h) is placed on a
microscope object glass warmed to 40.degree. C. and investigated at
400 X magnification. No significant change is seen.
[0260] o) [comparative] One drop of the dispersion of
polymer/gelatin microparticles from Example 1(i) is placed on a
microscope object glass warmed to 50.degree. C. and investigated at
400 X magnification. No significant change is seen.
[0261] p) One drop of a dispersion of human serum
albumin-stabilised air microbubbles prepared as described in U.S.
Pat. No. 4,718,433 and one drop of the 2-methylbutane emulsion from
Example 1(j) were placed on a microscope object glass at 20.degree.
C. and investigated at 400 X magnification. Significant growth of
microbubbles was observed as the drops mixed.
[0262] q) A sample of the perfluorobutane gas dispersion from
Example 1(a) was diluted with purified water (1:1) and cooled to
0.degree. C. A drop of the perfluorodecalin/perfluorobutane
emulsion from Example 1(z) was added to the diluted microbubble
dispersion on a microscope object glass maintained at 0.degree. C.
by means of a heating/cooling stage and covered with a cover glass,
also at 0.degree. C. The temperature of the object glass was
gradually increased to 40.degree. C. using the heating/cooling
stage. Rapid and substantial microbubble growth was observed by
microscopy.
[0263] r) A sample of the perfluorobutane gas dispersion from
Example 1(a) was diluted with purified water (1:1) and cooled to
0.degree. C. A drop of the perfluorodecalin/perfluoropropane
emulsion from Example 1(aa) was added to the diluted microbubble
dispersion on a microscope object glass maintained at 0.degree. C.
by means of a heating/cooling stage and covered with a cover glass,
also at 0.degree. C. The temperature of the object glass was
gradually increased to 40.degree. C. using the heating/cooling
stage. Rapid and substantial microbubble growth was observed by
microscopy.
[0264] s) A sample of the perfluorobutane gas dispersion from
Example 1(a) was diluted with purified water (1:1) and cooled to
0.degree. C. A drop of the perfluorodecalin/sulphur hexafluoride
emulsion from Example 1(ab) was added to the diluted microbubble
dispersion on a microscope object glass maintained at 0.degree. C.
by means of a heating/cooling stage and covered with a cover glass,
also at 0.degree. C. The temperature of the object glass was
gradually increased to 40.degree. C. using the heating/cooling
stage, whereupon an increase in the size of the microbubbles was
observed after 4-5 minutes, although the increase was less heavy
and less rapid then that observed in Examples 2(q) and 2(r).
[0265] t) A sample of the perfluorobutane gas dispersion from
Example 1(a) was diluted with purified water (1:1) and cooled to
0.degree. C. A drop of the Pluronic F68-stabilised perfluoropentane
emulsion from Example 1(ad) was added to the diluted microbubble
dispersion on a microscope object glass maintained at 0.degree. C.
by means of a heating/cooling stage and covered with a cover glass,
also at 0.degree. C. The temperature of the object glass was
gradually increased to 40.degree. C. using the heating/cooling
stage. Rapid and substantial microbubbble growth was observed by
microscopy.
[0266] u) One drop of the perfluorobutane gas dispersion from
Example 1(a) and one drop of the Brij58:Fluorad FC-170C-stabilised
perfluoropentane emulsion from Example 1(aj) were placed on a
microscope object glass warmed to 40.degree. C. and investigated at
400 X magnification. After a while slow microbubble growth was
observed.
[0267] v) One drop of the perfluorobutane gas dispersion from
Example 1(a) and one drop of the Brij58:Fluorad FC-170C-stabilised
perfluoropentane emulsion from Example 1(ak) were placed on a
microscope object glass warmed to 40.degree. C. and investigated at
400 X magnification. After a while microbubble growth was
observed.
[0268] w) One drop of the perfluorobutane gas dispersion from
Example 1(a) and one drop of the perfluoro-4-methylpent-2-ene
emulsion from Example 1(al) were placed on a microscope object
glass warmed to 40.degree. C. and investigated at 400 X
magnification. After a while slow microbubble growth was
observed.
[0269] x) One drop of the perfluorobutane gas dispersion from
Example 1(a) and one drop of the 1 H, 1 H, 2
H-heptafluoropent-1-ene emulsion from Example 1(am) were placed on
a microscope object glass warmed to 40.degree. C. and investigated
at 400 X magnification. Significant and rapid microbubble growth
was observed as the drops mixed.
[0270] y) One drop of the perfluorobutane gas dispersion from
Example 1(a) and one drop of the perfluorocyclopentene emulsion
from Example 1(an) were placed on a microscope object glass warmed
to 40.degree. C. and investigated at 400 X magnification.
Significant, rapid and heavy microbubble growth was observed as the
drops mixed.
[0271] z) 400 .mu.l of a perfluorobutane gas dispersion prepared as
in Example 1(b) was transferred to a 2 ml vial at room temperature,
and 100 .mu.l of the azeotropic emulsion of Example 1(ap) was
added. One droplet of the microbubble/emulsion mixture was placed
on a microscope object glass maintained at 20.degree. C. by means
of a heating/cooling stage. The temperature of the object glass was
rapidly raised to 37.degree. C. using the heating/cooling stage. A
substantial, spontaneous and rapid microbubble growth was
observed.
[0272] aa) One drop of biotinylated microbubbles prepared as
described in Example 1(bq) was added to one drop of emulsion
prepared as described in Example 1(bh) on a microscope object glass
warmed up to 60.degree. C. and investigated at 400 X magnification.
Significant growth of microbubbles and accumulation of microbubbles
at the aggregated emulsion droplets was seen.
[0273] ab) Microbubbles prepared as described in Example 1(bq) may
be analysed by flow cytometry, e.g. by employing a fluorescent
streptavidin emulsion prepared as described in Example 1(bk) to
detect attachment of streptavidin to the biotinylated
microbubbles.
[0274] ac) One drop of the Echovist dispersion prepared as
described in Example 1(bo) was placed on an object glass for
microscopy investigation and kept at 37.degree. C. using a
heating/cooling stage. The sample was covered with a cover glass
and placed under a microscope. Significant bubble growth was
observed.
[0275] ad) One drop of the aerogel dispersion from Example 1(br)
was placed on an object glass for microscopy investigation and kept
at 37.degree. C. using a heating/cooling stage. The sample was
covered with a cover glass and placed under a microscope. A droplet
of 2-methylbutane emulsion (from Example 1(c) above, except that
100 .mu.l 2-methylbutane was used instead of 200 .mu.l) was added
to the edge of the cover glass so that the emulsion penetrated into
the aerogel dispersion. On increasing the temperature to
approximately 60.degree. C., microbubbles occurred from the aerogel
particles.
[0276] ae) [comparative] One drop of the aerogel dispersion from
Example 1(br) was placed on an object glass for microscopy
investigation and kept at 20.degree. C. using a heating/cooling
stage. The sample was covered with a cover glass and placed under a
microscope and the temperature was raised to 60.degree. C. No
microbubble growth was observed.
[0277] One drop of the microbubble dispersion from Example 1(bs)
was placed on an object glass for microscopy investigation. The
sample was covered with a cover glass and placed under a microscope
fitted with a heating/cooling stage keeping the sample temperature
at 20.degree. C. One droplet of 2-methylbutane emulsion from
Example 1(c) above was added to the edge of the cover glass so that
the emulsion penetrated the microbubble dispersion. No microbubble
growth was observed during the mixing stage. The temperature was
then raised to 40.degree. C., whereupon substantial microbubble
growth was observed.
[0278] ag) [comparative] One drop of the microbubble dispersion
from Example 1(bs) was placed on an object glass for microscopy
investigation. The sample was covered with a cover glass and placed
under a microscope fitted with a heating/cooling stage keeping the
sample temperature at 20.degree. C. When the temperature was raised
to 40.degree. C., no microbubble growth was observed.
[0279] ah) To Echovist granulate (Schering AG) on a microscope
object glass was added one drop of solvent for Echovist granulate
at ambient temperature. One drop of 2-methylbutane emulsion
prepared as Example 1(bw) was added and investigated at 100 X
magnification. Significant growth of microbubbles was observed as
the drops mixed.
[0280] ai) One drop of Levovist.RTM. prepared for injection and one
drop of 2-methylbutane emulsion prepared as in Example 1(bw) were
placed on a microscope object glass at ambient temperature and
investigated at 400 X magnification. Significant, rapid and heavy
growth of microbubbles was observed as the drops mixed.
[0281] aj) One drop of perfluorobutane gas dispersion from Example
1(br) and one drop of 2-methylbutane emulsion prepared as Example
1(bw) were placed on a microscope object glass at ambient
temperature and investigated at 400 X magnification. Significant,
rapid and heavy microbubble growth was observed as the drops
mixed.
[0282] ak) One drop of 2-methylbutane emulsion prepared as Example
1(by) was added to one drop of Buckminsterfullerene C.sub.60
dispersion from Example 1(bu) on a microscope object glass at
40.degree. C. Significant, heavy and rapid growth of microbubbles
was observed as the drops mixed.
[0283] al) One drop of 2-methylbutane emulsion prepared as Example
1(bw) was added to one drop of sulphur hexafluoride gas dispersion
from Example 1(bv) on a microscope object glass at 40.degree. C.
Significant, rapid and heavy microbubble growth was observed as the
drops mixed.
[0284] am) One drop of 0.5 M hydrochloric acid was added to one
drop of perfluorobutane gas dispersion in aqueous sodium
bicarbonate from Example 1(bx) on a microscope object glass at
ambient temperature. Rapid, heavy andshort lived microbubble growth
was observed as the drops mixed.
[0285] an) One drop of 2-methylbutane emulsion prepared as in
Example 1(bw) was added to one drop of the perfluorobutane gas
dispersion with iron oxide particles from Example 1(bz) on a
microscope object glass at ambient temperature. Significant, heavy
and rapid microbubble growth was observed as the drops mixed.
[0286] ao) One drop of 2-methylbutane emulsion prepared as in
Example 1(bw) was added to one drop of the perfluorobutane gas
dispersion with iron oxide particles from Example 1(ca) on a
microscope object glass at ambient temperature. Significant, heavy
and rapid microbubble growth was observed as the drops mixed.
[0287] ap) [comparative] One drop of 2-methylbutane emulsion
prepared as Example 1(bw) was added to one drop of the iron oxide
particle dispersion from Example 1(cb) on a microscope object glass
at ambient temperature. No microbubble formation was observed.
[0288] aq) One drop of 2-methylbutane emulsion prepared as in
Example 1(bw) was added to one drop of the perfluorobutane gas
dispersion with oleic acid-coated iron oxide particles from Example
1(cc) on a microscope object glass at ambient temperature.
Significant, heavy and rapid microbubble growth was observed as the
drops mixed.
[0289] ar) 1 ml of the microbubble dispersion prepared as described
in Example 1(bp) was diluted with 50 ml water. One drop of the
diluted dispersion was added to one drop of soda water on a
microscope object glass at ambient temperature. Spontaneous
microbubble growth was observed as the drops mixed.
[0290] as) 0.4 .mu.l of a biotinylated microbubble dispersion
prepared according to Example 1(bq) and 0.02 ml of
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(bh) are added sequentially to a beaker containing 200 ml
of Isoton at 37.degree. C. with continuous stirring. The mixture is
incubated for 20 seconds. A beam of pulsed ultrasound (10 kHz
repetition frequency, 100 .mu.J in each pulse) at 2.5 MHz is aimed
through the solution, which is obsreved in sharp side light against
a black background. A bright streak of larger bubbles is
immediately observed in the beam path.
[0291] at) One drop of the microbubble dispersion prepared as in
Example 1(bl) is placed on an object glass for microscopy
examination. The sample is covered with a cover glass and placed
under a microscope fitted with a heating/cooling stage, keeping the
temperature at 20.degree. C. One droplet of a
perfluorodimethylcyclobutane emulsion prepared as in Example 1(as)
is added to the edge of the cover glass so that the emulsion can
penetrate the microbubble dispersion. On increasing the temperature
to approximately 60.degree. C., substantial microbubble growth is
observed.
[0292] Example 3--In vitro microbubble size and distribution
characterisation
[0293] a) Measurements using Malvern Mastersizer
[0294] Microbubble growth and the change in size distribution
following mixture with diffusible component were analysed using a
Malvern Mastersizer 1002 fitted with a 45 mm lens and having a
measuring range of 0.1-80 .mu.m. The sample cell contained Isoton
II (150 ml) and was connected to a thermostatted bath operable over
the temperature range 9-37.degree. C. A sample of the
perfluorobutane gas dispersion from Example 1(a) (110 .mu.l) was
added to the sample cell and after equilibration a portion of the
2-methylbutane emulsion from Example 1(c) (500 .mu.l) was added.
The Isoton II solution was pumped through the Mastersizer and the
thermostatted bath so as to pass the measuring cell every 30
seconds. Repeated measurements were carried out every 30 seconds
for 3 minutes. The temperature of the Isoton II solution was
gradually increased and further measurements were made. The
perfluorobutane gas dispersion and the 2-methylbutane emulsion were
also analysed separately using similar conditions.
[0295] Analysis of the perfluorobutane gas dispersion alone showed
that at 9.degree. C. 82% of the microbubbles were of size below 9.9
.mu.m; this proportion was reduced to 31% when the temperature had
increased to 37.degree. C. This temperature change was accompanied
by a correspnding increase in the proportion of microbubbles in the
size range 15-80 .mu.m, from 8% to 42%.
[0296] Following mixing of the perfluorobutane gas dispersion and
2-methylbutane emulsion at 9.degree. C. a slight increase in
microbubble size was observed. Increase of the temperature to
25.degree. C. led to strong microbubble growth, with about 81% of
the microbubbles having sizes in the range 15-80 .mu.m. Further
temperature increase led to microbubble growth to sizes beyond the
measuring range of the instrument.
[0297] Mixing of the perfluorobutane gas dispersion and
2-methylbutane emulsion at 37.degree. C. led to rapid microbubble
growth; after one 30 second measuring cycle 97% of the microbubbles
had sizes in the range 15-80 .mu.m.
[0298] b) Measurements using Coulter Multisizer
[0299] Microbubble growth and the change in size distribution
following mixture with diffusible component were analysed using a
Coulter Multisizer II fitted with a 50 .mu.m aperture and having a
measuring range of 1-30 .mu.m. The two components of each sample
were added to the sample cell, which contained 200 ml Isoton II
preheated to 37.degree. C., at which temperature the measurements
were performed. The size distribution of the mixture was measured
immediately and 1.5 minutes after introduction of the samples.
Thereafter the sample cell was exposed to ultrasound for 1 minute,
using a 2.25 MHz transducer connected to a pulse generator; the
energy level was 100 .mu.J.
[0300] b) (i) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(1) led
to rapid and substantial microbubble growth after exposure to
ultrasound. The total volume concentration increased from 3% to
approximately 16%.
[0301] b)(ii) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluorobutane emulsion from Example 1(m) led to
rapid and substantial microbubble growth. The total volume
concentration increased from 1% to approximately 6%.
[0302] b) (iii) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(p) led
to rapid and substantial microbubble growth after exposure to
ultrasound. The total volume concentration increased from
approximately 1% to approximately 4%.
[0303] b) (iv) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(af) led
to rapid and substantial microbubble growth after exposure to
ultrasound. The total volume concentration increased from
approximately 2% to approximately 8%.
[0304] b) (v) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane/perfluoro-4-methylpent-2-ene
emulsion from Example 1(q) led to rapid and substantial microbubble
growth after exposure to ultrasound. The total volume concentration
increased from 2% to approximately 4%.
[0305] b) (vi) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane/1 H, 1 H, 2
H-heptafluoropent-1-ene emulsion from Example 1(r) led to rapid and
substantial microbubble growth. The total volume concentration
increased from 2% to approximately 4.5%.
[0306] b) (vii) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(s) led
to rapid and substantial microbubble growth after exposure to
ultrasound. The total volume concentration increased from 2% to
approximately 13%.
[0307] b) (viii) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(t) led
to rapid and substantial microbubble growth after exposure to
ultrasound. The total volume concentration increased from 2% to
approximately 13%.
[0308] b) (ix) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(u) led
to rapid and substantial microbubble growth after exposure to
ultrasound. The total volume concentration increased from 3% to
approximately 15%.
[0309] b) (x) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(v) led
to rapid and substantial microbubble growth after exposure to
ultrasound. The total volume concentration increased from 3% to
approximately 22%.
[0310] b) (xi) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(ai) led
to rapid and substantial microbubble growth after exposure to
ultrasound. The total volume concentration increased from
approximately 3% to approximately 8%.
[0311] b) (xii) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane:perfluoro-4-methylpent-2-ene
emulsion from Example 1(x) led to rapid and substantial microbubble
growth after exposure to ultrasound. The total volume concentration
increased from 2% to approximately 7.5%.
[0312] b) (xiii) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane:perfluoro-4-methylpent-2-ene
emulsion from Example 1(y) led to rapid and substantial microbubble
growth after exposure to ultrasound. The total volume concentration
increased from 2.5% to approximately 7%.
[0313] b) (xiv) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(ac) led
to rapid and substantial microbubble growth. The increase in the
size of the microbubbles was more heavy and more rapid then that
observed in Example 3(b) (xv). The total volume concentration
increased from 3.5% to approximately 53%.
[0314] b) (xv) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(ae) led
to rapid and substantial microbubble growth. The total volume
concentration increased from 7% to approximately 19%. Exposure to
ultrasound resulted in further microbubble growth indicated by an
increase in the total volume concentration to approximately
54.5%.
[0315] b) (xvi) Mixing of the perfluoropropane gas dispersion from
Example 1(ah) and perfluoropentane emulsion from Example 1(1) led
to rapid microbubble growth, although not so heavy as observed in
Example 3(b)(i). The total volume concentration increased from 3%
to approximately 4.5%.
[0316] b) (xvii) Mixing of the perfluorobutane gas dispersion from
Example 1(ag) and perfluoropentane emulsion from Example 1(o) led
to rapid and substantial microbubble growth after exposure to
ultrasound. The total volume concentration increased from
approximately 1% to approximately 8%.
[0317] b) (xviii) A sample of perfluorohexane emulsion prepared as
described in Example 1(ar) had a total concentration of droplets of
8.6 vol % and the droplet size was 2.6 .mu.m.
[0318] b) (xix) A sample of 2, 2, 3, 3, 3-pentafluoropropyl methyl
ether emulsion prepared as described in example 1(at) had a total
concentration of droplets of 4.3 vol % and the droplet size was 1.5
.mu.m.
[0319] b) (xx) A sample of 2 H, 3 H-decafluoropentane emulsion
prepared as described in Example 1(au) had a total concentration of
droplets of 5.6 vol % and the droplet size was 1.9 .mu.m.
[0320] b) (xxi) A sample of perfluoroheptane emulsion prepared as
described in Example 1(bj) had a total concentration of droplets of
8.5 vol % and the droplet size was 2.2 .mu.m.
[0321] (c) Measurements using Coulter Multisizer (140 .mu.m
aperture)
[0322] Microbubble growth and change of size distribution following
mixture with diffusible component emulsions were analysed using a
Coulter Multisizer II fitted with a 140 .mu.m aperture. The
measuring range was set to 10-80 .mu.m. The bubble dispersion and
emulsion droplets were added to the sample cell containing 200 ml
preheated Isoton II. The measurements were performed at 37.degree.
C. The size distribution of the mixture was measured immediately
and 3 minutes after mixing. Thereafter the sample solution was
exposed to ultrasound for 1 minute using a 2.25 MHz transducer
connected to a pulse generator. The energy level was 100 .mu.J. The
size distribution of the mixture was measured 1 minute and 3
minutes after exposure to ultrasound.
[0323] c) (i) Following addition of 182 .mu.l of the
heptafluoropent-1-ene emulsion prepared as described in Example
1(am) to 400 .mu.l of a perfluorobutane gas dispersion prepared as
described in Example 1(bl), the microbubbles immediately increased
in size and the total volume concentration in the size range 10-80
.mu.m was increased from insignificant to about 60 vol % within 1
minute.
[0324] c) (ii) Following addition of 70 .mu.l of the
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(av) to 330 .mu.l of perfluorobutane gas dispersion
prepared as described in Example 1(bl), the microbubbles increased
substantially in size after exposure to ultrasound. The total
volume concentration in the size range 10-80 .mu.m was increased
from insignificant to about 14 vol % within 3 minutes.
[0325] c) (iii) Following addition of 71 .mu.l of the
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(aw) to 330 .mu.l of perfluorobutane gas dispersion
prepared as described in Example 1(bl), the microbubbles increased
substantially in size after exposure to ultrasound. The total
volume concentration in the size range 10-80 .mu.m was increased
from insignificant to about 8.6 vol % within 3 minutes.
[0326] c) (iv) Following addition of 105 .mu.l of the
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(ax) to 300 .mu.l of perfluorobutane gas dispersion
prepared as described in Example 1(bl), the microbubbles increased
in size after exposure to ultrasound. The total volume
concentration in the size range 10-80 .mu.m was increased from 3.2
vol % to about 4.8 vol % within 3 minutes.
[0327] c) (v) Following addition of 105 .mu.l of the
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(ay) to 300 .mu.l of perfluorobutane gas dispersion
prepared as described in Example 1(bl), the microbubbles increased
in size after exposure to ultrasound. The total volume
concentration in the size range 10-80 .mu.m was increased from 1.5
vol % to about 2.2 vol % within 3 minutes.
[0328] c) (vi) Following redispersion of lyophilised
perfluorobutane microbubbles in perfluorodimethylcyclobutane
emulsion as described in Example 1(bg) an immediate increase in
microbubble size occurred. The total volume concentration in the
size range 10-80 .mu.m was increased from insignificant to about 60
vol % within 1 minute.
[0329] c) (vii) Following addition of 76 .mu.l of the 1
H-tridecafluorohexane emulsion prepared as described in Example
1(bi) to 400 .mu.l of a perfluorobutane gas dispersion prepared as
described in Example 1(bl), the microbubbles immediately increased
in size and the total volume concentration in the size range 10-80
.mu.m was increased from insignificant to about 20 vol % within 3
minures.
[0330] c) (viii) Following addition of 63 .mu.l of the
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(bm) to 741 .mu.l of a perfluorobutane gas dispersion
prepared as described in Example 1(bl) the microbubbles immediately
increased in size and the total volume concentration in the size
range 10-80 .mu.m was increased from insignificant to about 2 vol %
within 3 minutes.
[0331] c) (ix) Following addition of 67 .mu.l of the
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(aq) to 56 .mu.l of perfluoropropane gas dispersion
prepared as described in Example 1(bn) the microbubbles increased
in size after exposure to ultrasound. The total volume
concentration in the size range 10-80 .mu.m was increased from
insignificant to about 2.7 vol % within 1 minute.
[0332] Example 4--In vitro measurements of acoustic attenuation
[0333] a) A sample of the perfluorobutane gas dispersion from
Example 1(a) (1 .mu.l) was suspended in Isoton II (55 ml) at
37.degree. C. and acoustic attenuation was measured as a function
of time using two broadband transducers with centre-frequencies of
3.5 MHz and 5.0 MHz in a pulse-echo technique. After 20 seconds a
diffusible component was added to the suspension and measurements
were continued for a further 120 seconds.
[0334] a) (i) Following addition of 100 .mu.l of the 2-methylbutane
emulsion from Example 1(c) attenuation immediately increased by a
factor of more than 4; exact quantification was not possible since
the attenuation exceeded the maximum value measurable by the
system. The effect lasted for 50 seconds and was accompanied by a
complete change in the shape of the attenuation spectra indicating
a pronounced increase in microbubble size.
[0335] a) (ii) Addition of 20 .mu.l of the 2-methylbutane emulsion
from Example 1(c) led to a gradual increase in attenuation,
reaching a maximum of between three and four times the initial
value after 40 seconds and then decreasing rapidly. Again a
complete change in the shape of the attenuation spectra indicated a
pronounced increase in microbubble size.
[0336] a) (iii) Addition of 5 .mu.l of the 2-methylbutane emulsion
for Example 1(c) led to a gradual increase in attenuation, reaching
a maximum of about 50% above the initial value after 30 seconds and
then decreasing slowly towards the initial value. A shift towards
lower resonance frequencies in the attenuation spectra indicated a
moderate increase in microbubble size.
[0337] a) (iv) Addition of 500 .mu.l of the 2-chloro-1, 1,
2-trifluoroethyl difluoromethyl ether emulsion from Example 1(e)
led to a gradual increase in attenuation, reaching a maximum of
about 50% above the initial value after 20 seconds and then
decreasing slowly towards the initial value. A shift towards lower
resonance frequencies in the attenuation spectra indicated a
moderate increase in microbubble size.
[0338] a) (v) Addition of 500 .mu.l of the perfluoropentane
emulsion from Example 1(d) led to a small increase in attenuation.
A shift towards lower resonance frequencies in the attenuation
spectra indicated a small increase in microbubble size.
[0339] By way of control, addition of 500 .mu.l of water produced
no discernible change in attenuation.
[0340] b) A sample of the 2-methylbutane emulsion from Example 1(c)
(100 .mu.l) was added to the Isoton II (55 ml) at 37.degree. C. and
acoustic attenuation was measured as described in (a) above. After
20 seconds a sample of the perfluorobutane gas dispersion from
Example 1(a) (1 .mu.l) was added to the suspension and measurements
were continued for a further 120 seconds. Attenuation increased
rapidly following addition of the gas dispersion, reaching the
maximum measuring level of the system after 20 seconds, and
starting to decrease after 50 seconds. The attenuation spectra
indicated the presence of large microbubbles.
[0341] By way of control, when 100 .mu.l of water was used in place
of the 2-methylbutane emulsion, attenuation increased rapidly
following addition of the gas dispersion; after 40 seconds it
reached a stable level one quarter of that measured using the
2-methylbutane emulsion. Attenuation remained at this level
throughout the remainder of the 120 second measurement period. The
attenuation spectra indicated the presence of small
microbubbles.
[0342] Example 5--In vivo imaging of dog heart with perfluorobutane
gas dispersion [comparative]
[0343] An injection syringe containing an amount of the
perfluorobutane gas dispersion from Example 1(b) corresponding to 2
.mu.l of gas content was prepared and the contents were injected
into an open-chest 20 kg dog using a catheter inserted into an
upper limb vein. Imaging of the heart was performed with a Vingmed
CFM-750 scanner, using a midline short axis projection. The scanner
was adjusted to acquire images once in each end-systole by gating
to the ECG of the animal. Bright contrast was seen in the right
ventricle a few seconds after the injection, and contrast of
similar brightness appeared in the left ventricle some 4-5 seconds
later, however with a substantial attenuation transiently hiding
the posterior parts of the heart. Off-line digital backscatter
intensity analysis was performed based on cine-loop data recorded
by the scanner. A brief, transient peak of contrast enhancement
lasting some 10 seconds, beginning 3 seconds after the onset of
contrast enhancement within the left ventricle was evident in a
representative region of anterior left ventricle myocardium.
[0344] Example 6--In vivo imaging of dog heart with 2-methylbutane
emulsion [comparative]
[0345] An injection syringe containing 1.0 ml of the 2-methylbutane
emulsion from Example 1(c) was prepared and the contents were
injected into the animal as in Example 5. Imaging of the heart was
performed as described in Example 5. No contrast effects could be
seen.
[0346] Example 7--In viva imaging of dog heart with rerfluorobutane
gas dispersion and 2-methylbutane emulsion
[0347] Injection syringes were prepared as in Examples 5 and 6 and
the contents of both syringes were injected simultaneously into the
dog via a Y-piece connector and the catheter described in Example
5. Imaging of the heart was performed as described in Example 5.
The echo enhancement of the ventricles was similar to the
observations in Example 5. In the left ventricular myocardium there
was a monotonous rise in echo intensity in the 30 seconds following
arrival of the contrast bolus to the coronary circulation. The
contrast effects in the myocardium had completely vanished 5
minutes later.
[0348] Example 8--In vivo imaging of dog heart with
perfluoropentane emulsion [comparative]
[0349] An injection syringe containing 0.5 ml of the
perfluoropentane emulsion from Example 1(d) was prepared and the
contents were injected into the animal as in Example 5. Imaging of
the heart was performed as described in Example 5. No signs of echo
enhancement could be observed in any region of the image.
[0350] Example 9--Low intensity in vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluoropentane emulsion
[0351] Injection syringes were prepared as in Examples 5 and 8 and
the contents of both syringes were injected simultaneously into an
open-chest 20 kg mongrel dog via a Y-piece connector and a catheter
inserted into an upper limb vein. Imaging of the heart was
performed with a Vingmed CFM-750 sanner, using a midline short axis
projection. The scanner was adjusted to minimise acoustical output
by lowering the emitted power to a value of 1 (on a scale ranging
from 0 to 7), and by acquiring images only once in each end-systole
by gating to the ECG of the animal. The observed contrast
enhancement was as described in Example 5 with, however, a slightly
prolonged duration in the myocardium.
[0352] Example 10--High intensity in viva imaging of dog heart with
perfluorobutane gas dispersion and perfluoropentane emulsion
[0353] The experiment of Example 9 was repeated, except that the
scanner output was adjusted to maximise ultrasound exposure to the
imaged tissue region. This was done by using a combination of
continuous high frame rate imaging and the highest output power (7
on a scale ranging from 0 to 7). After the injection, intense and
bright contrast enhancement was seen in both ventricles of the
heart. A steady rise in contrast enhancement was seen in all
regions of the myocardium, up to an enhancement intensity
approaching the maximum white level on the screen. The duration of
tissue contrast was approximately 30 minutes, whilst contrast
effects in the blood-pool declined to near baseline within 5
minutes of the injection, leaving an image with almost no
blood-pool attenuation, and a complete and extremely bright
circumferential contrast enhancement of the myocardium. The
contrast effect in the myocardium close to the transducer did not
seem to fade despite continuous high intensity ultrasound
exposure.
[0354] Example 11--High intensity in vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluoronentane emulsion
[0355] The procedure of Example 10 was repeated except that the
perfluoropentane emulsion employed was prepared by cooling a
solution of polyethylene glycol 10000 methyl ether
16-hexadecanoyloxyhexadecanoate (200 mg, prepared as in Example
2(k) of WO-A-9607434) in purified water (20 ml), transferring a 1
ml portion of this solution to a 2 ml vial, adding perfluoropentane
(200 .mu.l), shaking the vial for 45 seconds using a CapMix.RTM.
and storing the emulsion at 0.degree. C. when not in use. The
observed contrast enhancements of blood and myocardial tissue were
as described in Example 5.
[0356] Example 12--In vivo imaging of dog kidney
[0357] The same substances and injection procedure as described in
Example 9 were used. The left kidney of the dog was imaged through
the intact abdominal wall using the same high output instrument
settings as in Example 10. Central structures of the kidney
containing the supplying arteries were included in the image. 20
seconds after the injection, the beginning of a steady rise in
kidney parenchymal contrast enhancement was seen, reaching an
intensity plateau of extreme brightness 1-2 minutes later. The
transducer was moved to image the right kidney 4 minutes after the
injection. At first, this kidney had a normal, non-enhanced
appearance. However, this application of high intensity ultrasound
was observed to generate a slight increase in echo intensity after
a few minutes, although not up to the level that was observed in
the left kidney.
[0358] Example 13--In vivo imaging of dog heart with
perfluorobutane gas dispersion and reduced amount of
perfluoropentane emulsion
[0359] The procedure of Example 10 was repeated except that the
dose of the perfluoropentane emulsion was reduced to one third. The
peak intensity of myocardial contrast enhancement was comparable to
that observed in Example 10, but the duration of tissue contrast
was reduced from 30 minutes to less than 10 minutes.
[0360] Example 14--Closed-chest in vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluoropentane emulsion
[0361] The procedure of Example 10 was repeated in a closed-chest
experiment. The myocardial contrast enhancement was comparable to
that observed in Example 10.
[0362] Example 15--Colour Doppler in vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluoropentane emulsion
[0363] The procedure of Example 10 was repeated except that the
scanner (in the colour Doppler mode) was applied to the left heart
ventricle during the first minute after injection in order to
initiate microbubble growth. Thereafter the myocardial contrast
enhancement was more intense than that observed in Example 10.
[0364] Example 16--In vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluoro-4-methylpent-2-ene
emulsion
[0365] 0.5 ml of isotonically reconstituted perfluorobutane gas
dispersion prepared in accordance with Example 1(ag) and 66 .mu.l
of the perfluoro-4-methylpent-2-ene emulsion from Example 1(al)
were injected as described in Example 10.
[0366] The resulting myocardial contrast enhancement was comparable
in intensity to that observed in Example 10, but had a duration of
6-8 minutes.
[0367] Example 17--In vivo imaging of hyperemic region of dog heart
with perfluorobutane gas dispersion and perfluoropentane
emulsion
[0368] A branch of the circumflex coronary artery of the dog was
temporarily ligated for 2 minutes, whereafter contrast agent was
injected as described in Example 10. Contrast enhancement of the
now hyperaemic myocardium was substantially more intense than that
of surrounding normal tissue.
[0369] Example 18--In vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluorodimethylcyclobutane
emulsion
[0370] 0.5 ml of isotonically reconstituted perfluorobutane gas
dispersion prepared in accordance with Example 1(ag) and 66 .mu.l
of the perfluorodimethylcyclobutane emulsion from Example 1(ao)
were injected as described in Example 10. The resulting intense
myocardial contrast enhancement was comparable to that observed in
Example 16.
[0371] Example 19--In vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluorodimethylcyclobutane
emulsion
[0372] 0.5 ml of isotonically reconstituted perfluorobutane gas
dispersion prepared in accordance with Example 1(bl) and 66 .mu.l
of the perfluorodimethylcyclobutane emulsion from Example 1(aq)
were injected as described in Example 10. The resulting intense
myocardial contrast enhancement was comparable to that observed in
Example 16.
[0373] Example 20--In vivo "particle-to-particle" targeting
[0374] 0.02 .mu.l/kg of avidinylated perfluorobutane microbubbles
prepared according to Example 1(bq), and 0.02 .mu.l/kg of
perfluorodimethylcyclobu- tane emulsion prepared as described in
Example 1(bh) are simultaneously intravenously injected into a 20
kg anaesthetised mongrel dog, while the heart is imaged by
ultrasound as described in Example 10. Myocardial echo enhencement
was similar to that observed in Example 10, except that the peak of
attenuation in the left ventriclar blood was far less
pronounced.
[0375] Example 21--In viva imaging of rabbit heart with is
perfluorobutane gas dispersion and perfluorodimethylcyclobutane
emulsion
[0376] An injection syringe containing an amount of the
perfluorobutane microbubble dispersion prepared as in Example 1(bl)
(volume median diameter 3.0 .mu.m) corresponding to 1 .mu.l of gas
content and a further injection syringe containing 105 .mu.l of the
perfluorodimethylcyclobutan- e emulsion from Example 1(aq) were
prepared. The contents of both syringes were injected
simultaneously into a 5 kg rabbit using a catheter inserted into an
ear vein. B-mode imaging of the heart was performed using an ATL
HDI-3000 scanner with a P5-3 probe, using an open thorax
parasternal short axis projection. The results were comparable to
those observed in Example 18.
[0377] Example 22--Ultrasonication-induced drug delivery
[0378] A 3 kg anaesthetised New Zealand Black rabbit was injected
intravenously with 0.04 ml of perfluorodimethylcyclobutane emulsion
prepared as described in Example 1(aq) and simultaneously with 0.12
ml of perfluorobutane gas suspension prepared as described in
Example 1(bl), while the left kidney was imaged with an ATL
HDI-3000 scanner with a P5-3 probe, the scanner being adjusted for
maximum output power. Significant bubble growth and accumulation
within the kidney parenchyma was observed. Then 160 mg of
FITC-dextran (mw 2,000,000) was dissolved in 5 ml of water and
injected intravenously, and ultrasound imaging at the same site was
continued for another 5 minutes, now swithcing the scanner to Power
Doppler mode to maximise acoustical output. The animal was then
sacrificed, and both kidneys were removed and examined in
ultraviolet light. An increased amount of fluorescence was observed
as 50-100 .mu.m spots in the interstitium within the regions of the
left kidney that were exposed to imaging ultrasound in the presence
of microbubbles. Associated with each such spot was a nephron
devoid of intravascular fluorescence.
[0379] Example 23--Albunex.RTM. as gas dispersion
[0380] 0.3 ml/kg of Albunex.RTM. and 1.5 .mu.l/kg of
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(aq) were injected intravenously into a 20 kg
anaesthetised male mongrel dog and imaged by ultrasound as
described in Example 10. The myocardial enhancement was as
described in Example 10.
[0381] Example 24--Targeted microbubbles in imagina of rabbit
heart
[0382] 0.1 .mu.l/kg of microbubbles prepared as described in
Example 1(az) were injected intravenously into a rabbit, while
imaging the rabbit's heart by ultrasound using an ATL HDI-3000
scanner with a P5-3 probe. A faint but lasting myocardial echo
enhancement was seen. Three minutes later, 1.5 .mu.l/kg of
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(aq) was injected. A slight increase in the echo intensity
from the insonified myocardium was observed.
[0383] Example 25--In vivo imaging of rat heart with
perfluorobutane gas dispersion and perfluorodimethylcyclobutane
emulsion
[0384] The experiment described in Example 19 was performed on a
rat, with comparable results.
[0385] Example 26--In vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluorohexane emulsion
[0386] 0.1 .mu.l/kg of perfluorohexane emulsion prepared as
described in Example 1(ar) and 0.2 .mu.l/kg of the perfluorobutane
microbubble suspension prepared as decribed in Example 1(bl) were
injected simultaneously into a dog as described in Example 10. The
myocardial contrast effect was comparable to that observed in
Example 10.
[0387] Example 27--In vivo imaging of dog heart with
perfluorobutane gas dispersion and heptafluoropent-1-ene
emulsion
[0388] 0.3 .mu.l/kg of the perfluorobutane microbubble suspension
prepared as described in Example 1(bl) and 0.15 ml of the
heptafluoropent-1-ene emulsion described in Example 1(am) were
injected simultaneously into a dog as described in Example 10. A
relatively weak myocardial contrast effect was observed, which was
however more intense and more long-lasting than that which was
observed in Example 5.
[0389] Example 28--In vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluorodimethylcyclobutane
emulsion stabilised with sterilised phospholipid
[0390] 0.3 .mu.l/kg of a perfluorobutane microbubble suspension
prepared as described in Example 1(bl) and 0.3 .mu.l/kg of the
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(bm) were injected simultaneously into a dog as described
in Example 19. A myocardial contrast effect comparable to that
described in Example 19 was observed.
[0391] Example 29--In vivo imaging of dog heart with
perfluoropropane gas dispersion and perfluorodimethylcyclobutane
emulsion
[0392] 0.17 ml of the perfluoropropane microbubble suspension
prepared as described in Example 1(bn) and 0.3 .mu.l/kg of the
perfluorodimethylcyclobutane emulsion prepared as described in
Example 1(aq) were injected simultaneously into a dog as described
in Example 19. A myocardial contrast effect comparable to that
described in Example 19 was observed.
[0393] Example 30--In vivo imaging of dog gastrointestinal tract
with perfluorobutane gas dispersion and
perfluorodimethylcyclobutane emulsion
[0394] 20 ml of an emulsion of perfluorodimethylcyclobutane,
prepared as described in Example 1(aq) is given via a gastric tube
to an anaesthetised dog. Thereafter a small amount (dose range
0.1-0.2 .mu.l gas/kg) of a perfluorobutane microbubble dispersion
prepared as in Example 1(a) is injected intravenously. An
ultrasound imaging transducer is applied onto the abdominal wall,
and localised microbubble growth in the gastric wall capillary
system provides enhanced contrast with improved delineation of the
mucosal contours.
[0395] Example 31--In vivo imaging of dog gastrointestinal tract
with perfluorobutane gas dispersion and
perfluorodimethylcyclobutane emulsion
[0396] A perfluorobutane microbubble dispersion prepared as in
Example 1(a) is given via a gastric tube to an anaesthetised dog.
The dispersion is allowed to distribute evenly inside the gastric
ventricle, as verified by ultrasound imaging. A small amount of an
emulsion of perfluorodimethylcyclobutane, prepared as described in
Example 1(aq) (dose range 0.2-1 .mu.l perfluorocarbon/kg), is
injected intravenously. The ultrasound transducer is maintained on
the region of interest; microbubble growth in the gastric fluid
layers proximal to the mucosal surfaces provides enhanced contrast
with improved delineation of the mucosal contours.
[0397] Example 32--In vivo imaging of dog heart with
perfluorobutane gas dispersion and perfluorodimethylcyclobutane
emulsion and coadministered adenosine
[0398] An occluding snare was placed around a major branch of the
left anterior descending coronary artery of an open-chest 22 kg dog
and an ultrasound transit time flowmeter was placed immediately
downstream of the occluder, which was then adjusted to produce a
steady 25% flow reduction from about 14 to 10 ml/min. The contents
of three syringes, respectively containing (i) an amount of a
perfluorobutane microbubble dispersion prepared as in Example 1(bl)
corresponding to 4.4 .mu.l of gas content, (ii) an amount of the
perfluorodimethylcyclobutane emulsion from Example 1(aq)
corresponding to 33 .mu.l of the dispersed
perfluorodimethylcyclobutane phase, and (iii) 3.0 mg adenosine
dissolved in 0.9% saline, were then intravenously injected as a
simultaneous bolus; commencing 10 seconds later a further 3.0 mg of
adenosine dissolved in 0.9% saline was injected slowly over 20
seconds. Imaging of the left ventricle of the heart was performed
using an ATL HDI-3000 scanner with a P5-3 probe; continuous
ultrasonication at maximum power was applied for 1 minute to induce
microbubble growth, whereafter the myocardium was examined using
B-mode imaging. A clearly evident difference in gray scale levels
could be seen between stenotic areas (brighter than baseline
recordings) and normal areas (very much brighter than baseline
recordings).
* * * * *