U.S. patent application number 09/783793 was filed with the patent office on 2001-08-02 for contrast agents.
Invention is credited to Braenden, Jorunn, Dugstad, Harald, Klaveness, Jo, Rongved, Pal, Skurtveit, Roald.
Application Number | 20010010811 09/783793 |
Document ID | / |
Family ID | 27268137 |
Filed Date | 2001-08-02 |
United States Patent
Application |
20010010811 |
Kind Code |
A1 |
Dugstad, Harald ; et
al. |
August 2, 2001 |
Contrast agents
Abstract
Microbubble dispersions stabilized by phospholipids
predominantly comprising molecules which individually have an
overall net charge exhibit advantageous stability, rendering them
useful as efficacious contrast agents. An improved process for
preparing microbubble-containing contrast agents is also disclosed,
this comprising lyophilising an aqueous dispersion of gas
microbubbles stabilized by one or more membrane-forming lipids to
yield a dried product which may be reconstituted in an injectable
carrier liquid to generate a microbubble-containing contrast
agent.
Inventors: |
Dugstad, Harald; (Oslo,
NO) ; Klaveness, Jo; (Oslo, NO) ; Rongved,
Pal; (Oslo, NO) ; Skurtveit, Roald; (Oslo,
NO) ; Braenden, Jorunn; (Oslo, NO) |
Correspondence
Address: |
Amersham Pharmacia Biotech, Inc.
800 Centennial Avenue
Piscataway
NJ
08855
US
|
Family ID: |
27268137 |
Appl. No.: |
09/783793 |
Filed: |
February 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09783793 |
Feb 15, 2001 |
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09136410 |
Aug 19, 1998 |
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6221337 |
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09136410 |
Aug 19, 1998 |
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PCT/GB97/00459 |
Feb 19, 1997 |
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Current U.S.
Class: |
424/9.52 ;
424/450 |
Current CPC
Class: |
A61K 49/227 20130101;
A61K 49/22 20130101; Y10T 428/2982 20150115; A61K 49/223
20130101 |
Class at
Publication: |
424/9.52 ;
424/450 |
International
Class: |
A61K 049/22 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 19, 1996 |
GB |
9603466.5 |
Jun 7, 1996 |
GB |
9611894.8 |
Dec 11, 1996 |
GB |
9625663.1 |
Claims
1. An aqueous dispersion of gas microbubbles stabilised by
amphiphilic material consisting essentially of phospholipid
predominantly comprising molecules which individually have an
overall net charge.
2. A size fractionated microbubble dispersion according to claim
1.
3. A microbubble dispersion according to claim 1 or claim 2 wherein
at least 75% of the phospholipid consists of molecules which
individually have an overall net charge.
4. A microbubble dispersion according to claim 3 wherein
substantially all of the phospholipid consists of molecules which
individually have an overall net charge.
5. A microbubble dispersion according to any of the preceding
claims wherein the phospholipid is selected from naturally
occurring, semisynthetic and synthetic phosphatidylserines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids,
cardiolipins, lyso forms of any of the foregoing and mixtures of
any of the foregoing.
6. A microbubble dispersion according to claim 5 wherein acyl
groups present in the phospholipid each contain about 14-22 carbon
atoms.
7. A microbubble dispersion according to claim 5 or claim 6 wherein
one or more phosphatidylserines constitute at least 70% of the
phospholipid.
8. A microbubble dispersion according to claim 7 wherein said
phosphatidylserine or phosphatidylserines are selected from
synthetic phosphatidylserine, semisynthetic phosphatidylserine,
hydrogenated natural phosphatidylserine, hydrogenated semisynthetic
phosphatidylserine, synthetic distearoylphosphatidylserine,
synthetic dipalmitoylphosphatidylserine and synthetic
diarachidoylphosphatidylserin- e.
9. A microbubble dispersion according to any of the preceding
claims wherein the gas is selected from air, nitrogen, oxygen,
carbon dioxide, hydrogen, nitrous oxide, inert gases, sulphur
fluorides, selenium hexafluoride, silanes, halogenated silanes, low
molecular weight hydrocarbons, halogenated low molecular weight
hydrocarbons, ethers, ketones, esters and mixtures of any of the
foregoing.
10. A microbubble dispersion according to claim 9 wherein the gas
comprises sulphur hexafluoride or a fluorinated low molecular
weight hydrocarbon.
11. A microbubble dispersion according to claim 10 wherein said
hydrocarbon is perfluorinated.
12. A microbubble dispersion according to claim 11 wherein said
perfluorinated hydrocarbon comprises perfluoropropane,
perfluorobutane or perfluoropentane.
13. A microbubble dispersion according to claim 7 wherein the gas
is perfluorobutane.
14. A microbubble dispersion according to any of claims 10 to 13
characterised in that the microbubbles exhibit at least 90%
recovery of size distribution and echogenic properties following
exposure to an overpressure of 300 mm Hg for 90 seconds.
15. A contrast agent for use in diagnostic studies, comprising a
microbubble dispersion as claimed in any of the preceding claims in
an injectable aqueous carrier liquid.
16. A method of diagnostic imaging which comprises administering to
a subject a contrast-enhancing amount of a contrast agent according
to claim 15 and imaging at least a part of said subject.
17. A method according to claim 16 wherein an MR image of said
subject is generated.
18. A method according to claim 16 wherein an X-ray image of said
subject is generated.
19. A method according to claim 16 wherein a scintigraphic or light
image of said subject is generated.
20. A method according to claim 16 wherein an ultrasound image of
said subject is generated.
21. A method according to claim 20 wherein the contrast agent is
administered at a dose such that the amount of phospholipid
administered is in the range 0.1-10 .mu.g/kg body weight.
22. A method according to claim 21 wherein the dose is such that
the amount of phospholipid administered is in the range 1-5
.mu.g/kg body weight.
23. A process for the preparation of a contrast agent comprising
the steps: i) dispersing gas in an aqueous medium containing a
membrane-forming lipid to form a lipid-stabilised gas microbubble
dispersion; ii) lyophilising said dispersion to yield a dried
lipid-containing product; and iii) reconstituting said dried
product in an aqueous injectable carrier liquid.
24. A process as claimed in claim 23 wherein the gas employed in
step (i) is a fluorinated low molecular weight hydrocarbon.
25. A process as claimed in claim 24 wherein said hydrocarbon is
perfluorinated.
26. A process as claimed in claim 23 wherein the gas employed in
step (i) is sulphur hexafluoride.
27. A process as claimed in any of claims 23 to 26 wherein the
lipid-containing aqueous medium employed in step (i) further
contains one or more additives selected from viscosity enhancers
and solubility aids for the lipid.
28. A process as claimed in claim 27 wherein said additive or
additives are selected from alcohols and polyols.
29. A process as claimed in any of claims 23 to 28 wherein the
membrane-forming lipid comprises at least one phospholipid.
30. A process as claimed in any of claims 23 to 29 wherein the
membrane-forming lipid consists essentially of phospholipid and
predominantly comprises molecules which individually have an
overall net charge.
31. A process as claimed in any of claims 23 to 30 wherein the
lipid-stabilised dispersion formed in step (i) is washed prior to
being lyophilised.
32. A process as claimed in any of claims 23 to 31 wherein the
lipid-stabilised dispersion is size fractionated prior to being
lyophilised.
33. A process as claimed in any of claims 23 to 32 wherein a
cryoprotectant and/or lyoprotectant is added to the
lipid-stabilised dispersion formed in step (i) prior to it being
lyophilised.
34. A process as claimed in claim 33 wherein said cryoprotectant
and/or lyoprotectant is selected from alcohols, polyols,
aminoacids, carbohydrates and polyglycols.
35. A process as claimed in claim 34 wherein said cryoprotectant
and/or lyoprotectant is a physiologically tolerated sugar.
36. A process as claimed in any of claims 23 to 35 wherein said
dried product is reconstituted by hand-shaking the product in the
carrier liquid.
37. A lyophilised residue of a suspension of gas microbubbles in an
amphiphilic material-containing aqueous medium wherein the
amphiphilic material consists essentially of phospholipid
predominantly comprising molecules which individually have an
overall net charge.
38. A lyophilised residue according to claim 37 wherein at least
75% of the phospholipid consists of molecules which individually
have an overall net charge.
39. A lyophilised residue according to claim 38 wherein
substantially all of the phospholipid consists of molecules which
individually have an overall net charge.
40. A lyophilised residue according to any of claims 37 to 39
wherein the phospholipid is selected from naturally occurring,
semisynthetic and synthetic phosphatidylserines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids,
cardiolipins, lyso forms of any of the foregoing and mixtures of
any of the foregoing.
41. A lyophilised residue according to claim 40 wherein acyl groups
present in the phospholipid each contain about 14-22 carbon
atoms.
42. A lyophilised residue according to claim 40 or claim 41 wherein
one or more phosphatidylserines constitute at least 70% of the
phospholipid.
43. A lyophilised residue according to claim 42 wherein said
phosphatidylserine or phosphatidylserines are selected from
synthetic phosphatidylserine, semisynthetic phosphatidylserine,
hydrogenated natural phosphatidylserine, hydrogenated semisynthetic
phosphatidylserine, synthetic distearoylphosphatidylserine,
synthetic dipalmitoylphosphatidylserine and synthetic
diarachidoylphosphatidylserin- e.
44. A lyophilised residue according to any of claims 37 to 43
wherein the gas is selected from air, nitrogen, oxygen, carbon
dioxide, hydrogen, nitrous oxide, inert gases, sulphur fluorides,
selenium hexafluoride, silanes, halogenated silanes, low molecular
weight hydrocarbons, halogenated low molecular weight hydrocarbons,
ethers, ketones, esters and mixtures of any of the foregoing.
45. A lyophilised residue according to claim 44 wherein the gas
comprises sulphur hexafluoride or a fluorinated low molecular
weight hydrocarbon.
46. A lyophilised residue according to claim 45 wherein said
hydrocarbon is perfluorinated.
47. A lyophilised residue according to claim 46 wherein said
perfluorinated hydrocarbon comprises perfluoropropane,
perfluorobutane or perfluoropentane.
48. A lyophilised residue according to any of claims 37 to 47
derived from a size fractionated microbubble suspension.
49. A microbubble-releasing matrix containing gas-filled
substantially spherical cavities or vacuoles surrounded by layers
of membrane-forming lipid material.
50. A matrix according to claim 49 wherein the matrix structural
material is a carbohydrate.
51. A matrix according to claim 49 or claim 50 wherein the
membrane-forming lipid material comprises at least one
phospholipid.
52. A matrix according to any of claims 49 to 51 wherein the
membrane-forming lipid material consists essentially of
phospholipid predominantly comprising molecules which individually
have an overall net charge.
53. A matrix according to claim 52 wherein at least 75% of the
phospholipid consists of molecules which individually have an
overall net charge.
54. A matrix according to claim 53 wherein substantially all of the
phospholipid consists of molecules which individually have an
overall net charge.
55. A matrix according to any of claims 52 to 54 wherein the
phospholipid is selected from naturally occurring, semisynthetic
and synthetic phosphatidylserines, phosphatidylglycerols,
phosphatidylinositols, phosphatidic acids, cardiolipins, lyso forms
of any of the foregoing and mixtures of any of the foregoing.
56. A matrix according to claim 55 wherein acyl groups present in
the phospholipid each contain about 14-22 carbon atoms.
57. A matrix according to claim 55 or claim 56 wherein one or more
phosphatidylserines constitute at least 70% of the
phospholipid.
58. A matrix according to claim 57 wherein said phosphatidylserine
or phosphatidylserines are selected from synthetic
phosphatidylserine, semisynthetic phosphatidylserine, hydrogenated
natural phosphatidylserine, hydrogenated semisynthetic
phosphatidylserine, synthetic distearoylphosphatidylserine,
synthetic dipalmitoylphosphatidyl- serine and synthetic
diarachidoylphosphatidylserine.
59. A matrix according to any of claims 49 to 58 wherein the gas is
selected from air, nitrogen, oxygen, carbon dioxide, hydrogen,
nitrous oxide, inert gases, sulphur fluorides, selenium
hexafluoride, silanes, halogenated silanes, low molecular weight
hydrocarbons, halogenated low molecular weight hydrocarbons,
ethers, ketones, esters and mixtures of any of the foregoing.
60. A matrix according to claim 59 wherein the gas comprises
sulphur hexafluoride or a fluorinated low molecular weight
hydrocarbon.
61. A matrix according to claim 60 wherein said hydrocarbon is
perfluorinated.
62. A matrix according to claim 61 wherein said perfluorinated
hydrocarbon comprises perfluoropropane, perfluorobutane or
perfluoropentane.
63. A frozen microbubble-releasing aqueous dispersion comprising
gas microbubbles stabilised by amphiphilic material comprising at
least one phospholipid.
64. A frozen microbubble dispersion according to claim 63 wherein
said amphiphilic material consists essentially of phospholipid
predominantly comprising molecules which individually have an
overall net charge.
65. A size fractionated frozen microbubble dispersion according to
claim 63 or claim 64.
66. A frozen microbubble dispersion according to claim 64 or claim
65 wherein at least 75% of the phospholipid consists of molecules
which individually have an overall net charge.
67. A frozen microbubble dispersion according to claim 66 wherein
substantially all of the phospholipid consists of molecules which
individually have an overall net charge.
68. A frozen microbubble dispersion according to any of claims 64
to 67 wherein the phospholipid is selected from naturally
occurring, semisynthetic and synthetic phosphatidylserines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids,
cardiolipins, lyso forms of any of the foregoing and mixtures of
any of the foregoing.
69. A frozen microbubble dispersion according to claim 68 wherein
acyl groups present in the phospholipid each contain about 14-22
carbon atoms.
70. A frozen microbubble dispersion according to claim 68 or claim
69 wherein one or more phosphatidylserines constitute at least 70%
of the phospholipid.
71. A frozen microbubble dispersion according to claim 70 wherein
said phosphatidylserine or phosphatidylserines are selected from
synthetic phosphatidylserine, semisynthetic phosphatidylserine,
hydrogenated natural phosphatidylserine, hydrogenated semisynthetic
phosphatidylserine, synthetic distearoylphosphatidylserine,
synthetic dipalmitoylphosphatidylserine and synthetic
diarachidoylphosphatidylserin- e.
72. A frozen microbubble dispersion according to any of claims 63
to 71 wherein the gas is selected from air, nitrogen, oxygen,
carbon dioxide, hydrogen, nitrous oxide, inert gases, sulphur
fluorides, selenium hexafluoride, silanes, halogenated silanes, low
molecular weight hydrocarbons, halogenated low molecular weight
hydrocarbons, ethers, ketones, esters and mixtures of any of the
foregoing.
73. A frozen microbubble dispersion according to claim 72 wherein
the gas comprises sulphur hexafluoride or a fluorinated low
molecular weight hydrocarbon.
74. A frozen microbubble dispersion according to claim 73 wherein
said hydrocarbon is perfluorinated.
75. A frozen microbubble dispersion according to claim 74 wherein
said perfluorinated hydrocarbon comprises perfluoropropane,
perfluorobutane or perfluoropentane.
76. A microbubble-containing contrast agent prepared by a process
as claimed in any of claims 24 to 26 wherein the membrane-forming
lipid consists essentially of phospholipid and predominantly
comprises molecules which individually have an overall net charge,
characterised in that the microbubbles exhibit at least 90%
recovery of size distribution and echogenic properties following
exposure to an overpressure of 300 mm Hg for 90 seconds.
77. An aqueous dispersion of gas microbubbles stabilised by
amphiphilic material consisting essentially of phospholipid
predominantly comprising molecules which individually have an
overall net charge, said dispersion having been prepared by: i)
dispersing gas in an aqueous medium containing said phospholipid to
form a phospholipid-stabilised gas microbubble dispersion; ii)
lyophilising said dispersion to yield a dried
phospholipid-containing product; and iii) reconstituting said dried
product in an aqueous medium.
78. A lyophilised residue as claimed in claim 48 wherein the gas is
perfluorobutane and one or more phosphatidylserines constitute at
least 70% of the phospholipid.
79. A contrast agent composition comprising as a first component a
lyophilised residue as claimed in claim 78 and as a second
component an injectable aqueous carrier liquid, said first and
second components being contained respectively within first and
second chambers of dual chamber storage means.
80. An aqueous dispersion of gas microbubbles stabilised by
amphiphilic material consisting essentially of phospholipid
predominantly comprising molecules which individually have an
overall net charge.
81. A microbubble dispersion according to claim 80 wherein
substantially all of the phospholipid consists of molecules which
individually have an overall net negative charge.
82. A microbubble dispersion according to claim 81 wherein one or
more phosphatidylserines constitute at least 70% of the
phospholipid.
83. A microbubble dispersion according to claim 80 wherein the gas
comprises sulphur hexafluoride or a perfluorinated low molecular
weight hydrocarbon.
84. A microbubble dispersion according to claim 83 wherein the gas
is perfluorobutane.
85. An ultrasound contrast agent for use in diagnostic studies,
comprising a microbubble dispersion as claimed in claim 80 in an
injectable aqueous carrier liquid.
86. A process for the preparation of a contrast agent comprising
the steps: i) dispersing gas in an aqueous medium containing a
membrane-forming lipid to form a lipid-stabilised gas microbubble
dispersion; ii) lyophilising said dispersion in the presence of a
cryoprotectant and/or lyoprotectant to yield a dried product
comprising a cryoprotectant and/or lyoprotectant matrix containing
gas-filled substantially spherical cavities or vacuoles surrounded
by one or more layers of the membrane-forming lipid; and iii)
reconstituting said dried product in an aqueous injectable carrier
liquid.
87. A process as claimed in claim 86 wherein the gas employed in
step (i) is sulphur hexafluoride or a perfluorinated low molecular
weight hydrocarbon.
88. A process as claimed in claim 86 wherein the lipid-containing
aqueous medium employed in step (i) further contains one or more
additives selected from alcohols and polyols.
89. A process as claimed in claim 86 wherein the membrane-forming
lipid consists essentially of phospholipid and predominantly
comprises molecules which individually have an overall net
charge.
90. A process as claimed in claim 86 wherein, prior to step (ii),
the lipid-stabilised dispersion formed in step (i) is washed and
the microbubbles are size fractionated.
91. A process as claimed in claim 86 wherein the cryoprotectant
and/or lyoprotectant is a physiologically tolerated sugar.
92. A lyophilised residue of a microbubble dispersion as claimed in
claim 80.
93. A microbubble-releasing matrix comprising a matrix structural
material containing a plurality of gas-filled substantially
spherical cavities or vacuoles surrounded by layers of
membrane-forming lipid material.
94. A matrix according to claim 93 wherein the matrix structural
material is a carbohydrate.
95. A matrix according to claim 93 wherein the membrane-forming
lipid material consists essentially of phospholipid predominantly
comprising molecules which individually have an overall net
charge.
96. A matrix according to claim 95 wherein substantially all of the
phospholipid consists of molecules which individually have an
overall net negative charge.
97. A matrix according to claim 96 wherein one or more
phosphatidylserines constitute at least 70% of the
phospholipid.
98. A matrix according to claim 93 wherein the gas comprises
sulphur hexafluoride or a perfluorinated low molecular weight
hydrocarbon.
99. An ultrasound contrast agent composition comprising as a first
component a matrix as claimed in claim 97 wherein the matrix
material is sucrose and the gas is perfluorobutane, and as a second
component an injectable aqueous carrier liquid, said first and
second components being contained respectively within first and
second chambers of dual chamber storage means.
100. A method of diagnostic ultrasound imaging which comprises
administering to a subject a contrast-enhancing amount of an
ultrasound contrast agent as claimed in claim 85 and generating an
ultrasound image of at least a part of said subject.
Description
[0001] This invention relates to novel gas-containing contrast
agents of use in diagnostic imaging, more particularly to such
contrast agents comprising phospholipid-stabilised gas microbubbles
and to a novel method for the preparation of gas-containing
contrast agents.
[0002] It is well known that ultrasonic imaging comprises a
potentially valuable diagnostic tool, for example in studies of the
vascular system, particularly in cardiography, and of tissue
microvasculature. A variety of contrast agents has been proposed to
enhance the acoustic images so obtained, including suspensions of
solid particles, emulsified liquid droplets, gas bubbles and
encapsulated gases or liquids. It is generally accepted that low
density contrast agents which are easily compressible are
particularly efficient in terms of the acoustic backscatter they
generate, and considerable interest has therefore been shown in the
preparation of gas-containing and gas-generating systems.
[0003] Gas-containing contrast media are also known to be effective
in magnetic resonance (MR) imaging, e.g. as susceptibility contrast
agents which will act to reduce MR signal intensity.
Oxygen-containing contrast media also represent potentially useful
paramagnetic MR contrast agents.
[0004] Furthermore, in the field of X-ray imaging it has been
observed that gases such as carbon dioxide may be used as negative
oral contrast agents or intravascular contrast agents.
[0005] The use of radioactive gases, e.g. radioactive isotopes of
inert gases such as xenon, has also been proposed in scintigraphy,
for example for blood pool imaging.
[0006] Initial studies involving free gas bubbles generated in vivo
by intracardiac injection of physiologically acceptable substances
have demonstrated the potential efficiency of such bubbles as
contrast agents in echography; such techniques are severely limited
in practice, however, by the short lifetime of the free bubbles.
Interest has accordingly been shown in methods of stabilising gas
bubbles for echocardiography and other ultrasonic studies, for
example using emulsifiers, oils, thickeners or sugars, or by
entraining or encapsulating the gas or a precursor therefor in a
variety of systems, e.g. as porous gas-containing microparticles or
as encapsulated gas microbubbles.
[0007] There is a body of prior art regarding use of phospholipids
as components of gas-containing ultrasound contrast agents. Thus,
for example, the use as ultrasound contrast media of phospholipid
liposomes in which a lipid bilayer surrounds a confined composition
including a gas or gas precursor is disclosed in U.S. Pat. No.
4,900,540. The encapsulated material is typically a gas precursor
such as aqueous sodium bicarbonate, which is said to generate
carbon dioxide following administration through exposure to body
pH. The cores of the resulting liposomes will therefore tend to
comprise liquid containing extremely small microbubbles of gas
which will exhibit only limited echogenicity by virtue of their
small size.
[0008] WO-A-9115244 discloses ultrasound contrast media comprising
microbubbles of air or other gas formed in a suspension of
liquid-filled liposomes, the liposomes apparently stabilising the
microbubbles. Such systems are differentiated from those of the
above-mentioned U.S. Pat. No. 4,900,540 in which the air or other
gas is inside the liposomes.
[0009] WO-A-9211873 describes aqueous preparations designed to
absorb and stabilise microbubbles and thereby serve as ultrasound
contrast agents, the compositions comprising
polyoxyethylene/polyoxypropylene polymers and negatively charged
phospholipids. The weight ratio of polymer to phospholipid is
typically about 3:1.
[0010] Ultrasound contrast agents comprising gas-filled liposomes,
i.e. liposomes which are substantially devoid of liquid in the
interior thereof, and their preparation by a vacuum drying gas
instillation method are described in WO-A-9222247. The preparation
of such gas-filled liposomes by a gel state shaking gas
instillation method is described in WO-A-9428780. A report on
gas-filled lipid bilayers composed of
dipalmitoylphosphatidylcholine as ultrasound contrast agents is
presented by Unger et al. in Investigative Radiology 29, Supplement
2, S134-S136 (1994).
[0011] WO-A-9409829 discloses injectable suspensions of gas
microbubbles in an aqueous carrier liquid comprising at least one
phospholipid stabiliser, the concentration of phospholipids in the
carrier being less than 0.01% w/w but equal to or above the amount
at which phospholipid molecules are present solely at the gas
microbubble-liquid interface. The amount of phospholipid may
therefore be as low as that necessary for formation of a single
monolayer of surfactant around the gas microbubbles, the resulting
film-like structure stabilising the bubbles against collapse or
coalescence. Microbubbles with a liposome-like surfactant bilayer
are said not to be obtained when such low phospholipid
concentrations are used.
[0012] A further body of prior art concerns selection of gases for
gas microbubble-containing ultrasound contrast media in order to
enhance properties such as their stability and duration of
echogenic effect. Thus, for example, WO-A-9305819 proposes use of
free microbubbles of gases having a coefficient Q greater than 5
where
Q=4.0.times.10.sup.-7.times.p/C.sub.sD
[0013] (where p is the density of the gas in kg.m.sup.-3, C.sub.s
is the water solubility of the gas in moles.l.sup.-1 and D is the
diffusivity of the gas in solution in cm.sup.3.sec.sup.-1). An
extensive list of gases said to fulfill this requirement is
presented.
[0014] EP-A-0554213 suggests that one may impart resistance against
collapse under pressure to gas-filled microvesicles by introduction
thereto of at least one gas whose solubility in water, expressed in
liters of gas/liters of water under standard conditions, divided by
the square root of its molecular weight does not exceed 0.003.
Preferred gases are said to include sulphur hexafluoride, selenium
hexafluoride and various Freons.RTM.. Such gases may, inter alia,
be used in phospholipid-containing compositions of the type
described in the above-mentioned WO-A-9215244.
[0015] Schneider et al. in Investigative Radiology 30(8),
pp.451-457 (1995) describe a new ultrasonographic contrast agent
based on sulphur hexafluoride-filled microbubbles apparently
stabilised by a combination of polyethyleneglycol 4000 and a
mixture of the phospholipids distearoylphosphatidylcholine and
dipalmitoylphosphatidylglycerol. The use of sulphur hexafluoride
rather than air is said to provide improved resistance to pressure
increases such as occur in the left heart during systole.
[0016] WO-A-9503835 proposes use of microbubbles containing a gas
mixture the composition of which is based on considerations of gas
partial pressures both inside and outside the microbubbles, so as
to take account of osmotic effects on microbubble size.
Representative mixtures comprise a gas having a low vapour pressure
and limited solubility in blood or serum (e.g. a fluorocarbon) in
combination with another gas which is more rapidly exchanged with
gases present in normal blood or serum (e.g. nitrogen, oxygen,
carbon dioxide or mixtures thereof).
[0017] WO-A-9516467 suggests use of ultrasound contrast media
containing a mixture of gases A and B, where gas B is present in an
amount of 0.5-41% v/v, has a molecular weight greater than 80
daltons and has aqueous solubility below 0.0283 ml/ml water under
standard conditions, the balance of the mixture being gas A.
Representative gases A include air, oxygen, nitrogen, carbon
dioxide and mixtures thereof. Representative gases B include
fluorine-containing gases such as sulphur hexafluoride and various
perfluorinated hydrocarbons. Preferred stabilisers in such contrast
media include phospholipids.
[0018] Phospholipids said to be useful in prior art contrast agents
include lecithins (i.e. phosphatidylcholines), for example natural
lecithins such as egg yolk lecithin or soya bean lecithin and
synthetic or semisynthetic lecithins such as
dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or
distearoylphosphatidylcholine; phosphatidic acids;
phosphatidylethanolamines; phosphatidylserines;
phosphatidylglycerols; phosphatidylinositols; cardiolipins;
sphingomyelins; mixtures of any of the foregoing and mixtures with
other lipids such as cholesterol. Lecithin derivatives generally
appear to be the most commonly used phospholipids, possibly by
virtue of their ready availability from natural sources. The use of
additives such as cholesterol in amounts of up to 50% w/w is
disclosed in WO-A-9115244 and WO-A-9409829, whilst the
incorporation of at least a small amount (e.g. ca. 1 mole % of
negatively charged lipid (e.g. phosphatidylserine or a fatty acid)
to enhance stability is suggested in WO-A-9222247. A preferred
phospholipid composition according to WO-A-9428780 comprises
dipalmitoylphosphatidylcholine, polyethylene-glycol
5000-substituted dipalmitoylphosphatidylethanol-amine and
dipalmitoylphosphatidic acid in molar proportions of about 87:8:5.
Typical mixed phospholipid compositions according to WO-A-9409829
and WO-A-9516467 comprise diarachidoylphosphatidylcholine and
dipalmitoylphosphatidic acid in weight proportions of about 100:4,
although the latter specification also exemplifies use of equal
amounts by weight of distearoylphosphatidylcholi- ne and
dipalmitoylphosphatidylglycerol.
[0019] It will be apparent from the foregoing that in existing
phospholipid-containing microbubble suspensions proposed for use as
contrast media, at least 50% of the phospholipid content comprises
neutral phospholipids such as lecithins. Most commonly only a minor
proportion, e.g. ca. 5%, of charged phospholipids is present.
[0020] The present invention is based on the finding that the use
of predominantly charged phospholipids as essentially the sole
amphiphilic component of microbubble-containing contrast agents may
convey valuable and unexpected benefits in terms of parameters such
as product stability and acoustic properties. Whilst we do not wish
to be bound by theoretical considerations it is believed that
electrostatic repulsion between charged phospholipid membranes
encourages the formation of stable and stabilising monolayers at
microbubble-carrier liquid interfaces; the flexibility and
deformability of such thin membranes will enhance the echogenicity
of products according to the invention relative to gas-filled
liposomes comprising one or more lipid bilayers.
[0021] We have also found that the use of charged phospholipids may
enable the provision of microbubble contrast agents with
advantageous properties regarding, for example, stability,
dispersibility and resistance to coalescence without recourse to
additives such as further surfactants and/or viscosity enhancers,
thereby ensuring that the number of components administered to the
body of a subject upon injection of the contrast agents is kept to
a minimum. Thus, for example, the charged surfaces of the
microbubbles may minimise or prevent their aggregation as a result
of electrostatic repulsion.
[0022] Thus, according to one embodiment of the present invention,
there is provided a contrast agent for use in diagnostic studies
comprising a suspension in an injectable aqueous carrier liquid of
gas microbubbles stabilised by phospholipid-containing amphiphilic
material characterised in that said amphiphilic material consists
essentially of phospholipid predominantly comprising molecules with
net charges.
[0023] Desirably at least 75%, preferably substantially all of the
phospholipid material in the contrast agents of the invention
consists of molecules bearing a net overall charge under conditions
of preparation and/or use, which charge may be positive or, more
preferably, negative. Representative positively charged
phospholipids include esters of phosphatidic acids such as
dipalmitoylphosphatidic acid or distearoylphosphatidic acid with
aminoalcohols such as hydroxyethylenediamine. Examples of
negatively charged phospholipids include 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 cardiolipins. The fatty acyl groups of such phospholipids will
typically each contain about 14-22 carbon atoms, for example as in
palmitoyl and stearoyl groups. Lyso forms of such charged
phospholipids are also useful in accordance with the invention, the
term "lyso" denoting phospholipids containing only one fatty acyl
group, this preferably being ester-linked to the 1-position carbon
atom of the glyceryl moiety. Such lyso forms of charged
phospholipids may advantageously be used in admixture with charged
phospholipids containing two fatty acyl groups.
[0024] Phosphatidylserines represent particularly preferred
phospholipids of use in contrast agents according to the invention
and preferably constitute a substantial part, e.g. at least 80% of
the initial phospholipid content thereof, for example 85-92%,
although this may subsequently be reduced somewhat, e.g. to ca.
70%, in subsequent processing such as heat sterilisation. It will
be appreciated that such processing may lead to formation of
non-phospholipid degradation products such as free fatty acids,
e.g. at levels of up to 10%; references herein to amphiphilic
material consisting essentially of phospholipid are to be construed
as embracing phospholipids containing such free fatty acids. While
we do not wish to be bound by theoretical considerations, it may be
that ionic bridging between the carboxyl and amino groups of
adjacent serine moieties contributes to the stability of
phosphatidylserine-contai- ning systems, for example as evidenced
by their good pressure stability. Preferred phosphatidylserines
include saturated (e.g. hydrogenated or synthetic) natural
phosphatidylserine and synthetic or semi-synthetic
dialkanoylphosphatidylserines such as distearoylphosphatidylserine,
dipalmitoylphosphatidylserine and
diarachidoylphosphatidylserine.
[0025] An important advantage of the use of such
phosphatidylserine-based contrast agents is that the body
recognises aged red blood cells and platelets by high
concentrations of phosphatidylserine on their surface and so may
eliminate such contrast agents from the blood stream in a manner
similar to the elimination of red blood cells. Furthermore, since
the surface of such contrast agents may be registered as endogenous
by the body, they may avoid induction of adverse systemic side
effects such as haemodynamic effects and other anaphylactic
reactions which may accompany administration of some liposome
preparations (see e.g. WO-A-9512386). In support of this, no acute
toxic effects such as changes in blood pressure or heart rate have
been observed in animal tests on dogs injected with intravenous
boluses of contrast agents according to the invention at doses of
up to ten times a normal imaging dose.
[0026] Any biocompatible gas may be employed in the contrast agents
of the invention, it being appreciated that the term "gas" as used
herein includes any substances (including mixtures) 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;
nitrous oxide; 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
tetramethylsilane; 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 cyclobutane or cyclopentane, an alkene such as propene or a
butene, or an alkyne such as acetylene; an 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. At least some of the halogen atoms in halogenated gases
advantageously are fluorine atoms. Thus biocompatible halogenated
hydrocarbon gases may, for example, be selected from
bromochlorodifluoromethane, chlorodifluoromethane,
dichlorodifluoromethane, bromotrifluoromethane,
chlorotrifluoromethane, chloropentafluoroethane,
dichlorotetrafluoroethane and perfluorocarbons, e.g.
perfluoroalkanes such as perfluoromethane, perfluoroethane,
perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane,
optionally in admixture with other isomers such as
perfluoroisobutane), perfluoropentanes, perfluorohexanes and
perfluoroheptanes; perfluoroalkenes such as perfluoropropene,
perfluorobutenes (e.g. perfluorobut-2-ene) and perfluorobutadiene;
perfluoroalkynes such as perfluorobut-2-yne; and
perfluorocycloalkanes such as perfluorocyclobutane,
perfluoromethylcyclobutane, perfluorodimethylcyclob- utanes,
perfluorotrimethylcyclobutanes, perfluorocyclopentane,
perfluoromethylcyclopentane, perfluorodimethylcyclopentanes,
perfluorocyclohexane, perfluoromethylcyclohexane and
perfluorocycloheptane. Other halogenated gases include fluorinated,
e.g. perfluorinated, ketones such as perfluoroacetone and
fluorinated, e.g. perfluorinated, ethers such as perfluorodiethyl
ether.
[0027] It may be advantageous in contrast agents of the invention
to employ fluorinated gases such as sulphur fluorides or
fluorocarbons (e.g. perfluorocarbons) which are known to form
particularly stable microbubble suspensions (see, for example, the
article by Schneider et al. referred to above). Gas mixtures based
on considerations of partial pressures both inside and outside the
microbubbles and consequent osmotic effects on microbubble size,
e.g. as described in WO-A-9503835, may if desired be employed, for
example a mixture of a relatively blood-soluble gas such as
nitrogen or air and a relatively blood-insoluble gas such as a
perfluorocarbon.
[0028] We have found, however, that contrast agents of the
invention, for example comprising microbubbles of a perfluoroalkane
such as perfluorobutane stabilised by phosphatidylserine, are
surprisingly stable in size following intravenous administration to
a subject, and do not exhibit the previously described tendency of
microbubbles of such gases to grow uncontrollably as a result of
inward diffusion of blood gases such as oxygen, nitrogen and carbon
dioxide, instead rapidly reaching a maximum size beyond which
further growth is not observed. This avoidance of unlimited size
increases which could lead to undesirable and potentially highly
dangerous blocking of blood vessel capilliaries is a major
advantage of contrast agents according to the invention.
[0029] Contrast agents of the invention comprising perfluoroalkanes
such as perfluorobutane have also been found to exhibit
surprisingly high stability under pressures similar to those
typically encountered in vivo, for example showing substantially
complete (e.g. at least 90%) recovery to normal size distribution
and echogenic properties after exposure to overpressures (e.g. of
air) of up to 300 mm Hg for 90 seconds.
[0030] The contrast agents of the invention may be used in a
variety of diagnostic imaging techniques, including scintigraphy,
light imaging, ultrasound, MR and X-ray (including soft X-ray)
imaging. Their use in diagnostic ultrasound imaging and in MR
imaging, e.g. as susceptibility contrast agents, constitute
preferred features of the invention. A variety of imaging
techniques may be employed in ultrasound applications, for example
including fundamental and harmonic B-mode imaging and fundamental
and harmonic Doppler imaging; if desired three-dimensional imaging
techniques may be used. The contrast agent may also be used in
ultrasound imaging methods based on correlation techniques, for
example as described in U.S. Pat. No. 5,601,085 and International
Patent Application No. PCT/GB96/02413.
[0031] In vivo ultrasound tests in dogs have shown that contrast
agents according to the invention may produce an increase in
backscattered signal intensity from the myocardium of 15-25 dB
following intravenous injection of doses as low as 1-20 nl
microbubbles/kg body weight. Signals may be observed at even lower
doses using more sensitive techniques such as colour Doppler or
Doppler-derived techniques, e.g. amplitude based Doppler or
non-linear techniques such as are described by Tucker et al. in
Lancet (1968) p. 1253, by Miller in Ultrasonics (1981) pp. 217-224,
and by Newhouse et al. in J. Acoust. Soc. Am. 75, pp. 1473-1477
(1984). At these low doses attenuation in blood-filled compartments
such as the heart chambers has been found to be sufficiently low to
permit visualisation of regions of interest in the myocardial
vasculature. Tests have also shown such intravenously injected
contrast agents to be distributed throughout the whole blood pool,
thereby enhancing the echogenicity of all vascularised tissues, and
to be recirculated. They have also been found useful as general
Doppler signal enhancement aids, and may additionally be useful in
ultrasound computed tomography and in physiologically triggered or
intermittent imaging.
[0032] For ultrasound applications such as echocardiography, in
order to permit free passage through the pulmonary system and to
achieve resonance with the preferred imaging frequencies of about
0.1-15 MHz, it may be convenient to employ microbubbles having an
average size of 0.1-10 .mu.m, e.g. 1-7 .mu.m. We have found that
contrast agents according to the invention may be produced with a
very narrow size distribution for the microbubble dispersion within
the range preferred for echocardiography, thereby greatly enhancing
their echogenicity as well as their safety in vivo, and rendering
the contrast agents of particular advantage in applications such as
blood pressure measurements, blood flow tracing and ultrasound
tomography. Thus, for example, products in which over 90% (e.g. at
least 95%, preferably at least 98%) of the microbubbles have
diameters in the range 1-7 .mu.m and less than 5% (e.g. not more
than 3%, preferably not more than 2%) of the microbubbles have
diameters above 7 .mu.m may readily be prepared.
[0033] In ultrasound applications the contrast agents of the
invention may, for example, be administered in doses such that the
amount of phospholipid injected is in the range 0.1-10 .mu.g/kg
body weight, e.g. 1-5 .mu.g/kg in the case of fundamental B-mode
imaging. It will be appreciated that the use of such low levels of
phospholipid is of substantial advantage in minimising possible
toxic side effects. Furthermore, the low levels of phospholipids
present in effective doses may permit dosage increases to prolong
observation times without adverse effects.
[0034] The overall concentration of phospholipid in injectable
forms of contrast agents according to the invention may
conveniently be in the range 0.01-2% w/w, for example 0.2-0.8% w/w,
advantageously about 0.5% w/w.
[0035] In general we have found it unnecessary to incorporate
additives such as emulsifying agents and/or viscosity enhancers
which are commonly employed in many existing contrast agent
formulations into contrast agents of the invention. As noted above
this is of advantage in keeping to a minimum the number of
components administered to the body of a subject and ensuring that
the viscosity of the contrast agents is as low as possible. Since
preparation of the contrast agents typically involves a freeze
drying step as discussed in further detail hereinafter it may,
however, be advantageous to include one or more agents with
cryoprotective and/or lyoprotective effect and/or one or more
bulking agents, for example an alcohol, e.g. an aliphatic alcohol
such as t-butanol; a polyol such as glycerol; an aminoacid such as
glycine; a carbohydrate, e.g. a sugar such as sucrose, mannitol,
trehalose, glucose, lactose or a cyclodextrin, or a polysaccharide
such as dextran; or a polyglycol such as polyethylene glycol. A
substantial list of agents with cryoprotective and/or lyoprotective
effects is given in Acta Pharm. Technol. 34(3), pp. 129-139 (1988),
the contents of which are incorporated herein by reference. The use
of physiologically well-tolerated sugars such as sucrose, e.g. in
an amount such as to render the product isotonic or somewhat
hypertonic, is preferred.
[0036] Prior art microbubble-containing contrast agents, for
example as described in WO-A-9409829, are typically prepared by
contacting powdered surfactant, e.g. freeze-dried preformed
liposomes or freeze-dried or spray-dried phospholipid solutions,
with air or other gas and then with aqueous carrier, agitating to
generate a microbubble suspension which must then be administered
shortly after its preparation. Such processes, however, suffer the
disadvantages that substantial agitational energy must be imparted
to generate the required dispersion and that the size and size
distribution of the microbubbles are dependent on the amount of
energy applied and so cannot in practice be controlled.
[0037] We have now found that contrast agents according to the
invention may advantageously be prepared by generating a gas
microbubble dispersion in an appropriate phospholipid-containing
aqueous medium, which may if desired previously have been
autoclaved or otherwise sterilised, and thereafter subjecting the
dispersion to lyophilisation to yield a dried reconstitutable
product. Such products, e.g. comprising the lyophilised residue of
a suspension of gas microbubbles in an amphiphilic
material-containing aqueous medium wherein the amphiphilic material
consists essentially of phospholipid predominantly comprising
molecules with net charges, constitute a further feature of the
present invention. Where the dried product contains one or more
cryoprotective and/or lyoprotective agents it may, for example,
comprise a microbubble-releasing cryoprotectant and/or
lyoprotectant (e.g. carbohydrate) matrix containing gas-filled
substantially spherical cavities or vacuoles surrounded by one or
more layers of the amphiphilic material.
[0038] More particularly we have found that dried products so
prepared are especially readily reconstitutable in aqueous media
such as water, an aqueous solution such as saline (which may
advantageously be balanced so that the final product for injection
is not hypotonic), or an aqueous solution of one or more
tonicity-adjusting substances such as salts (e.g. of plasma cations
with physiologically tolerable counterions), or sugars, sugar
alcohols, glycols and other non-ionic polyol materials (e.g.
glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene
glycols, propylene glycols and the like) requiring only minimal
agitation such as may, for example, be provided by gentle
hand-shaking. The size of the microbubbles so generated is
consistently reproducible and in practice is independent of the
amount of agitational energy applied, being determined by the size
of the microbubbles formed in the initial microbubble dispersion,
this size parameter surprisingly being substantially maintained in
the lyophilised and reconstituted product. Thus, since the size of
the microbubbles in the initial dispersion may readily be
controlled by process parameters such as the method, speed and
duration of agitation, the final microbubble size may readily be
controlled.
[0039] Lyophilised products according to the invention have proved
to be storage stable for several months under ambient conditions.
The microbubble dispersions generated upon reconstitution in water
or an aqueous solution may be stable for at least 12 hours,
permitting considerable flexibility as to when the dried product is
reconstituted prior to injection.
[0040] The above-described process for the preparation of contrast
agents according to the invention is generally applicable to the
preparation of contrast agents comprising suspensions in an
injectable aqueous carrier liquid of gas microbubbles stabilised by
membrane-forming lipids, including both neutral and charged lipids
(e.g. phospholipids) as well as mixtures thereof. Such a process,
comprising the steps:
[0041] i) generating a dispersion of gas microbubbles in an aqueous
medium containing a membrane-forming lipid;
[0042] ii) lyophilising the thus-obtained lipid-stabilised gas
dispersion to yield a dried lipid-containing product; and
[0043] iii) reconstituting the said dried product in an injectable
aqueous carrier liquid, constitutes a further feature of the
present invention, as does a reconstitutable dried product
obtainable in accordance with steps (i) and (ii) of this process,
for example a product comprising a microbubble-releasing matrix
(e.g. of cryoprotectant/lyoprotectant) containing gas-filled
substantially spherical cavities or vacuoles surrounded by layers
of membrane-forming lipid material.
[0044] Step (i) may, for example, be effected by subjecting the
lipid-containing aqueous medium to any appropriate
emulsion-generating technique, for example sonication, shaking,
high pressure homogenisation, high speed stirring or high shear
mixing, e.g. using a rotor-stator homogeniser, in the presence of
the selected gas. The aqueous medium may, if desired, contain
additives which serve as viscosity enhancers and/or as solubility
aids for the lipid, such as alcohols or polyols, e.g. glycerol
and/or propylene glycol.
[0045] The gas employed in the emulsification step need not be that
desired in the final product. Thus most of this gas content may be
removed during the subsequent lyophilisation step and residual gas
may be removed by evacuation of the dried product, to which an
atmosphere of the desired end product gas may then be applied. The
emulsification gas may therefore be selected purely to optimise the
emulsification process parameters, without regard to end product
considerations. We have found emulsification in the presence of a
sulphur fluoride such as sulphur hexafluoride or a fluorinated
hydrocarbon gas such as a perfluoroalkane or perfluorocycloalkane,
preferably containing 4 or 5 carbon atoms, to be particularly
advantageous in terms of ultimately yielding end products with
consistent and narrowly distributed microbubble sizes.
[0046] The emulsification is conveniently effected at about ambient
temperature, e.g. at ca. 25 .+-.10.degree. C. It may be necessary
initially to heat the aqueous medium to facilitate hydration and
thus dispersion of the phospholipid and then allow it to
equilibrate to ambient temperature prior to emulsification.
[0047] Gas dispersions obtainable according to step (i), especially
aqueous dispersions of gas microbubbles stabilised by amphiphilic
material consisting essentially of phospholipid predominantly
comprising molecules with net charges, constitute a feature of the
invention. Certain such dispersions are disclosed in our
International Patent Publication No. WO-A-9640275 as intermediates
for use in the preparation of diagnostic contrast agents comprising
microbubbles of gas stabilised by one or more membrane-forming
lipids crosslinked or polymerised in the hydrophilic portion
thereof. These intermediate dispersions, in which the amphiphilic
material comprises dipalmitoylphosphatidylserine, more particularly
in the form of its sodium salt, either alone or in combination with
dipalmitoylphosphatidylcholine, and the gas is a mixture of air
with perfluoropentane, a mixture of air with perfluorohexane or a
mixture of perfluorobutane with perfluorohexane, are hereby
disclaimed.
[0048] It will be appreciated that, by virtue of being
intermediates, these dispersions will not have been prepared in
sterile, physiologically acceptable form, whereas gas dispersions
obtainable according to step (i) in accordance with the present
invention will be prepared in sterile, physiologically acceptable
form (e.g. using sterile, pyrogen-free water or saline as the
aqueous carrier liquid) if they are intended for use as contrast
agents per se.
[0049] Dispersions produced according to step (i) may
advantageously be subjected to one or more washing steps prior to
contrast agent use or to lyophilisation step (ii), in order to
separate and remove additives such as viscosity enhancers and
solubility aids, as well as unwanted material such as
non-gas-containing colloidal particles and undersized and/or
oversized microbubbles; the washed microbubble dispersions so
obtained constitute a feature of the invention. Such washing may be
effected in per se known manner, the microbubbles being separated
using techniques such as flotation or centrifugation. The ability
to remove additives in this way and also to obtain microbubble
dispersions with a particularly narrow size distribution represent
important advantages of the process of the invention especially
since, as noted above, the resulting size distribution is
substantially retained after lyophilisation and reconstitution.
Accordingly it is particularly preferred to use a process
comprising gas dispersion, washing/separation, lyophilisation and
reconstitution steps.
[0050] Size-fractionated microbubble dispersions may be prepared
wherein at least 90% of the microbubbles have sizes within a 2
.mu.m range, the microbubbles preferably having a volume mean
diameter within the range 2-5 .mu.m. Such dispersions and frozen
and lyophilised products derived therefrom, e.g. as described
hereinafter, represent further features of the invention.
[0051] Where one or more cryoprotective and/or lyoprotective agents
are employed these may advantageously be added after the washing
steps, prior to lyophilisation.
[0052] Lyophilisation of the gas dispersion may, for example, be
effected by initially freezing it and thereafter lyophilising the
frozen gas dispersion, for example in per se generally known
manner. Such frozen gas dispersions, i.e. frozen
microbubble-releasing aqueous dispersions comprising gas
microbubbles stabilised by amphiphilic material consisting
essentially of phospholipid predominantly comprising molecules
which individually have an overall net charge, constitute a further
feature of the invention. The microbubbles may preferably be size
fractionated prior to freezing, the released microbubbles
preferably having a volume mean diameter within the range 2-5
.mu.m. Such products may be stored frozen and thawed when desired,
e.g. by simple warming and/or by addition of a carrier liquid, to
regenerate microbubble dispersions useful as contrast agents in
accordance with the invention.
[0053] Since the dried product will normally be reconstituted in
accordance with step (iii) above prior to administration, the gas
dispersion may advantageously be filled into sealable vials prior
to lyophilisation so as to give vials each containing an
appropriate amount, e.g. a single dosage unit, of lyophilised dried
product for reconstitution into an injectable form. By lyophilising
the gas dispersion in individual vials rather than in bulk,
handling of the delicate honeycomb-like structure of the
lyophilised product and the risk of at least partially degrading
this structure are avoided. Following lyophilisation and any
optional further evacuation of gas and introduction into the
headspace of gas desired to be present as microbubbles in the
ultimately formulated contrast agent, the vials may be sealed with
an appropriate closure. It will be appreciated that the ability to
select the end product gas content, coupled with the ability
independently to control the end product microbubble size by
selection of appropriate process parameters during the initial
dispersion step and any ensuing washing/separation step, enable the
independent selection of microbubble size and gas content, thereby
permitting the products to be matched to particular
applications.
[0054] In general the frozen gas dispersion or the dried product
from step (ii), e.g. after any necessary and/or desired
supplementation or exchange of gas content, may be reconstituted by
addition of an appropriate sterile aqueous injectable carrier
liquid such as sterile pyrogen-free water for injection, an aqueous
solution such as saline (which may advantageously be balanced so
that the final product for injection is not hypotonic), or an
aqueous solution of one or more tonicity-adjusting substances (e.g.
as hereinbefore described). Where the dried product is contained in
a vial this is conveniently sealed with a septum through which the
carrier liquid may be injected using an optionally prefilled
syringe; alternatively the dried product and carrier liquid may be
supplied together in a dual chamber device such as a dual chamber
syringe. It may be advantageous to mix or gently shake the product
following reconstitution. However, as noted above, in the
stabilised contrast agents according to the invention the size of
the gas microbubbles may be substantially independent of the amount
of agitational energy applied to the reconstituted dried product.
Accordingly no more than gentle hand-shaking may be required to
give reproducible products with consistent microbubble size.
[0055] The following non-limitative Examples serve to illustrate
the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0056] In the accompanying drawings:
[0057] FIG. 1 represents a plot of percentage survival of volume
concentration following lyophilisation and reconstitution against
relative amount of charged phospholipid in the membranes of
contrast agents according to Example 1;
[0058] FIG. 2 represents plots of attenuation spectra for the
frequency range 1.5-8 MHz of contrast agent according to Example
2(a) measured a) before pressure testing, b) during pressure
testing, and c) after pressure testing, as described in Example
6;
[0059] FIG. 3 shows the percentage recovery in attenuation at 3.5
MHz of contrast agent according to Example 2(a) following 90 second
applications of overpressures of 0-300 mm Hg as described in
Example 6; and
[0060] FIG. 4 shows volume size distributions for contrast agent
according to Example 2(a) measured by Coulter analysis a) without
application of overpressure (.diamond-solid.), b) after 90 seconds
of overpressure at 150 mm Hg (.DELTA.), and c) after 90 seconds of
overpressure at 300 mm Hg (.box-solid.), as described in Example
6.
EXAMPLE 1
[0061] Effects of relative amounts of charged phospholipids
[0062] Dispersions of microbubbles stabilised by different
phospholipids or phospholipid mixtures were made according to the
general procedure described below, using the process parameters
given in Table 1.1 below.
[0063] Solutions of the selected phospholipids or phospholipid
mixtures in water containing 5.4% (w/w) of a mixture of propylene
glycol and glycerol (3:10 w/w) giving a phospholipid concentration
of 2-5 mg/ml were prepared (for phosphatidylethanolamine the water
was adjusted to pH=10.5 with sodium hydroxide), the phospholipids
being hydrated by ultrasonic treatment and/or heating to
approximately 80.degree. C. for the stated time (Table 1.1) and
cooled to room temperature prior to use. A given volume of this
solution was divided between several 2 ml chromatography vials,
using 0.8-1 ml solution per vial. The head space of each vial was
filled with perfluorobutane gas, and the vials were securely capped
and shaken for 45 seconds using an Espe CapMix.RTM. (mixer for
dental materials). The resulting microbubble dispersions were
transferred to a larger vial and centrifuged at 2000 rpm for 5
minutes, giving a turbid infranatant below a floating layer of
microbubbles. The infranatant was removed by a syringe and replaced
with an equal volume of water at neutral pH. The washing step was
repeated, but now the infranatant was replaced by 10% (w/w)
sucrose. 2 ml portions of the washed dispersion were divided
between 10 ml flat-bottomed vials specially designed for
lyophilisation, and the vials were cooled to -47.degree. C. and
lyophilised for approximately 48 hours, giving a white fluffy solid
substance. The vials were transferred to a vacuum chamber, and air
was removed by a vacuum pump and replaced by perfluorobutane gas.
Prior to use, water was added and the vials were gently hand-shaken
for several seconds, giving microbubble dispersions suitable as
ultrasound contrast agents.
[0064] The size distribution and volume concentration of the
microbubbles were measured using a Coulter Counter Mark II
apparatus fitted with a 50 .mu.m aperture with a measuring range of
1-30 .mu.m. 20 .mu.l samples were diluted in 200 ml saline
saturated with air at room temperature and allowed to equilibrate
for 3 minutes prior to measurement. The measurements were made on
microbubble dispersions prior to lyophilisation (washed bubble
dispersion) and after lyophilisation (reconstituted with water to
the same volume as before lyophilisation). The data is presented in
Table 1.2 below.
[0065] The efficiency of lyophilisation for the different
phospholipid stabilised microbubble dispersions was calculated as
the percentage survival of the volume concentration following
lyophilisation and reconstitution. A plot (see FIG. 1 of the
drawings) shows how this parameter varies with the relative amount
of charged phospholipid in the membrane. As can be seen, the
efficiency of lyophilisation increases with increased amount of
charged phospholipid in the membrane, being highest for membranes
containing charged phospholipids only.
1TABLE 1.1 Composition and process parameters used in production of
phospholipid stabilised perfluoro-n-butane gas bubble dispersions
as described in Example 1 Amount Heat Vol. PLs and Amount aqueous
Sonication treat- Batch per ratios PL solvent bath ment size vial
(by weight) [mg/ml] [ml] [min] [min] [ml] [ml] DPPE 20 10 -- 30 10
0.8 H-PC/H-PS 45.5 9.1 10 2 9 0.9 (9:1) H-PC/H-PS 14.0 7 10 2 7 1
(4:1) DSPC/DSPS 10.4 5.2 10 2 4 1 (4:1) DSPC/DSPG 15.2 7.6 10 2 7 1
(1:1) DPPS 24.9 12.5 -- 30 11 1 DSPS 24.8 12.5 -- 30 11 1 DSPG/DPPA
20.2 10 -- 10 10 0.8 (10:1) DSPG/DPPA 52.0 10.4 -- 10 8 0.8 (1:1)
Legend: PL = phospholipid DPPE =
dipalmitoylphosphatidylethanolamine H-PC = hydrogenated egg
phosphatidylcholine H-PS = hydrogenated egg phosphatidylserine DSPC
= distearoylphosphatidylcholine DSPS = distearoylphosphatidylserine
DSPG = distearoylphosphatidylglycero- l DPPS =
dipalmitoylphosphatidylserine DPPA = dipalmitoylphosphatidic
acid.
[0066]
2TABLE 1.2 Yield measured as volume concentration of bubbles (in
percent of total dispersion volume) (i) after washing the
dispersion and (ii) after lyophilisation and reconstitution Amount
surviving Vol. conc. Vol. conc. lyophilis- PLs and % charged (%)
(%) ation ratios lipid in prior to after [% initial (by weight)
membrane lyophilisation lyophilisation vol. conc.] DPPE 0 0.7 0.1
16.4 H-PC/H-PS 10 6.4 0.9 14.1 (9:1) H-PC/H-PS 20 1.0 0.2 20.0
(4:1) DSPC/DSPS 20 4.8 1.0 20.8 (4:1) DSPC/DSPG 50 0.3 0.1 33.3
(1:1) DPPS 100 0.7 0.4 57.1 DSPS 100 1.0 0.5 50.0 DSPG/DPPA 100 1.4
0.7 52.9 (10:1) DSPG/DPPA 100 4.3 1.8 41.9 (1:1) Legend: See TABLE
1.1
EXAMPLE 2
[0067] a) Preparation of perfluorobutane microbubble dispersions by
shaking
[0068] 25.3 mg hydrogenated egg phosphatidylserine was added to
12.5 ml water containing 5.4% (w/w) of a mixture of propylene
glycol and glycerol (3:10 w/w). The phospholipid material was
hydrated by heating to 70.degree. C. for approximately 30 minutes,
followed by cooling to room temperature. 11 ml of the dispersion
was divided in 1 ml portions between eleven 2 ml vials, and the
head space of the vials was filled with perfluoro-n-butane gas. The
vials were securely capped and shaken for 45 seconds using an Espe
CapMix.RTM. (mixer for dental materials). The resulting microbubble
dispersions were combined in four larger vials and centrifuged at
2000 rpm for 5 minutes, giving a turbid infranatant below a
floating layer of microbubbles. The infranatant was removed by a
syringe and replaced with an equal volume of water at neutral pH.
The washing step was repeated, but now the infranatant was replaced
by 10% (w/w) sucrose. 2 ml portions of the resulting dispersion
were divided between 10 ml flat-bottomed vials specially designed
for lyophilisation, and the vials were cooled to -47.degree. C. and
lyophilised for approximately 48 hours, giving a white fluffy solid
substance. The vials were transferred to a vacuum chamber, and air
was removed by a vacuum pump and replaced by perfluoro-n-butane
gas. Prior to use, water was added and the vials were gently
hand-shaken for several seconds, giving microbubble dispersions
suitable as ultrasound contrast agents.
[0069] b) Preparation of perfluorobutane microbubble dispersions by
rotor stator mixing
[0070] 500.4 mg hydrogenated egg phosphatidylserine was added to
100 ml water containing 5.4% (w/w) of a mixture of propylene glycol
and glycerol (3:10 w/w). The mixture was shaken and heated to
80.degree. C. for five minutes, allowed to cool to room
temperature, shaken again and left standing overnight prior to
use.
[0071] 50 ml of the resulting solution was transferred to a
round-bottomed flask with a conical neck. The flask was fitted with
a glass jacket having a temperature control inlet and outlet
connected to a water bath maintained at 25.degree. C. A rotor
stator mixing shaft was introduced into the solution and to avoid
gas leakage the space between the neck wall and the mixing shaft
was sealed with a specially designed metal plug fitted with a gas
inlet/outlet connection for adjustment of gas content and pressure
control. The gas outlet was connected to a vacuum pump and the
solution was degassed for one minute. An atmosphere of
perfluoro-n-butane gas was then applied through the gas inlet.
[0072] The solution was homogenised at 23000 rpm for 10 minutes,
keeping the rotor stator mixing shaft such that the openings were
slightly above the surface of the liquid. A white coloured creamy
dispersion was obtained, which was transferred to a sealable
container and flushed with perfluoro-n-butane. The dispersion was
then transferred to a separating funnel and centrifuged at 12000
rpm for 30 minutes, yielding a creamy layer of bubbles at the top
and a turbid infranatant. The infranatant was removed and replaced
with water. The centrifugation was then repeated twice, but now at
12000 rpm for 15 minutes. After the last centrifugation, the
supernatant was replaced by 10% (w/w) sucrose. 2 ml portions of the
resulting dispersion were divided between 10 ml flat-bottomed vials
specially designed for lyophilisation, and the vials were cooled to
-47.degree. C. and lyophilised for approximately 48 hours, giving a
white fluffy solid substance. The vials were now transferred to a
vacuum chamber, and air was removed by a vacuum pump and replaced
by perfluoro-n-butane gas. Prior to use, water was added and the
vials were gently hand-shaken for several seconds, giving
microbubble dispersions suitable as ultrasound contrast agents.
[0073] c) Preparation of perfluorobutane microbubble dispersions by
sonication
[0074] 500.4 mg hydrogenated egg phosphatidylserine was added to
100 ml water containing 5.4% (w/w) of a mixture of propylene glycol
and glycerol (3:10 w/w). The mixture was shaken and heated to
80.degree. C. for five minutes, allowed to cool to room
temperature, shaken again and left standing overnight prior to
use.
[0075] This solution was pumped through a 4 ml sonicator
flow-through cell and exposed to ultrasound at 20 kHz with an
amplitude of 90 .mu.m. The diameter of the sonicator horn was 1.3
cm, the inner diameter of the cell was 2.1 cm and the distance
between the horn and the bottom of the cell was 1 cm. The lipid
solution was mixed with perfluoro-n-butane at a ratio of 1:2 v/v
before it entered the sonicator cell (20 ml/min lipid solution and
40 ml/min perfluoro-n-butane gas). The temperature was kept at
33.degree. C. A white and creamy dispersion was obtained which was
filled into a container and flushed with perfluoro-n-butane.
[0076] Characterisation
[0077] The size distribution and volume concentration of the
microbubbles were measured using a Coulter Counter Mark II
apparatus fitted with a 50 .mu.m aperture with a measuring range of
1-30 .mu.m. 20 .mu.l samples were diluted in 200 ml saline
saturated with air at room temperature, and allowed to equilibrate
for 3 minutes prior to measurement.
[0078] Ultrasound characterisation was performed on a experimental
set up slightly modified from de Jong, N. and Hoff, L. as described
in "Ultrasound scattering properties of Albunex microspheres",
Ultrasonics 31(3), pp. 175-181 (1993). This instrumentation
measures the ultrasound attenuation efficacy in the frequency range
2-8 MHz of a dilute suspension of contrast agent. During the
attenuation measurement a pressure stability test was performed by
exposing the sample to an overpressure of 120 mm Hg for 90 seconds.
Typically 2-3 .mu.l of sample was diluted in 55 ml Isoton II and
the diluted sample suspension was stirred for 3 minutes prior to
analysis. As primary response parameter the attenuation at 3.5 MHz
was used, together with the recovery attenuation value at 3.5 MHz
after release of the overpressure.
3TABLE 2.1 In vitro characteristics of bubble dispersions produced
according to Example 2(a)-(c) (number and volume weighted
concentrations and volume mean diameters, as well as acoustic
properties measured according to description above) Production
Number Vol. Atten. Survival after Freq. at method conc. Vol. mean
at over- max (Example [10.sup.6/ conc. diam. 3.5 Mhz pressure
atten. No.) ml] [%] [.mu.m] [dB/cm] [%] [MHz] 2(a) 1519 1.45 3.91
30.46 100 4.1 2(b) 10518 6.51 3.16 150.4 96 4.3 2(c) 23389 9.57
3.83 117 100 3.5
EXAMPLE 3
[0079] Effects of gas exchange
[0080] The gas contents of five samples prepared according to
Example 2(b) above were replaced with air, perfluorobutane, sulphur
hexafluoride, trifluoromethylsulphur pentafluoride and
tetramethylsilane respectively, according to the following
procedure:
[0081] Two samples containing lyophilised product from Example 2(b)
were placed in a desiccator having a gas inlet and a gas outlet.
The desiccator was connected to a Buchi 168 vacuum/distiller
controller which permitted controlled evacuation of the samples and
inlet of a selected gas. The samples were evacuated at
approximately 10 mbar for 5 minutes, whereafter the pressure was
increased to atmospheric by inlet of the selected gas, followed by
careful capping of the vials. The procedure was repeated using
further pairs of samples for each of the selected gases.
[0082] 2 ml distilled water was added to each vial and the vials
were gently hand-shaken prior to use. The resulting microbubble
dispersions were characterised with respect to size distribution
measurements as described in Example 2. The results are summarised
in Table 3.1.
4TABLE 3.1 In vitro characteristics of phosphatidylserine-
stabilised microbubble dispersions produced according to Example 3
- number and volume weighted concentrations and volume mean
diameters Number Vol. Number mean Vol. mean conc. diam. conc. diam.
Gas [10.sup.6/ml] [.mu.m] [%] [.mu.m] Perfluorobutane 9756 1.8 4.9
5.8 Trifluoromethyl- 10243 1.9 5.9 3.5 sulphur pentafluoride
Sulphur hexafluoride 9927 1.9 5.7 3.2 Tetramethylsilane 9947 1.9
6.1 3.7 Air 9909 1.9 6.4 4.0
[0083] As will be seen from the above results there was no
significant change in size distribution upon gas exchange.
[0084] In vivo results
[0085] One batch prepared with each of the five gases was evaluated
in vivo for Doppler enhancement properties at 10 MHz. The
dispersions were injected into chinchilla rabbits via an ear vein
and measured using a Doppler technique where an ultrasound probe
was placed directly on a carotid artery. Signal intensities and
duration were recorded and the integral of the Doppler curve was
calculated. The results obtained (see Table 3.2 below) showed that
microbubbles containing perfluorobutane gave the strongest Doppler
intensity enhancement. Microbubbles containing sulphur
hexafluoride, trifluoromethylsulphur pentafluoride or
tetramethylsilane were only slightly less efficacious as Doppler
enhancers than those containing perfluorobutane, giving integrals
in the range 60-80% of the figure for perfluorobutane.
5TABLE 3.2 Results for i.v. injections of Example 3 products into
rabbits (values are adjusted for drift in baseline; the Doppler
unit is defined as the increase in Doppler signal relative to that
of blood) Integrated Arterial Doppler Gas Enhancement (NDU.s)
Perfluorobutane* 10361 Trifluoromethylsulphur 8006 pentafluoride
Tetramethylsilane 6370 Sulphur hexafluoride 6297 Air 1024 *Average
of two injections
EXAMPLE 4
[0086] Frozen dispersions and lyophilised products
[0087] 250 mg hydrogenated egg phosphatidylserine was added to 50
ml water for injection containing 5.4% (w/w) of a mixture of
propylene glycol and glycerol (7:20 w/w). The mixture was shaken
and heated to 80.degree. C. for five minutes, allowed to cool to
room temperature, shaken again and left standing overnight prior to
use.
[0088] 100 ml of the resulting solution was transferred to a
round-bottomed flask with a conical neck and processed according to
the procedure described in Example 2(b). A white coloured creamy
dispersion was formed. This dispersion was transferred to a
separating funnel and centrifuged at 12000 rpm for 30 minutes,
yielding a creamy layer of microbubbles at the top and a turbid
infranatant. The infranatant was removed and replaced with 50 ml
water for injection. The centrifugation was then repeated twice,
but now at 12000 rpm for 15 minutes. To 6 ml of the resulting
dispersion was added 6 ml 30% (w/w) trehalose; 2 ml portions of
this dispersion were divided between 10 ml flat-bottomed vials
specially designed for lyophilisation, and the vials were cooled to
-47.degree. C. and stored at this temperature for one day.
[0089] Half of the vials were thawed after one day at -47.degree.
C., giving homogeneous creamy white dispersions of gas microbubbles
suitable as ultrasound contrast agents. The thawed dispersions were
characterised by measuring size distribution as described in
Example 2 above (see Table 4.1). The remaining vials were
lyophilised for approximately 48 hours, giving a white fluffy solid
substance. The vials were transferred to a vacuum chamber, and air
was removed by a vacuum pump and replaced by perfluoro-n-butane
gas. Prior to use, water was added and the vials were gently
hand-shaken for several seconds, giving bubble dispersions suitable
as ultrasound contrast agents. The reconstituted products were
characterised by measuring size distribution and acoustic
attenuation using the methods as described in Example 2 above. The
results are presented in Table 4.1.
6TABLE 4.1 Bubble concentration, size data and acoustic data of
perfluoro-n-butane gas bubble dispersions stabilised by
hydrogenated phosphatidylserine, treated by freeze- thawing and
lyophilisation Survival Vol. Atten. after Freq. Number Vol. mean at
over- at max. Sample conc. conc. diam. 3.5 Mhz pressure atten.
treatment [10.sup.6/ml] [%] [.mu.m] [dB/cm] [%] [MHz] Washed 10390
10.4 3.8 n.a. n.a. n.a. Freeze- 10142 9.9 3.6 n.a. n.a. n.a. thawed
Lyophilised 7780 4.6 3.1 58.0 89 5.3 Legend: n.a. = not
analysed
EXAMPLE 5
[0090] Exposure of perfluorobutane microbubble dispersion to
air-saturated fluid
[0091] A vial containing lyophilised material under an atmosphere
of perfluorobutane was prepared as described in Example 2(b). Water
was added to the vial just before use to give a microbubble
dispersion.
[0092] 200 ml Isoton II fluid was exposed to air for several days
at room temperature to give a fully air-saturated solution. Another
200 ml of the fluid was degassed in a vacuum flask at 60.degree. C.
for one hour and cooled to room temperature while maintaining the
vacuum. Air was admitted to the flask immediately prior to use.
[0093] 10 .mu.l portions of the microbubble suspension were added
to each of the fluids and the resulting mixtures were incubated for
5 minutes prior to size characterisation (Coulter Multisizer Mark
II).
[0094] In the degassed environment, where no diffusion of gases
from the fluid into the microbubbles would be expected, the mean
microbubble diameter was 1.77 .mu.m and 0.25% of the microbubbles
were larger than 5 .mu.m. In the air-saturated fluid the
corresponding values were 2.43 .mu.m and 0.67%; repeated
measurements made after a further 5 minutes indicated that the
microbubble sizes had reached a stable value.
[0095] These findings show that the average diameter of the
microbubbles increased by only 37% when they were exposed to an
air-saturated fluid analogous to arterial blood, with very few
microbubbles reaching a size which might cause blockage of
capillary blood vessels. This may be contrasted with the doubling
in size of air/perfluorohexane-containing microbubbles in a similar
environment (i.e. a highly diluted dispersion of microbubbles in
water containing dissolved air) reported in Example II of
WO-A-9503835.
EXAMPLE 6
[0096] Pressure stability of perfluorobutane microbubble
dispersion
[0097] Vials containing lyophilised material under an atmosphere of
perfluorobutane were prepared as described in Example 2(a). Water
(2 ml) was added to the vials just before use to give microbubble
dispersions.
[0098] Attenuation spectra were recorded for 1.5-8 MHz before,
during and after application of an overpressure of air at 300 mm
Hg; the pressure was released after 90 seconds. The results are
shown in FIG. 2 of the drawings, and indicate that although
attenuation at 4 MHz (the peak for unpressurised contrast agent)
fell to less than one third under pressure, it was almost fully
(85%-95%) restored when the pressure was released.
[0099] Overpressures of air at up to 300 mm Hg were applied for 90
seconds duration and attenuation was measured at 3.5 MHz. The
results are shown in FIG. 3 of the drawings and indicate good
recovery of attenuation (at least about 95%) following pressure
release for all the overpressures used.
[0100] Size distributions were determined by Coulter analysis for a
non-pressurised sample and for samples subjected to overpressures
of air at 150 and 300 mm Hg applied for durations of 90 seconds.
The results are shown in FIG. 4 of the drawings and indicate that
there were no significant differences between the distribution
curves in the range 1-10 .mu.m.
* * * * *