Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ 338~07
The invention relates to contrast media useful ~or
ultrasonic diagnostics made up of small gas bubbles and
microparticles; these media are characterized in that they
contain as the microparticles a mixture of at least one
(C10-C20)-fatty acid with at least one solid that is not a
surfactant, ~ucp~n~ in a liquid vehicle.
The ultrasonic examination of organs (sonography) is a
diagnostic method that has been popular and has been
practiced for several years. Ultrasonic waves in the
10 megahertz range (above 2 megahertz with wavelengths of
between 1 and 0.2 mm~ are reflected on interfaces of varlous
type3 of tissue. The thus-produced echoes are amplified and
made visible. In this connection, the examination of the
heart with the use of this method, called echocardiography,
is of special significance (Haft, J. I . et al . ~
Echocardiography", Futura, Mount Kisco, New York 1978;
Kohler, E., "Klinische Echokardiographie" [Clinical
Echocardiography], Enke, Stuttgart 1979: Stefan, G., et al.:
"Echokardiographie" [Echocardiography], Thieme, Stuttgart-
28 New York 1981; G Biamino, 1.. Lange: "Echokardiographie",
Hoechst AG, 1983).
Since liquids -- blood as well -- yield ultrasonic
contrast in the B-scan image only if there are differences
in density with respect to the surroundings, ~c
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~ ~ 338507
2 --
possibilities were investigated for making the blood and
its flow visible for ultrasonic B-scan image analysis.
This has indeed been made feasible by the addition of
extremely fine gas bubbles.
The blood flow can be imaged even without contrast
media by using, during ultrasonic investigation of the
heart or the vessels, the weak reflections of ultrasound
on the red blood corpuscles and by utilizing the so-
called doppler phenomenon. Elowever, in this method the
addition of small gas bubbles into the blood flow is
likewise advantageous because the stronger reflections
on the small gas bubbles permit better utilization of
the image [Z. Kardiol, 77: 227-232 (1988) ] .
Several methods for the production and
stabilization of the small gas bubbles have been known
from the literature. They can be produced, for example,
prior to in; ection into the bloodstream by vigorous
shaking or stirring of solutions, such as saline
solutions, dye solutions, or previously drawn blood.
Although ultrasonic contrast imaging has thereby
been attained, these methods display grave disadvantages
manifesting themselves in poor reproducibility, greatly
fluctuating size of the small gas bubbles, and -- due to
a proportion of visible, large bubbles -- a certain risk
of embolism.
These drawbacks have been overcome, in part, by
other manufacturing methods, such as, for example, by
the process disclosed in U.S. Patent 3,640,271 wherein
small bubbles of reproducible size are formed by
filtration or by the use of an electrode unit under
direct surrent. The advantage of having the possibility
of being able to prepare small gas bubbles of
reproducible size must be weighed against the
disadvantage of considerable technical expenditure.
U S. Patent 4,276,885 describes the production of
small gas bubbles having a de~inite size, these bubbles
- 3 - 1 3 ~ 8 5 ~ 7
being YuLL~-ul.ded by a gelatin envelop protecting against
coalescence. The finished bubbles can only be stored in
the "frozen" condition, for example by storage at
refrigerator temperature; for use, they must again be
brought to body temperature.
U.S. Patent 4,265,251 discloses the manufacture
and use of small gas bubbles with a solid ~uLL~ ull-iing
wall of saccharides which can be filled with a
pressurized gas. If the bubbles are under normal
~r~ ,uL~,they can be utilized as ultrasonic contrast
media; when using increased internal pressure, the
bubbles serve for measuring tke blood ~Les,.uLe.
Although here the storage of the solid gas bubbles does
not present a problem, t~rhn~ expenditure during
production represents a considerable cost factor.
The risks inherent in these and other contrast
media available in accordance with the state of the art
are evoked by two factoræ: size and number of the solid
particles as well a6 of the small gas bubbles.
The state of the art as r~is--llssl~d thus far permits
production of ultra60nic contrast media which in all
instances exhibit merely some of the required
properties:
~1) Elimination of risk of embolism
- small gas bubbles (size and number);
- solid particles (size and number).
( 2 ) Rep roduc ib il ity .
(3) Adequately long stability.
(4) Ability of passing through the lungs, for
example to obtain ultrasonic contrast of the
left portion of the heart.
(5) Ability to pa6s through capillaries.
(6) Sterility and freedom from pyrogen6 to be
displayed by the preparation.
(7) Ease of manufacture at tolerable cost.
(8) Storage without problems.
1 338507
European Patent Application, Publication No.
52575 ~C;~n;~ n patent 1,170, 569) does disclose the
production of small gas bubbles supposedly exhibiting
these necessary properties. For their manufacture,
microparticles of a solid crystalline ~ ~ollnfl, e.g.,
galactose, are suspended in a liquid vehicle; the gas
that is adsorbed on the particle surface is occluded in
cavities between the particles or in inter-crystalline
cavities, forming the gas bubbles. The resultant
10 suspension of small gas bubbles and microparticles is
in~ected within 10 minutes. Although it is asserted in
European Patent Application 52575 that the suspension
prepared according to the disclosed method is suitable
for appearing, after in~ ection into a peripheral vein,
on the right side of the heart as well as, after passing
through the lungs, in the left side of the heart, and
for rendering the blood and its flow visible at that
location upon ultrasonic examination, this assertion
collapses when subjected to analysis. Thus, it has been
20 found that the contrast medium produced according to the
method described in European Application 52575 and
in~ ected into a peripheral vein did not evoke any
ultrasonic echoes in the left portion of the heart.
EP-A-77752 (t~;~n;ltl; ;In patent l, l99, 577) likewise
discloses the preparation of a liquid mixture for use as
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~ 1 338507
contrzst medium consisting, in turn, of a mixture of atenside or an aqueous solution of the tenside, and an
aqueous, viscous carrier liquid.
In a p-lhl; ched European patent application
(Publication No. 0122624 (t~n~l;An patent 1,23g,092) ),
an agent enhancing ultrasonic contrast and containing
microparticles and small gas bubbles is described which
is suitable for ~nh~n~-i n~, after intravenous
administration and passage through the lungs, contrast
10 imaging of the left side of the heart, of the
myocardium, as well as other organs, such as the liver,
the spleen, and the kidneys. Although this application
also cites fatty acids [nsaturated or unsaturated (C4-
C20)-fatty acidsn] as being suitable for the production
of the microparticles, ~nnfi rr~tion by example has only
been provided for their esters or salts as surfactant
substances, such as, for example, ascorbyl palmitate or
sucrose monopalmitate. These, however, exhibit the
drawback that they are relatively quickly decomposed in
20 the formulation even when stored under normal
conditions (25C) (see the table below~. This is
deleterious in regard to a commercial preparation and
its purity requirements. The related imaging agent of
USP 4, 442, 843 is also deficient.
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~ l 338507
~ ccordingly, this invention provides, inter alia,
media as well as methods that do not exhibit this
disadvantage. This invention employs free fatty acids
Isaturated) as the surfactant c u--ds, instead of
their salts or esters, for the production of
microparticles used in contrast media. In this
connection, the fatty acids containing 10-20 carbon
atoms are especially suitable, such as, for example,
lauric, myristic, palmi-tic, stearic, or arachic acid,
10 or mixtures thereof. Thus, the ultrasound contrast
media are made up of small gas bubbles and micro-
particles, characterized in that they contain, as the
microparticles, a mixture of at least one (C10-C20)-fatty
acid with at least one solid that is not a surfactant,
suspended in a liquid vehicle.
The ultrasonic contrast media according to this
invention, obtained by suspending the microparticles of this
invention in a liquid vehicle, are capable of v~sl-sl1;7in~
for ultrasound, upon intravenous ad~inistration, the blood
20 and its flow characteristics not only on the right-hand side
of the heart, but also, after passing through the capillary
bed of the lungs, on the left-hand side of the heart.
Moreover, they surprisingly show a higher intensification
effect,
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- 6 - 1 3 3 8~ ~ 7
stability and better reproducibility of the ultrasonic
contrast than the ultrasonic contrast media of the prior
art .
TABLE -- STABILITY STUDY
Formulation: A Galactose + 0.134% Ascorbyl
Palmitate
B Galactose + 0.1% Palmitic Acid
Chemical Stability of Additives in Dependence on
Storage Temperature and Time
Storage Period F O R M U L A T I o N
A
% (m/m) Ascorbyl % (m/m) Pal-
p~l~nitate mitic Acid
St~rt 100% 10Q%
6 weeks
Room Temperature 84.3% Not analyzed
20 40rc 67.9% Not analyzed
50-C 33.6% 97.4%
12 weeks
Room Temperature 79 . 8% 98 . 0%
25 40 C 53 . 7% 98 . 4%
50 C 18 . 7% 95 . 1%
Contrast Intensity: A reduction in additive content
is a~ n; ed by decrease in left-heart cDntrast.
The surfactant compound in the microparticles is
usually utilized in a concentration of 0 . 01-5% by
weight, preferably 0 . 04-1% by weight.
The microparticles comprise a mixture of at
least one of the surfactant compounds with at least
one physiologically compatible solid. Organic and
inorganic materials can be utilized fDr this purpose,
e.g., salts, such as sodium chloride, sodium
_ _ _ _ _
- 7 - l 338507
citrate, sodlum acetate or sodium tartrate, monosaccha-
rides, such as glucose, fructose or galactose, disaccha-
rides, such as sucrose, lactose or maltose, pentoses,
such as arabinose, xylose or ribose, or cyclodextrins,
such as ~-, B- or Y-cyclodextrin, where galactose, fruc-
tose, glucose, lactose and d-cyclodextrin are preferred.
They are contained in the microparticles in a concentra-
tion of 95-99.99% by weight, preferably, 99-99.96%.
In order to prepare the microparticles, the com-
pounds intended therefor are recrystallized under asep-
tic conditions. Subsequently, they are comminuted under
aseptic conditions , e . g ., by grinding in an air ~ et
mill, until the desired particle size has been obtained.
A desirable particle size is one comparable to erythro-
cytes, e.g., of < 10 llm, preferably < 8 ~lm. The median
value of the micronized product is 1-3 ~lm. The particle
size is determined in suitable conventional measuring
devices. The thus-produced microparticles consist of a
mixture of a surfactant compound and a solid that is not
2 0 a surfactant .
The size of the microparticles attained by the
comminuting method as well as the size of the small gas
bubbles contained in the contrast medium of this
invention ensure hazardless passage of the capillary
system and the capillary bed of the lungs, and preclude
the formation of embolisms.
The gas volume required for contrast imaging is
transported by the microparticles. This volume is, in
part, adsorbed on the surface of the microparticles,
present in the cavities between the microparticles, or
occluded in intercrystalline fashion. The gas (e.g.,
air, nitrogen or argon) volume transported by the
microparticles in the form of small gas bubbles is 0 . 02
- 0. 6 ml per gram of microparticles.
The liquid vehicle can be water, aqueous solutions
of one or more inorganic salts, such as physiological
_ _ _ .
~33
- 8 - ~35i~7
sodium chloride solution and buffer solutions, aqueous
solutions of mono- or disaccharides, such as galactose,
glucose or lactose, mono- or polyhydride alcohols inso-
far as they are physiologically compatible, such as
ethanol, propanol, isopropyl alcohol, polyethylene
glycol, ethylene glycol, glycerol, propylene glycol,
propylene glycol methyl ether, or their aqueous solu-
tions. Preferred vehicles are water and physiological
electrolyte solutions, such as physiological sodium
chloride solution, as well as aqueous sugar solutions,
such as, for example, galactose, glucose, fructose and
lactose. If solutions are employed, the concentration
of the dissolved compound is 0.1-30% by weight, prefer-
ably 0.5-15% by weight; in particular, water, 0.9%
aqueous sodium chloride solution, or 5-6% aqueous
galactose solution is utilized. In general, 1-500,
preferably 10-400 mg of microaprticles are used per ml
of suspension.
The invention also relates to a process for the
preparation of the agent of this invention characterized
in that microparticles comprising at least one (C10-
C20)-fatty acid and at least one solid that is not a
surfactant are combined with a liquid vehicle and shaken
until a homogeneous suspension has been obtained.
In order to produce the ready-for-use ultrasonic
contrast medium, the sterile liquid vehicle is added to
the sterile combination, present in the form of micro-
particles, of at least one (C10-C20)-fatty acid with at
least one material that is not a surfactant, and this
mixture is shaken until a homogeneous suspension has
been formed, for which purpose about 5-10 seconds are
required. The resultant suspellsion is injected immedi-
ately af ter its preparation, but at the latest 5 minutes
thereafter, in the form of a bolus into a peripheral
vein or an already inserted catheter, typically admini-
stering 0 . 01 ml to 1 ml per kg of body weight.
_ 9 _ 1 338~a7
For practical reasons, the components required for
preparing the medium of this invention, such as the
liquid vehicle (A) and microparticles of the combination
(C10-C20)-fatty acid and soli~ that is not a surfactant
(B) are preferably stored sterile in the quantity
required for one examination in two vessels. Both
vessels (vials) preferably have seals permitting
withdrawal and filling by means of an injection syringe
under sterile conditions. The size of vessel B is such
that the content of vessel A can be transferred by
in~ ection syringe into B and the combined components can
be shaken.
The use of the contrast medium according to this
invention will be demonstrated by performing an
echocardiographic examination on a baboon weighing 10
kg:
8.5 ml of liquid vehicle (see preparation examples)
is withdrawn with an injection syringe from a vial and
added to 3 g of microparticles present in a second vial,
and shaken for about 5-10 seconds until a homogeneous
suspension has been formed. Of this suspension, 2 ml if
injected into a pprirhpral vein (V. jugularis,
br~C~h;Al ;~ or gaphena) by way of a three-way valve at an
infusion rate of at least 1 ml/sec, better at 2-3
ml/sec. The in; ection of 10 ml of physiological sodium
chloride solution immediately follows the in~ection of
contrast medium at the same rate, 80 that the contrast
medium bolus remains intact as long as possible.
Before, during, and after injection, a commercially
available transducer for echocardiography is held
against the thorax of the test animal 80 that a typical
cros6 section is obtained through the right and left
heart. Thi6 testing arrangement corresponds to the
state of the art and is known to those skilled in the
3 5 art .
Once the ultrasonic contrast medium has reached the
- lo _ ~ 3385~7
right heart, an observation can be made in the 2-D echo
image or in the N-mode echo image of how the blood
labeled by the contrast medium f irst reaches the level
of the right atrium, then the level of the right
5 ventricle and the p~ ry artery, homogeneous filling
prevailing for a time period adequate for diagnostic
examination. While the cavities of the right heart
become empty again in the ultrasonic image, the blood
labeled with contrast medium reappears, after passing
10 through the lungs, in the plll- -ry veins, fills the
left atrium, the left ventricle, and the aorta in a
homogeneous fashion, the contrast l ~nln~ longer than
on the right side of the heart. In addition to imaging
of the blood flow through the cavities of the left
15 heart, a contrast image of the myocardium is likewise
obtained, reflecting blood circulation.
The use of the ultrasonic contrast medium of this
invention is, however, not limited to rendering the
bloodstream visible in the arterial portion of the heart
20 after venous administration; rather, with excellent
success, the contrast medium is also employed in the
examination of the right heart and other organs by
contrast medium.
The use and administration of the contrast medium
25 of thi6 invention is analogous to the procedures
described, e.g., in the disclosures cited above.
Without further elaboration, it is believed that
one skilled in the art can, using the preceding
description; utilize the present invention to its
30 fullest extent. The following preferred specific
embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the remainder of the
disclosure in any way whatsoever.
In the foregoing and in the following examples, all
35 temperatures are set forth uncorrected in degrees
Celsius and unless otherwise indicated, all parts and
1 338507
percentages are by weight.
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~ 1 3385C7
EXAMPLE 1
(A) Liquid Vehicle; Water for purposes of
in j ection
(B) Preparation of Microparticles:
I 1,998 g of galactose in 1,080 g of water is purified,
dissolved, filtered under sterile conditions, and
cooled under aseptic conditions to 6-10 C.
II 2 g of palmitic acid is dissolved in 120 g of ethanol,
filtered under sterile conditions, and added to I
under agitation.
III The combined solutions are dried under aseptic condi-
tions at about 40 C and under a vacuum of 50 mbar.
IV The recrystallized product is comminuted under aseptic
conditions by means of an air jet mill to the
following grain size distribution:
D10 c 1 llm
D50 2.5 um
D90 ~ 5 llm
Determination of the grain size distribution takes
20 place after suspending the micronized product in alcohol
with the use of a particle measuring device (e.g. Cilas
Granulometer 715).
V Packaging of the microparticles is effected into
20 ml vials at respectively 3 g.
~ - 12 - I 3 3 8 5 07
(C) Production of Ready-For-Use Ultrasonic
Contrast Medium
By means of an injection syringe, 8.5 ml of
water for injection purposes is transferred into the
5 20 ml vial containing 3 g of microparticles, and the
vial is shaken until a homogeneous suspension is obtained
(5-10 seconds).
EXAMPLE 2
~A) Liquid Vehicle: Water for injection purposes
lB) Preparation of Microparticles:
1,998 g of galactose in 1,080 g of water is purified,
dissolved, filtered under sterile conditions, and
cooled under aseptic conditions to 6-10 C.
II 2 g of myristic acid is dissolved in 120 g of ethanol,
filtered under sterile conditions, and added to I
under agitation.
III The combined solutions are dried under aseptic condi-
tions at about 4D C and under a vacuum of 50 mbar.
IV The recrystallized product is comminuted under aseptic
conditions with an air ]et mill to the following grain
size distribution:
D l o ~ m
D50 ~ 2.5 ~m
9 0 ~ 11
Determination of grain size distribution takes place
after suspending the micronized product in alco~lol with
_ _ _, . _ .. .. .. . . .. . ... . .. . . . . .. .... . . .. .. .. .... . . ..
~ - 13 - 1 3 3 8 5 û 7
a particle measuring instrument (e . g. Cilas Granulometer
715) .
V Packaging of the microparticles takes place into
20 ml vials with respectively 3 g.
(C) Production of ~eady-For-Use Ultrasonic
Contrast Medium
Using an injection syringe, 8.5 ml of water
for injection purposes is transferred into the Z0 ml
vial containing 3 g of microparticles, and the vial is
10 shaken until a homogeneous suspension is produced
( 5-10 seconds ) .
EXAMPLE 3
(A~ Liquid Vehicle: Water for injection purposes
(B) Preparation of Microparticles:
I 1,998 g of galactose in 1,080 g of water is purified,
dissolved, filtered in sterile state, and cooled under
aseptic conditions to 6-10 C.
II 2 g of stearic acid is dissolved in 120 g o~ ethanol,
filtered under sterile conditions, and added to I
with stirring.
III The combined solutions are brought to the dry condi-
tion in an aseptic environment at about 40 C and
under a vacuum of 50 mbar.
IV The recrys~l 1; 7~d product is comminuted under
aseptic conditions by means of an air jet mill to
the following grain size distributlon:
~ - 14 - 1 3~850~
D 10 ~ m
D50 ~ 2.5 ,um
g O ~ m
Determination of grain size distribution is made
5 after suspending the micronized product in alcohol
using a particle measuring device~(e.g. C~ilas
Granulometel 715 ) .
V The microparticles are packaged into 20 ml vials with
respectively 3 g.
(C) Production of Ready-For-Use Ultrasonic
Contrast Medium
With the use of an injection syringe, 8.5 ml
of water for injection purposes is transferred into the
20 ml vial containing 3 g of microparticles, and the vial is
15 shaken until a homogeneous suspension is obtained
(5-10 seconds).
EXAMPLE 4
(A) Liquid ~lehicle: Water for injection purposes
(s) Preparation of Microparticles:
I 1,998 g of galactose in 1,080 g of water is purified,
dissolved, filtered in the sterile state, and cooled
to 6-10 C under aseptic conditions.
II 1 g of myristic acid + 1 g of arachic acid are dis-
solved in 120 g of ethanol, filtered under sterile
conditions, and added to I under stirring.
III The combined solutions are brought to dryness under
aseptic conditions at about 40 C and under a
vacuum of 50 mbar.
_ _ _ _ .
- 15 - 1 3 3 8 5 0 7
IV The recrystallized product is aseptically comminuted
with the use of a jet air mill to the following grain
size distribution:
D ~ 1
D50 ~ 2. 5 llm
Dgo ~ 5 ~m
Determination of the grain size distribution takes
place after suspending the micronized product in alcohol
using a particle measuring device (e . g . Cilas Granulometer
715 ) .
V The microparticles are packaged into 20 ml vials
with respectively 3 g.
(C) Production of Ready-For-Use Ultrasonic
Contrast Medium
By means of an injection syringe, 8.5 ml
of water fqr injection purposes is transferred into the
20 ml vial containing 3 g of microparticles, and the vial
is shaken until a homogeneous suspension is obtained
(5-10 seconds).
2 0 EXAMPLE 5
(A) Production of Liquid Vehicle:
55 g of galactose is dissolved in water
for injection purposes, filled up to a volume of 1,000 ml,
filte~ed through a 0,2 llm filter, respectively 10 ml of
25 the filtered solution .is d~spensed into 10 ml vials, and
sterilized for 15 minutes at 121 C.
- 16 - 1 3 3 8 5 0 7
(s) Preparation of Microparticles:
1,998 g of galactose in 1,080 g of water is purified,
dissolved, filtered under sterile conditions, and
aseptically cooled to 6-10 C.
5 II 1 g of palmitic acid + 1 g of stearic acid are
dissolved in 120 g of ethanol, filtered under sterile
conditions, and added to I under agitation.
II The eombined solutions are aseptically dried at about
40 C and under a vaeuum of 50 mbar.
10 IV The reerystallized produet is eomminuted under aseptie
conditions with an air ]et mill to the following grain
size distribution:
D l o ~ m
D50 ~ 2-5 um
Dgo _ 5 llm
Determination of the grain size distribution is
effeeted after suspending the mieronized produet in
alcohol, using a particle size measuring device (e.g.
Cilas Granulometer 715).
V The microparticles are packaged into 20 ml vials
with respectively 3 g.
~ - 17 - 1 3 3 8 5 07
.
(C) Production of Ready-For-Use Ultrasonic
Contrast Medium
Using an injection syringe, 8.5 ml of
galactose solution A is transferred into the 20 ml vial
5 which contains 3 g of microparticles, and the vial is
shaken until a homogeneous suspension is obtained
l 5-10 seconds ) .
