U.S. patent application number 10/962460 was filed with the patent office on 2005-03-17 for long-lasting aqueous dispersions or suspensions of pressure-resistant gas-filled microvesicles and methods for the preparation thereof.
Invention is credited to Barrau, Marie-Bernadette, Garcel, Nadine, Grenier, Pascal, Puginier, Jerome, Schneider, Michel, Yan, Feng.
Application Number | 20050058605 10/962460 |
Document ID | / |
Family ID | 8211861 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050058605 |
Kind Code |
A1 |
Schneider, Michel ; et
al. |
March 17, 2005 |
Long-lasting aqueous dispersions or suspensions of
pressure-resistant gas-filled microvesicles and methods for the
preparation thereof
Abstract
One can impart outstanding resistance against collapse under
pressure to gas-filled microvesicle used as contrast agents in
ultrasonic echography by using as fillers gases whose solubility in
water, expressed in liter of gas by liter of water under standard
conditions, divided by the square root of the molecular weight does
not exceed 0.003.
Inventors: |
Schneider, Michel; (Troinex,
CH) ; Yan, Feng; (Geneva, CH) ; Grenier,
Pascal; (Segny, FR) ; Garcel, Nadine; (Segny,
FR) ; Puginier, Jerome; (Valleiry, FR) ;
Barrau, Marie-Bernadette; (Geneva, CH) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Family ID: |
8211861 |
Appl. No.: |
10/962460 |
Filed: |
October 13, 2004 |
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Current U.S.
Class: |
424/9.52 |
Current CPC
Class: |
A61K 49/223
20130101 |
Class at
Publication: |
424/009.52 |
International
Class: |
A61K 049/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 1992 |
EP |
92 810046.0 |
May 18, 1990 |
EP |
90 810367.4 |
Apr 2, 1990 |
EP |
90 810262.7 |
Apr 2, 1991 |
WO |
PCT/EP91/00620 |
Claims
1.-18. Canceled.
19. A method for making dry precursors for gas filled microbubbles
for ultrasonic imaging comprising the steps of: dissolving a
mixture of hydrogenated soya lecithin and dicetyl-phosphate in a
chloroform solution; evaporating said solution to yield a dried
mixture; adding an aqueous liquid to the dried mixture to form a
suspension; extruding said suspension to form a liposome
suspension; diluting said liposome suspension with a lactose
solution to form a diluted suspension; freezing said diluted
suspension to form a frozen suspension; and freeze-drying said
frozen suspension in a vessel to form dried powder, said vessel
filled with a gas selected from the group consisting of CF.sub.4,
CBrF.sub.3, CF.sub.4 and CHClF.sub.2.
Description
TECHNICAL FIELD
[0001] The present invention concerns stable dispersions or
compositions of gas filled microvesicles in aqueous carrier
liquids. These dispersions are generally usable for most kinds of
applications requiring gases homogeneously dispersed in liquids.
One notable application for such dispersions is to be injected into
living beings, for instance for ultrasonic echography and other
medical applications. The invention also concerns the methods for
making the foregoing compositions including some materials involved
in the preparations, for instance pressure-resistant gas-filled
microbubbles, microcapsules and microballoons.
BACKGROUND OF INVENTION
[0002] It is well known that microbodies or microglobules of air or
gas (defined here as microvesicles), e.g. microbubbles or
microballoons, suspended in a liquid are exceptionally efficient
ultrasound reflectors for echography. In this disclosure the term
of "microbubble" specifically designates hollow spheres or
globules, filled with air or a gas, in suspension in a liquid which
generally result from the introduction therein of air or gas in
divided form, the liquid preferably also containing surfactants or
tensides to control the surface properties and the stability of the
bubbles. The term of "microcapsule" or "microballoon" designates
preferably air or gas-filled bodies with a material boundary or
envelope, i.e. a polymer membrane wall. Both microbubbles and
microballoons are useful as ultrasonic contrast agents. For
instance injecting into the bloodstream of living bodies
suspensions of air-filled microbubbles or microballoons (in the
range of 0.5 to 10 .mu.m) in a carrier liquid will strongly
reinforce ultrasonic echography imaging, thus aiding in the
visualization of internal organs. Imaging of vessels and internal
organs can strongly help in medical diagnosis, for instance for the
detection of cardiovascular and other diseases.
[0003] The formation of suspensions of microbubbles in an
injectable liquid carrier suitable for echography can be produced
by the release of a gas dissolved under pressure in this liquid, or
by a chemical reaction generating gaseous products, or by admixing
with the liquid soluble or insoluble solids containing air or gas
trapped or adsorbed therein.
[0004] For instance, in U.S. Pat. No. 4,446,442 (Schering), there
are disclosed a series of different techniques for producing
suspensions of gas microbubbles in a sterilized injectable liquid
carrier using (a) a solution of a tenside (surfactant) in a carrier
liquid (aqueous) and (b) a solution of a viscosity enhancer as
stabilizer. For generating the bubbles, the techniques disclosed
there include forcing at high velocity a mixture of (a), (b) and
air through a small aperture; or injecting (a) into (b) shortly
before use together with a physiologically acceptable gas; or
adding an acid to (a) and a carbonate to (b), both components being
mixed together just before use and the acid reacting with the
carbonate to generate CO.sub.2 bubbles; or adding an
over-pressurized gas to a mixture of (a) and (b) under storage,
said gas being released into microbubbles at the time when the
mixture is used for injection.
[0005] EP-A-131,540 (Schering) discloses the preparation of
microbubble suspensions in which a stabilized injectable carrier
liquid. e.g. a physiological aqueous solution of salt, or a
solution of a sugar like maltose, dextrose, lactose or galactose,
is mixed with solid microparticles (in the 0.1 to 1 .mu.m range) of
the same sugars containing entrapped air. In order to develop the
suspension of bubbles in the liquid carrier, both liquid and solid
components are agitated together under sterile conditions for a few
seconds and, once made, the suspension must then be used
immediately, i.e. it should be injected within 5-10 minutes for
echographic measurements; indeed, because they are evanescent, the
bubble concentration becomes too low for being practical after that
period.
[0006] In an attempt to cure the evanescence problem,
microballoons, i.e. microvesicles with a material wall, have been
developed. As said before, while the microbubbles only have an
immaterial or evanescent envelope, i.e. they are only surrounded by
a wall of liquid whose surface tension is being modified by the
presence of a surfactant, the microballoons or microcapsules have a
tangible envelope made of substantive material. e.g. a polymeric
membrane with definite mechanical strength. In other terms, they
are microvesicles of material in which the air or gas is more or
less tightly encapsulated.
[0007] For instance, U.S. Pat. No. 4,276,885 (Tickner et al.)
discloses using surface membrane microcapsules containing a gas for
enhancing ultrasonic images, the membrane including a multiplicity
of non-toxic and non-antigenic organic molecules. In a disclosed
embodiment, these microbubbles have a gelatine membrane which
resists coalescence and their preferred size is 5-10 .mu.m. The
membrane of these microbubbles is said to be sufficiently stable
for making echographic measurements.
[0008] Air-filled microballoons without gelatin are disclosed in
U.S. Pat. No. 4,718,433 (Feinstein). These microvesicles are made
by sonication (5 to 30 kHz) of protein solutions like 5% serum
albumin and have diameters in the 2-20 .mu.m range, mainly 2-4
.mu.m. The microvesicles are stabilized by denaturation of the
membrane forming protein after sonication, for instance by using
heat or by chemical means. e.g. by reaction with formaldehyde or
glutaraldehyde. The concentration of stable microvesicles obtained
by this technique is said to be about 8.times.10.sup.6/ml in the
2-4 .mu.m range, about 10.sup.6/ml in the 4-5 .mu.m range and less
than 5.times.10.sup.5 in the 5-6 .mu.m range. The stability time of
these microvesicles is said to be 48 hrs or longer and they permit
convenient left heart imaging after intravenous injection. For
instance, the sonicated albumin microbubbles when injected into a
peripheral vein are capable of transpulmonary passage. This results
in echocardiographic opacification of the left ventricle cavity as
well as myocardial tissues.
[0009] Recently, still further improved microballoons for injection
ultrasonic echography have been reported in EP-A-324.938 (Widder).
In this document there are disclosed high concentrations (more than
10.sup.8/ml) of air-filled protein-bounded microvesicles of less
than 10 .mu.m which have life-times of several months or more.
Aqueous suspensions of these microballoons are produced by
ultrasonic cavitation of solutions of heat denaturable proteins.
e.g. human serum albumin, which operation also leads to a degree of
foaming of the membrane-forming protein and its subsequent
hardening by heat. Other proteins such as hemoglobin and collagen
were also said to be convenient in this process. The high storage
stability of the suspensions of microballoons disclosed in
EP-A-324.938 enables them to be marketed as such. i.e. with the
liquid carrier phase, which is a strong commercial asset since
preparation before use is no longer necessary.
[0010] Similar advantages have been recently discovered in
connection with the preparation of aqueous microbubble suspensions.
i.e. there has been discovered storage-stable dry pulverulent
composition which will generate long-lasting bubble suspensions
upon the addition of water. This is being disclosed in Application
PCT/EP 91/00620 where liposomes comprising membrane-forming lipids
are freeze-dried, and the freeze-dried lipids, after exposure to
air or a gas for a period of time, will produce long-lasting bubble
suspensions upon simple addition thereto of an aqueous liquid
carrier.
[0011] Despite the many progresses achieved regarding the stability
under storage of aqueous microbubble suspensions, this being either
in the precursor or final preparation stage, there still remained
until now the problem of vesicle durability when the suspensions
are exposed to overpressure, e.g. pressure variations such as that
occurring after injection in the blood stream of a patient and
consecutive to heart pulses, particularly in the left ventricle.
Actually, the present inventors have observed that, for instance in
anaesthetised rabbits, the pressure variations are not sufficient
to substantially alter the bubble count for a period of time after
injection. In contrast, in dogs and human patients, typical
microbubbles or microballoons filled with common gases such as air,
methane or CO.sub.2 will collapse completely in a matter of seconds
after injection due to the blood pressure effect. This observation
has been confirmed by others: For instance, S. GOTTLIEB et al. in
J. Am. Soc. of Echocardiography 3 (1990) 238 have reported that
cross-linked albumin microballoons prepared by the sonication
method were losing all echogenic properties after being subjected
to an overpressure of 60 Torr. It became hence important to solve
the problem and to increase the useful life of suspensions of
microbubbles and membrane bounded microballoons under pressure in
order to ensure that echographic measurements can be performed in
vivo safely and reproducibly.
[0012] It should be mentioned at this stage that another category
of echogenic image enhancing agents has been proposed which resist
overpressures as they consist of plain microspheres with a porous
structure, such porosity containing air or a gas. Such microspheres
are disclosed for instance in WO-A-91/12823 (DELTA BIOTECHNOLOGY),
EP-A-327 490 (SCHERING) and EP-A-458 079 (HOECHST). The drawback
with the plain porous microspheres is that the encapsulated
gas-filled free space is generally too small for good echogenic
response and the spheres lack adequate elasticity. Hence the
preference generally remains with the hollow microvesicles and a
solution to the collapsing problem was searched.
DISCLOSURE OF THE INVENTION
[0013] This problem has now been solved by using gases or gas
mixtures in conformity with the criteria outlined in the claims.
Briefly, it has been found that when the echogenic microvesicles
are made in the presence of a gas, respectively are filled at least
in part with a gas, having physical properties in conformity with
the equation below, then the microvesicles remarkably resist
pressure >60 Torr after injection for a time sufficient to
obtain reproducible echographic measurements: 1 s gas s air .times.
Mw air Mw gas 1
[0014] In the foregoing equation. "s" designates the solubilities
in water expressed as the "BUNSEN" coefficients, i.e. as volume of
gas dissolved by unit volume of water under standard conditions (1
bar, 25.degree. C.), and under partial pressure of the given gas of
1 atm (see the Gas Encyclopaedia. Elsevier 1976). Since, under such
conditions and definitions, the solubility of air is 0.0167, and
the square root of its average molecular weight (Mw) is 5.39, the
above relation simplifies to:
s.sub.gas/{square root}Mw.sub.gas.ltoreq.0.0031
[0015] In the Examples to be found hereafter there is disclosed the
testing of echogenic microbubbles and microballoons (see the
Tables) filled with a number of different gases and mixtures
thereof, and the corresponding resistance thereof to pressure
increases, both in vivo and in vitro. In the Tables, the water
solubility factors have also been taken from the aforecited Gas
Encyclopaedia from "L'Air Liquide". Elsevier Publisher (1976).
[0016] The microvesicles in aqueous suspension containing gases
according to the invention include most microbubbles and
microballoons disclosed until now for use as contrast agents for
echography. The preferred microballoons are those disclosed in
EP-A-324.938, PCT/EP91/01706 and EP-A-458 745; the preferred
microbubbles are those of PCT/EP91/00620: these microbubbles are
advantageously formed from an aqueous liquid and a dry powder
(microvesicle precursors) containing lamellarized freeze-dried
phospholipids and stabilizers; the microbubbles are developed by
agitation of this powder in admixture with the aqueous liquid
carrier. The microballoons of EP-A-458 745 have a resilient
interfacially precipitated polymer membrane of controlled porosity.
They are generally obtained from emulsions into microdroplets of
polymer solutions in aqueous liquids, the polymer being
subsequently caused to precipitate from its solution to form a
filmogenic membrane at the droplet/liquid interface, which process
leads to the initial formation of liquid-filled microvesicles, the
liquid core thereof being eventually substituted by a gas.
[0017] In order to carry out the method of the present invention.
i.e. to form or fill the microvesicles, whose suspensions in
aqueous carriers constitute the desired echogenic additives, with
the gases according to the foregoing relation, one can either use,
as a first embodiment, a two step route consisting of (1) making
the microvesicles from appropriate starting materials by any
suitable conventional technique in the presence of any suitable
gas, and (2) replacing this gas originally used (first gas) for
preparing the microvesicles with a new gas (second gas) according
to the invention (gas exchange technique).
[0018] Otherwise, according to a second embodiment, one can
directly prepare the desired suspensions by suitable usual methods
under an atmosphere of the new gas according to the invention.
[0019] If one uses the two-step route, the initial gas can be first
removed from the vesicles (for instance by evacuation under
suction) and thereafter replaced by bringing the second gas into
contact with the evacuated product, or alternatively, the vesicles
still containing the first gas can be contacted with the second gas
under conditions where the second gas will displace the first gas
from the vesicles (gas substitution). For instance, the vesicle
suspensions, or preferably precursors thereof (precursors here may
mean the materials the microvesicle envelopes are made of, or the
materials which, upon agitation with an aqueous carrier liquid,
will generate or develop the formation of microbubbles in this
liquid), can be exposed to reduced pressure to evacuate the gas to
be removed and then the ambient pressure is restored with the
desired gas for substitution. This step can be repeated once or
more times to ensure complete replacement of the original gas by
the new one. This embodiment applies particularly well to precursor
preparations stored dry, e.g. dry powders which will regenerate or
develop the bubbles of the echogenic additive upon admixing with an
amount of carrier liquid. Hence, in one preferred case where
microbubbles are to be formed from an aqueous phase and dry
laminarized phospholipids. e.g. powders of dehydrated lyophilized
liposomes plus stabilizers, which powders are to be subsequently
dispersed under agitation in a liquid aqueous carrier phase, it is
advantageous to store this dry powder under an atmosphere of a gas
selected according to the invention. A preparation of such kind
will keep indefinitely in this state and can be used at any time
for diagnosis, provided it is dispersed into sterile water before
injection.
[0020] Otherwise, and this is particularly so when the gas exchange
is applied to a suspension of microvesicles in a liquid carrier
phase, the latter is flushed with the second gas until the
replacement (partial or complete) is sufficient for the desired
purpose. Flushing can be effected by bubbling from a gas pipe or,
in some cases, by simply sweeping the surface of the liquid
containing the vesicles under gentle agitation with a stream
(continuous or discontinuous) of the new gas. In this case, the
replacement gas can be added only once in the flask containing the
suspension and allowed to stand as such for a while, or it can be
renewed one or more times in order to assure that the degree of
renewal (gas exchange) is more or less complete.
[0021] Alternatively, in a second embodiment as said before, one
will effect the full preparation of the suspension of the echogenic
additives starting with the usual precursors thereof (starting
materials), as recited in the prior art and operating according to
usual means of said prior art, but in the presence of the desired
gases or mixture of gases according to the invention instead of
that of the prior art which usually recites gases such as air,
nitrogen. CO.sub.2 and the like.
[0022] It should be noted that in general the preparation mode
involving one first type of gas for preparing the microvesicles
and, thereafter, substituting the original gas by a second kind of
gas, the latter being intended to confer different echogenic
properties to said microvesicles, has the following advantage: As
will be best seen from the results in the Examples hereinafter, the
nature of the gas used for making the microvesicles, particularly
the microballoons with a polymer envelope, has a definitive
influence on the overall size (i.e. the average mean diameter) of
said microvesicles: for instance, the size of microballoons
prepared under air with precisely set conditions can be accurately
controlled to fall within a desired range, e.g. the 1 to 10 .mu.m
range suitable for echographying the left and right heart
ventricles. This not so easy with other gases, particularly the
gases in conformity with the requirements of the present invention;
hence, when one wishes to obtain microvesicles in a given size
range but filled with gases the nature of which would render the
direct preparation impossible or very hard, one will much
advantageously rely on the two-steps preparation route, i.e. one
will first prepare the microvesicles with a gas allowing more
accurate diameter and count control, and thereafter replace the
first gas by a second gas by gas exchange.
[0023] In the description of the Experimental part that follows
(Examples), gas-filled microvesicles suspended in water or other
aqueous solutions have been subjected to pressures over that of
ambient. It was noted that when the overpressure reached a certain
value (which is generally typical for a set of microsphere
parameters and working conditions like temperature, compression
rate, nature of carrier liquid and its content of dissolved gas
(the relative importance of this parameter will be detailed
hereinafter), nature of gas filler, type of echogenic material,
etc.), the microvesicles started to collapse, the bubble count
progressively decreasing with further increasing the pressure until
a complete disappearance of the sound reflector effect occurred.
This phenomenon was better followed optically, (nephelometric
measurements) since it is paralleled by a corresponding change in
optical density, i.e. the transparency of the medium increases as
the bubble progressively collapse. For this, the aqueous suspension
of microvesicles (or an appropriate dilution thereof) was placed in
a spectrophotometric cell maintained at 25.degree. C. (standard
conditions) and the absorbance was measured continuously at 600 or
700 nm, while a positive hydrostatic overpressure was applied and
gradually increased. The pressure was generated by means of a
peristaltic pump (GILSON's Mini-puls) feeding a variable height
liquid column connected to the spectrophotometric cell, the latter
being sealed leak-proof. The pressure was measured with a mercury
manometer calibrated in Torr. The compression rate with time was
found to be linearly correlated with the pump's speed (rpm's). The
absorbance in the foregoing range was found to be proportional to
the microvesicle concentration in the carrier liquid.
[0024] FIG. 1 is a graph which relates the bubble concentration
(bubble count), expressed in terms of optical density in the
aforementioned range, and the pressure applied over the bubble
suspension. The data for preparing the graph are taken from the
experiments reported in Example 4.
[0025] FIG. 1 shows graphically that the change of absorbance
versus pressure Is represented by a sigmoid-shaped curve. Up to a
certain pressure value, the curve is nearly flat which indicates
that the bubbles are stable. Then, a relatively fast absorbance
drop occurs, which indicates the existence of a relatively narrow
critical region within which any pressure increase has a rather
dramatic effect on the bubble count. When all the microvesicles
have disappeared, the curve levels off again. A critical point on
this curve was selected in the middle between the higher and lower
optical readings. i.e. intermediate between the "full"-bubble (OD
max) and the "no"-bubble (OD min) measurements, this actually
corresponding where about 50% of the bubbles initially present have
disappeared. i.e. where the optical density reading is about half
the initial reading, this being set, in the graph, relative to the
height at which the transparency of the pressurized suspension is
maximal (base line). This point which is also in the vicinity where
the slope of the curve is maximal is defined as the critical
pressure PC. It was found that for a given gas, PC does not only
depend on the aforementioned parameters but also, and particularly
so, on the actual concentration of gas (or gases) already dissolved
in the carrier liquid: the higher the gas concentration, the higher
the critical pressure. In this connection, one can therefore
increase the resistance to collapse under pressure of the
microvesicles by making the carrier phase saturated with a soluble
gas, the latter being the same, or not, (i.e. a different gas) as
the one that fills the vesicles. As an example, air-filled
microvesicles could be made very resistant to overpressures
(>120 Torr) by using, as a carrier liquid, a saturated solution
of CO.sub.2. Unfortunately, this finding is of limited value in the
diagnostic field since once the contrast agent is injected to the
bloodstream of patients (the gas content of which is of course
outside control), it becomes diluted therein to such an extent that
the effect of the gas originally dissolved in the injected sample
becomes negligible.
[0026] Another readily accessible parameter to reproducibly compare
the performance of various gases as microsphere fillers is the
width of the pressure interval (.DELTA.P) limited by the pressure
values under which the bubble counts (as expressed by the optical
densities) is equal to the 75% and 25% of the original bubble
count. Now, it has been surprisingly found that for gases where the
pressure difference DP=P.sub.25-P.sub.75 exceeds a value of about
25-30 Torr, the killing effect of the blood pressure on the
gas-filled microvesicles is minimized. i.e. the actual decrease in
the bubble count is sufficiently slow not to impair the
significance, accuracy and reproducibility of echographic
measurements.
[0027] It was found, in addition, that the values of PC and AP also
depend on the rate of rising the pressure in the test experiments
illustrated by FIG. 1, i.e. in a certain interval of pressure
increase rates (e.g. in the range of several tens to several
hundreds of Torr/min), the higher the rate, the larger the values
for PC and .DELTA.P. For this reason, the comparisons effected
under standard temperature conditions were also carried out at the
constant increase rate of 100 Torr/min. It should however be noted
that this effect of the pressure increase rate on the measure of
the PC and AP values levels off for very high rates: for instance
the values measured under rates of several hundreds of Torr/min are
not significantly different from those measured under conditions
ruled by heart beats.
[0028] Although the very reasons why certain gases obey the
aforementioned properties, while others do not, have not been
entirely clarified, it would appear that some relation possibly
exists in which, in addition to molecular weight and water
solubility, dissolution kinetics, and perhaps other parameters, are
involved. However these parameters need not be known to practise
the present invention since gas eligibility can be easily
determined according to the aforediscussed criteria.
[0029] The gaseous species which particularly suit the invention
are, for instance, halogenated hydrocarbons like the freons and
stable fluorinated chalcogenides like SF.sub.6. SeF.sub.6 and the
like.
[0030] It has been mentioned above that the degree of gas
saturation of the liquid used as carrier for the microvesicles
according to the invention has an importance on the vesicle
stability under pressure variations. Indeed, when the carrier
liquid in which the microvesicles are dispersed for making the
echogenic suspensions of the invention is saturated at equilibrium
with a gas, preferably the same gas with which the microvesicles
are filled, the resistance of the microvesicles to collapse under
variations of pressure is markedly increased. Thus, when the
product to be used as a contrast agent is sold dry to be mixed just
before use with the carrier liquid (see for instance the products
disclosed in PCT/EP91/00620 mentioned hereinbefore), it is quite
advantageous to use, for the dispersion, a gas saturated aqueous
carrier. Alternatively, when marketing ready-to-use microvesicle
suspensions as contrast agents for echography, one will
advantageously use as the carrier liquid for the preparation a gas
saturated aqueous solution; in this case the storage life of the
suspension will be considerably increased and the product may be
kept substantially unchanged (no substantial bubble count
variation) for extended periods, for instance several weeks to
several months, and even over a year in special cases. Saturation
of the liquid with a gas may be effected most easily by simply
bubbling the gas into the liquid for a period of time at room
temperature.
EXAMPLE 1
[0031] Albumin microvesicles filled with air or various gases were
prepared as described in EP-A-324 938 using a 10 ml calibrated
syringe filled with a 5% human serum albumin (HSA) obtained from
the Blood Transfusion Service, Red-Cross Organization. Bern,
Switzerland. A sonicator probe (Sonifier Model 250 from Branson
Ultrasonic Corp. USA) was lowered into the solution down to the 4
ml mark of the syringe and sonication was effected for 25 sec
(energy setting=8). Then the sonicator probe was raised above the
solution level up to the 6 ml mark and sonication was resumed under
the pulse mode (cycle=0.3) for 40 sec. After standing overnight at
4.degree. C., a top layer containing most of the microvesicles had
formed by buoyancy and the bottom layer containing unused albumin
debris of denatured protein and other insolubles was discarded.
After resuspending the microvesicles in fresh albumin solution the
mixture was allowed to settle again at room temperature and the
upper layer was finally collected. When the foregoing sequences
were carried out under the ambient atmosphere, air filled
microballoons were obtained. For obtaining microballoons filled
with other gases, the albumin solution was first purged with a new
gas, then the foregoing operational sequences were effected under a
stream of this gas flowing on the surface of the solution: then at
the end of the operations, the suspension was placed in a glass
bottle which was extensively purged with the desired gas before
sealing.
[0032] The various suspensions of microballoons filled with
different gases were diluted to 1:10 with distilled water saturated
at equilibrium with air, then they were placed in an optical cell
as described above and the absorbance was recorded while increasing
steadily the pressure over the suspension. During the measurements,
the suspensions temperature was kept at 25.degree. C.
[0033] The results are shown in the Table 1 below and are expressed
in terms of the critical pressure PC values registered for a series
of gases defined by names or formulae, the characteristic
parameters of such gases. i.e. Mw and water solubility being given,
as well as the original bubble count and bubble average size (mean
diameter in volume).
1TABLE 1 Bubble Bubble Solubi- count size S.sub.gas/ Sample Gas Mw
lity (10.sup.8/ml) (.mu.m) PC(Torr) {square root}Mw AFre1 CF.sub.4
88 .0038 0.8 5.1 120 .0004 AFre2 CBrF.sub.3 149 .0045 0.1 11.1 104
.0004 ASF1 SF.sub.6 146 .005 13.9 6.2 150 .0004 ASF2 SF.sub.6 146
.005 2.0 7.9 140 .0004 AN1 N.sub.2 28 .0144 0.4 7.8 62 .0027 A14
Air 29 .0167 3.1 11.9 53 .0031 A18 Air 29 .0167 3.8 9.2 52 -- A19
Air 29 .0167 1.9 9.5 51 -- AMe1 CH.sub.4 16 .032 0.25 8.2 34 .008
AKr1 Kr 84 .059 0.02 9.2 86 .006 AX1 Xe 131 .108 0.06 17.2 65 .009
AX2 Xe 131 .108 0.03 16.5 89 .009
[0034] From the results of Table 1, it is seen that the critical
pressure PC increases for gases of lower solubility and higher
molecular weight. It can therefore be expected that microvesicles
filled with such gases will provide more durable echogenic signals
in vivo. It can also be seen that average bubble size generally
increases with gas solubility.
EXAMPLE 2
[0035] Aliquots (1 ml) of some of the microballoon suspensions
prepared in Example 1 were injected in the jugular vein of
experimental rabbits in order to test echogenicity in vivo. Imaging
of the left and right heart ventricles was carried out in the grey
scale mode using an Acuson 128-XP5 echography apparatus and a 7.5
MHz transducer. The duration of contrast enhancement in the left
ventricle was determined by recording the signal for a period of
time. The results are gathered in Table 2 below which also shows
the PC of the gases used.
2 TABLE 2 Duration of Sample (Gas) contrast (sec) PC (Torr) AMe1
(CH.sub.4) zero 34 A14 (air) 10 53 A18 (air) 11 52 AX1 (Xe) 20 65
AX2 (Xe) 30 89 ASF2(SF.sub.6) >60 140
[0036] From the above results, one can see the existence of a
definite correlation between the critical pressure of the gases
tried and the persistence in time of the echogenic signal.
EXAMPLE 3
[0037] A suspension of echogenic air-filled galactose
microparticles (Echovist.RTM. from SCHERING AG) was obtained by
shaking for 5 sec 3 g of the solid microparticles in 8.5 ml of a
20% galactose solution. In other preparations, the air above a
portion of Echovist.RTM. particles was evacuated (0.2 Torr) and
replaced by an SF.sub.6 atmosphere, whereby, after addition of the
20% galactose solution, a suspension of microparticles containing
associated sulfur hexafluoride was obtained. Aliquots (1 ml) of the
suspensions were administered to experimental rabbits (by injection
in the jugular vein) and imaging of the heart was effected as
described in the previous example. In this case the echogenic
microparticles do not transit through the lung capillaries, hence
imaging is restricted to the right ventricle and the overall signal
persistence has no particular significance. The results of Table 3
below show the value of signal peak intensity a few seconds after
injection.
3 TABLE 3 Signal peak Sample No Gas (arbitrary units) Gal1 air 114
Gal2 air 108 Gal3 SF.sub.6 131 Gal4 SF.sub.6 140
[0038] It can be seen that sulfur hexafluoride, an inert gas with
low water solubility, provides echogenic suspensions which generate
echogenic signals stronger than comparable suspensions filled with
air. These results are particularly interesting in view of the
teachings of EP-A-441 468 and 357 163 (SCHERING) which disclose the
use for echography purposes of micropartcles, respectively,
cavitate and clathrate compounds filled with various gases
including SF6; these documents do not however report particular
advantages of SF6 over other more common gases with regard to the
echogenic response.
EXAMPLE 4
[0039] A series of echogenic suspensions of gas-filled microbubbles
were prepared by the general method set forth below:
[0040] One gram of a mixture of hydrogenated soya lecithin (from
Nattermann Phospholipids GmbH. Germany) and dicetyl-phosphate
(DCP), in 9/1 molar ratio, was dissolved in 50 ml of chloroform,
and the solution was placed in a 100 ml round flask and evaporated
to dryness on a Rotavapor apparatus. Then. 20 ml of distilled water
were added and the mixture was slowly agitated at 75.degree. C. for
an hour. This resulted in the formation of a suspension of
multilamellar liposomes (MLV) which was thereafter extruded at
75.degree. C. through, successively. 3 .mu.m and 0.8 .mu.m
polycarbonate membranes (Nuclepore.RTM.). After cooling, 1 ml
aliquots of the extruded suspension were diluted with 9 ml of a
concentrated lactose solution (83 g/l), and the diluted suspensions
were frozen at -45.degree. C. The frozen samples were thereafter
freeze-dried under high vacuum to a free-flowing powder in a vessel
which was ultimately filled with air or a gas taken from a
selection of gases as indicated in Table 4 below. The powdery
samples were then resuspended in 10 ml of water as the carrier
liquid, this being effected under a stream of the same gas used to
fill the said vessels. Suspension was effected by vigorously
shaking for 1 min on a vortex mixer.
[0041] The various suspensions were diluted 1:20 with distilled
water equilibrated beforehand with air at 25.degree. C. and the
dilutions were then pressure tested at 25.degree. C. as disclosed
in Example 1 by measuring the optical density in a
spectrophotometric cell which was subjected to a progressively
increasing hydrostatic pressure until all bubbles had collapsed.
The results are collected in Table 4 below which, in addition to
the critical pressure PC, gives also the .DELTA.P values (see FIG.
1).
4TABLE 4 Bubble Sample Solubility count PC .DELTA.P No Gas M.sub.w
in H.sub.2O (10.sup.8/ml) (Torr) (Torr) LFre1 CF.sub.4 88 .0038 1.2
97 35 LFre2 CBrF.sub.3 149 .0045 0.9 116 64 LSF1 SF.sub.6 146 .005
1.2 92 58 LFre3 C.sub.4F.sub.8 200 .016 1.5 136 145 L1 air 29 .0167
15.5 68 17 L2 air 29 .0167 11.2 63 17 LAr1 Ar 40 .031 14.5 71 18
LKr1 Kr 84 .059 12.2 86 18 LXe1 Xe 131 .108 10.1 92 23 LFre4
CHClF.sub.2 86 .78 -- 83 25
[0042] The foregoing results clearly indicate that the highest
resistance to pressure increases is provided by the most
water-insoluble gases. The behavior of the microbubbles is
therefore similar to that of the microballoons in this regard.
Also, the less water-soluble gases with the higher molecular
weights provide the flattest bubble-collapse/pressure curves (i.e.
.DELTA.P is the widest) which is also an important factor of
echogenic response durability in vivo, as indicated
hereinbefore.
EXAMPLE 5
[0043] Some of the microbubble suspensions of Example 4 were
injected to the jugular vein of experimental rabbits as indicated
in Example 2 and imaging of the left heart ventricle was effected
as indicated previously. The duration of the period for which a
useful echogenic signal was detected was recorded and the results
are shown in Table 5 below in which C.sub.4F.sub.8 indicates
octafluorocyclobutane.
5 TABLE 5 Contrast duration Sample No Type of gas (sec) L1 Air 38
L2 Air 29 LMe1 CH.sub.4 47 LKr1 Krypton 37 LFre1 CF.sub.4 >120
LFre2 CBrF.sub.3 92 LSF1 SF.sub.6 >112 LFre3 C.sub.4F.sub.8
>120
[0044] These results indicate that, again in the case of
microbubbles, the gases according to the criteria of the present
invention will provide ultrasonic echo signal for a much longer
period than most gases used until now.
EXAMPLE 6
[0045] Suspensions of microbubbles were prepared using different
gases exactly as described in Example 4, but replacing the lecithin
phospholipid ingredient by a mole equivalent of
diarachidoyl-phosphatidyl- choline (C.sub.20 fatty acid residue)
available from Avanti Polar Lipids, Birmingham, Ala. USA The
phospholipid to DCP molar ratio was still 9/1. Then the suspensions
were pressure tested as in Example 4; the results, collected in
Table 6A below, are to be compared with those of Table 4.
6TABLE 6A Solubility Bubble Sample Type of Mw of in count PC
.DELTA.P No gas gas water (10.sup.8/ml) (Torr) (Torr) LFre1
CF.sub.4 88 .0038 3.4 251 124 LFre2 CBrF.sub.3 149 .0045 0.7 121 74
LSF1 SF.sub.6 146 .005 3.1 347 >150 LFre3 C.sub.4F.sub.8 200
.016 1.7 >350 >200 L1 Air 29 .0167 3.8 60 22 LBu1 Butane 58
.027 0.4 64 26 LAr1 Argon 40 .031 3.3 84 47 LMe1 CH.sub.4 16 .032
3.0 51 19 LEt1 C.sub.2H.sub.6 44 .034 1.4 61 26 LKr1 Kr 84 .059 2.7
63 18 LXe1 Xe 131 .108 1.4 60 28 LFre4 CHClF.sub.2 86 .78 0.4 58
28
[0046] The above results, compared to that of Table 4, show that,
at least with low solubility gases, by lengthening the chain of the
phospholipid fatty acid residues, one can dramatically increase the
stability of the echogenic suspension toward pressure increases.
This was further confirmed by repeating the foregoing experiments
but replacing the phospholipid component by its higher homolog.
i.e. di-behenoyl-phosphatidylcholine (C.sub.22 fatty acid residue).
In this case, the resistance to collapse with pressure of the
microbubbles suspensions was still further increased.
[0047] Some of the microbubbles suspensions of this Example were
tested in dogs as described previously for rabbits (imaging of the
heart ventricles after injection of 5 ml samples in the anterior
cephalic vein). A significant enhancement of the useful in-vivo
echogenic response was noted, in comparison with the behavior of
the preparations disclosed in Example 4, i.e. the increase in chain
length of the fatty-acid residue in the phospholipid component
increases the useful life of the echogenic agent in-vivo.
[0048] In the next Table below, there is shown the relative
stability in the left ventricle of the rabbit of microbubbles
(SF.sub.6) prepared from suspensions of a series of phospholipids
whose fatty acid residues have different chain lengths
(<injected dose: 1 ml/rabbit).
7TABLE 6B Duration of Chain length PC .DELTA.P contrast
Phospholipid (C.sub.n) (Torr) (Torr) (sec) DMPC 14 57 37 31 DPPC 16
100 76 105 DSPC 18 115 95 120 DAPC 20 266 190 >300
[0049] It has been mentioned hereinabove that for the measurement
of resistance to pressure described in these Examples, a constant
rate of pressure rise of 100 Torr/min was maintained. This is
justified by the results given below which show the variations of
the PC values for different gases in function to the rate of
pressure increase. In these samples DMPC was the phospholipid
used.
8 PC (Torr) Gas Rate of pressure increase (Torr/min) sample 40 100
200 SF.sub.6 51 57 82 Air 39 50 62 CH.sub.4 47 61 69 Xe 38 43 51
Freon 22 37 54 67
EXAMPLE 7
[0050] A series of albumin microballoons as suspensions in water
were prepared under air in a controlled sphere size fashion using
the directions given in Example 1. Then the air in some of the
samples was replaced by other gases by the gas-exchange sweep
method at ambient pressure. Then, after diluting to 1:10 with
distilled water as usual, the samples were subjected to pressure
testing as in Example 1. From the results gathered in Table 7
below, it can be seen that the two-steps preparation mode gives, in
some cases, echo-generating agents with better resistance to
pressure than the one-step preparation mode of Example 1.
9TABLE 7 Initial bubble Sample Type of Mw of the Solubility count
PC No gas gas in water (10.sup.8/ml) (Torr) A14 Air 29 .0167 3.1 53
A18 Air 29 .0167 3.8 52 A18/SF.sub.6 SF.sub.6 146 .005 0.8 115
A18/C.sub.2H.sub.6 C.sub.2H.sub.6 30 .042 3.4 72 A19 Air 29 .0167
1.9 51 A19/SF.sub.6 SF.sub.6 146 .005 0.6 140 A19/Xe Xe 131 .108
1.3 67 A22/CF.sub.4 CF.sub.4 88 .0038 1.0 167 A22/Kr Kr 84 .059 0.6
85
EXAMPLE 8
[0051] The method of the present invention was applied to an
experiment as disclosed in the prior art, for instance Example 1
WO-92/11873. Three grams of Pluronic.RTM. F68 (a copolymer of
polyoxyethylene-polyoxypropyle- ne with a molecular weight of
8400), 1 g of dipalmitoylphosphatidylglycero- l (Na salt. AVANTI
Polar Lipids) and 3.6 g of glycerol were added to 80 ml of
distilled water. After heating at about 80.degree. C., a clear
homogenous solution was obtained. The tenside solution was cooled
to room temperature and the volume was adjusted to 100 ml. In some
experiments (see Table 8) dipalmitoylphosphatidyl-glycerol was
replaced by a mixture of diarachidoylphosphatidylcholine (920 mg)
and 80 mg of dipalmitoylphosphatidic acid (Na salt. AVANTI Polar
lipids).
[0052] The bubble suspensions were obtained by using two syringes
connected via a three-way valve. One of the syringes was filled
with 5 ml of the tenside solution while the other was filled with
0.5 ml of air or gas. The three-way valve was filled with the
tenside solution before it was connected to the gas-containing
syringe. By alternatively operating the two pistons, the tenside
solutions were transferred back and forth between the two syringes
(5 times in each direction), milky suspensions were formed. After
dilution (1:10 to 1:50) with distilled water saturated at
equilibrium with air, the resistance to pressure of the
preparations was determined according to Example 1. The pressure
increase rate was 240 Torr/min. The following results were
obtained:
10 TABLE 8 Phospholipid Gas Pc (mm Hg) DP (mm Hg) DPPG air 28 17
DPPG SF.sub.6 138 134 DAPC/DPPA 9/1 air 46 30 DAPC/DPPA 9/1
SF.sub.6 269 253
[0053] It follows that by using the method of the invention and
replacing air with other gases e.g. SF.sub.6 even with known
preparations a considerable improvements i.e. increase in the
resistance to pressure may be achieved. This is true both in the
case of negatively charged phospholipids (e.g. DPPG) and in the
case of mixtures of neutral and negatively charged phospholipids
(DAPC/DPPA).
[0054] The above experiment further demonstrates that the
recognised problem sensitivity of microbubbles andicroballoons to
collapse when exposed to pressure i.e. when suspensions are
injected into living beings, has advantageously been solved by the
method of the invention. Suspensions with microbubbles or
microballoons with greater resistance against collapse and greater
stability can advantageously be produced providing suspensions with
better reproducibility and improved safety of echographic
measurements performed in vivo on a human or animal body.
* * * * *