U.S. patent application number 11/055544 was filed with the patent office on 2005-09-08 for contrast agents.
Invention is credited to Klaveness, Jo, Rongved, Pal, Stubberud, Lars.
Application Number | 20050196342 11/055544 |
Document ID | / |
Family ID | 10708317 |
Filed Date | 2005-09-08 |
United States Patent
Application |
20050196342 |
Kind Code |
A1 |
Klaveness, Jo ; et
al. |
September 8, 2005 |
Contrast agents
Abstract
Contrast agents comprising microbubble-generating carbohydrate
microparticles having a surfactant admixed within the
microparticulate structure, with the proviso that the surfactant is
not a C.sub.10-20 fatty acid, are disclosed. Processes for
preparing contrast agents also are disclosed.
Inventors: |
Klaveness, Jo; (Oslo,
NO) ; Rongved, Pal; (Hellvik, NO) ; Stubberud,
Lars; (Sodertalje, SE) |
Correspondence
Address: |
KENYON & KENYON
ONE BROADWAY
NEW YORK
NY
10004
US
|
Family ID: |
10708317 |
Appl. No.: |
11/055544 |
Filed: |
February 10, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11055544 |
Feb 10, 2005 |
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10820428 |
Apr 7, 2004 |
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10820428 |
Apr 7, 2004 |
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10635415 |
Aug 6, 2003 |
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10635415 |
Aug 6, 2003 |
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10072655 |
Feb 8, 2002 |
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10072655 |
Feb 8, 2002 |
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09231351 |
Jan 13, 1999 |
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09231351 |
Jan 13, 1999 |
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08939165 |
Sep 29, 1997 |
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08939165 |
Sep 29, 1997 |
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08478037 |
Jun 7, 1995 |
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5827502 |
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08478037 |
Jun 7, 1995 |
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08256149 |
Dec 9, 1994 |
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5558856 |
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Current U.S.
Class: |
424/9.51 ;
424/9.52 |
Current CPC
Class: |
A61K 49/1818 20130101;
A61K 49/1821 20130101; A61K 49/225 20130101; A61K 49/223
20130101 |
Class at
Publication: |
424/009.51 ;
424/009.52 |
International
Class: |
A61K 049/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 9, 1992 |
GB |
9200388.8 |
Claims
1-13. (canceled)
14. The method of generating an enhanced echocardiographic image of
a human or non-human body comprising: administering into the
pulmonary system of said body an echocardiographic contrast
enhancing amount of a contrast agent comprising
gasmicrobubble-generating aggregates of microparticles; applying to
a part of said body ultrasound at a frequency of 0.1 to 15 MHZ; and
generating said image; said microparticles comprising a water
soluble matrix material and a surfactant, the microbubbles
generated by said aggregates comprising SF.sub.6 or a fluorinated
low molecular weight hydrocarbon, said aggregates being 20-125
.PHI.m in size and said microparticles having an average size of
0.1 to 50 .PHI.m.
15. The method as claimed in claim 14 in which the surfactant is
selected from the group consisting of straight chain aliphatic
carboxylic acids and salts, sorbitan esters and mono- and
di-glycerides thereof; aralkanoic acids and the salts thereof;
steroid acids; sterols; straight chain aliphatic alcohols;
phospholipids; alkali metal alkyl sulphates and sulphonated esters;
polyoxyethylene-polyoxypropylene copolymers; polyoxyethylated
sorbitan esters; and mixtures of any of the foregoing.
16. The method as claimed in claim 14 in which the surfactant
comprises a lipophilically modified carbohydrate.
17. The method as claimed in claim 14 in which the surfactant is
present in an amount of 0.1-2.0% w/w relative to the water soluble
matrix.
18. The method as claimed in claim 14 for which the microbubbles
generated by said aggregates contain air in admixture with said
SF.sub.6 or fluorinated hydrocarbon.
19. The method as claimed in claim 14 for which the microbubbles
generated by said aggregates comprise carbon tetrafluoride.
20. The method as claimed in claim 14 in which the water soluble
matrix is a carbohydrate.
21. The method as claimed in claim 20 in which the carbohydrate is
a polysaccharide.
22. The method as claimed in claim 20 in which the carbohydrate is
a sugar alcohol.
23. The method as claimed in claim 14 which is non-contrast giving
before use, but which becomes effective on administration.
24. The method as claimed in claim 15 which is non-contrast giving
before use, but which becomes effective on administration.
25. The method as claimed in claim 16 which is non-contrast giving
before use, but which becomes effective on administration.
26. The method as claimed in claim 17 which is non-contrast giving
before use, but which becomes effective on administration.
27. The method as claimed in claim 18 which is non-contrast giving
before use, but which becomes effective on administration.
28. The method as claimed in claim 19 which is non-contrast giving
before use, but which becomes effective on administration.
Description
[0001] This invention relates to novel contrast agents, more
particularly to new microparticulate contrast agents of use in
diagnostic imaging.
[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 microbubbles 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] Initial studies involving free gas microbubbles generated in
vivo by intracardiac injection of physiologically acceptable
substances have demonstrated the potential efficiency of such
bubbles as contrast agents in echocardiography; such techniques are
severely limited in practice, however, by the short lifetime of the
free bubbles. Interest has accordingly been shown in methods of
generating and/or stabilising gas microbubbles for echocardiography
and other ultrasonic studies, for example using emulsifiers, oils,
thickeners or sugars.
[0004] Techniques involving the use of sugars in ultrasound
contrast agents are described in, for example, U.S. Pat. No.
4,681,119, U.S. Pat. No. 4,442,843 and U.S. Pat. No. 4,657,756,
which disclose the use of particulate solids having a plurality of
gas-filled voids and preferably also a plurality of nuclei for
microbubble formation. EP-A-0123235 and EP-A-0122624 suggest
ultrasound contrast agents consisting of surfactant-coated or
surfactant-containing gas-containing microparticles which may
include a variety of sugars. Where surfactant-containing
microparticles are described, these are prepared simply by
commingling the surfactant with the microparticulate materials,
e.g. by trituration.
[0005] DE-A-3834705 proposes the use of suspensions containing
microparticles of mixtures of at least one C.sub.10-20, tatty acid
with at least one non-surface active substance, including sugars
such as cyclodextrins, monosaccharides, disaccharides or
trisaccharides, as well as other polyols and inorganic and organic
salts; in practice only the use of galactose as the non-surface
active material and only the use of saturated fatty acids are
exemplified. The microparticulate materials are typically prepared
by coprecipitating the fatty acid and non-surface active substance
and comminuting the resulting product, e.g. using an air-jet
mill.
[0006] One material of, the type described in DE-A-3834705, SHU 508
(Levovist.RTM.), is described in the following publications:
[0007] Schlief, R. et al., Circulation Supplement III (1990) 82, p.
28; Schartl, M. et al., Circulation Supplement III (1990) 82, p.
261; Fritzsch, T. et al., Invest. Radiol. (1990) 25 (Suppl), pp.
160-161; Schlief, R. et al., Echocardiography (1990) 7, pp. 61-64;
Loughery, E. J. et al., Echocardiography (1990) 7, pp. 279-292; and
Smith, M. D. et al., JACC (1989) 13, pp. 1622-1628.
[0008] 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.
[0009] 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.
[0010] A general disadvantage of most of the existing
gas-containing/gas-generating particulate contrast agents such as
the sugar-based agents discussed above is their relative lack of
stability in vivo. This is a particular problem in applications
such as echocardiography, where there is a need for improved
contrast agents combining sufficient stability and small
microbubble size (typically less than about 10 .mu.m, preferably
less than about 7 .mu.m) to permit passage through the pulmonary
capillary bed and so allow enhanced visualisation of the left side
of the heart, it preferably for more than one passage of
circulation. There is accordingly a need for contrast agents which
generate microbubble systems exhibiting good stability while still
providing an effective level of contrast efficiency.
[0011] The present invention is based on our finding that contrast
agents comprising microparticles of a carbohydrate having a
surfactant admixed therewith (but excluding the previously
disclosed mixtures of galactose and saturated C.sub.10-20 fatty
acids) may be used to generate microbubble systems exhibiting
enhanced contrast effect and/or stability relative to previously
proposed carbohydrate-based contrast agents. In the ultrasound
field this may be demonstrated by, for example, in vitro
measurements of initial attenuation levels and the half lives of
the attenuative effect; a useful indication of the combined effect
of these properties is the integral obtained by determining the
area under the curve of a plot of attenuation against time.
[0012] The term "surfactant" as used herein means any compound
having amphiphilic properties capable of modifying surface
tension.
[0013] Thus, according to one aspect of the present invention,
there are provided contrast agents comprising
microbubble-generating carbohydrate microparticles having a
surfactant admixed within the microparticulate structure, with the
proviso that the surfactant is not a saturated C.sub.10-20 fatty
acid when the microparticulate carbohydrate is galactose.
[0014] The microparticulate carbohydrate is preferably water
soluble, and subject to the foregoing proviso may for example be
selected from hexoses such as glucose, fructose or galactose;
disaccharides such as sucrose, lactose or maltose; pentoses such as
arabinose, xylose or ribose; and polysaccharides such as .alpha.-,
.beta.- and .gamma.-cyclodextrins, maltodextrin and glycogen; the
term "carbohydrate" as used herein is also intended to embrace
sugar alcohols, e.g. alditols such as mannitol or sorbitol.
Microparticles of the above carbohydrates will normally have gas
present as an inclusion in the voids of their crystal structure
and/or adhered to their surface, which gas may generate
microbubbles when, for example, the microparticles are suspended or
dissolved in an injectable carrier liquid, for example water for
injection, an aqueous solution of one or more inorganic salts (e.g.
physiological saline or a physiological buffer solution), an
aqueous solution of a monosaccharide (e.g. glucose or galactose) or
disaccharide (e.g. lactose), or an aqueous solution of a
physiologically tolerable monohydric or polyhydric alcohol (e.g.
ethanol, propanol, isopropanol, ethylene glycol, propylene glycol,
glycerine or polyethylene glycol).
[0015] In addition to or alternatively to air, any biocompatible
gas may be employed in the contrast agents of the invention, for
example nitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide,
helium, argon, sulphur hexafluoride and low molecular weight
optionally fluorinated hydrocarbons such as methane, acetylene or
carbon tetrafluoride. The term "gas" as used herein includes any
substance in the gaseous form at 37.degree. C. The gas may be
contained in the contrast agent in such a way that before use the
product is non-contrast giving but becomes effective on
administration, e.g. as a result of the gas forming microbubbles as
a soluble carbohydrate matrix dissolves.
[0016] Additionally or alternatively the carbohydrate may
incorporate one or more gas precursors, including carbonates and
bicarbonates (e.g. sodium or ammonium bicarbonate) and
aminomalonate esters.
[0017] Subject to the foregoing proviso a wide variety of
surfactants may be used in the ultrasound contrast agents of the
invention; it will of course be appreciated that the surfactant is
required to be biocompatible, i.e. that it should be
physiologically tolerable in the quantities in which it is to be
administered. The surfactant is advantageously biodegradable in
vivo or otherwise readily eliminable from the system.
[0018] The surfactant may, for example, be an amphiphilic lipid,
e.g. selected from fatty acids and salts (e.g. alkali metal salts)
thereof, steroid acids, sterols, phospholipids and glycolipids.
Such lipids-include high molecular weight (e.g. C.sub.10-50)
straight chain saturated and unsaturated aliphatic acids, such as
capric, palmitic, hexadecanedioic, stearic, linolenic, behenic,
docosanedioic and melissic acids; aralkanoic acids, e.g. phenyl
lower alkanoic acids such as 2-phenylbutyric acid; salts of any of
the foregoing acids; mono- and di-glycerides, for example glyceryl
esters of high molecular weight (e.g. C.sub.10-50) aliphatic acids,
such as glyceryl monolaurate; cholanic acids such as
5.beta.-cholanic acid; cholesterol; sorbitan esters of fatty acids
such as Span-type materials; high molecular weight (e.g.
c.sub.10-50) straight chain aliphatic alcohols such as stearyl
alcohol and cetyl alcohol; phospholipids such as phosphatidyl
choline (lecithin) and dioleoylphosphatidyl ethanolamine (DOPE);
and mixtures thereof.
[0019] Other surfactants which may be employed include anionic
surfactants, for example alkali metal alkyl sulphates such as
sodium lauryl sulphate and sulphonated esters such as sodium
dioctyl sulphosuccinate (docusate); and non-ionic surfactants, for
example polyoxyethylene-polyoxyproplyene copolymers (e.g.
poloxamers such as Pluronic F68) and polyoxyethylated sorbitan
esters (e.g. polysorbates such as Tween-type materials).
[0020] The surfactant moiety may if desired be covalently linked to
a substrate such as a carbohydrate prior to its admixture with the
principal microparticulate carbohydrate. Thus, for example, a fatty
acid such as palmitic acid (preferably in the form of a reactive
derivative such as a corresponding acyl halide) may be used to
esterify a (preferably appropriately O-protected) sugar such as
galactose and the resulting lipophilically modified carbohydrate
used as the surfactant in accordance with the invention.
[0021] The surfactant may, for example, be present in an amount of
0.01-5.0 wt. %, preferably 0.1-2.0 wt. %, relative to the
microparticulate carbohydrate.
[0022] The contrast agents of the invention may be used in a
variety of diagnostic imaging techniques, including ultrasound, MR
and X-ray imaging. Their uses in diagnostic ultrasonic imaging and
MR imaging, e.g. as susceptibility contrast agents, constitute
preferred features of the invention.
[0023] The contrast agents of the invention may be prepared by any
convenient method which leads to physical admixture of the
surfactant within the microparticulate structure of the
carbohydrate and to production of microparticles of the desired
size.
[0024] In one preferred method according to the invention the
carbohydrate and the surfactant are each dissolved in appropriate
mutually miscible solvents (e.g. water in the case or the
carbohydrate and a lower alkanol such as ethanol in the case of
lipid surfactants such as fatty acids), the resulting solutions are
mixed, the solvents are removed (e.g. by evaporation under reduced
pressure), and the resulting solid mixture is micronised to yield
the desired microparticles. It will be appreciated that all such
operations should be effected under sterile conditions.
[0025] In an alternative method according to the invention a
(preferably aqueous) solution of the carbohydrate is mixed with a
liposome-forming material (e.g. a thin film of a lipid such as
lecithin formed on the inner surface of the mixing vessel by
evaporating the solvent from a solution of the lipid in an
appropriate organic solvent, for example a chlorinated hydrocarbon
such as chloroform) so as to form a liposome-containing
carbohydrate solution from which the solvent may be removed (e.g.
by freeze-drying) to yield a product comprising
carbohydrate-containing liposomes; this product may be micronised
to given microparticles of the desired size.
[0026] In, general conventional micronisation techniques such as
grinding or milling may be employed in processes according to the
invention. Ball-milling of the solid mixture has been found to be
particularly advantageous, permitting the preparation of
microparticles in the form of aggregates, (for example having an
aggregate size of 20-125 micrometers, such as 30-50 micrometers) of
particles having a particle size of, for example, 1-50 micrometers,
such as 1-10 micrometers. Such aggregates will tend to contain a
substantial volume of air adsorbed on their surfaces and entrained
in voids such as interparticle cavities or at grain boundaries
between the crystallites. The particle size may, for example, be
selected to be substantially commensurate with the desired
microbubble size. In ultrasonic 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 and microparticles having an average size of
0.1-10 .mu.m, e.g. 1-7 .mu.m; the use of microparticles of average
size 1-4 .mu.m to generate microbubbles with an average size of 4-7
.mu.m is generally advantageous. Substantially larger bubbles and
particles, e.g. with average sizes up to 500 .mu.m, may however be
useful in other applications, for example gastrointestinal
imaging.
[0027] Ultrasound contrast agents in the form of microparticles
comprising a microbubble-generating carbohydrate in admixture with
an amphiphilic organic acid containing in excess of 20 carbon atoms
are the subject matter of our international patent application
cofiled herewith and claiming priority from British patent
application No. 9200387.0.
[0028] The following non-limitative Examples serve to illustrate
the invention:--
EXAMPLES 1-18
[0029] General Procedure
[0030] D-(+)-galactose (10.0 g) was dissolved in distilled water
(14.2 g) at 50.degree. C., sterile filtered and cooled on ice to a
temperature of 4.about.8.degree. C. The stated amounts of the
surfactants (in % w/w relative to the galactose) listed in Table I
were each dissolved in the amount of 96% ethanol (or water in
Examples 5 and 6) shown in the Table, at 50.about.78.degree. C.,
and the resulting solution was sterile filtered and then
aseptically added to the cold aqueous galactose solution under
stirring. The resulting mixture was evaporated to dryness under
reduced pressure (10 torr, 40.degree. C.), and the resulting solid
product was dried in a desiccator overnight and then ground for 10
minutes under aseptic conditions in a stainless steel ball mill
having a 50 ml grinding cup and 3.times.20 mm bails (Retsch
centrifugal ball mill, Sl). The ground product was dried in a
desiccator for 24 hours.
1TABLE I Amount of Amount of ethanol Example Surfactant (or water)
No. Surfactant (% w/w) (g) 1 Lecithin 1.0 1.2 2 " 0.2 1.2 3 Sodium
Lauryl Sulphate 1.0 1.0 (water) 4 " 0.1 1.0 (water) 5 Span 80 1.0
1.2 6 " 0.1 1.2 7 Span 85 1.0 1.2 8 " 0.1 1.2 9 Pluronic F68 1.0
1.2 10 " 0.1 1.2 11 Sodium Docusate 1.0 1.2 12 " 0.1 1.2 13 DOPE
1.0 1.2 14 " 0.1 1.2 15 .alpha.-Glyceryl Monolaurate 0.2 3.2
Glyceryl Tripalmitate 0.2 Cholesterol 0.2 Cholesterol Acetate 0.2
Cholesterol Benzoate 0.2 16 .alpha.-Glyceryl Monolaurate 0.02 1.2
Glyceryl Tripalmitate 0.02 Cholesterol 0.02 Cholesterol Acetate
0.02 Cholesterol Benzoate 0.02 17 Hexadecanedioic Acid 0.2 1.2 18
Linolenic Acid 1.0 1.2
EXAMPLES 19-22
[0031] The general procedure for Examples 1-18 was repeated except
that the D-(+)-galactose was replaced by the carbohydrates listed
in Table II, in the amounts and using the quantities of water
shown, and that the surfactant used was palmitic acid (0.2% w/w
relative to the carbohydrate) dissolved in 96% ethanol (1.2 g).
2TABLE II Amount of Amount of Example Microbubble-generating
Carbohydrate water No. Carbohydrate (g) (g) 19 Xylose (BDH) 10.0
14.2 20 Maltodextrin 10.0 14.2 21 Glycogen (Merck) 5.0 17.2 22
.alpha.-Cyclodextrin (Sigma) 5.0 12.2
EXAMPLE 23
6-O-Palmitoyl-D-galactopyranose/galactose mixtures
(A) 6-O-Palmitoyl-1,2,3,4-diisopropylidene-D-galactopyranose
[0032] 1,2,3,4-Diisopropylidene-D-galactopyranose (Sigma, 13.4 g,
51.3 mmol) and triethylamine (7.15 ml, 51.3 mmol) were dissolved in
methylene chloride (150 ml) and cooled to 0.degree. C. Palmitoyl
chloride (Aldrich, 14.1 g, 51.3 mmol) dissolved in methylene
chloride (100 ml) was added dropwise with stirring over 1 h. The
cooling bath was removed and the reaction mixture was stirred
overnight. Precipitated triethylamine hydrochloride was removed by
filtration, the filtrate was transferred to a separating funnel and
extracted with water (3.times.50 ml), dried over MgSO.sub.4 and the
solvent was removed in vacuo. The residue was a light brownish oil
which solidified to waxy crystals. Crude yield: 23 g. The crude
product was used without further purification. A small aliquot was
recrystallized for characterisation FT-IR:CO-1734 cm.sup.-1.
[0033] .sup.13C-NMR: CO-ester 179. Mp. 124-127.degree. C.
(B) 6-O-Palmitoyl-D-galactopyranose
[0034] 6-O-Palmitoyl-1,2,3,4-diisopropylidene-D-galactopyranose (6
g) was dissolved in acetic acid (25 ml) and heated to 100.degree.
C. under nitrogen for 6 h. During subsequent cooling to room
temperature, the product precipitated from the solvent, and was
left at room temperature overnight. The crystals were collected by
filtration and dried under vacuum. Yield:3.3 g. The product was
characterized by FT-IR:CO-1734 cm.sup.-1; OH-3464 cm.sup.-1.
(C) 6-O-Palmitoyl-D-galactopyranose/galactose mixtures
[0035] (i) D-(+)-galactose (2 g) was dissolved in purified water
(2.879) and sterile filtered. 6-O-Palmitoyl-D-galactopyranose (0.25
g) prepared as described in (B) above was dissolved in ethanol (3
g) and sterile filtered. The solution of the
palmitoyl-galactopyranose was added to the galactose solution under
stirring and the whole mixture was taken to dryness under vacuum
(10 torr, 50.degree. C.). The product was dried in a desiccator
overnight.
[0036] (ii) The procedure of (i) was repeated using
6-O-palmitoyl-D-galactopyranose (0.50 g) dissolved in ethanol (6
g).
EXAMPLE 24
[0037] Freeze-dried liposomes containing D-(+)-galactose
particles
[0038] 1 ml 100 mg/ml phosphatidylcholine was dissolved in 10 ml
chloroform. The mixture was poured into a round bottom flask, and
the organic phase was evaporated at 40.degree. C. in such a way
that a thin film of the 35' phosphatidylcholine was, formed on the
inner surface of the flask. 10 ml of a sterile, pyrogen free 40%
aqueous D-(+)-galactose solution was then added at 40.degree. C.
and the flask was kept rotating for 1 hour. The aqueous solution
containing liposomes and dissolved galactose was then freeze-dried
for 24 hours, and the resulting product consisting of freeze-dried
galactose and freeze dried galactose-filled liposomes was then
ground in a ball-mill to yield a product with a particle size
distribution of 1-20 .mu.m.
EXAMPLE 25
[0039] Echogenicity In Vitro
[0040] 10 ml of propylene glycol mixed with 90 ml of 5% dextrose in
water was used as a carrier liquid for determining the echogenicity
of products according to the Examples. 1.0 g of each product to be
tested was dispersed in 3.0 ml of the carrier liquid and shaken for
seconds. The resulting mixture was added to 52 ml of 5% human serum
albumin infusion solution in the measurement cell and the acoustic
effects of the products were investigated by measuring the acoustic
transmission through the samples using a 5 MHz broadband transducer
in a pulse-reflection technique. The temperature in the measurement
cell was stabilised to 37.degree. C. and circulation of the liquid
was maintained by means of stirring at a constant rate. Ultrasound
transmission through the samples was measured as a function of time
over a duration of 390 seconds. Results were normalized to
measurements on a reference consisting of, 55 ml of 59 human serum
albumin infusion solution.
[0041] Results for representative exemplified products and
comparative results for unmodified milled D-(+)-galactose are shown
in the accompanying drawing as FIG. 1. It will be apparent that
these products exhibit a strong effect on ultrasonic attenuation in
vitro, an effect which persisted for several minutes.
* * * * *