U.S. patent application number 10/449832 was filed with the patent office on 2004-07-08 for contrast agents.
This patent application is currently assigned to Amersham Health AS. Invention is credited to Klaveness, Jo, Priebe, Hanno, Rongved, Pal, Stubberud, Lars.
Application Number | 20040131548 10/449832 |
Document ID | / |
Family ID | 10692381 |
Filed Date | 2004-07-08 |
United States Patent
Application |
20040131548 |
Kind Code |
A1 |
Klaveness, Jo ; et
al. |
July 8, 2004 |
Contrast agents
Abstract
The present invention provides contrast agents for use in
diagnostic ultrasound studies comprising microbubbles of gas or a
gas precursor encapsulated by non-proteinaceous crosslinked or
polymerised amphiphilic moieties.
Inventors: |
Klaveness, Jo; (Oslo,
NO) ; Priebe, Hanno; (Oslo, NO) ; Rongved,
Pal; (Oslo, NO) ; Stubberud, Lars; (Oslo,
NO) |
Correspondence
Address: |
KENYON & KENYON
ONE BROADWAY
NEW YORK
NY
10004
US
|
Assignee: |
Amersham Health AS
|
Family ID: |
10692381 |
Appl. No.: |
10/449832 |
Filed: |
May 29, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10449832 |
May 29, 2003 |
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10236076 |
Sep 5, 2002 |
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10236076 |
Sep 5, 2002 |
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09927919 |
Aug 10, 2001 |
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09927919 |
Aug 10, 2001 |
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09255112 |
Feb 22, 1999 |
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09255112 |
Feb 22, 1999 |
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08950731 |
Oct 15, 1997 |
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08950731 |
Oct 15, 1997 |
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08734167 |
Oct 21, 1996 |
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08734167 |
Oct 21, 1996 |
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08466615 |
Jun 6, 1995 |
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5567413 |
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08466615 |
Jun 6, 1995 |
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08119217 |
Oct 29, 1993 |
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5536490 |
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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 |
Mar 28, 1991 |
GB |
9106673.8 |
Claims
1. Contrast agents for use in diagnostic ultrasound studies
comprising microbubbles of gas or a gas precursor encapsulated by
non-proteinaceous crosslinked or polymerised amphiphilic
moieties.
2. Contrast agents as claimed in claim 1 in the form of gas-filled
microballoons.
3. Contrast agents as claimed in claim 1 or claim 2 containing
biodegradable linkages selected from amide, imide, imine, ester,
anhydride, acetal, carbamate, carbonate, carbonate ester and
disulphide groups.
4. Contrast agents as claimed in claim 3 containing biodegradable
crosslinking groups.
5. Contrast agents as claimed in claim 4 wherein the biodegradable
crosslinking groups include units of
formula--(Y).sub.n.CO.O.C(R.sup.1R.s- up.2).O.CO.(Z).sub.n-(where Y
and Z, which may be the same or different, are --O--, --S-- or
--NR.sup.3--; R.sup.1 and R.sup.2, which may be the same or
different, are hydrogen atoms or carbon-attached monovalent organic
groups or together represent a carbon-attached divalent organic
group; R.sup.3 is a hydrogen atom or an organic group; and the
symbols n, which may be the same or different, are zero or 1).
6. Contrast agents as claimed in claim 1 or claim 2 obtained from
polymerisable amphiphilic moieties containing unsaturated
lipophilic chains.
7. Contrast agents as claimed in claim 6 wherein the unsaturated
lipophilic chains are oleyl or linoleyl groups or contain
diacetylene groupings or acryloyl or methacryloyl groupings.
8. Contrast agents as claimed in any of the preceding claims
wherein the hydrophilic portion of the amphiphile contains one or
more groups selected from quaternary ammonium, hydroxyl, carboxy,
carboxylate ion, amide, phosphate, sulphate and sulphonate.
9. Contrast agents as claimed in claim 8 wherein the hydrophilic
portion of the amphiphile is the triglyceryl moiety of a
phospholipid, an iodinated X-ray contrast agent, a carbohydrate, or
a choline, ethanolamine, serine or glycerol residue.
10. Contrast agents as claimed in any of claims 1 to 7 wherein the
hydrophilic portion of the amphiphile is an optionally etherified
polyoxyethylene glycol residue.
11. Contrast agents as claimed in claim 10 wherein the amphiphile
is tetraethylene glycol mono-12-(methacryloyloxy)dodecanoate.
12. Contrast agents as claimed in claim 10 wherein the amphiphile
is polyethylene glycol (550) methyl ether
12-(methacryloyloxy)dodecanoate.
13. Contrast agents as claimed in claim 10 wherein the amphiphile
is polyethylene glycol (2000) methyl ether
12-(methacryloyloxy)dodecanoate.
14. Contrast agents as claimed in claim 10 wherein the amphiphile
is tetraethylene glycol mono-16-(methacryloyloxy)hexadecanoate.
15. Contrast agents as claimed in claim 10 wherein the amphiphile
is polyethylene glycol (350) methyl ether
16-(methacryloyloxy)hexadecanoate.
16. Contrast agents as claimed in claim 10 wherein the amphiphile
is tetraethylene glycol mono-12-(acryloyloxy)dodecanoate.
17. Contrast agents as claimed in any of the preceding claims
further containing an inorganic particulate stabiliser.
18. A process for the preparation of a contrast agent as claimed in
claim 1 which consists of forming a fluid dispersion of vesicles
comprising a gas or gas precursor encapsulated by amphiphilic
material and thereafter crosslinking or polymerising said
amphiphilic material.
19. A process as claimed in claim 11 wherein the fluid dispersion
is prepared by sonication to generate an oil-in-water emulsion in
which a volatile hydrocarbon is encapsulated by the amphiphilic
material and said volatile hydrocarbon is partially or completely
removed from the vesicles after crosslinking or polymerisation of
the amphiphilic material.
Description
[0001] This invention relates to novel contrast agents, more
particularly to new gas-containing or gas-generating contrast
agents of use in diagnostic ultrasonic 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 bubbles and
encapsulated gases or liquids. It is generally accepted that low
density contrast agents which are easily compressible are
particularly efficient in terms of the acoustic backscatter they
generate, and considerable interest has therefore been shown in the
preparation of gas-containing and gas-generating systems.
[0003] Initial studies involving free gas bubbles generated in vivo
by intracardiac injection of physiologically acceptable substances
have demonstrated the potential efficiency of such bubbles as
contrast agents in 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
stabilising gas bubbles for echocardiography and other ultrasonic
studies, for example using emulsifiers, oils, thickeners or
sugars.
[0004] WO 80/02365 discloses the use of gelatin-encapsulated gas
microbubbles for enhancing ultrasonic images. Such microbubbles do
not, however, exhibit adequate stability at the dimensions
preferred for use in echocardiography (1-10 .mu.m) in view of the
extreme thinness of the encapsulating coating.
[0005] U.S. Pat. No. 4,774,958 discloses the use of microbubble
dispersions stabilised by encapsulation in denatured protein, e.g.
human serum albumin. Such systems permit the production of
microbubble systems having a size of e.g. 2-5 .mu.m but still do
not permit efficient visualisation of the left heart and
myocardium.
[0006] EP-A-0327490 discloses, inter alia, ultrasonic contrast
agents comprising a microparticulate synthetic biodegradable
polymer (e.g. a polyester of a hydroxy carbonic acid, a polyalkyl
cyanoacrylate, a polyamino acid, a polyamide, a polyacrylated
saccharide or a polyorthoester) containing a gas or volatile fluid
(i.e. having a boiling point below 60.degree. C.) in free or bonded
form. Emulsifiers may be employed as stabilisers in the preparation
of such agents, but such emulsifiers do not chemically interact
with the polymer.
[0007] We have now found that particularly effective ultrasonic
contrast agents may be obtained by encapsulating gas bubbles or gas
generating systems with polymers containing chemically linked
surface active, i.e. amphiphilic, moieties. Thus the surface active
properties of the amphiphilic groups stabilise the microbubble
system by reducing surface tension at the gas-liquid interfaces,
e.g. by forming monolayers or one or more bilayers (alternatively
known by the terms micelles, vesicles, liposomes and niosomes) at
said interfaces, while the linking of the groups through the
polymer system generates further stability. Flexibility of the
encapsulating materials also enhances the image density afforded by
such contrast agents. For simplicity the terms "vesicle" is used
herein to denote all such microbubble structures prior to or after
crosslinking or polymerisation. It should be noted that under some
conditions irregularly shaped structures may be formed, e.g.
microtubules which may join with or even entrap spherical
structures.
[0008] Thus according to one aspect of the present invention there
are provided contrast agents for use in diagnostic ultrasound
studies comprising microbubbles of gas or a gas precursor
encapsulated by non-proteinaceous crosslinked or polymerised
amphiphilic moieties.
[0009] The term "crosslinked" is used herein to denote that the
amphiphilic moieties are linked to each other to form a polymeric
structure which may incorporate one or more polymer systems
(including copolymers).
[0010] A major advantage of contrast agents according to the
invention is that they may be designed to a particular desired
level of biodegradability in vivo by selecting appropriate
biodegradable linkages at appropriate positions. It will be
appreciated that in order to be effective the contrast agents must
be stable throughout the ultrasonic examination but are preferably
metabolised or removed safely from the circulation system shortly
thereafter. Contrast agents in accordance with the invention should
thus preferably have a half-life in vivo of not more than 48 hours,
for example 1-12 hours.
[0011] Biodegradable linkages which may be present in contrast
agents according to the invention include amide, imide, imine,
ester, anhydride, acetal, carbamate, carbonate, carbonate ester and
disulphide groups. At least one such group should preferably be
present in the amphiphilic moiety, in the hydrophilic and/or
lipophilic portion; it may be advantageous to position the group in
the hydrophilic part to facilitate enzymic interaction in vivo. It
is further preferred that biodegradable linkages be present in the
polymer backbone to ensure substantial breakdown of the polymer in
the body.
[0012] Any biocompatible gas may be employed in the contrast agents
of the invention, for example air, 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 gas may be free
within the microbubble, advantageously in the form of a gas-filled
"microballoon" since the echogenicity of such products may be
enhanced by virtue of their relatively flexible nature.
Alternatively the gas may be trapped or entrained within a
containing substance. The term "gas" as used herein includes any
substances in gaseous form at 37.degree. C.
[0013] Gas precursors include carbonates and bicarbonates, e.g.
sodium or ammonium bicarbonate and aminomalonate esters. The term
"gas precursor" as used herein also embraces substances such as
volatile hydrocarbons which may initially be encapsulated but
thereafter are partially or completely removed from the vesicles,
e.g. by evaporation or freeze-drying, to be replaced by gas.
[0014] For applications in echocardiography, in order to permit
free passage through the pulmonary system and to achieve resonance
with the preferred imaging frequency of about 0.1-15 MHz, it may be
convenient to employ microbubbles having an average size of 0.1-10
.mu.m, e.g. 1-7 .mu.m. Substantially larger bubbles, e.g. with
average sizes of up to 500 .mu.m, may however be useful in other
applications, for example gastrointestinal imaging or
investigations of the uterus or Fallopian tubes.
[0015] If desired the microbubbles may incorporate particulate
stabilisers, for example inorganic materials such as silica or iron
oxide which are only partially wetted by the solvent system
employed, e.g. having a particle size of 1-500 nm. Colloidal silica
having a particle size of 5-50 nm may advantageously be employed
for this purpose.
[0016] Polymer systems which may be employed in the contrast agents
of the invention include carbohydrates such as dextrans and
starches, chitin, chitosan, carboxymethylchitosan, alginate,
hyaluronic acid, polyacrylamides, polycyanoacrylates,
hydroxyalkylpolycyanoacrylates, polyhydroxy acids such as
polylactic acids, polyhydroxybutyrates, polyglycolic acids,
polylactide-glycolides, polyorthoesters, polyanhydrides,
polyurethanes, polyester imides, polyimides, polyacetals,
poly-epsilon-caprolactones, polydioxanones, polyaminotriazoles,
poly(amide-enamines), poly(amide-urethanes), polyphosphazenes,
polyvinyl alcohols, organo-polysiloxanes, poly(enol-ketones) and
copolymers of these materials, modified as necessary to introduce
hydrophilic or lipophilic moieties.
[0017] The microbubbles according to the invention may be prepared
by forming a fluid dispersion of vesicles comprising a gas or gas
precursor encapsulated by amphiphilic material followed by
crosslinking or polymerisation of the amphiphilic material.
[0018] The vesicles will normally comprise a substantially
spherical monolayer or multilayer of the amphiphilic material. The
hydrophilic moieties of the amphiphiles will be physically
associated to form a contiguous layer while the lipophilic moieties
will also form a layer which may be inside or outside the
hydrophilic layer. In bilayers, two layers of the amphiphilic
material may be superimposed: thus, for example, a first layer of
amphiphilic material may form in which the lipophilic groups are on
the outside A second layer of amphiphilic material may then overlay
the first layer with the lipophilic groups adjacent to the
lipophilic groups of the first layer and the hydrophilic groups on
the outside. Similarly, a bilayer may have the lipophilic groups on
the outside and inside and the hydrophilic groups sandwiched
between.
[0019] Where the fluid in which the vesicles are dispersed is
polar, for example aqueous, the hydrophilic groups of the vesicles
will tend to be on the outside of the micelles and the lipophilic
groups will be on the inside forming a monolayer. On the other
hand, if the dispersing fluid is apolar, the lipophilic groups will
be on the outside, particularly if the encapsulated material is
hydrophilic, e.g. a gas precursor or a solid material containing
absorbed or entrained gas, possible in association with a polar
liquid. Bilayers may form when the encapsulated material is of the
same type, i.e. hydrophilic or lipophilic, as the dispersing
fluid.
[0020] The amphiphiles used in accordance with the present
invention will carry functional groups permitting crosslinking or
polymerisation. These may in some instances be groups imparting
hydrophilic or lipophilic character or they may be independent of
the amphiphilic groupings.
[0021] The amphiphiles may be considered in three categories:
[0022] 1. The amphiphiles may carry at least two simple reactive
groups such as hydroxyl, amino or carboxyl groups which are capable
of reacting with polyvalent reactive monomers or preformed
polymers. For example, if the amphiphile carries two hydroxyl
groups (in the hydrophilic moiety), a dicarboxylic acid such as
suberic acid may be reacted with the vesicles after encapsulation
of the gas or gas precursor to provide a crosslinked or polymerised
structure. Diamino-amphiphiles may similarly be reacted with
dicarboxylic acids while dicarboxylic amphiphiles may be reacted
with diamines or diols. Additional crosslinking may be provided by
trifunctional reagents. A catalyst will normally be present to
assist reaction.
[0023] The crosslinkinq agent may itself be amphiphilic so that the
vesicle will form with the lipophilic and hydrophilic groups of the
first amphiphile and the amphiphilic crosslinking agent in
alignment, whereupon crosslinking between the reactive functional
groups may be initiated.
[0024] As indicated above, it is particularly advantageous for the
polymerised or crosslinked amphiphile to be biodegradable,
especially into relatively simple water soluble units. In the case
of the ester and amide bonds referred to above, esterase and
amidase enzymes will commonly be available in the vascular system
and can degrade the encapsulating material back to separate
amphiphile molecules and the diamine, diol or diacid reagents which
under physiological conditions will not recombine.
[0025] If desired, even more biolabile crosslinking groups such as
carbonate ester groups may be introduced e.g. using orthoester
crosslinking agents. Another useful class of crosslinking agents
have the formula (I)
A.sup.1.R.sup.8.(Y).sub.n.CO.O.C(R.sup.1R.sup.2).O.CO.(Z).sub.n.R.sup.9.A.-
sup.2 (I)
[0026] (where Y and Z, which may be the same or different, are
--O--, --S-- or --NR.sup.3--;
[0027] R.sup.1 and R.sup.2, which may be the same or different, are
hydrogen atoms or carbon-attached monovalent organic groups or
together represent a carbon-attached divalent organic group;
[0028] R.sup.3 is a hydrogen atom or an organic group;
[0029] the symbols n, which may be the same or different, are zero
or 1;
[0030] R.sup.8 and R.sup.9, which may be the same or different are
divalent organic groups, for example alkylene or alkylidene groups
having 1-12 carbon atoms; and
[0031] A.sup.1 and A.sup.2 are functional groups, for example
reactive with hydroxyl, amino or carboxyl groups), since the
crosslinking groups so introduced contain units of formula
--(Y).sub.n.CO.O.C(R.sup.1R.sup.2).O.CO.(Z).sub.n-
[0032] (where Y, Z, each n, R.sup.1 and R.sup.2 are as defined
above) which are particularly readily degraded by common esterases,
while exhibiting stability in the absence of enzymes.
[0033] R.sup.1, R.sup.2 and R.sup.3 may each be a hydrocarbyl or
heterocyclic group, for example having 1-20 carbon atoms, e.g. an
alkyl or alkenyl group (preferably having up to 10 carbon atoms), a
cycloalkyl group (preferably having up to 10 carbon atoms), an
aralkyl group (preferably having up to 20 carbon atoms), an acyl
group (preferably having up to 20 carbon atoms) or a heterocyclic
group having up to 20 carbon atoms and one or more heteroatoms
selected from O, S and N; such a hydrocarbyl or heterocyclic
grouping may carry one or more functional groups such as halogen
atoms or groups of the formulae --NR.sup.4R.sup.5,
--CONR.sup.4R.sup.5, --OR.sup.6, --SR.sup.6 and --COOR.sup.7, where
R.sup.4 and R.sup.5, which may be the same or different, are
hydrogen atoms, acyl groups or hydrocarbyl groups as defined for
R.sup.1 and R.sup.2; R.sup.6 is a hydrogen atom or an acyl group or
a group as defined for R.sup.1 or R.sup.2 and R.sup.7 is a hydrogen
atom or a group as defined for R.sup.1 or R.sup.2; where R.sup.1
and R.sup.2 represent a divalent grouping, this may for example be
an alkylene or alkenylene group (preferably having up to 10 carbon
atoms) which may carry one or more functional groups as defined
above. In general R.sup.1 and R.sup.2 are preferably hydrogen or
small groups such as C.sub.1-4 alkyl groups.
[0034] 2. The amphiphile may contain polymerisable groupings which
can be caused to polymerise after vesicle formation. Such
polymerisable groupings may, for example, include unsaturated
lipophilic chains, e.g. alkenyl or alkynyl groupings containing up
to 50 carbon atoms, for example 10-30 carbon atoms, such as oleyl
or linoleyl groups or groups containing diacetylene, acryloyl or
methacryloyl groupings. Polymerisation of such groupings will, in
general, yield hydrocarbon backbone polymers the backbones of which
are not readily biodegradable, although such polymers may be
designed so that the backbone residue resulting from biodegradation
is water-soluble, e.g. by virtue of the presence of hydrophilic
substituents such as carboxyl or hydroxyl groups, to enhance its
dispersibility. The chain length of such polymers is in general
preferably such that their molecular weight does not exceed
40,000.
[0035] Where a greater degree of biodegradability is required, it
may be preferable to avoid formation of polymeric hydrocarbon
chains which cannot readily be degraded and to effect
polymerisation or crosslinking exclusively through biodegradable
groups such as ester, carbonate, carbamate, amide or imide bonds of
the type referred to above. In general, the functional groups
leading to such bonds will be hydrophilic and thus lead to
crosslinking between the hydrophilic parts of the amphiphiles.
[0036] However, polymerisation of lipophilic hydrocarbon chains may
be used to yield a biodegradable polymer if the amphiphile
comprises a biodegradable hydrophilic moiety carrying two such
chains; where the lipophilic chains of adjacent amphiphile
molecules become crosslinked, e.g. via unsaturated carbon-carbon
bonds, the extended lipophilic groupings so formed will be
separated by the biodegradable hydrophilic groups; on
biodegradation, the polymeric structure will thus break up into
relatively small lipophilic molecules carrying the residues of the
degraded hydrophilic moieties.
[0037] 3. A soluble amphiphilic polymer carrying appropriate
functional groups may be further polymerised or crosslinked after
vesicle formation. Such substances include polyamino acids and
carbohydrates carring lipophilic groups, as well as low molecular
weight polyesters, polyamides etc carrying appropriate groups
providing amphiphilic character. Thus, for example, hydrophilic
polymers, such as those listed above, may be provided with
lipophilic chains, e.g. C.sub.10-30 alkyl, alkenyl or alkynyl
groups, to provide suitable amphiphiles for use in accordance with
the invention. Chemical methods for the attachment of such
lipophilic chains include partial esterification of the hydroxyl
groups of polyhydroxy acids, salt formation of anionic surfactants
on the amino groups of chitosan or covalent derivatisation of such
groups, and attachment of hydrophobic groups to carbohydrates or
cyclodextrins by way of ester bonds.
[0038] The soluble polymer for further polymerisation may also be
an amphiphile polymerised or crosslinked in accordance with (1) or
(2) above.
[0039] Polymerisable or crosslinkable amphiphiles which may be used
in accordance with the invention thus include compounds of the
general formula (II).
[(X).sub.p(R.sup.10).sub.q]B.sub.r (II)
[0040] where X is an anionic, cationic or non-ionic hydrophilic
moiety;
[0041] R.sup.10 is a lipophilic group;
[0042] B is a group capable of polymerisation or crosslinking;
[0043] p and q are integers; and
[0044] r is zero or, when neither X or R.sup.10 is capable of
polymerisation or crosslinking, is an integer.
[0045] The groups X and R.sup.10 may be joined in various ways.
Thus, for example, a hydrophilic group X may carry one or several
lipophilic groups R.sup.10 or a lipophilic group R.sup.10 may carry
one or several hydrophilic groups X. One or more hydrophilic groups
X may also join separate lipophilic groups R.sup.10 as long as the
amphiphile can adopt a configuration in which the hydrophilic and
lipophilic moieties of adjacent molecules are aligned.
[0046] Similarly, the group(s) B (where present) may be attached to
one or more of the groups X and R.sup.10.
[0047] To provide or enhance biodegradability, one or more
biodegradable groupings W may connect the groups X, R.sup.10 and
B.
[0048] The group X may, for example, be a quaternary ammonium
grouping --N(R.sup.11).sub.3V where the groups R.sup.11 (which may
be the same or different) may be, for example, alkyl, aralkyl or
aryl groups containing, for example, up to 20 carbon atoms, and V
is an anion. It will be appreciated that one or more of the groups
R.sup.11 may be a lipophilic group R.sup.10.
[0049] Other useful hydrophilic groups X include, hydroxyl,
carboxylate, amide, phosphate, sulphate and sulphonate groups.
Further examples of hydrophilic groups X include:
[0050] O.CH.sub.2.CH.sub.2.N.sup.+(CH.sub.3).sub.3 (choline)
[0051] O.CH.sub.2.CH.sub.2.N.sup.+H.sub.3 (ethanolamine)
[0052] O.CH(NH.sub.3.sup.+).COO.sup.- (serine)
[0053] O.CH.sub.2.CH(OH).CH.sub.2OH (glycerol)
[0054] hexoses and pentoses such as inositol.
[0055] The group R.sup.10 may, for example, be a saturated or
unsaturated, straight or branched hydrocarbon chain, which may
contain, for example, 6-50 carbon atoms and may be interrupted by
one or more biodegradable groups W and may carry one or more
functional groups permitting chains R.sup.10 on adjacent
amphiphiles to crosslink to form a biodegradable group. Useful
groups R.sup.10 include oleyl and linoleyl groups and chains
containing diacetylene groupings.
[0056] The group(s) B may be, for example, orthoester groups which
form carbonate ester linkages with hydroxyl groups, or hydroxyacid
groups (or separate hydroxyl and carboxyl groups) which form ester
linkages.
[0057] It will be appreciated that the hydrophilic group X may
comprise a moiety which is not itself directly responsible for
hydrophilic properties, as in the case of a group R.sup.11 of a
quaternary ammonium grouping as defined above, which may for
example be a lower alkyl group too small to impart lipophilic
character; such groups may also form part of the connection between
the groups X and R.sup.10. In other words, there may be
transitional regions between groups X and R.sup.10 which are not
strictly either lipophilic or hydrophilic in themselves but can be
regarded as part of either X or R.sup.10.
[0058] Thus, in a special case of the amphiphiles of formula (II),
the groups X, R.sup.10 and B may be attached to a preformed polymer
which may be regarded as part of X or of R.sup.10 according to its
chemical and physical character. Such a polymer may be a known
hydrophilic polymer on to which lipophilic groups (as discussed
above) have been attached, or a lipophilic polymer, e.g. a
polyolefin, carrying hydrophilic groups. Alternatively, such a
polymer may be obtained by partial polymerisation of an amphiphile
of formula (II). In all such cases, the preformed polymer should be
sufficiently soluble to permit vesicle formation and should be so
functionalised as to permit covalent, ionic or coordinate
crosslinking to stablise the vesicles.
[0059] Particularly useful monomeric amphiphiles include
cyanoacrylate esters carrying lipophilic esterifying groups (which
may also have hydrophilic moieties). Thus, for example, U.S. Pat.
No. 4,329,332 describes the micellar polymerisation of lower alkyl
cyanoacrylates, a technique which may be extendable to the
polymerisation of acrylates of the formula
CH.sub.2.dbd.C(CN).CO.O.(C.sub.6-20 aliphatic). Similarly, a
di-acrylate of the formula
CH.sub.2.dbd.CH.CO.O.(CH.sub.2.CH.sub.2.O).sub.98.(CH.sub.2.CH(Me).O).sub.-
67.(CH.sub.2.CH.sub.2.O).sub.98.CO.CH.dbd.CH
[0060] has been used by Ping et al (Int. J. Pharm, 61 (1990)
79-84). Corresponding cyanoacrylates may also be used.
[0061] Amphiphilic materials of use in accordance with the
invention include the following classes of substances derivatised
with lipophilic groups:
[0062] lecithin derivatives,
[0063] polyglycerol,
[0064] polyoxyethylene glycol and ethers thereof,
[0065] polyoxyethylene derivatives of steroids,
[0066] glycosides,
[0067] galactosides,
[0068] hydroxyacids or polyhydroxyacids (including carboxylic,
phosphonic, sulphonic and sulphinic acids),
[0069] carbohydrates and derivatives thereof,
[0070] aminoalcohols and derivatives thereof,
[0071] cyanoacrylates,
[0072] acrylamides, and
[0073] hydroxyamides.
[0074] Polymerisable Amphiphiles
[0075] A number of classes of useful polymerisable amphiphiles are
listed below:
[0076] 1.
CH.sub.2(OB.sub.1).CH(OB.sub.2).CH.sub.2.O.PO(O.sup.-)O(CH.sub.2-
).sub.2N.sup.+(CH.sub.3).sub.3
[0077] where B.sub.1 and B.sub.2 may be
--CO--(CH.sub.2).sub.8--C.ident.C--C.ident.C--(CH.sub.2).sub.n-CH.sub.3
[0078] (where n is an integer e.g. 9, 12 or 13) as described in WO
85/04326. Such compounds can be made by conventional phospholipid
chemistry as described in Hirth et al (Helv. Chem. Acta 40, 1957,
1928) and Pfeiffer et al (J. Org. Chem. 35, 1970, 221).
[0079] Such compounds may thus be prepared by procedures described
in EP-A-0032622. The zwitterionic group may be introduced by
subjecting the appropriate phosphonic or phosphinic acid or an
esterifiable derivative thereof to reaction with glycerol or an
esterifiable derivative thereof. The groups B.sub.1 and B.sub.2 may
be introduced into the molecule by esterification using the
carboxylic acid of B.sub.1 and B.sub.2 or an ester-forming
derivative thereof. These reactions can be carried out between the
glycerol or derivatives thereof on the one hand, and the carboxylic
acid and the phosphorus ester on the other, either simultaneously
or optionally in steps. Other known methods for the synthesis may
equally well be used.
[0080] Polymerisation of these compounds may, for example, be
obtained by irradiation at 254 nm using a xenon lamp after
formation of gas containing liposomes or formation of monolayers of
the amphiphiles at the gas/liquid interface.
[0081] 2. Phospholipids such as phosphodiglycerides and
sphingolipids carrying polymerisable groups.
[0082] 3. Unsaturated oils having hydrophilic groups such as corn
oil, sunflower seed oil, soybean oil, safflower oil, peanut oil,
cottonseed oil and olive oil.
[0083] 4. Saturated and unsaturated fatty acid derivatives with
hydroxyl groups, for example castor oil and ergot oil which are
triglycerides of d-12-hydroxyoleic acid.
[0084] 5. Compounds as described in "Polymerised Liposomes"
(Technical Insights Inc 1988) and Hub et al (J. Macromol. Sci.
Chem. A15, (5), 1981, 701-715). These may have the structures:
12
[0085] 6. Compounds of the formula:
[(CH.sub.3--(CH.sub.2).sub.12--C.ident.C--C.ident.C--(CH.sub.2).sub.8--CO--
L-(CH.sub.2).sub.2].sub.2M
[0086] where L and M may be --O--, --S-- or --NR.sup.12-- (where
R.sup.12 is H or an alkyl group), for example the compounds in
which
[0087] L=M=--O--; L=--O--, M=--N(CH.sub.3)--; L=--NH--,
M=--O--;
[0088] L=--O--, M=--N.sup.+(CH.sub.3).sub.2--B{overscore (r)}
and
[0089] L=--O--, M=--N(CH.sub.2.CH.sub.2.SO.sub.3H)--
[0090] Such compounds may be prepared by reacting a reactive
derivative of hexacosane-10,12-diynoic acid (e.g. the acid
chloride) with the appropriate compound (HLCH.sub.2CH.sub.2).sub.2M
in dry chloroform at 0.degree. C. in the presence of pyridine, if
necessary followed by quaternisation.
[0091] Synthesis of hexacosane-10,12-diynoic acid is described by
Singh et al (Polym. Prep.: Am. Chem. Soc. Div. Polym. Chem; 26 (2),
1985, 184-5). The acid chloride may be prepared by reaction with
oxalylchloride.
[0092] 7. Compounds as described by Paleos (Chem. Soc. Rev. 14,
1985, 45-67), for example of the following structures: 3
[0093] 8. Esters of .alpha.-amino fatty acids which may be self
condensed as described by Folda et al (Rapid. Commun. 3, 1982,
167-174) e.g. methyl 2-aminooctadecanoate, docosanyl
2-aminooctadecanoate, methyl 2-aminohexcosanoate and docosanyl
2-amino-hexacosanoate.
[0094] These esters of the long chain amino acids may be
synthesized from the saturated carboxylic acids by
.alpha.-bromination using the Hell-Volhard-Zelinsky reaction. The
resulting .alpha.-bromo acids are converted to the corresponding
amino acid by the method of Cheronis et al (J. Org. Chem 6 (1949)
349). The methyl esters of the amino acid hydrochlorides are
prepared by passing dry HCl-gas through a suspension of the amino
acid in refluxing methanol. The docosanyl ester of the amino acid
hydrochlorides are synthesized by passing dry HCl-gas through a 1:1
mixture of amino acid and docosanol at 110.degree. C. The ester
hydrochlorides are then suspended in dry chloroform and converted
to the free amine by passing dry ammonia through the
suspension.
[0095] 9. Long chain esters of sulphosuccinic acid carrying
polymerisable functions.
[0096] 10 Long chain esters of pyridinum dicarboxylic acids (e.g.
3,5-dicarboxy 1-methyl pyridinum iodide) carrying polymerisable
functions.
[0097] 11. Iodinated X-ray contrast agents carrying long chain
ether or ester groups having polymerisable functions. Thus, for
example, an X-ray contrast agent derived from iothalamic acid may
have multiple N-dihydroxyalkyl groups one or two of which may be
esterified with long chain fatty acids. Thus, for example, iohexol
may be partially protected by forming an acetonide derivative of
two of the three dehydroxy alkyl groups, followed by reaction with
an activated fatty acid, e.g. the acid chloride, and deprotection
to remove the acetonide groups. Such an amphiphile may readily be
cross-linked by reaction with a dicarboxylic acid after vesicle
formation.
[0098] 12. Di-fatty acid esters of sorbitan. The multiple free
hydroxyl groups which are present permit crosslinking by diacids.
Alternatively, the esterifying fatty acid groups may be unsaturated
to permit olefinic addition polymerisation.
[0099] 13. Diesters of the formula
R.sup.13.CO.O.CH(R.sup.14).O.CO.R.sup.13
[0100] where R.sup.14 is a hydrophilic group and each R.sup.13 is a
lipophilic group, at least one of R.sup.13 and R.sup.14 carrying a
polymerisable group and/or functional groups permitting
crosslinking. Such compounds may be synthesised by reaction of a
dihalide of the formula R.sup.14.CH.Hal.sub.2 with a salt of an
acid R.sup.13.COOH. They are particularly readily
biodegradable.
[0101] It may also be beneficial to include in the encapsulating
material one or more further amphiphiles such as cholesterol which
are not bonded or polymerised but serve to improve the stability
and/or flexibility of the microbubbles.
[0102] As indicated above the microbubbles may be stabilised by
incorporation of particulate material together with the
encapsulated gas. Such particles include, for example, silica and
iron oxide. The preferred particle size for such stabilising
particles is in the range 1 to 500 nm, depending on the size of the
microbubbles. The particles should be such that they are only
partially wetted by the fluid medium used to disperse the micelles,
i.e. the contact angle between the material of the particles and
the fluid should be about 90 degrees.
[0103] The stabilising particles may carry functional groups which
will interact with the amphiphiles to form covalent or other
linkages. Particles of the polymerised amphiphiles of formula (II)
may be useful in this context. Colloidal silica particles may have
a particle size in the range 5-50 nm and may carry silanol groups
on the surface which are capable of interaction with the amphiphile
by hydrogen bonding or by forming covalent bonds.
[0104] The amphiphiles may stabilize the gas or gas precursor by
forming a monolayer at the interface between the liquid medium and
the gas or gas precursor system, or by forming vesicles consisting
of one or more layers containing the gas or gas precursor. The
liquid medium may be water or an any non-aqueous liquid with polar,
protic, aprotic or apolar characteristics.
[0105] The stabilisation or the system by monolayers or multilayers
or the formation of the vesicles may be activated, as fully
described in the literature, by sonication or even shaking of the
amphiphilic material mixture in the appropriate medium, or the
vesicles may be formed by any conventional liposome/vesicle-forming
principle.
[0106] The amphiphiles may form conventional micelles, or inverse
micelles when using an apolar non-aqueous medium. The stabilized
systems may be dried or freeze-dried or the non-aqueous phase may
be evaporated. The resulting dried system may be resuspended in any
physiological acceptable solvent such a saline or phosphate buffer,
optionally using a suspending or emulsifying agent.
[0107] The methods of polymerization used for the stabilisation of
the vesicles, are well established methods in polymer chemistry,
i.e. as described in "Comprehensive Polymer Science", Vol 1-7,
Pergamon Press, Oxford 1989, or "Methoden der Organischen Chemie",
Houben-Weyl, Makromolekulare Stoffe Band E20/1-3, Georg Thieme
Verlag, Stuttgart 1987. Examples of suitable methods may be chain
polymerization methods such as ionic or radical polymerisation or
metal catalysed polymerisation, or the systems may polymerize
spontaneously by step polymerisation when monolayers or vesicles
are formed. Initiators may be UV-irradiation or simple pH-change,
or radical initiators. Particularly interesting here may be
encapsulation of a substance which, by slight increase in
temperature develops a gas, and simultaneously generates free
radicals which initiates polymerisation of the surrounding shell.
Such a substance is described in "Comprehensive Polymer Science",
Vol 3, Pergamon Press, Oxford 1989, p.p. 99, i.e.
azo-bis-isobutyronitrile (AIBN), which by UV-irradiation, or by
warming to 40.degree. C. starts generating N.sub.2 while generating
two molecules of cyano-isopropyl radicals which may initiate
polymerisation or rapidly pair. Polymerisation of amphiphiles
containing unsaturated groupings may also be initiated by
sonication (see Price et al., Brit. Polym. J. 23 (1990), 63-66),
e.g. when this is used to generate a gas-in-liquid emulsion as
described in greater detail hereafter.
[0108] A gas entrapped system may be obtained by using a gas
precursor or the gas itself may be entrapped. The gas may be
entrapped into the amphiphile mixture simply by vigorously shaking
the mixture in the presence of air, i.e. creating a gas-in-liquid
emulsions as described in U.S. Pat. No. 4,684,479. Another well
established method, described e.g. in U.S. Pat. No. 4,774,958 for
creating a gas containing bubble is by sonication of the mixture in
the presence of air. Another well known method comprises passing
gas through a syringe into a mixture of amphiphile and liquid. As
described in U.S. Pat. No. 3,900,420 the microgas-emulsion may be
created by using an apparatus for introducing gas rapidly into a
fast-flowing liquid. A region of low pressure is created in a
liquid containing the amphiphile. The gas is then introduced to the
region of low pressure and the gas-in-liquid system is obtained by
pumping the liquid through the system.
[0109] By using the principle of electrolysis it is possible to
generate the gas to be entrapped directly in a container containing
the amphiphiles. The electrolytes necessary for the electrolysis
may even help to further stabilize the amphiphiles to make the
polymerisation possible. An aqueous solution containing
electrolytes may generate hydrogen gas at the cathode and oxygen at
the anode. The electrodes may be separated by a salt bridge. On
adding hydrazine nitrogen gas may be generated at the anode. Using
the Kolbe reaction, one may also generate CO.sub.2 from carboxylic
acids using electrolysis.
[0110] As described above, gas entrapped vesicles may be obtained
by forming liposomes or vesicles consisting of one or more
bilayers. These vesicles may be formed at elevated pressure
conditions in such a way that the gas is entrapped in the
vesicles.
[0111] It is also possible to form a liquid-liquid (e.g.
oil-in-water emulsion in the presence of amphiphile systems as
discussed above, e.g. by sonication, to form liquid-containing
vesicles which can then be polymerised. The polymerised vesicles
may then be treated to remove the liquid (conveniently a volatile
hydrocarbon) therefrom by evaporation, where the boiling point of
the liquid is relatively low, or by extraction with a low-boiling
solvent which can itself be removed by evaporation. Evaporation of
low-boiling liquid cores may also occur spontaneously during
sonication. Where the liquid in the vesicles is water, it can be
removed by freeze drying.
[0112] The following Examples are given by way of illustration
only;
[0113] Bis-linoleyl-lecithin is commercially available from Lipids
Products, Surrey, UK:--
EXAMPLE 1
[0114] A saturated solution of the bis-linoleyl-lecithin in an
aqueous medium is obtained by mixing 100 mg of the amphiphile in
100 ml of sterile, pyrogen free water. The saturated solution is
filtered through a 0.45 .mu.m filter, and the resulting solution is
sonicated for 1-10 minutes in the presence of air. During the
sonication, air is entrapped into the solution and a gas-in-liquid
emulsion is formed. Polymerization of the monolayer of the
amphiphile at the gas-liquid interphase is achieved by
UV-irradiation of the solution at 254 .mu.m using a xenon lamp, or
by addition of a radical initiator.
[0115] The resulting product contains microspheres with gas
entrapped. The microspheres are separated from excess polymerised
amphiphiles using a separating funnel. The resulting microspheres
are resuspended in sterile, pyrogen-free saline, and filled into 10
ml vials. The product is produced using aseptic techniques in a
"clean room" (LAF-station) to obtain a sterile, pyrogen free
product. The particle sizes of the microspheres are in the range of
0.5-10 .mu.m.
EXAMPLE 2
[0116] Example 1 is repeated using as polymerisable amphiphile the
compound bis-(trieicoso-10,12-diynoyl) phosphatidyl choline (Hirth
et al; Helv Chim Acta 40, 957, 1928).
EXAMPLE 3
[0117] 100 mg of bis-linoleyl-lecithin are dissolved in a mixture
of chloroform/methanol. The mixture is poured into a round bottom
flask, and the organic phase is evaporated using a rotavapor in
such a way that a thin film of the lecithin derivative is formed at
the inner surface of the flask. 10 ml of sterile, pyrogen-free-free
water are added and the lipids are dispersed in the solution by
sonication at the air/liquid interphase for 5-15 minutes. Gas
entrapped vesicles are formed, and the gas-containing microspheres
are polymerised by UV-irradiation of the solution at 254 nm using a
xenon-lamp or by addition of a radical initiator under continuous
stirring Polymerised gas-entrapped vesicles are separated from
excess polymerised amphiphiles using a separating funnel. The
resulting vesicles are suspended in sterile, pyrogen free saline
and filtered to obtain a product which contains microspheres in the
range of 0.5-5 .mu.m. The product is produced using aseptic
techniques in a "clean room" (LAF-station) to obtain a sterile,
pyrogen free product. The final product is filled into 10 ml
vials.
EXAMPLE 4
[0118] Example 3 is repeated using as polymerisable amphiphile the
compound bis-(trieicoso-10,12-diynoyl) phosphatidyl choline (Hirth
et al; Helv Chim Acta 40, 957, 1928).
[0119] Preparation of Polymerisable Amphiphiles
EXAMPLE 5
Tetraethylene Glycol Mono-12-(Methacryloyloxy)Dodecanoate
[0120] 12-(Methacryloyloxy)dodecanoic acid (Regen et al., J. Am.
Chem. Soc. 1982, 104, 795) (2.75 g, 9.65 mmol) was dissolved in
tetrahydrofuran (45 ml) and a solution of oxalyl chloride (2.1 ml,
24.2 mmol) in tetrahydrofuran (5 ml) was added dropwise. The
mixture was stirred for 24 hours at room temperature, and then the
solvent was evaporated under reduced pressure. The residue was
dissolved in tetrahydrofuran (25 ml) and added dropwise to a
solution of tetraethylene glycol (1.88 g, 9.65 mmol) and pyridine
(0.92 g, 11.7 mmol) in tetrahydrofuran (35 ml). The mixture was
stirred for 24 hours at room temperature. The precipitated
pyridinium salt was filtered off and the solvent evaporated.
Chromatographic purification on a silica gel column (ethyl acetate)
afforded 1.67 g (38%) of the title compound. .sup.1H NMR (60 MHz,
CDCl.sub.3): .delta. 1.3 (br s, 18H, (CH.sub.2).sub.9), 1.95 (m,
3H, C.dbd.CCH.sub.3), 2.1-2.6 (m, 2H, CH.sub.2COO), 3.5-3.8 (m,
14H, 3.times.CH.sub.2OCH.sub.2CH.sub.2+COOCH.sub.2CH.sub.2),
4.0-4.4 (m, 4H, COOCH.sub.2), 5.52 (m, 1H, vinyl), 6.10 (m, 1H,
vinyl).
EXAMPLE 6
Polyethylene Glycol (550) Methyl Ether
12-(Methacryloyloxy)Dodecanoate
[0121] 12-(Methacryloyloxy)dodecanoic acid (1.90 g, 6.69 mmol) was
dissolved in tetrahydrofuran (20 ml) and a solution of oxalyl
chloride (2.12 g, 16.7 mmol) in tetrahydrofuran (10 ml) was added
dropwise. The mixture was stirred for 24 hours at room temperature,
and then the solvent was evaporated under reduced pressure. The
residue was dissolved in tetrahydrofuran (10 ml) and added dropwise
to a solution of polyethylene glycol (550) monomethyl ether (3.68
g, 6.69 mmol) and pyridine (0.53 g, 6.69 mmol) in tetrahydrofuran
(25 ml). The mixture was stirred for 24 hours at room temperature.
The precipitated pyridinium salt was filtered off and the solvent
evaporated. Chromatographic purification on a silica gel column
(chloroform) afforded 2.31 g (42.3%) of the title compound. .sup.1H
NMR (60 MHz, CDCl.sub.3): .delta. 1.3 (br s, 18H,
(CH.sub.2).sub.9), 1.95 (m, 3H, C.dbd.CCH.sub.3), 2.1-2.5 (m, 2H,
CH.sub.2COO), 3.11 (s, 3H, CH.sub.3O), 3.5-3.8 (m, 25H (average),
CH.sub.2OCH.sub.2CH.sub.2+COOCH.sub.2CH.sub.2), 3.9-4.4 (m, 4H,
COOCH.sub.2), 5.52 (m, 1H, vinyl), 6.10 (m, 1H, vinyl).
EXAMPLE 7
Polyethylene Glycol (2000) Methyl Ether
12-(Methacryloyloxy)Dodecanoate
[0122] 12-(Methacryloyloxy)dodecanoic acid (2.84 g, 0.01 mol) in
tetrahydrofuran (20 ml) was reacted with oxalyl chloride (3.0 g,
0.024 mol) to obtain the corresponding acid chloride. This acid
chloride (3.0 g, 0.01 mol) dissolved in anhydrous tetrahydrofuran
(10 ml) was added dropwise to a mixture of polyethylene glycol
(2000) monomethyl ether (20.0 g, 0.01 mol) and anhydrous pyridine
(0.83 g, 0.01 mol) in anhydrous tetrahydrofuran (300 ml). The
mixture was stirred for 48 hours at room temperature. The resulting
liquid was purified by flash chromatography (silica gel/ethyl
acetate) to give 16.5 g (75%) of the title compound. .sup.1H NMR
(60 MHz, CDCl.sub.3): .delta. 1.20 (s, 18H, CH.sub.2), 2.15 (m, 2H,
CH.sub.2COOH), 3.5 (s, 3H, CH.sub.3O), 3.6 (s, 180H,
90.times.CH.sub.2O), 4.0 (m, 4H, 2.times.COOCH.sub.2), 5.7-6.0 (m,
3H, CH.sub.2.dbd. and .dbd.CH).
EXAMPLE 8
a) 16-(Methacryloyloxy)Hexadecanoic Acid
[0123] 16-Hydroxyhexadecanoic acid (6.81 g, 25.0 mmol) was
dissolved in tetrahydrofuran (150 ml) and the solution was cooled
to 0.degree. C. before adding pyridine (2.73 g, 34.5 mmol).
Methacryloyl chloride (2.61 g, 25.0 mmol) was dissolved in
tetrahydrofuran (75 ml) and added dropwise. The mixture was stirred
for 1 hour at 0.degree. C., and then at room temperature for 24
hours. The solvent was removed under reduced pressure (room
temperature), the residue suspended in ether (100 ml) and the
mixture washed with distilled water. The ether layer was dried
(MgSO.sub.4) and the ether evaporated. Chromatographic purification
on a silica gel column (1:2 ethyl acetate/hexane) afforded 5.0 g
(64%) of the title compound. .sup.1H NMR (60 MHz, CDCl.sub.3):
.delta. 1.3 (br s, 26H, (CH.sub.2).sub.13), 1.95 (m, 3H,
C.dbd.CCH.sub.3), 2.1-2.6 (m, 2H, CH.sub.2COO), 4.0-4.4 (m, 2H,
COOCH.sub.2), 5.52 (m, 1H, vinyl), 6.10 (m, 1H, vinyl).
b) Tetraethylene Glycol Mono-16-(Methacryloyloxy)Hexadecanoate
[0124] 16-(Methacryloyloxy)hexadecanoic acid (2.05 g, 6.57 mmol)
was dissolved in tetrahydrofuran (25 ml) and a solution of oxalyl
chloride (1.4 ml, 16.5 mmol) in tetrahydrofuran (10 ml) was added
dropwise. The mixture was stirred for 24 hours at room temperature,
and then the solvent was evaporated under reduced pressure. The
residue was dissolved in tetrahydrofuran (10 ml) and added dropwise
to a solution of tetraethylene glycol (1.07 g, 5.50 mmol) and
pyridine (0.44 g, 5.50 mmol) in tetrahydrofuran (25 ml). The
mixture was stirred for 24 hours at room temperature. The
precipitated pyridinium salt was filtered off and the solvent
evaporated. Chromatographic purification on a silica gel column
(2:1 ethyl acetate/hexane) afforded 0.84 g (30%) of the title
compound. .sup.1H NMR (60 MHz, CDCl.sub.3): .delta. 1.3 (br s, 26H,
(CH.sub.2).sub.13), 1.95 (m, 3H, C.dbd.CCH.sub.3); 2.1-2.6 (m, 2H,
CH.sub.2COO), 3.5-3.8 (m, 14H,
3.times.CH.sub.2OCH.sub.2CH.sub.2+COOCH.su- b.2CH.sub.2), 4.0-4.4
(m, 4H, COOCH.sub.2), 5.52 (m, 1H, vinyl), 6.10 (m, 1H, vinyl).
EXAMPLE 9
Polyethylene Glycol (350) Methyl Ether
16-(Methacryloyloxy)Hexadecanoate
[0125] The product was prepared from
16-(methacryloyloxy)-hexadecanoic acid (prepared as described in
Example 8(a)), and polyethylene glycol (350) monomethyl ether using
the procedure given in Example 6.
EXAMPLE 10
a) 12-(Acryloyloxy)Dodecanoic Acid
[0126] 12-Hydroxydodecanoic acid (5.0 g, 0.023 mol) dissolved in
tetrahydrofuran (100 ml) and pyridine (2.16 g, 0.027 mol) was
cooled to 0.degree. C. Acryloyl chloride (3.15 g, 0.023 mol) in
tetrahydrofuran (75 ml) was then added dropwise to the solution.
The mixture was stirred for 5 hours at 0.degree. C. then stirred
overnight at room temperature. The precipitated pyridinium salt was
filtered off and the solvent removed under vacuum. The resulting
liquid was purified by flash chromatography (silica gel/chloroform)
to give 2.5 g (40%) of the title compound. .sup.1H NMR (60 MHz,
CDCl.sub.3): .delta. 1.20 (s, 18H, CH.sub.2), 2.15 (m, 2H,
CH.sub.2COOH), 4.0 (m, 2H, COOCH.sub.2), 5.7-6.0 (m, 3H,
CH.sub.2.dbd. and .dbd.CH).
b) Tetraethylene Glycol Mono-12-(Acryloyloxy) Dodecanoate
[0127] 12-Acryloyloxydodecanoic acid (2.00 g, 0.007 mol) in diethyl
ether (20 ml) was reacted with oxalyl chloride (2.40 g, 0.019 mol)
to obtain the corresponding acid chloride. This acid chloride (1.80
g, 0.006 mol) dissolved in anhydrous chloroform (10 ml) was added
dropwise to a mixture of tetraethylene glycol (1.20 g, 0.006 mol)
and anhydrous pyridine (0.50 g, 0.006 mol) in anhydrous chloroform
(30 ml). The mixture was stirred overnight at room temperature. The
resulting liquid was purified by flash chromatography (silica
gel/ethyl acetate) to give 1.10 g (40%) of the title compound as a
colourless oil. .sup.1H NMR (60 MHz, CDCl.sub.3): .delta. 1.20 (s,
18H, CH.sub.2), 2.15 (m, 2H, CH.sub.2COOH), 3.50 (s, 3H,
CH.sub.3O), 3.6 (s, 14H, 7.times.CH.sub.2O), 4.0 (m, 5H,
2.times.COOCH.sub.2 and OH), 5.7-6.0 (m, 3H, CH.sub.2.dbd. and
.dbd.CH).
EXAMPLE 11
Tetraethylene Glycol Mono-10,12-Tricosadiynoate
[0128] 10,12-Tricosadiynoic acid (2.50 g, 0.007 mol) in
tetrahydrofuran (30 ml) was reacted with oxalyl chloride (2.25 g,
0.017 mol) to obtain the corresponding acid chloride. This acid
chloride (2.45 g, 0.007 mol) dissolved in anhydrous tetrahydrofuran
(10 ml) was added dropwise to a mixture of tetraethylene glycol
(1.32 g, 0.007 mol) and anhydrous pyridine (0.83 g, 0.01 mol) in
anhydrous tetrahydrofuran (40 ml). The mixture was stirred
overnight at room temperature. The precipitated pyridinium salt was
filtered off and the solvent removed under vacuum. The resulting
liquid was purified by flash chromatography (silica gel/ethyl
acetate) to give 1.50 g (41%) of the title compound as a colourless
oil. .sup.1H NMR (60 MHz, CDCl.sub.3) .delta. 0.88 (m, 3H,
CH.sub.3CH.sub.2), 1.30 (m, 28H, CH.sub.2), 2.20 (m, 6H, CH.sub.2),
3.65 (s, 14H, 7.times.CH.sub.2O), 4.20 (m, 2H, CH.sub.2CO).
EXAMPLE 12
Polyethylene Glycol (550) Methyl Ether 10,12-Tricosadiynoate
[0129] 10,12-Tricosadiynoic acid (2.50 g, 0.007 mol) in
tetrahydrofuran (30 ml) was reacted with oxalyl chloride (2.25 g,
0.017 mol) to obtain the corresponding acid chloride. This acid
chloride (2.45 g, 0.007 mol) dissolved in anhydrous tetrahydrofuran
(10 ml) was added dropwise to a mixture of polyethylene glycol
(550) monomethyl ether (3.85 g, 0.007 mol) and anhydrous pyridine
(0.83 g, 0.01 mol) in anhydrous tetrahydrofuran (30 ml). The
mixture was stirred overnight at room temperature. The precipitated
pyridinium salt was filtered off and the solvent removed under
vacuum. The resulting liquid was purified by flash chromatography
(silica gel/ethyl acetate) to give 2.72 g (41%) of the title
compound as a colourless oil. .sup.1H NMR (60 MHz, CDCl.sub.3):
.delta. 0.88 (m, 3H, CH.sub.3CH.sub.2), 1.30 (m, 28H, CH.sub.2),
2.20 (m, 6H, CH.sub.2), 3.65 (s, 48H, 24.times.CH.sub.2CO), 3.50
(s, 3H, CH.sub.3O), 4.20 (m, 2H, CH.sub.2CO).
EXAMPLE 13
a) Methyl 10.12-Tricosadiynoate
[0130] 10,12-Tricosadiynoic acid (3.0 g, 0.0084 mol), methanol (15
ml) and concentrated sulfuric acid (0.8 ml) were heated to reflux
and stirred for 1 hour. The cooled mixture was taken up in ether
(40 ml) and washed with 10% NaHCO.sub.3 (20 ml) and water (20 ml),
and the organic phase was dried (MgSO.sub.4). Evaporation of the
solvent gave 2.68 g (74%) of the title compound. .sup.1H NMR (60
MHz, CDCl.sub.3): .delta. 0.98 (m, 3H, CH.sub.3CH.sub.2), 1.28 (m,
28H, CH.sub.2), 2.25 (m, 6H, CH.sub.2), 3.70 (s, 3H,
CH.sub.3O).
b) N-(2',3'-Dihydroxypropyl)-10,12-Tricosadiynamide
[0131] Methyl 10,12-tricosadiynoate (1.69 g, 4.67 mmol) was
dissolved in methanol. 3-Amino-1,2-propanediol (0.509 g, 5.6 mmol)
and sodium methoxide 2.5% solution in methanol (0.146 g, 3 mol %)
was added. The mixture was refluxed for 3 hours and the solvent
evaporated. The crude product was recrystallized from
chloroform.
[0132] Yield: 1.00 g (51%). .sup.1H NMR (60 MHz, CDCl.sub.3):
.delta. 0.7-1.0 (m, 3H, CH.sub.3CH.sub.2), 1.3 (s, br, 28H,
CH.sub.2), 2.0-2.4 (m, 6H, CH.sub.2), 3.3-3.8 (m, 5H,
2.times.CH.sub.2+CH (propanediol)), 6.0-6.3 (m, 1H, NH).
EXAMPLE 14
N,N'-bis(2,3-Dihydroxypropyl)
2,4,6-Triiodo-5-(Tricosa-10,12-Diynoylamino) Isophthalamide
[0133]
5-Amino,N,N'-bis(2,3-diacetoxypropyl)-2,4,6-triiodoisophthalamide
(2.19 g, 2.5 mmol) and 10,12-tricosadiynoyl chloride (1.82 g, 5
mmol) were dissolved in 20 ml dichloromethane. The solution was
stirred for 3 days at ambient temperature under a nitrogen
atmosphere. TLC (ethyl acetate) indicated that the reaction was
complete. The reaction mixture was evaporated and dissolved in a
mixture of methanol (30 ml) and 1M sodium hydroxide solution (15
ml). After 1 hour TLC (methanol/chloroform) indicated that the
reaction was complete. The solution was neutralized with
concentrated hydrochloric acid. The residue was dissolved in
chloroform and filtered. The solvent was removed and the reaction
mixture was purified through silica gel with methanol/chloroform
(1:3) to give the title compound. .sup.1H NMR (300 MHz, DMSO):
.delta. 0.8 (CH.sub.3, t), 1.2-1.7 (17.times.CH.sub.2, m), 2.2-2.3
(2.times.CH.sub.2, t), 3.1-3.2 (2.times.CH.sub.2NH, m), 3.3-3.5
(2.times.CH.sub.2OH, m), 3.6-3.8 (2.times.CHOH), 4.4-4.7
(4.times.OH, m), 8.4-8.5 (2.times.CONH, m), 9.8 (2.times.ArNHCO,
s).
EXAMPLE 15
N-(3',4',5'-Trihydroxy-6'-Hydroxymethyltetrahydropyran-2'-yl)-10,12-Tricos-
adiynamide
[0134] 1-Amino-1-deoxy-.beta.-D-galactose (180 mg, 1 mmol),
10,12-tricosadiynoic acid (350 mg, 1 mmol) and
1-ethyl-3-(3-dimethylamino- propyl)carbodiimide were dissolved in
25 ml dry dimethylformamide and stirred at room temperature
overnight. The solvent was removed in vacuo, the residue
redissolved in chloroform/methanol (1:1), filtered and purified by
straight phase chromatography on a CHROMATOTRON. The relevant
fractions were collected, concentrated in vacuo, and the product
was characterised by NMR.
EXAMPLE 16
6-(2',6'-Diaminohexanoylamino)-3,4,5-Trihydroxytetra-Hydropyran-2-ylmethyl
10,12-Tricosadiynoate
[0135] 1-Amino-1-deoxy-.beta.-D-galactose (180 mg, 1 mmol), and
Fmoc-Lys(Boc)-OPfp (650 mg, 1 mmol) were dissolved in 4 ml dry
dimethylformamide and stirred at room temperature overnight. The
solvent was removed in vacuo, the residue was redissolved in
acetonitrile/water (1:1), filtered and purified by reversed phase
chromatography (Lobar RP8B, acetonitrile/water 50:50 and 65:35).
The relevant fractions were collected, concentrated in vacuo, and
the product was characterised by NMR. The purified product (1 g, 1
mmol), 10,12-tricosadiynoic acid (350 mg, 1 mmol) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are dissolved in 10
ml dry dimethylformamide and stirred at room temperature overnight
The solvent is removed in vacuo, the residue redissolved in
chloroform/methanol (95:5), filtered and purified by straight phase
chromatography on a CHROMATOTRON. The relevant fractions are
collected, concentrated in vacuo, and the product is characterised
by NMR. The protecting groups of the .alpha.-.epsilon. amino groups
are removed by standard reactions. Boc is removed by treatment with
trifluoroacetic acid/methylene chloride for 30 minutes. The solvent
is removed in vacuo. Fmoc is removed by treating the residue with
20% piperidine in dimethylformamide for 30 minutes, and the solvent
is removed in vacuo. The final product is purified by reversed
phase chromatography (Lobar RP8B).
EXAMPLE 17
(3,4,5,6-Tetrahydroxytetrahydropyran-2-ylmethyl)
10.12-Tricosadiynoate
[0136] 1,2;3,4-di-O-isopropylidene-D-galactopyranose (2.6 g, 10
mmol) and 10,12-tricosadiynoic acid (3.5 g, 10 mmol) were dissolved
in 25 ml methylene chloride.
1-Ethyl-3(3-dimethylaminopropyl)carbodiimide (2 g, >10 mmol) was
added neat. The reaction mixture was stirred overnight at room
temperature. The reaction mixture was diluted to 100 ml, extracted
with water (2.times.25 ml), dried over MgSO.sub.4 and the solvent
was removed in vacuo. The crude product was treated with
trifluoroacetic acid (10 ml) at room temperature for 30 minutes,
evaporated in vacuo, and purified by straight phase chromatography
on a CHROMATOTRON, eluted with methanol/chloroform (5:95). The
product was characterised by NMR.
[0137] Preparation of Ultrasound Contrast Agents
EXAMPLES 18-41
i) General Preparative Procedure
[0138] The polymerisable amphiphile was dissolved in a minimum of
methanol and added to a mixture of water and a hydrocarbon. A
comonomer and/or 2,2'-azobisisobutyronitrile (AIBN) dissolved in a
minimum of methylene chloride were optionally added and nitrogen
was bubbled through the mixture for 1 minute, whereafter the
mixture was sonicated under a nitrogen atmosphere using a LABSONIC
2000 apparatus, the sonication probe (length 127 mm, diameter 9.5
mm) being placed 2-3 cm below the surface of the mixture and the
energy used being "full scale" or "half scale" in the low position.
The resulting emulsions were optionally irradiated with UV light
under a nitrogen atmosphere or treated with a redox initiator
comprising potassium metabisulphite (0.05 g, 0.22 mmol) in water (1
ml) and potassium peroxosulphate (0.0023 g, 3.3.times.10.sup.-3
mmol) in water (1 ml). The procedure was modified in Example 31 in
that AIBN was added and the mixture was then shaken by hand,
whereafter a first portion of comonomer was added and sonication
was effected while nitrogen gas was bubbled through the mixture. A
further portion of comonomer was then added and the resulting
emulsion subjected to UV irradiation.
[0139] The specific reaction conditions employed in each Example
are set out in Table 1. Similar conditions, e.g. involving
sonication for 5 minutes using the full scale setting and
irradiating for 1 hour or adding the above-described redox
initiator system and stirring carefully for 30 minutes, may be
employed to treat the amphiphiles prepared in Examples 14-17.
1TABLE 1 Reaction conditions Example in which amphiphile prepared
and Volume Hydrocarbon Comonomer Quantity Sonication level Duration
of ample quantity used of water and volume and quantity of AIBN and
duration UV Irradiation Redox No (g/mmol) (ml) (ml) (g/mmol)
(g/mmol) (minutes) (hours) system 5 MM 0.039/0.084 50 PE-5
0.018/0.18 -- ls-5 -- -- 5 MM 0.037/0.080 50 IP-5 0.018/0.18 --
hs-3 -- -- 5 MM 0.383/0.83 500 IP-50 0.18/1.8 0.20/1.21 ls-6 1.5 --
5 MM 0.042/0.091 50 IP-5 0.09/0.9 0.02/0.12 ls-3 1 -- 5 MM
0.040/0.086 50 PE-2.5 0.018/0.18 0.02/0.12 ls-3 1 -- 6 MM
0.053/0.065 50 PE-5 0.018/0.18 0.02/0.12 ls-4 -- -- 6 MM 0.530/0.65
500 PE-50 0.180/1.80 0.200/1.20 ls-8 1 -- 6 MM 0.500/0.61 500 PE-50
0.180/1.80 0.200/1.20 ls-8 2.5 -- 6 MM 0.200/0.245 20 PE-2
0.018/0.18 0.020/0.12 ls-3 1(Ex 26a) {square root}(Ex 26b) 6 MM
0.053/0.065 50 PE-25 0.018/0.18 0.020/0.12 ls-3 -- {square root} 6
MM 0.054/0.066 50 PE-1 0.018/0.18 0.020/0.12 ls-3 -- -- 6 MM
0.054/0.066 50 TO-5 0.018/0.18 0.020/0.12 ls-3 -- -- 6 ST
0.056/0.069 50 PE-5 0.042/0.41 0.020/0.12 ls-3 1 -- 6 ST
0.057/0.070 50 PE-5 0.042/0.41 + 0.099/0.95 0.020/0.12 ls-3 1 -- 6
0.054/0.066 50 IP-5 -- 0.020/0.12 ls-6 -- -- 7 MM 0.193/0.090 50
PE-5 0.018/0.18 0.02/0.12 ls-3 -- -- 8(b) MM 0.042/0.081 50 IP-5
0.018/0.18 0.020/012 hs-3 1 -- 8(b) ST 0.046/0.089 50 PE-5
0.042/0.41 0.020/0.12 ls-3 -- -- 9 0.052/0.077 50 PE-5 -- -- ls-3
-- -- 10(b) MM 0.036/0.08 50 PE-5 0.018/0.18 0.02/0.12 ls-3 1 -- 11
0.047/0.09 50 PE-5 -- 0.02/0.12 ls-6 -- -- 11 0.080/0.15 50 PE-5 --
0.02/0.12 ls-3 1 -- 12 0.057/0.06 50 PE-5 -- 0.02/0.12 ls-3 --
{square root} 13(b) 0.046/0.11 50 PE-5 -- 0.02/0.12 ls-3 -- --
.multidot. PE = petroleum ether (b.p. 40-80.degree. C.); IP =
Isopentane; TO = toluene; MM = methylmethacrylate; ST = styrene; fs
= full scale; hs = hall scale Amount of potassium peroxosulphate
reduced to 0.002 g (0.003 mmol)
ii) Acoustic Characterisation
[0140] The acoustic effects of the products of Examples 18-41 were
investigated by measuring their ultrasonic transmission as a
function of time, over a period of 90 seconds. The tests were
performed on samples of emulsified material as formed immediately
after sonication and, where appropriate, on the material after
subjection to UV irradiation or redox initiation. In the case of
Example 25 the sample removed after irradiation was retested after
dilution with water (1:1). In the case of Example 31 a sample
removed after the manual shaking was also tested. A 3.5 MHz
broadband transducer was used in a pulse-reflection technique. All
the readings were stable during the 90 seconds measurement period,
so that a single value (in dB/cm) is sufficient to describe each 90
second measurement. In certain cases the measurements were repeated
at time intervals to investigate further the stability of the
ultrasound contrast agents. The results are presented in Table 2,
the time intervals (in minutes from sonication) to acoustic
characterisation are given in brackets for each reading.
2TABLE 2 Acoustic characterisations Acoustic effect after UV
Acoustic effect irradiation/redox Example No. after sonication
initiation 18 2.6 (0) 19 3.7 (0) 20 3.7 (0) 1.4 (90) 1.7 (90) 21
0.6 (0) 0 (60) 22 0.7 (0) 0.5 (60) 23 0.9 (5) 0 (120) 5.9 (0) 4.3
(104) 24 6.0 (0) 4.1 (60) 25 4.4 (0) 4.2 (30) 2.9 (150) 2.9 (150)
1.4 (150) diluted 26 4.0 (0) 2.8 (20, redox) 1.8 60) 0.4 (60, UV)
27 3.6 (0) 2.9 (10) 3.2 (10) 2.3 (60) 3.6 (60) 0.6 (720) 28 0.9 (0)
29 0.6 (0) 30 5.7 (0) 4.1 (60) 3.2 (60) 3.2 (150) 2.6 (150) 31 2.5
(after shaking) 5.4 (0) 4.0 (60) 2.2 (60) 3.3 (150) 1.7 (150) 32
4.9 (0) 33 5.5 (0) 4.7 (20) 3.5 (60) 2.4 (60) 3.1 (100) 34 2.2 (0)
0 (60) 35 1.1 (0) 36 2.1 (0) 37 1.7 (0) 0 (60) 38 4.5 (0) 39 5.6
(0) 4.7 (60) 4.9 (60) 4.5 (120) 4.3 (120) 40 3.6 (0) 0 (60) 41 5.3
(0)
iii) Microscopy Analysis
[0141] A selection of the products from Examples 18-41 were
investigated using a light microscope (Nikin UFX-II) with a
micrometer scale. The investigations were generally performed by
taking out samples of emulsified material as formed immediately
after sonication, except for Example 31 (where the sample was
withdrawn after manual shaking), Example 39 (where the sample was
withdrawn after UV irradiation) and Example 40 (where samples were
withdrawn both immediately after sonication and after redox
initiation), and placing each sample between two glass plates. The
results of these investigations are presented in Table 3; the time
intervals (in minutes from sonication) to microscopy analysis are
given for each sample.
3TABLE 3 Microscopy analysis Time after sonication Size Comments
(shape, Example No. (min) (diam., .mu.m) size distribution) 25 10 4
spheres, narrow size distribution 26 10 10-25 spheres 27 10 4
spheres, narrow size distribution 28 10 4-6 spheres, narrow size
distribution 29 10 variable various shapes, broad size distribution
30 10 4-6 spheres, narrow size distribution 31 10 10-100 large
bubbles, unli (after the sonicated sampl shaking) 33 10 2-3 spheres
35 10 10-15 spheres 36 10 8-15 spheres, broad size distribution 38
10 5-10 spheres 39 10 5-10 spheres, also larger bubbles 40 30 5-10
spheres 40 30 variable bubbles of irregula (after shape redox) 41
10 4 spheres, narrow size distribution
iv) Size Exclusion Chromatography
[0142] Size Exclusion Chromatography (SEC) was performed on the
freeze dried product from Example 25 using tetrahydrofuran
(Rathburn HPLC quality) as eluant and refractive index as detector
(Knauer, Germany). The column set used consisted of 3.times.30 cm
columns containing 5 .mu.m styrogel with pore sizes of 10.sup.5,
10.sup.4, and 500 .ANG. (Polymer Laboratories Ltd., England).
Calibration was made against polystyrene standards (Polymer
Laboratories Ltd., England). The amphiphilic monomer starting
material gave a peak molecular weight of 1,600 Daltons and the
polymer product gave a peak molecular weight of 22,000 Daltons,
both given in polystyrene equivalents. Using the conversion factor
of 0.59 for converting from polystyrene equivalents to "real"
molecular weights (the value for PEG given by Dawkins et al., J.
Liq. Chromatog. 7, 1739, (1984), these correspond to molecular
weights of 944 Daltons for the monomer and 13,000 Daltons for the
polymer respectively.
* * * * *