U.S. patent application number 11/448601 was filed with the patent office on 2007-01-04 for contrast agents.
Invention is credited to Jo Klaveness, Pal Rongved, Per Strande.
Application Number | 20070003485 11/448601 |
Document ID | / |
Family ID | 10692392 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070003485 |
Kind Code |
A1 |
Klaveness; Jo ; et
al. |
January 4, 2007 |
Contrast agents
Abstract
The invention relates to ultrasound contrast agents comprising
vesicles comprising a protein capable of formation of
gas-containing vesicles, wherein the vesicles contain gas which
comprises sulphur hexafluoride or a low molecular weight
fluorinated hydrocarbon. These contrast agents exhibit stability in
vivo upon administration so as to permit ultrasound visualization
while allowing rapid subsequent elimination from the system.
Inventors: |
Klaveness; Jo; (Oslo,
NO) ; Rongved; Pal; (Nesoddtaugen, NO) ;
Strande; Per; (Oslo, NO) |
Correspondence
Address: |
KENYON & KENYON LLP
ONE BROADWAY
NEW YORK
NY
10004
US
|
Family ID: |
10692392 |
Appl. No.: |
11/448601 |
Filed: |
June 6, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11055543 |
Feb 10, 2005 |
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11448601 |
Jun 6, 2006 |
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10820448 |
Apr 7, 2004 |
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11055543 |
Feb 10, 2005 |
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10640504 |
Aug 13, 2003 |
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10820448 |
Apr 7, 2004 |
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10071863 |
Feb 8, 2002 |
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10640504 |
Aug 13, 2003 |
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09406100 |
Sep 27, 1999 |
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10071863 |
Feb 8, 2002 |
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09260561 |
Mar 2, 1999 |
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09406100 |
Sep 27, 1999 |
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09114045 |
Jul 13, 1998 |
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09260561 |
Mar 2, 1999 |
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08840646 |
Aug 25, 1997 |
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09114045 |
Jul 13, 1998 |
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08462404 |
Jun 5, 1995 |
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08840646 |
Aug 25, 1997 |
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08119218 |
Oct 29, 1993 |
5529766 |
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08462404 |
Jun 5, 1995 |
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Current U.S.
Class: |
424/9.52 |
Current CPC
Class: |
A61K 47/26 20130101;
A61K 49/223 20130101; A61K 47/42 20130101; A61K 49/225 20130101;
C07C 69/96 20130101 |
Class at
Publication: |
424/009.52 |
International
Class: |
A61K 49/00 20060101
A61K049/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 1991 |
GB |
9106686.0 |
Claims
1. Contrast agents for use in diagnostic ultrasound studies
comprising microbubbles of gas or a gas precursor encapsulated in a
protein shell characterised in that the said protein is crosslinked
with crosslinking groupings containing biodegradable linkages.
2. Contrast agents as claimed in claim 1 wherein the crosslinking
groupings contain biodegradable linkages selected from amide,
imide, imine, ester, anhydride, acetal, carbamate, carbonate,
carbonate ester and disulphide groups.
3. Contrast agents as claimed in claim 2 wherein the crosslinking
groups contain biodegradable linkages of formula
--(Y).sub.n--CO--O--C(R.sup.1R.sup.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).
4. Contrast agents as claimed in any of the preceding claims
wherein the protein is albumin, gelatin or globulin.
5. Contrast agents as claimed in claim 4 wherein the protein is
human serum albumin.
6. Contrast agents as claimed in any of the preceding claims
further containing an inorganic particulate stabiliser.
7. A process for the preparation of a contrast agent as claimed in
claim 1 which comprises encapsulating a gas or gas precursor in a
protein and crosslinking the protein with crosslinking groups
containing biodegradable linkages before, during or after said
encapsulation.
8. A process as claimed in claim 7 wherein crosslinking is effected
after encapsulation.
9. A process as claimed in claim 7 or claim 8 wherein crosslinking
is effected using a crosslinking agent of formula (I)
A.sup.1-X-A.sup.2 (I) (where X is a linking group containing one or
more biodegradable linkages as defined in claim 2 or claim 3 and
A.sup.1 and A.sup.2, which may be the same or different, are
functional groups reactive with proteins).
10. A process as claimed in claim 9 in which A.sup.1 and A.sup.2
are both aldehyde groups.
11. A process as claimed in any of claims 8 to 10 wherein
encapsulation is effected by agitation or sonication of the protein
in an aqueous medium to yield a protein foam which is dried and
thereafter suspended in a solution of the crosslinking agent in a
polar organic solvent 12. A process as claimed in claim 11 in which
the crosslinking agent is a compound of formula (I) as defined in
claim 9 in which A.sup.1 and A.sup.2 are both O-linked sulphonated
N-hydroxysuccinimidyl residues.
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] 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
saccharine 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.
[0006] U.S. Pat. No. 4,774,958 discloses the use of microbubble
dispersions stabilised by encapsulation in denatured protein, e.g.
human serum albumin (HSA). 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.
[0007] Other ultrasound contrast agents using proteins as
encapsulating agents have been described in the literature, for
example in EP 0359 246 (Molecular Biosystems), U.S. Pat. No.
4,832,941 (Max-Planck Gessellschaft), U.S. Pat. No. 4,844,882
(Molecular Biosystems), WO 84/02838 (Feinstein), U.S. Pat. No.
4,572,203 (Feinstein), EP 0077 752 (Schering), U.S. Pat. No.
4,747,610 (The Regents of the University of California), WO
80/02365 (Rasor), U.S. Pat. No. 4,774,958 (Feinstein), U.S. Pat.
No. 4,718,433 (Feinstein), EP 0224 934 (Feinstein).
[0008] The only protein-based ultrasound contrast agent under
commercial development consists of a suspension of gas-filled
albumin, Albunex.RTM., prepared by sonication of a solution of
albumin.
[0009] Albumin based ultrasound contrast agents are described in
the following publications:
[0010] Feinstein et al. in Circulation 78S, 565 (1988), Reisner et
al. in Circulation 78S, 565 (1988), Dick et al. in Circulation 78S,
565 (1988), Armstrong et al. in Circulation 78S, 565 (1988), Desir
et al. in Circulation 78S, 566 (1988), Heidenreich et al. in
Circulation 78S, 566 (1988), Keller et al. in Circulation 78S, 567
(1988), Barnhart et al. in Contrast Media Research (1989),
Silverman et al. in Circulation 80S, 369 (1989), Silverman et al.
in Circulation 80S, 349 (1989), Segar at al. in Clin.Res. 37, 294
(1989), Heidenreich et al. in Circulation 80S, 370 (1989), Reiser
et al. in Circulation 80S, 370 (1989), Heidenreich et al. in
Circulation 80S, 566 (1989), Shandas et al. in Circulation 82, 95
(1990), Geny et al. in Circulation 82, 95 (1990), Ten-Cate et al.
in Eur Heart J. 19, 389 (1989), Feinstein et al. in
Echocardiography 6, 27 (1989), Zotz et al. in Eur Heart J. 11, 261
(1990), Ten-cate et al. in Eur Heart J. 11, 261 (1990), Barnhart et
al. in Invest Radiol 25S, 162 (1990), Keller et al. in J. Am Soc
Echo 2, 48 (1989), Bleeker et al. in J. Acoust Soc Am 87, 1792
(1990), Feinstein et al. in J. Am. Coll. Cardiol 16, 316 (1990),
Kaul et al. in J. Am Coll. Cardiol 15, 195 (1990), Bleeker et al in
J. Ultrasound Med 9, 461 (1990), Hilpert et al. in Radiology 173,
361 (1989), and Shapiro et al. in J. Am. Coll. 16, 1603 (1990).
[0011] However, as indicated above, ultrasound contrast agents
based on gas-filled protein microspheres are unstable in vivo, and
there is room for improvement of such products. Segar et al. have,
in Advances in Echocardiography (Sep. 21-22, 1989), concluded that
batch, mixing pressure, mixing time and medium all affect the left
atrium contrast with such protein based products.
[0012] Feinstein et al. have in J. Am. Coll. Cardiol 16, 316 (1990)
published that irrespective of dose group, a cavity opacification
with albumin microspheres was seen in the right ventricle in 88% of
the injections and in the left ventricle in 63% of the injections.
Shandas et al. have in Circulation 82, 95 (1990) raised questions
about the pressure related stability of gas filled albumin
microspheres and Shapiro et al. have recently published in J. Am.
Coll. Cardiol 16, 1603 (1990) lack of ultrasound myocardial
contrast enhancement after administration of sonicated albumin.
[0013] Feinstein has in EP 0224 934 on page 4,8 and claim 9, U.S.
Pat. No. 4,718,433 columns 3 and 5 and U.S. Pat. No. 4,774,958
columns 3 and 5 suggested chemical denaturation to stabilize
albumin gas bubbles: [0014] "The microbubbles formed from 5%
albumin may, in the alternative, be stabilized to form a
commercially, clinically usable contrast agent by treatment with
various chemical agents which chemically denature, or "fix", the
protein, and derivatives thereof. Chemical denaturation of the
protein (or derivatives) may be accomplished by either binding the
protein with a protein-reactive aldehyde, such as glutaraldehyde.
For the latter procedure of stabilizing the invented microbubble
contrast agent, the microbubbles may be reacted with 0.25 grams of
50% aqueous glutaraldehyde per gram of protein at pH4.5 for 6
hours. The treated contrast agent is then gently and extensively
washed to remove as much of the unreacted glutaraldehyde as
possible."
[0015] Various denaturing chemicals or cross linking agents for
proteins have been described in the literature. (See for example
Methods Enzymol 172, 584 (1989) and Chemical Reagents for Protein
Modification, Volume II, page 123, CRC Press Inc.)
[0016] However it is important that any contrast agent should be
rapidly eliminated from the subject in a short term after use, e.g.
preferably having a half life of not more than 48 hours.
Crosslinking by glutaraldehyde or formaldehyde may not always be
effective in providing an adequate balance between stability during
ultrasound visualisation and rapid elimination. The protein itself,
being human serum albumin, is not rapidly degraded by vascular
enzymes and reagents such as glutaraldehyde do not form readily
biodegradable bonds with the protein.
[0017] The present invention is based on the concept of
crosslinking the protein shells of microbubbles to introduce
biodegradable linking groups, thus providing ultrasound contrast
agents with adequate stability for the duration of ultrasound
visualisation but sufficient biodegradability to permit rapid
elimination subsequently.
[0018] According to the present invention, therefore, we provide
ultrasound contrast agents comprising microbubbles of gas or a gas
precursor encapsulated in a shell of protein crosslinked with
biodegradable crosslinking groupings.
[0019] Biodegradable linkages which may be used 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 crosslinking grouping. In
general, any esters will be biodegradable particularly those
containing the grouping --CO.O-- or --O.CO.O--. One particularly
useful class of biodegradable ester groupings has the structure
--(Y).sub.n.CO.O.C(R.sup.1R.sup.2).O.CO.(Z).sub.n- (where Y and Z,
which may be the same or different, are --O--, --S-- or
--NR.sup.3--; the symbols n, which may be the same or different,
are zero or 1; R.sup.1 and R.sup.2, which may be the same or
different, are hydrogen atoms or carbon-attached monovalent groups
or together represent a carbon-attached divalent organic group; and
R.sup.3 is a hydrogen atom or an organic group. Y and Z are
preferably --O--. Such groups generally degrade to eliminate a
compound R.sup.1R.sup.2CO and either form carboxyl groups on the
residue or, in the case of carbonate esters, may eliminate carbon
dioxide to form hydroxyl groups on the residue.
[0020] 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.
[0021] The protein component can be any protein or derivative
thereof including polyamino acids. Albumin, gelatin and
.gamma.-globulin are representative compounds. The protein, for
instance albumin, can be obtained from biological sources, for
example from human or animal blood, or produced by a lower organism
using recombinant technology. A typical method for preparation of
human serum albumin by fermentation is described in WO 9002808
(Delta Biotechnology Ltd.).
[0022] According to a further feature of the invention, we provide
a process for the preparation of microbubble ultrasound contrast
agents in which a gas or a gas precursor is encapsulated in a
protein which is crosslinked with biodegradable crosslinking
groups.
[0023] The crosslinking of the protein can be effected before,
during or after encapsulation. It is preferred to encapsulate, e.g.
by forming microbubbles, first and to effect crosslinking
subsequently.
[0024] The crosslinking agent may be a compound of the formula (I)
A.sup.1-X-A.sup.2 (I) where X is a linking group containing one or
more biodegradable linkages and the groups A.sup.1 and A.sup.2,
which may be the same or different, are functional groups reactive
with proteins.
[0025] The group X may carry further groups reactive with proteins
to provide an even greater degree of crosslinking.
[0026] Preferably, the group X should have a chain length of not
more than 30 atoms.
[0027] The group X may thus be of the form --R.sup.8-E-R.sup.9--
where 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, which may carry groups reactive with
proteins and/or further inert groups, and the group E is an ester
grouping, for example of the formula --O.CO--, --O.CO.O-- or
--(Y).sub.n.CO.0.C(R.sup.1R.sup.2).O.CO.(Z).sub.n- as defined
above.
[0028] Crosslinking agents of the formula
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 where A.sup.1, A.sup.2, R.sup.1, R.sup.2, R.sup.8, R.sup.9,
n, Y and Z have the above meanings may be prepared by reaction of
an acid of the formula A.sup.1.R.sup.8.(Y).sub.n.CO.OH or a form
thereof in which A.sup.1 and any other reactive groups are
protected (or a functional derivative thereof) with a compound of
the formula L.sup.1.C(R.sup.1R.sup.2).L.sup.2 where L.sup.1 is a
leaving group such as a halogen atom or mesyloxy or tosyloxy and
L.sup.2 is a group as defined for L.sup.1 (giving a symmetrical
di-ester) or a group of the formula
--O.CO.(Z).sub.n.R.sup.9.A.sup.2 or a protected form thereof, if
necessary followed by deprotection. The functional derivative of
the acid may for example be a salt, e.g. the potassium salt. The
reaction will normally be carried out in solution, for example in a
polar solvent such as dimethylformamide. Protecting groups for
A.sup.1 and A.sup.2 may be those conventional in the art. Preferred
protecting groups for aldehydes include acetals, e.g. cyclic
acetals such as dioxolan.
[0029] The compound
L.sup.1.C(R.sup.1R.sup.2).O.CO.(Z).sub.n.R.sup.9.A.sup.2, where
L.sup.1 is halogen, may be prepared from R.sup.1R.sup.2.CO by
reaction with a compound of the formula
Hal.CO.(Z).sub.n.R.sup.9.A.sup.2 (where Hal represents a halogen
atom) in the presence of a base such as pyridine.
[0030] Apart from aldehyde groups, which are preferred, the groups
A.sup.1 and A.sup.2 may be activated carboxyl groups, such as
N-hydroxysuccinimidyl groups (especially water solubility-enhanced
sulphonated N-hydroxysuccinimidyl derivatives), imidoesters,
halo-nitroaryl groups, nitrene precursor groups such as
azidophenyl, carbene precursor groups, ketone groups,
isothiocyanate groups etc.
[0031] Any biocompatible gas maybe 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 maybe free
within the microbubble or may be trapped or entrained within a
containing substance. The term "gas" as used herein includes any
substance in the gaseous form at 37.degree. C.
[0032] Gas precursors include carbonates and bicarbonates, e.g.
sodium or ammonium bicarbonate and aminomalonate esters.
[0033] 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.
[0034] 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.
[0035] The stabilising particles may carry functional groups which
will interact with the protein to form covalent or other linkages.
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 protein by hydrogen bonding or by
forming covalent bond.
[0036] The protein 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 bilayers containing the gas or gas precursor.
[0037] The stabilisation of the system by monolayers or the
formation of the vesicles may be activated, as fully described in
the literature, by sonication or even shaking of the protein
material mixture in the appropriate medium, or the vesicles may be
formed by any conventional liposome/vesicle-forming principle.
[0038] The stabilized microbubbles 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.
[0039] 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
emulsion as described in U.S. Pat. No. 4,684,479. Another well
established method, described i.e. 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 is passing the gas
through a syringe into the mixture of the protein and the 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 protein material. 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.
[0040] By using the principle of electrolysis it is possible to
generate the gas to be entrapped directly in a container containing
the protein material. The electrolytes necessary for the
electrolysis may even help to further stabilize the protein
material. 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.
[0041] As described above, microbubbles 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.
[0042] In one procedure according to the invention, encapsulation
is effected by agitation or sonication of the protein in an aqueous
medium to yield a protein foam which is dried and thereafter
suspended in a solution of the crosslinking agent in a polar
organic solvent (e.g. a sulphoxide such as dimethyl sulphoxide)
which is capable of wetting the protein foam.
[0043] The following Examples are given by way of illustration
only:
Preparation 1
Methylene bis (.alpha.-formylacetate)
[0044] The preparation of the starting material, the
dioxolan-protected aldehyde methyl .alpha.-formylacetate, is
described by T. Hosokawa et al. J. Org. Chem. Soc. 52, (1987)
1758-1764. The protected aldehyde (6.0 g, 3.75 mmol) is treated
with a mixture of 2N aqueous potassium hydroxide and
tetrahydrofuran 20:80 (v/v) at reflux for 8 hours. The pH is
adjusted to 8 using diluted HCl, and the mixture is evaporated to
dryness. The solid is mixed with 100 ml freshly distilled and dried
dimethylformamide, and after 30 minutes at 60.degree. C. the
undissolved material is filtered off. Diiodomethane (150 .mu.l,
1.87 mmol) is added dropwise during 5 minutes to the solution at
60.degree. C. as described in WO 89/00988 page 13 (NYCOMED AS). The
precipitate is removed by filtration after stirring for 4 days, and
the solvent removed at reduced pressure. The dioxolan protection is
removed as described by P. A. Grieco et al. J. Am. Chem. Soc. 99,
(1977) 5773-5780--the residue is dissolved in tetrahydrofuran (60
ml), 5% aqueous HCl (20 ml) is added and the mixture is stirred for
20 hours at ambient temperature. The reaction mixture is evaporated
to dryness under reduced pressure to yield the title compound.
Preparation 2
Methylene dimethacrylate
[0045] A solution of potassium hydroxide (1.00 M, 40.00 ml) is
added to methacrylic acid (3.44 g, 40.00 mmol) at 0.degree. C. and
the solution freeze dried for 16 hours. Dry dimethylformamide (230
ml) is added and the suspension heated to 60.degree. C. under a dry
nitrogen atmosphere. Diiodomethane (1.61 ml, 20.00 mmol) is added
in two portions during 10 min. and the reaction mixture left for 4
days at 60.degree. C. The solvent is removed under reduced pressure
(0.05 mm Hg), before diethyl ether (140 ml), saturated aqueous
sodium hydrogen carbonate (50 ml) and water (50 ml) are added. The
aqueous layer is extracted with diethyl ether (6.times.60 ml) and
the combined ether extracts washed with water (4.times.50 ml),
dried (MgSO.sub.4), and evaporated to give 2.63 g (72%) of the
title compound. .sup.1H NMR (60 MHz, CDCl.sub.3): .delta. 1.97
(2.times.CH.sub.3, m), 5.63 (2.times.H--C.dbd., m), 5.88 (CH.sub.2,
S), 6.18 (2.times.H--C.dbd., m). IR (film, c=.sup.-1): 2987 (w),
2962 (w), 2930 (w), 1732 (str), 1638 (w), 1454 (w), 1315 (w), 1295
(w), 1158 (w), 1100 (str), 1012 (m), 989(m). This product may be
used in accordance with the invention, for example to crosslink
acrylamide polymers.
Preparation 3
Methylene diacrylate
[0046] A solution of potassium hydroxide (1.00 M, 40.00 ml) is
added to acrylic acid (2.88 g, 40.00 mmol) at 0.degree. C. and the
solution freeze dried for 16 hours. Dry dimethylformamide (200 ml)
is added and the suspension heated to 60.degree. C. under a dry
nitrogen atmosphere. Diiodomethane (1.61 ml, 20.00 mmol) is added
in two portions during 10 min. and the reaction mixture left for 4
days at 60.degree. C. The solvent is removed under reduced pressure
(0.05 mm Hg), before diethyl ether (140 ml), saturated aqueous
sodium hydrogen carbonate (50 ml) and water (50 ml) are added. The
aqueous layer is extracted with diethyl ether (6.times.60 ml) and
the combined ether extracts washed with water (4.times.50 ml),
dried (MgSO.sub.4), and evaporated to give 1.06 g (34%) of the
title compound. .sup.1H NMR (60 MHz, CDCl.sub.3): .delta. 5.81-6.61
(2.times.CH2=CH--, m), 5.84 (CH.sub.2, s). This product may be used
in accordance with the invention, for example to crosslink acrylic
acid and methyl acrylate polymers.
Preparation 4
Chloromethyl (2-methacryloyloxy)ethyl carbonate
[0047] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a
solution of chloromethyl chloroformate (0.89 ml, 11.00 mmol) and
2-hydroxyethyl methacrylate (1.22 ml, 10.00 mmol) in
dichloromethane (12 ml) at 0.degree. C. under a dry nitrogen
atmosphere. After 21 hours at 20.degree. C. the reaction mixture is
washed with hydrochloric acid (1.00 M, 10 ml), saturated aqueous
sodium hydrogen carbonate (10 ml) and water (10 ml). The organic
phase is dried (MgSO.sub.4) and the solvent evaporated under
reduced pressure (10 mm Hg) to give 1.97 g (88%) of the title
compound. .sup.1H NMR (60 MHz, CDCl.sub.3): .delta.1.88 (CH.sub.3,
d, J=2 Hz), 4.35 (O--CH.sub.2--CH.sub.2--O, m), 5.47 (H--C.dbd.,
m), 5.63 (CH.sub.2--Cl, s), 6.00 (H--C.dbd., m).
Preparation 5
(2-Methacryloyloxy)ethyl methacryloyloxymethyl carbonate
[0048] A solution of potassium hydroxide (1.00 M, 5.00 ml) is added
to methacrylic acid (0.43 g, 5.00 mmol) at 0.degree. C. and the
solution freeze dried during 16 hours. Dry dimethylformamide (50
ml) is added and to the resulting suspension is added chloromethyl
(2-methacryloyloxy) ethyl carbonate (1.11 g, 5.00 mmol). 18-Crown-6
(0.066 g, 0.25 mmol) is added as a catalyst and the reaction left
under a dry nitrogen atmosphere. After 24 hours at 20.degree. C.
and 6 days at 4.degree. C. the solvent is removed under reduced
pressure (0.05 mm Hg) and diethyl ether (30 ml) and water (20 ml)
added. The aqueous layer is extracted with diethyl ether
(3.times.20 ml) and the combined ether extracts washed with water
(20 ml), dried (MgSO.sub.4) and evaporated to give 1.26 g (93%) of
the title compound. .sup.1H NMR (60 MHz, CDCl.sub.3): .delta.1.97
(2.times.CH.sub.3, m), 4.38 (O--CH.sub.2--CH.sub.2--O, m), 5.53
(2.times.H--C.dbd., m), 5.77 (CH.sub.2, s), 6.07
(2.times.H--C.dbd., m).
Preparation 6
Ethylene bis(chloromethyl carbonate)
[0049] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a
solution of chloromethyl chloroformate (1.32 ml, 14.83 mmol) and
ethylene glycol (0.28 ml, 5.00 mmol) in dichloromethane (10 ml) at
7.degree. C. with good stirring under a dry N.sub.2 atmosphere.
After 15 min. at 7.degree. C. and 6 hours at 20.degree. C. the
reaction mixture is transferred to a separating funnel with the aid
of dichloromethane (10 ml). The reaction mixture is washed with
hydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium
hydrogen carbonate (10 ml) and water (10 ml). The organic phase is
dried (MgSO.sub.4) and the solvent evaporated under reduced
pressure to give 1.12 g (90%) of the title product. .sup.1H NMR
(300 MHz, CDCl.sub.3): .delta. 4.48 (s, O--CH.sub.2CH.sub.2--O),
5.75 (s, 2.times.Cl--CH.sub.2--O). .sup.13C NMR (75 MHz,
CDCl.sub.3): .delta. 65.8 (O--CH.sub.2CH.sub.2--O), 72.2
(2'Cl--CH.sub.2--O), 153.0 (2.times.C.dbd.O).
Preparation 7
Bis (2-chloromethoxycarbonyloxyethyl)ether
[0050] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a
solution of chloromethyl chloroformate (1.32 ml, 14.83 mmol) and
diethylene glycol (0.47 ml, 5.00 mmol) in dichloromethane (10 ml)
at 7.degree. C. with good stirring under a dry N.sub.2 atmosphere.
After 10 min. at 7.degree. C. and 6 hours at 20.degree. C. the
reaction mixture is transferred to a separating funnel with the aid
of dichloromethane (10 ml). The reaction mixture is washed with
hydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium
hydrogen carbonate (10 ml) and water (10 ml). The organic phase is
dried (MgSO.sub.4) and the solvent evaporated under reduced
pressure (10 mm Hg) to give 1.26 g (86%) title product. .sup.1H NMR
(300 MHz, CDCl.sub.3): .delta. 3.72 (m, 2.times.CH.sub.2--O), 4.34
(m, 2.times.CH.sub.2--O--C.dbd.O), 5.71 (s,
2.times.Cl--CH.sub.2--O). .sup.13C NMR (75 MHz, CDCl.sub.3):
.delta. 67.6 (2.times.CH.sub.2--O), 68.5
(2.times.CH.sub.2--O--C.dbd.O), 72.1 (2.times.Cl--CH.sub.2--O),
153.2 (2.times.C.dbd.O).
Preparation 8
1-Chloroethyl 2-methacryloyloxyethyl carbonate
[0051] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a
solution of 1-chloroethyl chloroformate (1.20 ml, 11.00 mmol) and
2-hydroxyethyl methacrylate (1.22 ml, 10.00 mmol) in
dichloromethane (12 ml) at 3.degree. C. under a dry N.sub.2
atmosphere. After 15 min. at 3.degree. C. and 17 hours at
20.degree. C. the reaction mixture is transferred to a separating
funnel with the aid of dichloromethane (10 ml). The reaction
mixture is washed with hydrochloric acid (1.00 M, 10 ml), saturated
aqueous sodium hydrogen carbonate (10 ml) and water (2.times.10
ml). The organic phase is dried (MgSO.sub.4) and the solvent
evaporated under reduced pressure to give 1.76 g (74%) of the title
product. .sup.1H NMR (60 MHz, CDCl.sub.3): .delta. 1.85 (3 H, d,
J=6 Hz, CH.sub.3--CH), 1.96 (3 H,d, J=2 Hz, CH.sub.3--C.dbd.), 5.55
(1 H, m, CH.dbd.), 6.10 (1 H, m, CH.dbd.), 6.38 (1 H, k, J=6 Hz,
CH--CH.sub.3).
Preparation 9
Chloromethyl 4-acryloyloxybutyl carbonate
[0052] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a
solution of chloromethyl chloroformate (0.98 ml, 11.00 mmol) and
4-hydroxybutyl acrylate (1.38 ml, 10.00 mmol) in dichloromethane
(12 ml) at 3.degree. C. under a dry N.sub.2 atmosphere. After 15
min. at 3.degree. C. and 17 hours at 20.degree. C. the reaction
mixture is transferred to a separating funnel with the aid of
dichloromethane (10 ml). The reaction mixture is washed with
hydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium
hydrogen carbonate (10 ml) and water (2.times.10 ml). The organic
phase is dried (MgSO.sub.4) and the solvent evaporated under
reduced pressure to give 1.76 g (74%) of the title product. .sup.1H
NMR (60 MHz, CDCl.sub.3): .delta. 1.82 (4 H, m,
CH.sub.2--CH.sub.2), 4.27 (4 H, m, 2.times.CH.sub.2--O), 5.77 (2 H,
s, Cl--CH.sub.2--O), 5.8-6.7 (3 H, m, CH.dbd.CH.sub.2).
Preparation 10
1-Chloroethyl 4-acryloyloxybutyl carbonate
[0053] Pyridine (0.89 ml, 11.00 mmol) is added dropwise to a
solution of 1-chloroethyl chloroformate (1.20 ml, 11.00 mmol) and
4-hydroxybutyl acrylate (1.38 ml, 10.00 mmol) in dichloromethane
(12 ml) at 3.degree. C. under a dry N.sub.2 atmosphere. After 15
min. at 3.degree. C. and 17 hours at 20.degree. C. the reaction
mixture is transferred to a separating funnel with the aid of
dichloromethane (10 ml). The reaction mixture is washed with
hydrochloric acid (1.00 M, 10 ml), saturated aqueous sodium
hydrogen carbonate (10 ml) and water (2.times.10 ml). The organic
phase is dried (MgSO.sub.4) and the solvent evaporated under
reduced pressure to give 2.26 g (90%) of the title product. .sup.1H
NMR (60 MHz, CDCl.sub.3): .delta. 1.80 (4 H, m,
CH.sub.2--CH.sub.2), 1.86 (3 H, d, J=5 Hz, CH.sub.3), 4.24 (4 H, m,
2.times.CH.sub.2--O), 5.7-6.6 (4 H, m, CH.dbd.CH.sub.2 and CH).
Preparation 11
1-Methacryloyloxyethyl 2-methacryloyloxyethyl carbonate
[0054] 1-Chloroethyl 2-methacryloyloxyethyl carbonate (1.183 g,
5.00 mmol) prepared as described in Preparation 8 is added to a
suspension of freeze dried potassium methacrylate (0.683 g, 5.50
mmol) and 18-crown-6 (0.066 g, 0.25 mmol) in dimethylformamide (50
ml) under a dry N.sub.2 atmosphere. After 5 days at 20.degree. C.
the solvent is removed under reduced pressure and the residue
dissolved by adding dichloromethane (60 ml) and water (30 ml).
After separating the phases the aqueous layer is extracted with
dichloromethane (3.times.30 ml) and the combined organic phase
washed with saturated aqueous sodium hydrogen carbonate (50 ml).
The organic phase is dried (MgSO.sub.4) and the solvent removed
under reduced pressure to give 1.10 g (77%) of the title product.
.sup.1H NMR (60 MHz, CDCl.sub.3): .delta. 1.63 (3 H, d, J=5 Hz,
CH.sub.3--CH), 1.98 (6 H, s, 2.times.CH.sub.3), 4.42 (4 H, s,
O--CH.sub.2--CH.sub.2--O), 5.62 (2 H, m, CH.dbd.), 6.15 (2 H, m,
CH.dbd.), 6.84 (1 H, k, J=5 Hz, CH--CH.sub.3).
Preparation 12
Acryloyloxymethyl 4-acryloyloxybutyl carbonate
[0055] Chloromethyl 4-acryloyloxybutyl carbonate (1.183 g, 5.00
mmol) prepared as described in Preparation 9 is added to a
suspension of freeze dried potassium acrylate (0.606 g, 5.50 mmol)
and 18-crown-6 (0.066 g, 0.25 mmol) in dimethylformamide (50 ml)
under a dry N.sub.2 atmosphere. After 5 days at 20.degree. C. the
solvent is removed under reduced pressure and the residue dissolved
by adding dichloromethane (60 ml) and water (30 ml). After
separating the phases the aqueous layer is extracted with
dichloromethane (3.times.30 ml) and the combined organic phase
washed with saturated aqueous sodium hydrogen carbonate (50 ml).
The organic phase is dried (MgSO.sub.4) and the solvent removed
under reduced pressure to give 1.24 g (91%) of the title product.
.sup.1H NMR (60 MHz, CDCl.sub.3): .delta. 1.82 (4 H, m,
CH.sub.2--CH.sub.2), 4.23 (4 H, m, 2.times.CH.sub.2--O) 5.88 (2 H,
s, O--CH.sub.2--O), 5.7-6.8 (6 H, 2.times.CH.dbd.CH.sub.2).
Preparation 13
1-Acryloyloxyethyl 4-acryloyloxybutyl carbonate
[0056] 1-Chloroethyl 4-acryloyloxybutyl carbonate (1.253 g, 5.00
mmol) prepared as described in Preparation 10 is added to a
suspension of freeze dried potassium acrylate (0.606 g, 5.50 mmol)
and 18-crown-6 (0.066 g, 0.25 mmol) in dimethylformamide (50 ml)
under a dry N.sub.2 atmosphere. After 5 days at 20.degree. C. the
solvent is removed under reduced pressure and the residue dissolved
by adding dichloromethane (60 ml) and water (30 ml). After
separating the phases the aqueous layer is extracted with
dichloromethane (3.times.30 ml) and the combined organic phase
washed with saturated aqueous sodium hydrogen carbonate (50 ml).
The organic phase is dried (MgSO.sub.4) and the solvent removed
under reduced pressure to give 1.28 g (89%) of the title product.
.sup.1H NMR (60 MHz, CDCl.sub.3): .delta. 1.58 (3 H, d, J=5 Hz,
CH.sub.3--CH), 1.80 (4 H, m, CH.sub.2--CH.sub.2), 4.24 (4 H, m,
2.times.CH.sub.2--O), 5.7-6.7 (6 H, m, 2.times.CH.dbd.CH.sub.2),
6.87 (1 H, k, J=5 Hz, CH--CH.sub.3).
Preparation 14
a) Methylene bis(3,3-dimethoxypropionate)
[0057] Cesium 3,3-dimethoxypropionate (19.95 g, 75 mmol) is added
to dry DMF (1000 ml). Diiodomethane (10.04 g, 37.5 mmol) is added
to the suspension and the reaction mixture is stirred for 2 days at
60.degree. C. under a dry N.sub.2 atmosphere. DMF is removed under
reduced pressure (0.01 mmHg). Diethyl ether (500 ml) is added to
the residue, which is then washed with saturated aqueous sodium
hydrogen carbonate (250 ml). The aqueous layer is extracted with
diethyl ether (5'75 ml). The combined ether extracts are washed
with water (2.times.100 ml), dried (MgSO.sub.4) and evaporated to
give 7.1 g (72%) product. .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 2.61 (CH.sub.2, d), 3.26 (CH.sub.3, S).
b) Methylene bis(3-methoxypropenoate)
[0058] Methylene bis(3,3-dimethoxypropionate) (14.01 g, 50 mmol)
prepared as described in (a) above and a catalytic amount of
p-toluene sulfonic acid is added to toluene (250 ml). The methanol
is removed by warming the reaction under an N.sub.2 atmosphere.
When the reaction is complete the toluene is distilled off under
reduced pressure. Diethyl ether (250 ml) is added and the mixture
is washed with saturated aqueous sodium hydrogen carbonate
(5.times.50 ml) and water (3.times.50 ml). The organic layer is
dried (MgSO.sub.4) before evaporation to give 8.52 g (79%) product.
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 3.65 (2.times.CH.sub.3,
s), 5.2 (2.times.CH, d), 5.8 (O--CH.sub.2--O), 7.65
(2.times.CH.sub.2, d).
Preparation 15
a) Methylene bis(10-undecenoate)
[0059] 10-Undecylenic acid (12.75 g, 75 mmol) is dissolved in 100
ml water. Cesium carbonate (13.04 g, 40 mmol) is added to the
mixture. The water is removed under reduced pressure and the salt
dried for 2 hours in vacuo. The cesium salt is mixed with 150 ml
DMF and diiodomethane is added to the solution. The reaction is
stirred for 3 days at 60.degree. C. under an N.sub.2 atmosphere.
DMF is then removed under reduced pressure. The residue is purified
through silica gel with hexane/ethyl acetate (8:2) as eluant. The
solvent is evaporated to give 7.18 g (54%) product. .sup.1H NMR
(300 MHz, CDCl.sub.3): .delta. 1.2-1.4 (10.times.CH.sub.2, m), 1.6
(2.times.CH.sub.2, m), 2.0 (2.times.CH.sub.2, m), 2.19
(2.times.CH.sub.2, t), 4.9 (2.times.H.sub.2 C.dbd., m), 5.88
(O--CH.sub.2--O, s), 5.9 (2.times.HC.dbd., m). .sup.13C NMR (300
MHz, CDCl.sub.3): .delta. 24.92-33.98 (8.times.CH.sub.2), 79.04
(O--CH.sub.2--O), 114.18 (.dbd.CH.sub.2), 139.11 (.dbd.CH), 172.48
(C.dbd.O).
b) Methylene bis(10-epoxyundecanoate)
[0060] Methylene bis(10-undecenoate) (8.8 g, 25 mmol) prepared as
described in (a) above is added under an N.sub.2 atmosphere to
methylene chloride and cooled to 0.degree. C. Metachloroperbenzoic
acid 55% (15.75 g, 50 mmol) is added to methylene chloride (150 ml)
and the organic layer is separated and dried (MgSO.sub.4). The
metachloroperbenzoic acid is then added dropwise to the diester.
After completed addition the temperature is increased to 25.degree.
C. After 5 hours the reaction is complete. The mixture is washed
with saturated aqueous sodium sulphite (75 ml) and saturated
aqueous sodium hydrogen carbonate (2.times.75 ml). The organic
layer is purified on neutral aluminium oxide. The solvent is
removed under reduced pressure to yield 8.45 g (82%) product.
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 1.2-1.7
(14.times.CH.sub.2, m), 2.35 (2.times.CH.sub.2CO, t), 2.45
(2.times.CH,q), 2.75 (2.times.CH,q), 2.90 (2.times.CH,m), 5.75
(O--CH.sub.2--O). .sup.13C NMR (300 MHz, CDCl.sub.3): .delta. 24.58
(CH.sub.2), 25.99 (CH.sub.2), 28.94 (CH.sub.2), 29.09 (CH.sub.2),
29.32 (2.times.CH.sub.2), 32.45 (CH.sub.2), 33.92 (CH.sub.2), 47.06
(CH.sub.2--O), 52.36 (CH--O), 79.06 (O--CH.sub.2--O), 172.2
(C.dbd.O).
Preparation 16
Methylene bis(4-epoxypentanoate)
[0061] Metachloroperbenzoic acid (15.68 g, 55%, 50 mmol) is
dissolved in methylene chloride (200 ml). Water is separated and
the organic layer is dried (MgSO.sub.4). The resulting
metachloroperbenzoic acid solution is added dropwise to methylene
bis(4-pentenoate) (4.10 g, 19 mmol) dissolved in methylene chloride
(50 ml). The mixture is stirred at ambient temperature under
nitrogen for 12 hrs, whereafter the reaction mixture is washed with
saturated aqueous sodium bicarbonate solution (50 ml), water (50
ml), dried (MgSO.sub.4) and evaporated to give 3.61 g (78%) of the
title compound as a crystalline product. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 1.70-1.85 (2.times.CH,m), 1.95-2.10
(2.times.CH,m), 2.50-2.55 (2.times.CH, 2.times.CH.sub.2,m), 2.75
(2.times.CH,t), 3.0 (2.times.CH,m), 5.8 (O--CH.sub.2--O, s).
.sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 27 (2.times.CH.sub.2),
30 (2.times.CH.sub.2), 47 (2.times.CH.sub.2), 51 (2.times.CH), 79.8
(O--CH.sub.2--O), 171.8 (2.times.C.dbd.O).
Preparation 17
Methylene bis(2-butenoate)
[0062] Vinylacetic acid (4.3 g, 50 mmol) is added to an aqueous
cesium carbonate solution (50 ml). The mixture is stirred for 5
min. and then evaporated, and the residue is dried under vacuum for
2 hrs. The resulting cesium salt and diiodomethane are added to
dimethylformamide (200 ml) and the mixture is stirred for 24 hrs.
at 50.degree. C. under nitrogen, whereafter the dimethylformamide
is removed under reduced pressure. The residue is dissolved in
diethyl ether (100 ml) and washed with saturated aqueous sodium
bicarbonate (25 ml) and water (25 ml). The organic layer is dried
(MgSO.sub.4) and evaporated to give 1.32 g (29%) product. .sup.1H
NMR (300 MHz, CDCl.sub.3): .delta. 1.9 (2.times.CH.sub.2,m),
5.8-5.9 (2.times.CH,m), 5.9 (OCH.sub.2O,s), 7.0-7.1 (2CH,m).
Preparation 18
Methylene bis(chloroacetate)
[0063] Chloroacetic anhydride (12.75 g, 75 mmol), paraformaldehyde
(2.25 g, 75 mmol) and conc. sulfuric acid (15 drops) are added to
methylene chloride (15 ml). The mixture is stirred for 24 hrs. at
50.degree. C. under nitrogen, whereafter the reaction mixture is
extracted with saturated aqueous potassium carbonate until carbon
dioxide emission ends. The organic layer is dried (MgSO.sub.4),
evaporated to dryness and the residue is distilled (80.degree. C.,
0.15 mmHg) to yield 10.2 g (57%) product. .sup.1H NMR (200 MHz,
CDCl.sub.3): .delta. 4.1 (2.times.CH.sub.2Cl,s), 5.9 (CH.sub.2,s).
.sup.13C NMR (200 MHz, CDCl.sub.3): .delta. 41.1 (CH.sub.2Cl), 81.4
(O--CH.sub.2--O), 166.4 (CO).
Preparation 19
Methylene bis(4-oxopentanoate)
[0064] 4-Oxopentanoic acid (11.6 g, 100 mmol) is dissolved in
acetonitrile (70 ml), and 1,8-diazabicyclo[5.4.0]undec-7-ene (15.25
g, 100 mmol) diluted with acetonitrile (30 ml) is added.
Diiodomethane (13.4 g, 50 mmol) is added in one batch, and the
reaction mixture is refluxed under a nitrogen atmosphere. After 2
hours, gas chromatography indicates full consumption of
diiodomethane. The solvent is removed in vacuo and the residual
brown oil is transferred to a separation funnel with ethyl acetate
(200 ml) and water (75 ml). The organic phase is washed with 1M
sodium bicarbonate (25 ml) and water (3.times.25 ml), dried over
MgSO.sub.4, and the solvent is removed in vacuo to yield the title
compound (10 g). .sup.1H NMR: .delta. 2.19 (2.times.CH.sub.2, s),
2.760-2.804 (2.times.CH.sub.2, t), 2.600-2.645 (2.times.CH.sub.2,
t), 5.735 (CH.sub.2 bridge, s).
Preparation 20
Methylene bis(succinimidylazelate)
[0065] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(1.49 g, 7.71 mmol) was added in portions to a stirred solution of
methylene bis (hydrogen azelate) from Example 25 (1.00 g, 2.57
mmol) and N-hydroxysuccinimide (0.89 g, 7.71 mmol) in dry
dimethylformamide at ambient temperature. After 20 hours stirring,
the reaction mixture was poured into ice-water and the product
precipitated as an oil. The colourless oil was,dissolved in
diethylether (50 ml), washed with water (3.times.10 ml) and dried
over MgSO.sub.4. The solvent was removed under reduced pressure and
hexane (5 ml) was added to the oily product. After seven days
storage at 4.degree. C. the oil had crystallized to a white, waxy
solid. Yield: 1.50 g (69%). m.p.: 45-47.degree. C. .sup.13C NMR (75
MHz, CDCl.sub.3) .delta.: 24.42, 24.46, 25.59, 28.48, 28.63, 30.85,
33.82, 79.61, 168.6, 169.30, 172.34.
Preparation 21
Methylene bis(sulphosuccinimidylazelate) sodium salt
[0066] Methylene bis(hydrogen azelate) (0.38 g, 1 mmol),
N-hydroxysuccinimide sodium salt (0.48 g, 2.2 mmol) and
dicyclohexylcarbodiimide (0.45 g, 2.2. mmol) were dissolved in
dimethylformamide (10 ml). The suspension was stirred overnight at
room temperature under an atmosphere of nitrogen. The reaction
mixture was filtered and purified by reversed phase chromatography
(RP-8) with water/acetonitrile (1:1) as eluant to give the title
compound.
Preparation 22
a) Methylene bis(10,11-dihydroxyundecanoate)
[0067] N-Methylmorpholine-N-oxide (13.5 g, 11 mmol) and methylene
bis(10-undecenoate) from Preparation 15(b) (19 g, 5 mmol) were
dissolved in 400 ml of a mixture of tetrahydrofuran and water (3:1
v/v). A catalytic amount of osmium tetroxide was added, and the
solution stirred at ambient temperature for 20 hours. TLC indicated
complete consumption of the starting material. Excess sodium
hydrogen sulphite and sodium chloride were then added to the
reaction mixture. The product was extracted from the resulting
mixture with ethyl acetate (400 ml) and the water phase was washed
with ethyl acetate (3.times.50 ml). The combined organic phases
were dried and evaporated, and the product recrystallised from
tetrahydrofuran to yield 14.5 g (68%) of the product as a white
solid. .sup.13C NMR (45 MHz) CD.sub.3OD: .delta. 24.6-34.0
(16.times.CH.sub.2), 66.6 (2.times.CH.sub.2OH), 72.3
(2.times.CHOH), 79.2 (O--CH.sub.2--O), 174.0 (2.times.C.dbd.O).
b) Methylene bis (10-oxodecanoate)
[0068] Methylene bis(10,11-dihydroxyundecanoate) (2.24 g, 5 mmol)
was dissolved in 150 ml tetrahydrfuran. Sodium metaperiodate (2.06
g, 10 mmol) was dissolved in 150 ml water and added dropwise to the
tetrahydrofuran solution. TLC indicated full consumption of the
diol after 60 minutes, whereupon sodium chloride was added to the
reaction mixture until the two phases separated. The water phase
was extracted with diethyl ether (3.times.50 ml). The combined
organic phases was dried with magnesium sulphate and evaporated to
give the title product as an oil, 1.43 g (74%). .sup.13C NMR (45
MHz) CDCl.sub.3: .delta. 21.9-43.9 (16.times.CH.sub.2), 79.1
(O--CH.sub.2--O), 173.0 (2.times.C.dbd.O), 202.6 (2.times.CHO).
EXAMPLE 1
[0069] 1. Gas-filled albumin microspheres are prepared according to
EP-A-0359 246 and resuspended to homogeneity by gentle rolling on a
vial roller.
[0070] 2. 25 ml of the suspension are poured into a 25 ml
separating funnel and left for 30 min. The bottom 20 ml are
discarded.
[0071] 3. To the remaining 5 ml is added 20 ml of a phosphate
buffer (20 mM NaPO.sub.4, pH 7.0), and the resulting suspension is
transferred to a vial with a cap septum.
[0072] 4. The vial is centrifuged upside down at 170.times.g for 5
min.
[0073] 5. The solution underneath the microsphere layer is
withdrawn using a syringe, and the microspheres are resuspended in
25 ml of the phosphate buffer by 10 min of gentle rolling.
[0074] 6. Points 4 and 5 are repeated twice.
[0075] 7. The resulting suspension is centrifuged as in point 4,
and the microspheres are resuspended in the phosphate buffer to a
final concentration of about 5.times.10.sup.8 particles per ml.
[0076] 8. The crosslinker methylene bis(.alpha.-formylacetate),
prepared as described in Preparation 1, is added to the suspension,
and the crosslinking reaction is allowed to proceed for the desired
time (usually 30-60 min) under gentle rolling.
[0077] 9. 1.5 M Tris-HCl-buffer (pH 8.8) is added to a final
concentration of 0.25 M, and the suspension is rolled gently for 10
min.
[0078] 10. The vial is centrifuged as in point 4, and the solution
underneath the microsphere layer is removed as in point 5.
[0079] 11. The microspheres are resuspended in phosphate buffer
(same volume as final volume in point 9), and the suspension is
rolled for 10 min.
[0080] 12. Points 10 and 11 are repeated twice.
[0081] 13. The resulting suspension is centrifuged as in point 4,
and the microspheres are resuspended in the phosphate buffer to a
final concentration of about 5.times.10.sup.8 particles per ml.
[0082] 14. This final suspension of crosslinked gas/albumin
microspheres is stored at 4.degree. C.
EXAMPLE 2-22
[0083] The procedure of Example 1 is repeated using crosslinking
agents prepared as described in Preparations 2-22, except that
dimethyl suplhoxide is used in place of phosphate buffer in the
processing of the gas-filled albumin microspheres according to
steps 3-7 and the crosslinking agent is added in step 8 as a
solution in dimethyl sulphoxide.
[0084] The number and size distribution of the products are
determined by Coulter counter analysis.
* * * * *