Note: Descriptions are shown in the official language in which they were submitted.
CA 02145505 2003-08-12
1
MICROPARTICLE PREPARATIONS MADE FROM
BIODEGRADABLE COPOLYMERS
The invention relates to microparticles produced from
biopolymers, monomers capable of polymerization, active
ingredients and/or diagnostically detectable components, a
process for their production as Well as their use in diagnosis
and treatment, especially as contrast media in ultrasonic
diagnosis.
It is known that particles, whose diameter is smaller or in
the range of the size of red blood cells, can circulate within
the circulatory system after injection in the circulatory
pathway. Pharmaceutical preparations of such microparticles are
thus suitable as vehicle systems injectable in the circulatory
system for active ingredients or diagnostic agents in medicine.
As vehicle materials, in principle all biodegradable, compatible
and non-water-soluble substances are suitable. So far, above all
fats, waxes, lipids (e. g., soybean lecithin), denatured
biopolymers (e.g., albumin, gelatin) and synthetically '
biodegradable polymers (e. g., polylactic acid, polyhydroxybutyric
acid, polyalkylcyanoacrylates, poly-L-lysine) are described.
There is.a difference in how quickly and in the numbers in
which the microparticles circulating in the circulatory pathway
are recognized, as a function of their physical and chemical
properties, by the cells of the monocytic phagocytizing system
,(MPS) and taken up (mainly in the liver, lung and spleen). The
particle charge, the particle size, the properties (molecular
weight, amphiphilia) on the particle surface of adsorbed
2_2145505
substances, as well as the affinity of the particle surface for
blood components such as fibronectin, albumin, etc. are
considered as essential factors, which determine the kinetics of
the absorption of the microparticles by the cells of the MPS. By
specific variation of the physicochemical surface properties of
microparticles, the kinetiics of the phagocytosis can be
influenced by the cells of the MPS and the extent of the
concentration of the particles within the corresponding organs
(i.a., liver, lung, spleen, bone marrow) (passive targeting): A
specific concentration of microparticles in target tissues or
body structures, which are not among the organs of the RES, is in
this way not possible. It can rather be achieved only by the
combination of the microparticles with substances which have
site-specific, structure-specific or-tissue-specific binding
properties (homing devices). But the particles previously
described for use in ultrasonic diagnosis are suitable only
insufficiently as preparations suitable for the combination with
homing devices.
Thus, it has to be accepted in the case of the contrast
media described in EP 0 458 079 and DE 38 03 972 that they can be
produced only, with the help of expensive processes, which make
necessary the use of organic solvents, whose use is harmful for
reasons of protection of the environment and work place safety.
In addition, before using the preparations, there has to be
assurance that the used organic solvents are no longer contained
in the product to be used pharmaceutically. Moreover, surface-
active adjuvants (e.g., surfactants) are necessary for the
j
CA 02145505 2003-08-12
3
production, which are frequently considered as harmful in the
case of injection preparations. Further, a control of the
concentration behavior in various organs is not controllable in
the case of these particles, a linkage of the particles of DE 38
03 972 with selectively accumulating compounds (so-called homing
devices, such as, e.g., monoclonal antibodies) is not possible.
The microparticles made of polymerized aldehydes described
in DE 40 04 430 are also not suitable as vehicles for substance-
specific or structure-specific substances because of the unclear
biodegradability. Another drawback is that also in this case
surface-active adjuvants are necessary for the production of the
particles.
The microparticles made of proteins, especially of albumin,
described in EP 0 224 934 exhibit an only very low in vitro and
in vivo.stability.
It was therefore an object of the invention to provide
microparticle (microcapsule) preparations especially for use
in ultrasonic diagnosis, which get by without the use of
physiologically harmful solvents or adjuvants (e. g.,
surfactants), are easily producible and biodegradable, which
either contain substances with site-specific, structure-
specific or tissue-specific binding properties in the wall
material or can be linked covalently with such and which
exhibit a sufficient in vitro and in vivo stability.
CA 02145505 2003-08-12
3a
The present invention provides microcapsules having a
core defined by a wall material comprising a copolymer of at
least one synthetic polymeric material and at least one
biopolymer, wherein a) the biopolymer exhibits site-specific,
structure-specific or tissue-specific properties, or b) the
biopolymer has functional groups, by which, optionally,
chelating ligands or their metal complexes and/or site
specific, structure-specific or tissue-specific substances
are bound, c) the synthetic polymeric material has a
hydrophobic alkylbackbone, and d) optionally, the wall
material contains one or more pharmaceutically active
ingredient(s), and wherein the core of the microcapsules
comprises a) a gas or gas mixture, b) one or more
pharmaceutically active ingredients, or c) the same material
as the capsule wall, provided that the synthetic polymeric
material is not made of polymerizable aldehydes, and wherein
the weight ratio of biopolymer to synthetic polymeric
material is in the range of 10:90 to 80:20 and wherein the
microcapsule size is 0.5 to 8 dun.
According to the invention, the above object is achieved by
mirxaparticles whose shell is formed from the comabination of
' ~ 2145~0~
biopolymers -- preferably polypeptides (also glycosylated) -- and
synthetic material polymerized during the production.
Therefore, microparticles made of a copolymer of at least
one synthetic polymer and at least one biopolymer are an object
of the invention, and polypeptides, preferably natural ones, or
produced synthetically or partially synthetically or obtained by
genetic engineering as biopolymers, such as, e.g., albumin,
collagen decomposition products, gelatin, fibrinogen,
fibronectin, polygeline, oxypolygelatin, their decomposition
products as well as poly-h-lysine are suitable. The biopolymers
can also be glycosylated. As polymerizable monomers, preferably
alkylcyanoacrylates, acrylic acid, acrylamide, acrylic acid
chloride and acrylic acid glycide ester are suitable.
The microparticles according to the invention are suitable
in the production in gas-saturated solution by the inclusion of~
the gas, especially as a contrast medium for ultrasonic studies.
They act as highly effective scatter elements in the ultrasonic
field because of the contained gas. In addition, they can be
excited by diagnostic ultrasound to radiate independent signals,
which can be evaluated, e.g., with the help of the color Doppler
technology.
As gases, air, nitrogen, carbon dioxide, oxygen, helium,
neon, argon, krypton or their mixtures are suitable. The charge
with the corresponding gas or gas mixture takes place by
_production of the particles in an aqueous solution saturated with
the respective gas or gas mixture.
' ' ~ 2145505
The microparticles can also (optionally in addition) contain
other substances, detectable with the help of medicinally-
diagnostic processes, such as magnetic resonance tomography,
magnetic resonance spectroscopy, scintigraphy or highly sensitive
magnetic field measurements with suitable magnetometers -
(biomagnetism), both microencapsulated and in the wall material
and (optionally with the help of suitable substances, such as,
e.g., chelating agents) coupled to the wall material. Thus, it
is possible, e.g., in using radioactive isotopes, to use the
microparticles according to the invention in scintigraphy.
Likewise, its use as contrast medium in magnetic resonance
tomography, magnetic resonance spectroscopy or in measurements of
the magnetic field is possible by the microencapsulation or
incorporation in the wall material of suitable para-, superpara-,
ferri- or ferromagnetic substances.
Surprisingly, it has been found that in the production of
the particles according to the invention (in maintaining
sufficient concentrations of biopolymers), the addition of '
surface-active substances, such as, e.g., surfactants, is not
necessary. This represents a decisive advantage in comparison
with the previously known production process for microparticles
based on synthetic polymers, since the surfactants usually
necessary for reducing the interfacial tension and for preventing
the particle aggregation are considered as physiologically
_harmful and therefore are to be removed again from the
preparations before the use in the organism up to compatible
residue contents.
6 21455U5
As a further advantage of the microparticle preparations
according to the invention, the varied particle properties that
can be matched to the respective use can be mentioned, which are
easily controllable by variation of various production
parameters. Thus, the pharmacokinetic parameters of the'
microparticle preparations (organ distribution, retention period
in the circulatory pathway) can be influenced by the selection of
the respectively used biopolymers or by changes of the~functional
groups of the biopolymer (e. g., by acylation with dicarboxylic
anhydrides, such as succinic acid, diglycolic acid, glutaric
acid, malefic acid or fumaric acid anhydride or by acetylation
with monocarboxylic anhydrides, such as acetic anhydride or
propionic acid anhydride).
Further, the content of the biopolymer in the wall material
can be varied in a wide scope, by which it is possible to
influence the period of the biodegradation of the capsule
material in vivo and to match it to the desired use. This
content can be controlled directly by the portion of the
biopolymer in the production solution. Thus, for example, the
wall material consists of microparticles according to the
invention, made of 55% (M/M) biopolymers, produced according to
example 1 from an autoclaved aqueous solution containing 1% (V/V)
butylcyanoacrylic acid and 5% gelatin, while with the same use of
butylcyanoacrylate with microparticles produced in 2.5% aqueous
.autoclaved gelatin solution, the wall material consists of 35%
(M/M) biopolymers, with microparticles produced in 7.5% aqueous
_. ' _214~~0~
autoclaved gelatin solution, the wall material consists of 65%
(M/M) biopolymers.
Surprisingly, the microparticles according to the invention
can be freeze-dried without adding other adjuvants such as
lactose, mannitol or sorbitol, as they.are usually used as
skeleton formers for freeze-drying. These skeleton formers are
responsible, after drying, for the mechanical destruction of a
considerable part of the microcapsules, which then is no longer
usable for the imaging. In contrast to this, in the case of the
microparticles according to the'invention, the biopolymer of the
wall material used in excess is used as a skeleton former, by
which surprisingly the ratio of intact to destroyed microcapsules
is drastically improved. Because of this more favorable ratio,
the dose necessary for imaging can clearly be reduced.
But the microparticles according to the invention can
also -- optionally in addition -- contain incorporated
pharmaceutical active ingredients, by, e.g., the opacifying agent
(in the case of contrast media for ultrasonic studies, a gas or
gas mixture is involved here) and one or more active ingredients
in the particles being microencapsulated. Preferably, the active
ingredients can also be incorporated in the wall material with
the methods described for the site-specific, structure-specific
or tissue-specific substances. If the active ingredients are
biopolymers, they can also partially form the wall material
themselves, by being used in the production either exclusively or
in a mixture with other suitable biopolymers (e. g., gelatin,
albumin, fibronectin, poly-L-lysine) as initial material for
214550
microparticle preparation with the addition of a polymerizable
monomer or oligomer. The special advantage of coupling active
ingredients to the biopolymer portion of the capsule material
lies in the fact that active ingredients, which, e.g., are bound
by peptide bonds to the biopolymer portion of the capsule
material, can be released by'enzymatic decomposition in vivo.
The microparticles according to the invention are used
especially to detect or to treat thromboses and atheroscierotic
changes. In this case, the use of antibodies or antibody
fragments against fibrin, fibrin-bonding plasma proteins or their
partial structures, tissue plasminogen activators or partial
structures of them (e. g., type I-homology and doughnut
sequences), protein components of lipoproteins (also partial
structures) as homing devices can be considered as especially
advantageous.
Other fields of use for the microparticles according to the
invention can be, e.g., also the diagnosis or the treatanent of
hormonal functions (in this case, the use of peptide hormones or
their modified products with the capability for receptor bonding
as homing devices is to be considered as especially
advantageous), or the diagnosis or treatment of lesions of
vascular endothelia (in this case, either the use of antibodies
or antibody fragments against substances of the integrin group,
especially the selectins such as, e.g., LAM-1, ELAM-1 and GMP-
_140, or the use of receptors or their bond-imparting fragments
for substances of the integrin group, especially the selectins
such as, e.g., LAM-1, ELAM-1 and GMP-140, as homing devices is to
CA 02145505 2003-08-12
9
be considered as especially advantageous). Moreover, the
microparticles according to the invention can also be used for
diagnosis or treatment of tumors, by antibodies or antibody
mixtures being used as horning devices against surface antigens of
tumors.
The production of mic~oparticles according to the invention
takes place by the polymerization of a suitable reactive monomer
or oligomer (e. g., cyanoacrylic acid butyl ester, cyanoacrylic
acid isobutyl ester, cyanoacrylic acid isopropyl ester,
cyanoacrylic acid propyl ester, cyanoacrylic acid isohexyl ester,
cyanoacrylic acid hexyl ester, cyanoacrylic acid methyl ester,
acrylic acid, acrylamide, acrylic acid glycide ester, acrylic
acid chloride) in a concentration relative to the total volume of
the production solution of 0.01-10% (m/V) (preferably 0.1-10%)
under suitable conditions (e.g., by selection of the pH, by
adding radicals, and by UV irradiation) with dispersion in
aqueous phase, which contains a biopolymer, e.g., albumin,
gelatin, oxypolygelatin, polygeline, fibronectin, poly-L-lysine -
dissolved in a concentration of 0.5-20% (m/V) (preferably 1%-15%
(m/V)). By using collagen decomposition products, such as, e.g.,
gelatin, polygeline or oxypolygelatin, it is often advantageous
to autoclave the solutions before the microparticle production.
After completion of the polymerization, the resulting
microparticles are separated depending on density and particle
size by one-time or repeated centrifuging, filtration,
sedimentation, or flotation, optionally further purified
by dialysis and suspended in a physiologically
compatible suspending agent (preferably
' '' ~° 2145~0~
water for injection purposes) until the desired concentration.
The suspensions can be isotonized by the addition of suitable
water-soluble substances, such as, e.g., glucose, mannitol,
sorbitol, common salt, galactose, lactose, fructose, trehalose.
The size distribution of the microparticles developing in
the production cair be controlled within wide ranges by the type
of stirring device used and the number of revolutions.
The production of gas-filled microparticles takes place by
the reaction being performed in a solution saturated with the
desired gas. In this connection, the density of the resulting
microparticles, i.e. the ratio between wall material and gas
portion, can be controlled both by the stirring conditions and
especially by the portion of biopoiymers during the production
process.
If microparticles are to be obtained, in which the core
consists of the same material as the shell, attention must be
paid in the production that by the selection of a suitable
stirring device and a suitable stirring speed, a foaming of the
reaction solution is avoided.
The required ability for combination with site-specific,
structure-specific or tissue-specific substances, which are to
assure an additional concentration of the microparticles in
target fields outside the organs of the RES (homing devices),
takes place either by the coupling of the substances to the
_polypeptides co-forming the shell material, performed before the
microparticle preparation or afterwards, with known methods of
biochemistry for coupling amino acids (e. g., W. Konig, R. Geiger:
y 11
Eine neue Methode zur Synthese von Peptiden: Aktivierung der
Carboxylgruppe mit Dicyclohexylcarbodiimid unter Zusatz von 1-
Hydroxy-benzotriazolen [A New Method to Synthesize Peptides:
Activation of the Carboxyl Group with Dicyclohexylcarbodiimide
while Adding 1-Hydroxy-benzotriazoles], Chem. Ber. 103, 788-798
(1970)), or in that the microparticles are produced in an aqueous
solution of the site-specific, structure-specific or tissue-
specific substance, if the latter represents a polypeptide, so
that the substance is used directly as a component of the shell
material.
As site-specific, structure-specific or tissue-specific
substances that can be coupled to the microparticles or co-
forming the shell material, preferably antibodies, conjugated
antibodies, hormones (especially peptide hormones), transferrin,
fibronectin, heparin, transcobalamin, epidermal growth factors,
lipoproteins, plasma proteins as well as their specificity-
imparting partial structures and oligopeptides such as RGD, RGDS,
RGDV and RGDT are suitable.
As cheiating ligands that can be coupled to the
microparticles, diethylenetriaminepentaacetic acid or its
derivatives are suitable. The linkage of these ligands with the
particles takes place in a way known in the art [Hanatowich et
al., Science 220 (1983) 613]. Then, the particles are reacted
with the desired metal ions to the respective particle-fixed
metal complex.
The selection of the used metal ion depends on the desired
area of use. In the field of NMR diagnosis, paramagnetic metal
CA 02145505 2003-08-12
12
ions, preferred according to the invention, of the elements of
atomic numbers 21-29 and 57-70, especially gadolinium(III) ions,
are used. For the use in scintigraphy, suitable emitters of
radioactive radiation, preferably ~i~In or ~"'Tc, ~Z3I and '3'I are
used.
The finished micropar~icle suspensions can be used directly
for the respectively predetermined use, but it has proven
advantageous to improve the storage stability, to freeze and then
to freeze-dry the suspensions while adding skeleton formers (such
as, e.g., trehalose, polyvinylpyrrolidone, lactose, mannitol,
sorbitol, glycine), which also can be used to set the tonicity.
It has proven especially advantageous to use the biopolymer used
in excess itself as skeleton former. In both cases, it is
suitable to move the suspensions during the freezing to prevent
uneven particle distributions in the frozen material by
sedimentation or flotation. The production of ready-to-use,
injectable suspensions from the freeze-dried preparations takes
place by resuspending the lyophilizate in a pharmaceutically
acceptable suspension medium such as, e.g., water p.i., aqueous
solutions of one or more inorganic salts such as physiological
electrolyte solutions, aqueous solutions of monosaccharides or
disaccharides such as glucose or lactose, sugar alcohols such as
mannitol, but preferably in water suitable for injection
purposes. Additionally, a multivalent alcohol can be included.
The total concentration of the optionally dissolved substances is
0-205 by weight.
The concentration of the ready-to-use contrast medium can be
set in the range of 0.01 to 20 mg, preferably 0.5 to 6 mg of
214550
13
particles/ml of suspension medium. The injected dose depends on
the respective use; in ultrasonic diagnostic studies in the study
of vessels, it lies in the range of 1 to 500 ug, preferably
between 10 and 100 ~g of particles/kg of body weight, in the
study of liver and spleen by color Doppler sonography in-the
range of 50 to 1000, preferably between 200 and 600 ~g/kg of body
weight. The invention is explained by the following examples:
14 _2145~0~
. _
Example 1
In 300 ml of distilled water, 15 g of gelatin (300 bloom) is
dissolved and adjusted to pH 3.0 with hydrochloric acid. The
solution is autoclaved for 30 minutes at 121°C. After cooling to
room temperature, the pH of the solution is corrected to pH 5.0
(with sodium hydroxide: solution) and.stirred in a 2000 ml beaker
with a quick-running stirrer at 10000 rpm. 3 ml of cyanoacrylic
acid butyl ester is slowly (10 minutes) instilled in the solution
with stirring. Stirring of the resulting microparticles is
continued for 60 minutes. Then, the suspension is floated in a
separatory funnel for 2 days. The subnatant is drained and the
supernatant is supplemented with distilled water to 100 ml. The
suspension contains gas-filled, sound-active microparticles in a
size of about 0.1-8 Vim, and by additional flotation or
filtration, if necessary, the particle sizes can be further
concentrated (e.g., to 0.5-3 um). The capsule wall of the
microparticle item consists to about 55% (M/M) of polypeptides
and to about 45% (M/M) of polycyanoacrylic acid butyl ester. The -
particles can be dispersed in water without the addition of
surface-active adjuvants. They do not tend toward aggregation.
By adding a suitable adjuvant (e. g., glucose, sodium chloride,
mannitol, lactose, galactose), the suspension can be isotonized.
The suspension can be freeze-dried, if necessary, to
increase the storage stability without losing its suitability as
_a contrast medium for ultrasonic studies, preferably after adding
a cryoprotector, such as, e.g., lactose, polyvinylpyrrolidone,
mannitol, glycine.
_ 15 _2145505
Example 2
500 mg of poly-L-lysine (MG 5000) is dissolved in 20 ml of
distilled water and adjusted to pH 4.5 with phosphate buffer.
100 mg of acrylic acid glycide ester is added, the mixture is
stirred with a quick-running stirrer under cooling at 20°C. 10
' . mg of ammonium peroxydisulfate and 0.1 ml of N,N,N',N'-
tetramethylenediamine are added. It is stirred for another 90
minutes. The resulting gas-filled microparticles are separated
by flotation. The particle size of the microparticles is between
0 . 2 and 6 ~,m .
Example 3
7.5 g of polygeline is dissolved in 200 ml of water for
injection purposes. The solution is adjusted to pH 3..0 with
phosphoric acid and supplemented with water for injection
purposes to 300 ml. The solution is filtered through a 0.22 ~m
filter for sterilization by filtration and stirred with a quick-
rotating dissolver at 6000 rpm. With stirring, a mixture of 1.5
ml of cyanoacrylic acid isopropyl ester and 1.5 ml of
cyanoacrylic acid butyl ester is slowly instilled. Stirring is
continued for,120 minutes. The resulting suspension is floated
for three days in a separatory funnel. The further course of
action corresponds to example 1. The resulting microparticles
contain gas. They are suitable as contrast media for ultrasonic
studies. Their wall material consists to about 22% (M/M) of
biopolymer, to about 40% (M/M) of polycyanoacrylic acid butyl
ester and to about 38% (M/M) of poiycyanoacrylic acid isopropyl
- 16 _2145505
ester. They can be dispersed in water without adding surface-
active adjuvants without in this connection aggregating. Their
particle size is about 0.2-6 ~.m.
Example 4
g of human serum albumin is dissolved in 200 ml of water
for injection purposes. The solution is adjusted to pH 4.0 with
hydrochloric acid and supplemented with water for injection
purposes to 300 ml. The solution is filtered through a 0.22 ~m
filter for sterilization by filtration and stirred with a quick-
rotating dissolves at 10000 rpm. With stirring, 2 ml of
cyanoacrylic acid isopropyl ester is slowly instilled. Stirring
is continued for 60 minutes. The resulting suspension is floated
for three days in a separatory funnel. The further course of
action corresponds to example 1. The resulting microparticles
contain gas. They are suitable as contrast media for ultrasonic
studies. Their wall material consists to about 30% (M/M) of
human serum albumin and to about 70% (M/M) of polycyanoacrylic
acid isopropyl ester. They can be dispersed in water without
adding surface-active adjuvants without in this connection
aggregating. ,Their particle size is on the average about 0.2-3
Vim.
',. 17 _ 214~5~~
Example 5
250 ml of oxypolygelatin solution is adjusted to pH 2.5 with
hydrochloric acid and supplemented with water for injection
purposes to 300 ml. The solution is filtered through a 0.22 ~Cm
filter for sterilization by filtration and stirred with a quick-
rotating rotor-stator-stirxer.at 80b0 rpm. With stirring, 3 ml
of cyanoacrylic acid butyl ester is slowly instilled. Stirring
is continued for 90 minutes. The resulting suspension is floated
for three days in a separatory funnel. The further course of
action corresponds to example 1. The resulting microparticles
contain gas. They are suitable as contrast media for ultrasonic
studies. Their wall material consists to about 25% (M/M) of
oxypolygelatin and to about 75% (M/M) of polycyanoacryiic acid
butyl ester. They can be dispersed in water without adding
surface-active adjuvants without in this connection aggregating.
Their particle size is about 0.2-4 ~Cm.
Example 6
500 mg of fibronectin is dissolved in 5 ml of distilled
water and adjusted to pH 3.5 with hydrochloric acid. The
solution is filtered through a 0.22 ~Cm filter for sterilization
by filtration and stirred with a quick-rotating rotor-stator-
stirrer in a cooled 15 m1 vessel (15°G.) at 8000 rpm. With
stirring, 0.3 ml of cyanoacrylic acid butyl ester is slowly
_instilled. Stirring is continued for 90 minutes. The resulting
suspension is floated for three days in a separatory funnel. The
18
' ' 21455th
supernatant is suspended in 2 mi of water for injection purposes,
which contains 100 mg of mannitol. The suspension is frozen at
-50°C with shaking and freeze-dried. Before use, the
microparticles are redispersed with 2 ml of water for injection
purposes. The particle size of the microparticles is on the
average 0.8 Wm. They are suitable as contrast media for
ultrasonic studies. The wall material of the microparticles
consists to about 35% (M/M) of fibronectin and to about 65% (M/M)
of polycyanoacrylic acid butyl ester.
Examble 7
100 mg of an antibody against fibrin is dissolved in 4 ml of
phosphate buffer (pH 4.5). The solution is.filtered through a
0.22 ~Cm filter for sterilization by filtration and stirred in a
double-walled stirrer vessel (10 ml capacity) with a quick-
rotating dissolver-stirrer under cooling at 6000 rpm. During the
stirring, 0.2 ml of cyanoacrylic acid butyl ester is slowly
instilled. Stirring is continued for 60 minutes. The resulting
suspension is floated for two days in a separatory funnel. The
subnatant is drained, the supernatant is mixed with 200 mg of
lactose and 2,m1 of water for injection purposes. The suspension
is frozen at -40°C with shaking in a cold bath and then freeze-
dried. Before use, the microparticles are resuspended with 2 ml
of water for injection purposes. They are gas-filled and
_suitable as contrast media for ultrasonic studies. Their
particle size is about 1 ~m on the average. The wall material of
19
' ' 21455U5
the microparticles consists to about 20% (M/M) of the antibody
and to about 80% (M/M) of polycyanoacrylic acid butyl ester.
Example 8
15 g of polygeline is dissolved in 50 ml of water for
injection purposes, 2 N sodium hydroxide solution is added drop
by drop under pH control. A total of 2 g of diglycolic acid
anhydride is added gradually, and the pH is kept between 7.5 and
8Ø After completion of the reaction, the excess diglycolic
acid is removed from the solution by repeated ultrafiltration
(exclusion limit MG 1000). The acylated polygeline solution is
supplemented with water for injection purposes to 300 ml and
filtered through a 0.22 ~m filter. 3 ml of cyanoacrylic acid
butyl ester is slowly added with stirring at 10000 rpm. After
completion of the addition, stirring is continued for 60 minutes.
The resulting gas-filled microparticles are separated by
centrifuging at 1500 rpm (30 minutes) and taken up in 50 ml of
water for injection purposes. They can be dispersed in water
without adding surface-active adjuvants without aggregating.
Their particle size is approximately 0.1-6 Vim. The wall material
of the microparticles consists to about 45% (M/M) of acylated
polygeline and to about 55% (M/M) of polycyanoacrylic acid butyl
ester.
Example 9
20 ml of the gas-containing microparticles produced
according to example 3 is taken up in 20 ml of phosphate buffer
2~ 214~5~0
of pH 4.5. The suspension is stirred at 4°C (100 rpm) and 25 mg
of (3-dimethylaminopropyl)-N'-ethylcarbodiimide-HC1 is added to
the mixture. After 60 minutes, 25 mg of fibronectin, which w.as
previously dissolved in 10 ml of phosphate buffer, is added to
the microparticle suspension. It is stirred for 60 minutes at
4°C and for another 120 minutes at room temperature. ~ Then., the
suspension is dialyzed three times against phosphate buffer of pH
4.5 (exclusion limit MG 1000) and floated for two days in a
separatory funnel. The supernatant is taken up in 20 ml of water
for injection purposes, mixed with 5% polyvinylpyrrolidone (m/V),
frozen at -40°C with shaking and freeze-dried.
Example 10
The lyophilizate of example 9 is resuspended with 20 ml of
5% glucose solution. 0.1 ml of it is added to 10 ml of PBS
solution of 37°C, which contains a freshly produced fibrin clot
(diameter 1 mm). After 10 minutes of incubation with shaking in
a water bath, the clot is removed, washed five times with 10 ml
of PBS (pH 7.4) each and then sonographically studied. In the
color Doppler, signals of clinging microparticles can be clearly
detected. The procedure is analogous with the particles of
example 3 (without the reaction with fibronectin shown in example
9). In the sonographic study of the clots, no clinging
microparticles can be detected (also in the color Doppler).
21
_ 214~~Q
Example 11
ml of the gas-containing microparticles produced according
to example 3 is taken up in 5 ml of phosphate buffer of pH 4.5.
The suspension is stirred at 4°C (100 rpm), and 10 mg of (3-
dimethylaminopropyl)-N~-ethylcarbodiimide-HC1 is added to the
mixture. After five minutes, 2.5 mg of an antibody against
fibrin (No. 0541 clone E8, Immunotech, Marseilles, France), which
was previously dissolved in 1 ml of phosphate buffer, is added to
the microparticle suspension. It is stirred for 60 minutes at
4°C and for another 120 minutes at room temperature. Then, the
suspension is dialyzed three times against phosphate buffer of pH
4.5 (exclusion limit MG 1000) and floated for two days in a
separatory funnel. The supernatant is taken up in 2 ml of water
for injection purposes, mixed with 5% polyvinylpyrrolidone (m/V),
frozen at -40°C with shaking and freeze-dried.
Example 12
The lyophilizate of example 1l is resuspended with 2 ml of -
5% glucose solution. 0.1 ml of it is added to 10 ml of PBS
solution of 37°C, which contains a freshly produced fibrin clot
(diameter 1 mm). After 10 minutes of incubation with shaking in
a water bath, the clot is removed, washed five times with 10 ml
of PBS (pH 7.4) each and then sonographically studied. In the
color Doppler, signals of clinging microparticles can be clearly
detected. With the particles of example 3 (without the reaction
with the antibody against fibrin shown in example 11), the
procedure is analogous. In the sonographic study of the clots,
22
- 214550
no clinging microparticles can be detected (also in the color
Doppler).
Example 13
0.1 ml of the resuspended particles of example 6 is-examined
for their fibrin bond in an experimental set-up analogous to
example 11. In the sonographic study, microparticles bound to
the clot can be clearly detected.
Example 14
0.1 ml of the resuspended particles of example 7 is examined
for their fibrin bond in an experimental set-up analogous to
example 11. In the sonographic study, microparticles bound to
the clot can be clearly detected.
Example 15
ml of the microparticles produced according to example 8
is taken up in 10 ml of phosphate buffer of pH 4.5, and 20 mg of
1-hydroxybenzotriazole is added. After cooling to 4°C, it is
stirred (100 rpm), and 10 mg of (3-dimethylaminopropyl)-N'-
ethylcarbodiimide-HC1 is added. Stirring is continued for 60
minutes at 4°C. Then, it is stirred for another 60 minutes at
room temperature. 10 mg of pancreozymin, which was previously
dissolved in 5 ml of phosphate buffer, is added to the suspension
_at room temperature. It is stirred for 120 minutes at room
temperature. Then, the suspension is dialyzed five times against
phosphate buffer of pH 4.5 (exclusion limit MG 1000) and floated
23
- 21455~~
for two days in a separatory funnel. The supernatant is taken up
in 10 ml of water for injection purposes, mixed with 5%
polyvinylpyrrolidone (m/V), frozen at -40°C with shaking and then
freeze-dried.
'Example i6
The lyophilizate of example 15 is resuspended with 10 ml of
water for injection purposes. 0.1 ml of the suspension is
injected in the caudal vein of a rat. After 10 minutes, the
pancreas is removed and sonographically studied in a water bath.
In the color Doppler, ultrasonic signals of the microparticles
can be detected.
Example 17
ml of the gas-containing microparticles produced according
to example 3 is taken up in 5 ml of phosphate buffer of pH 4.5.
The suspension is stirred at 4°C (100 rpm), and 10 mg of (3-
dimethylaminopropyl)-N~-ethylcarbodiimide-HC1 is added to the
mixture. After five minutes, 5 mg of tPA, which was previously
dissolved in 1 ml of phosphate buffer, is added to the
microparticle suspension. Stirring is continued for 24 hours at
4°C. Then, the suspension is dialyzed three times against
phosphate buffer of pH 4.5 (exclusion limit MG 1000) and floated
for two days in a separatory funnel. The supernatant is taken up
,in 2 ml of water for injection purposes.
i
_ 24 2145505
Example 18
Two fibrin clots (weight about 50 mg each) are produced,
which are added to 20 ml of plasma. 0.05 ml of the particle
suspension produced according to example 17 is added to the
clots. After 10 minutes, the clots are removed from the plasma,
in the.sonographic study, signals of clinging microparticles~
appear in the color Doppler.
Example 19
0.6 g of gelatin is dissolved in 20 ml of an aqueous
suspension of magnetite particles (about 20 mmol of iron/ml,
diameter of the particles about 20 nm). The solution is adjusted
to pH 3 with hydrochloric acid. With stirring (3000 rpm), 0.2 ml
of cyanoacrylic acid isobutyl ester is slowly added. After
completion of the addition, stirring is continued for 90 minutes.
The suspension is centrifuged (2000 rpm, 60 minutes). The
supernatant is discarded, the subnatant is taken up in 10 ml of
PBS of pH 7.4 (10 mmol). The suspension is cooled to 4°C and -
with stirring (100 rpm), l0 mg of (3-dimethylaminopropyl)-N'-
ethylcarbodiimide-HC1 is added. Stirring is continued for 60
minutes at 4°C. Then, 5 mg of an antibody against fibrin (no.
0541 clone E8, Immunotech, Marseilles, France) is added.
Stirring is continued for 60 minutes at 4°C and then for 120
minutes at room temperature. The suspension is ultrafiltered
_(exclusion limit MG 5000) five times against PBS of pH 7.4 (10
mmol). Then, the suspension is centrifuged (2000 rpm, 60
minutes). The subnatant is taken up in 5 ml of water for
y 25 2145~fl~
injection purposes, which contains 5% mannitol (m/V) and is
filtered through a 5 ~Cm membrane filter. The filtrate is frozen
at -40°C and then freeze-dried.
Example 20
15 g of .gelatin (300 bloom) is dissolved at .80°C in .150 ml
of water for injection purposes. After the cooling, the solution
is adjusted to pH 2.5 with 0.1 N HC1 and supplemented with water
for injection purposes to 300 ml. The solution is autoclaved
(process A 121, Deutsches Arzneibuch [German Pharmacopoeia] 9th
Edition). The pH of the autoclaved solution is controlled and if
necessary corrected to pH 2.5. 3 ml of cyanoacrylic acid
isobutyl ester is added to the solution with stirring. Stirring
is continued for 90 minutes. The resulting microparticle
suspension is centrifuged at 1000 rpm for 60 minutes, the -
supernatant is taken up in 50 ml of water for injection purposes
and again centrifuged at 1000 rpm for 60 minutes. This is
repeated a total of 5 times. The supernatant of the final
centrifuging is taken up in 50 ml of PBS (pH 7.0) and added with
stirring to 0.1 mg of solid diethylenetriaminepentaacetic acid
dianhydride (cf.: Hnatowich et al. (1983) Science 220: 613). It
is stirred for 5 minutes. The suspension is centrifuged at 1000
rpm for 60 minutes, the supernatant is taken up in 50 ml of water
for injection purposes. The centrifuging against water for
_injection purposes is repeated another 4 times. The supernatant
of the final centrifuging is taken up with 50 ml of water for
injection purposes and filtered through a filter column~from HDC-
26
pore filters of pore sizes 70, 40, 20 and 10 Vim. The filtrate
contains about 2 x 109 particles/ml, which have DTPA groups on
their surface. The average particle size is about 2 ~Cm. The
particles can be labeled with the known methods with radioactive
metal ions (e.g., In-111 ar Tc-99).
Example 21 .
Fibrin clots as examples for sound application lead to
detection only with a gamma counter instead of with Doppler.
Example 22
7.5 g of polygeline is dissolved at 80°C in 150 ml of water
for injection purposes. After cooling, the solution is adjusted
to pH 3 with 0.1 N HC1 and supplemented with water for injection
purposes to 300 ml. The solution is autoclaved (process A 121,-
German Pharmacopoeia 9th Edition). The pH of the autoclaved
solution is corrected to pH 2. 3 ml of cyanoacrylic acid butyl
ester is added to the solution with stirring. Stirring is
continued for 90 minutes. The resulting microparticle suspension
is centrifuged at 1000 rpm for 60 minutes, the supernatant is
taken up in 50 ml of water for injection purposes and again
centrifuged at 1000 rpm for 60 minutes. This is repeated a total
of 5 times. The supernatant of the final centrifuging is taken
up in 50 ml of PBS (pH 7.4) and cooled to 4°C. With stirring at
_4°C, 50 mg of streptavidin and 5 mg of EDC are added. Stirring
is continued for 1 hour. The suspension is centrifuged 3 times
(1000 rpm, 60 minutes). After each. centrifuging, the supernatant
'. ' 27 ~ 2145505
is taken up with 50 ml of PBS (pH 7.0, 10 mmol of phosphate).
The antibody against fibrin is labeled in a molar ratio of 1:5
with sulfosuccinimidyl-6-(biotinamido)-hexanoate (NHS-LC-biotin)
according to the method of D. J. Hnatovitch et al., J. Nucl. Med.
28 (1987), 1294-1302.
Example 23
0.5 mg of the biotin-labeled antibody against fibrin of
example 22 is intravenously injected in a rabbit fed with a diet
containing cholesterol. After 3 hours, the particles of example
22 are then injected. 10 minutes later, the carotid is removed
from the previously killed animal, and the atherosclerotic
arterial sections are examined in a water bath in the Doppler
mode for contrast media signals. Sound signals of clinging
particles can clearly be detected. -
Example 24
a) 20 ml of the microparticle suspension produced according
to example 8 is brought to a pH of 4.5 by 5 ml of phosphate
buffer and then mixed with 50 mg of a 125-iodine labeled antibody
against fibrin (5 ~Ci). With stirring at 4°C, 500 mg of (3-
dimethylaminopropyl)-N'-ethyicarbodii~ide hydrochloride is added
to the reaction mixture. Then, stirring is continued for 8 hours
under cooling, the microparticles are separated by centrifuging
and washed a total of three times with 20 ml each of water for
injection purposes, and each individual washing process takes
place in the way_that the particles are resuspended in water and
' 28 » 2145505
then centrifuged. After the final washing process, the particles
are resuspended in 20 ml of water for injection purposes.
The degree of bonding of the antibody to the particle is
determined with a gamma counter based on the 125-iodine activity.
Then, 93% of the originally used amount of antibodies is bound
permanently to the particle surface.
b) ~Microparticles produced according to example 4 of DE 38
03 972 are reacted in an amount corresponding to example 24a)
with 50 mg of a 125-iodine labeled antibody against fibrin (5
~CCi) under otherwise identical reaction conditions.
A comparison of the degree of bonding with the particles
according to example 24a) shows a considerably lower value of
only 1%.
