Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2~73~
HOECHST AKTIEMGESELLSCHAFT HOE 91/~ 277 JK Dr.TH/PL
Description
Echogenic particles, a process for theix production and
their use
The invention relates to echogenic particles which
comprise a gas and a shaping substance, wherein the
surface of the particles is essentially smooth and the
shape comprises no or 1 to 10 conca~e ~urface segments,
or the surface is not smooth and the shape comprises 1 to
10 concave surface segmen~s, and which can pass through
the lung in the blood circula~ion, a spray process for
their preparation and their use, in particular a3 diag-
nostic agents and therapeutic agents.
: Because it is easy to handle and has no complications,
ultrasonic diagnosis has found wide use in medicine.
Ultrasonic waves are reflected at the boundarie~ of
different types of tissue. The resulting echo 3ignal8 are
amplified and visualized electronically.
The visualization of blood vessels and internal organs by
ultrasound generally does not allow visualization of the
~: blood flow present therein. Fluids, especially blood,
produce an ultrasonic contrast only if there are density
: differences with respect ~o the environment. Contras~
media which are used in medical ultrasonic diagnosis are,
for example, substances which comprise gases or produce
gases, since the difference in impedance between the gas
and ~urrounding blood is considerably ~reater than that
between fluids or solids and blood (Le~ine R.A.f J. Am.
Coll. Cardiol 3: ~8, 1989; and Machi, i.J. CU 11: 3,
30 1983~.
Several methods for the preparation and stabilization
of gas bubbles are known in the literature.
2 o ~ rl ~ ~ 3
-- 2 ~
U.S. Patent 4,276,885 describes the prepara~ion of gas
bubbles of defined size, which are surrounded by a
gelatin shell which protects the ga~ bubbles from
merging. The finished gas bubbles can ~e stored only in
the frozen state, and ~he gas bubbles have ko be brought
to body temperature again before use.
EP-A2-0,123,235 and 0,122,624 describe gas-containing
ultrasonic contrast media which comprise mixtures of
surface-active substances with a solid in a liquid
carrier. The ultrasonic contras$ media are prepared by an
expensive grinding process with an air jet mill. The
particles thus produced have only a short u6eful life,
because they guickly lose the gases included.
EP-A2-0,224,934 describes ultrasonic contrast media in
the form of gas-filled hollow bodies of gelatin or
albumin. However, the use of proteins forei~n to the body
or denatured endogenous proteins i~ a disadvantage,
because of the associated allergenic risk.
The present invention is based on the object of
discovering echogenic particles which refle t, absorb,
diffract or scatter ultrasonic waves and in this way
produce a significant contrast to the surro~nding tissue,
which are so small and stable that, after intravenous
administration, they reach the left ventricle without
substantial 105s of gas and essentially guantitatively,
and which can be produced quickly and easily.
It has been found that the quality of echogenic particles
depends in particular on the shape and surface of ~hese
particles. It i~ found that echogenic particles having
concave surface segments can pass through the lung in the
blood circulation considerably better and effect a higher
contrast in ultrasound. These echogenic parti~les com-
prise an air/water boundary, the scattering cross-section
of which in ultrasonic examinations i8 very high.
~` .
2~7~3~3
Echogenic particl~s having an essentially smooth surface
can pass through the lungs of vertebrate~ in the blood
circulation without the particles being filtered ou~ to
a great extent.
The invention thus relates to echogenic particles which
comprise a gas and a shaping subs~ance or a substance
mixture, which is biologically degradable and/or excret-
able, and which can pa~s through the lung of vertebrates
in the blood circulation, wherein the ~urface of the
echogenic particle i~ essentially smooth and the shape of
the Pchogenic particle comprises no or 1 to 10 concave
surface segments, or the surface of the echogenic par-
ticle is no~ smooth and the ~hape of ~he echogenic
particle comprises 1 to 10 concave surface segments.
~chogenic par~icles are particle~ ha~ing a diameter which
: ranges from 0.1 ~m to 20 ~m, preferably 0.2 ~m to 7 ~m,
in particular from 0,5 ~m to 3 ~,m, in 90% of the par-
~ ticles. These paxticles furthermore ar~ capable of
; reflecting, absorbing, diffracting or ~cattering sound
waves.
Concave surface segment of an echogenic particle means a
particle which has at least one surface curved inwards.
The particles according to the invention include, for
example, particles in which the maximum curve inwards,
that is to say the depth of the conca~e surface segment,
is more than 10% of the smallest cross-sec~ion of the
particle, the curve inwards particularly preferably being
30 to 90%, in particular 50 to gO~0 This thus essentially
means particles which look cup~, hat- or pot-shaped.
The number of concave ~urface ~e~ments ranges from 1 to
10, preferably 1 to 5, in particular 1 ~o 3.
The paxticle~ can also have a smooth surface. An essen-
tially smooth surface of an echogenic particle means
- 4 - ~ ~7~3
surface~ which reveal no structural differences at all on
the surface under lO,OOO fold magnification, i.e. distur-
bances on the surface of these particles, such as eleva-
tions or depressions, are essentially at a distance of
less than 100 nm from the average fiurface of the
particle, in par~icular 90 to 50 nm, preferably 60 to
80 ~m. The average par~icle surface i8 obtained from the
average of the elevations and depressions of the surface.
Disturbances in the ~urface include changes in the
homogeneous surface which, in an electron microscopy
photograph, look like elevations, fractures, distortions,
a ladder-, step-, scale or layer-like build up of the
surface and holes, depressions or pores in the surfaGe.
The property of the particles according to the invention
of being able to pass through the lung of vertebrates in
the blood circulation is determined, for example, by
injecting the echogenic particles into a peripheral vein
of a test animal under s~erile conditions. An ultrasonic
head of an ultrasonic unit is held on the thorax of th~
test animal, so that a typical cross-section through the
right and left ventricle i6 obtained. As soon as the
ul~rasonic contrast medium reaches the right ventricle,
it can be seen on the monitor of the ultrasonic unit how
the blood labeled by the contrast medium reaches the
right auricle and then leaves the right ventricle and
then ~he heart again via the pulmonary axtery. After
passage through the lung, a significant increase in
contrast due to the contrast medium is detectable in the
left ventricle.
The echogenic particles compris~ gas, for example air,
nitrogen, a noble gas such as helium, neon, argon or
kryptonJ hydrogen, carbon dioxide, oxygen, or mi~ures
thereof. The echogenic particle~ are charged with a ga~
by, for example, storing the particles in an appropriate
gas atmosphere or obtaining the particles directly by
spray drying during production in an appropriate
2~77~83
-- 5 --
gas atmosphere.
Shaping ~ubs~ances or sub~tance mixtures are understood
as meaning all compounds with which particles ~an be
pr~duced O
These include, for example~ naturally occurring polymers
or polymers which can be prepared by synthesis, lipids
which form micelles in agueous solutions, or low mole~
cular weight amino acids, fats, or carbohydrates.
Biologically degradable and/or excretable shaping sub-
stance or substance mixtur~ is und~rstood as meaning
substances which are converted into non~toxic degradation
products in th~ body of anLmals, humans or plants, or are
excreted. The degradation can take place enzymaticallyJ
hydrolytically or by partial or complete solution of the
substances or disintegration of the particles into
smaller sub-units in water. The degradation product~ are
excreted via the kidneys, skin, lungs or in~estine. If
the su~stances dissolve completely or partly in water or
arP not degraded, for ex~mple in the ca~e of low mole-
cular weight carbohydrates, they are excreted via thekidneys without degradation.
Shaping substances or substance mixture~ which are
biologically degradable a~d/or excretable are chosen from
the following groups:
25 polyesters of an ~ or ~-hydroxycarbo~ylic
acid, for example poly-~-caprolactone,
polyhydroxybutyric acid, polylactic acid
or polyhydroxyvaleric acid,
copolymers of the abovementioned polyester% with one
another or with polyglycolic acid,
polycondeDsate~ which comprise 2,3-O-alkylidenetartaric
acid, 2,3-O-alkylidene-L~threitol, furo-
[2,5] group~ or terephthalates,
2~77~3
-- 6 --
polyaminodicarboxylic acid co-imides, such a8 polysuccin-
Lmide-co~ -(butoxycarbonylpropion
ethyl)-D,~-aspartamide,
polyamino acid6, such a~ polyglutamic acid, poly~spartic
; S acid or polylysine, which give a basic or
acid reac~ion in aqueous solution~ and
derivatives thereof,
polyamide~ t for axample polylysine mathyl ester-
fumaric acid-co glutar~mide or poly~
hydroxyethylaspartamide,
`: polyortho esters,
polyanhydride~ for example those which are compo~ed of
; sebacic acid and bis(p-carboxyphenoxy)-
propane,
15 gelatin, for example hydrophobically modified
gelatin,
albumins, for example bovine ~erum albumin,
poly~accharide~, for example dextrans, starch or basic or
:~ acid deriva~ives thereof,
polymethylene malonate,
: ~ugars, for example glucose, galactose, lacto~e or
Bucrose ~
fatty acids having 8 to 20 carbon ~toms,
amino acid~, ~uch as proteinogenic amino acids ~nd
hydrophobic derivatives thereof,
alcohols having 12 to 20 carbon atoms and
lipids, such as phospholipids.
The echoyenic particles according to the invention
particularly preferably compri~e biologically degradable
~ 30 andJor excretable polymers from the following group:
: polyamides, polycondensates, polyh~droxybutyric acid and
~ poly~minodicarboxylic acid co-imides.
: ~.
The abovementioned shaping substances or substance
mixture~ can be prepared by processe~ which are known
35 from the litarature (EP-0,327,490; EP-0,398,935;
~ .
,
2~773~3
-- 7 --
EP-0,458,079; EP-0,123,235; EP-0,~24,934; EP-0,245,8~0,
M. Bornschein et al., Die Pharmazie, 49, Volume 9, 1989,
page 585ff.; J.W. Freytag, J. Microencapsulation~ 2,
Volume 1, 1985, pages 31-38, Y. Kawashima et al. 7
J. Phanmaceutical Sciences, 81, Volume 2, lg92,
page 135ff., EP OtO77,752 and EP 0,131,540).
The abovementioned polycondensates essentially contain at
least 95 mol% of recurring structural units of the
fo~mula I
O O
[-C-R'-C-X-R2 X-] (I~
in which Rl is
: a) a compound of the formula II
--C--C--
0 \ / 0 (II)
R5 R~
in which Rs and R6 zre inert radicals,
b) straight-chain or branched alkyl, alkenyl,
~` cycloalkyl or cycloalkenyl, which can be
substituted b~ one or more inert radicals,
c) aryl which is mono- or polynuclear and mono- or
polysubstituted by inert radicals,
d) compounds of the formula V
~o~ (V)
~' .
- 8 20773~3
or
e) a heterocyclic radical; which can be mono- or
polynu~lear and c~n be mono- or polysubstituted
by inert radicals,
1 5 in which ~ is
a) O-,
b) -NH-- or
c ) --S--,
:
in which R2 iS
a) a compound of the formula III
- CH2-- C C-- CH2 -
\ / ~III)
R5 R6
in which R5 and R6 have the abovementioned
meaning,
b) straight-chain or branched alkyl, alkenyl,
cycloalkyl or cycloalkenyl, which can be
substituted by one or more inert radicals,
c) mono- or polynuclaar aryl, which i8 mono- or
polysubstituted by inert radical6,
d) a heterocyclic radical, which can be mono- or
polynuclear and can be mono- or polysubstituted
by inert radicals, or
~3 a compound of the formula VI
H H
--C - C--
o = c c o (YI~
~ I I
o o
~: 1 5 1 6
20~73~
_ 9 _
in which Rs and R6 have ~he abovementioned
meaning,
wi~h the proviso ~hat ~he polycondensa~e of the formula
I con~ains more than 5 mol% of the radicals R1 andior R2,
S in which Rl is a compound of the formula II and/or R2 i8
a compound of the formula III,
or that the polycondensate of the formula I comprises
more than S mol% of the radicals R1 and ~2, in which
a) R1 i5 a compound of the formula ~ and R2 is a
compound of ~he formula VI, or
b) R1 is phenyltl,2], phenylll,3] or phenyl[1,4]
and
R2 is a compound of the formula VI.
The term "inert radicals~ is understood as meaning
substances which do not react with one another or are
prevented from reacting with one another by protective
groups under the preparation and processing conditions
for the polycondensates according to the invention. Inert
radicals can be, for example, inorganic radicals, such as
halogen, or organic radicals, ~uch as alkyl, alkenyl,
cycloalkyl, cycloalkenyl, alkoxy or dialkylaminoalkyl.
:
Functional groups which are prevented from reaction by
protective groups ara, for example, amino or hydroxyl.
Xnown protective groups are, for example, the benzyloxy
or phenylsulfonyl group.
Heterocyclic radicals can be mono- or polynuclear and
contain, in particular, one or two oxygen, nitrogen or
sulfur atoms in the nucleus.
Aryl is an aromatic carbon ring. Polynuclear aryl
radicals can ba fused to one another or bonded to one
another linearly via C-C bon~s or via bridge groups, such
as, for axample, -O-, -COO-, -CH2-, -CO-NH-, -S~, -CO- or
2~738~
-- 10 --
an -SO~- group.
Examples of polynuclear aromatic aryl radicals are 4~4'-
biphenyl, 1,5- or 2,6-naphthalene gxoups or 1,8-naph-
thalene groups.
To prepare ~he abovementioned polycondensates, a dicar-
bo~lic acid dichloride of the formula X
ClOC - R1- COCl (X)
is reacted with one or more of the diols, di~min~s,
dithio co~pounds or diazines of the formulae XI, XII,
XIII or XIV
HO - R2 _ OH (XI)
H2N - R2 _ NH2
~S - R2 _ SH (XIII)
I
/ CH2 - CH \
HN NH (XIV)
CH2 CH
IB
in which Rl, R2, R7 and R~ have the abovementioned meaning.
The dicarbo~ylic acid dichloride of the ~ormula X and the
individual diol, diamine, dithio or diazine types of the
formulae XI, XII, ~III or ~IV can also be employed in the
form of mixtures with one another.
It is obvious to the expert that the 8um of all the
structural units derived from the dicarboxylic acid~ (A)
7 3 ~ 3
and the sum of all the structural units derived from the
diols, di~hio compounds, diamines and diazines (B) are
essentially the same, i.e. they di~er by not more than
about 1~, preferably by not more than 0.2%, and in
parti.cular are the same in the context of practical
measurement and metering possibilitie~.
The molecular weight of ~he polyamides fo~med can be
controlled, inter alia, ~ia the choice of the proportions
of ~ to B. These choice criteria are known to the expert
in the field of polycondensation.
Examples of suitable dicarboxylic acids from which the
dicarboxylic acid dichlorides of the formula ~ are
derived are 2,3-0-isopropylidene-L-tartaric acid,
2-chloroterephthalic acid, furandicarboxylic acid,
2-bromoterephthalic acid, 2-methylterephthalic acid and
in particular 2,3-0-isopropylidene-L-tartaric acid.
Examples of suitable diamines of the formula XII are
lysine methyl ester, naphthalene-1,4~diamiDe, naph-
thalene 1,5-diamine and naphthalene 2,6-diamine, and in
particular lysine methyl ester or diazines.
Examples of suitable diols of the formula XI are diethyl
tartrate, 2,3-0-isopropylidene-L threitol and diisopropyl
tartrate.
The condensation of the components described above is in
general carried out in solution.
For this, the monomeric compounds to be r~acted with one
another are as a rule dissolved in an organic solvent.
The organic solvent here preferably comprises at least
one solvent, such as, for example, N-methyl-2-pyrrol-
idone, ~,N dimethylacetamide, pyridine, tetramethylurea,N-methyl-2-piperidone, methylene chloride, N,N~-dimethyl-
ethyleneurea, N methylcaprolactam, N-acetylpyrrolidine
2~7~3
- 12 -
and N,N dimethylpropyleneurea. The preferred organic
solvents pyridine, methylene chloridet furan or a mixtuxe
of these compounds are of importance for the process
according to the invention.
S In a pxeferred form of carrying out the solution polymer-
i~ation, the monomeric 2,3-O-isopropylidene-L-tartaric
acid dichlorides and diethyl tartrate are mlxed in a
mixture of pyridine and methylene chloride, while
stirring vi~orously.
The polycondensation temperatures usually ran~e from
-20C to +120C, preferably from +10C to +100C, during
the solution polymerizakion. Particularly good results
are achieved at reaction temperatures of ~10C to +80C.
The 8Um of th~ concentrations of the monomeric compounds
in the polymerization mixture solu-tion can be ad~usted
taking into account the desired degree of polymerization,
the desired viscosity of the polymeri~ation mixture, the
nature of the monomeric compounds used, the nature of the
solvent used and the desired polymeri~ation temperature.
~O The most favorable sum of the concentra~ions can be
determined for the course of the polymerization on the
- basis of a series of prelLminary experiments.
~he polycondensation reactions are preferably carried out
such that, after the reaction, 2 to 15, preferably 5 to
15% by weight of polycondensate is pre ent in the 501u-
tion. Particularly good results are achieved at concen-
trations of 5.0 to 10% by weight.
In the course of the polycondensation, the molecular
weight of the pol~mer and therefore also the viscosity of
the reaction mixture increases. Molecular weights of 500
to 1,000,000, preferably 3000 to 100,000, are achieved.
Viscosities of more than 0.1 dl.g~l (Staudinger index,
dimethylformamide, 25C) are achieved as a rule.
13 2~73~3
-
~hen the polymex ~olution has reached the viscosity
required for further proc0ssing, the polyconden~ation can
be stopped in the customary manner by addition of mono-
functional compounds, such as, or example, acetyl
chloxide. The hydrogen chloxide formed, which is loo~ely
bonded to the amide solvent, can then he neutralized by
addition of basic substances. Substance~ which are
suitable for this are, for example, pyridine, triethyl-
amine and morpholine, bu~ in particular pyridine.
ln The 2,3~0-alkylidene-L-tartaric acid dichlorides which
can be employed as the ~tarting ~ubstance are obtained,
for example, by reaction of tartaric acid alkyl esters
with 2,2-dLmethoxypropane to give the corresponding
tartaric acid dialkyl ester-acetone ketals by a reaction
analogous to tho~e described by M. Carmack et al. (J.
Org. Chem. 33, 1968, pages 2171 - 2173). These tartaric
acid dialkyl ester-acetone ketals are converted into the
corresponding salts using alkali metal hydroxides, and
finally into the corresponding L-tartaric acid ~ichloride
derivatives by reac~ion with phosphorus trichloride,
phosphorus pentachloride, oxalyl chloride or thionyl
chloride.
The 2,3-0-alkylidene-L-threitol is prepared, for example,
by reaction of the tartaric acid dialkyl e~ter-acetone
ketal with LiAlH4 to give the corresponding 2,3-0 alkyl-
idene-2-threitol (P.W. Feit, J. Med. Chem. 7, 1964, pages
14 - 17).
Furandicarboxylic acid dichlorides are obtainabl~, for
example, by the process described by Moore and Xelley
30 (American Chemical Societyy 11, No. 3, 1978, page~ 568 -
573).
;` .
The invention furthermore relate~ to a process for the
production of the echogenic particles according to the
invention, which comprise5 di~olving the shaping
14- 2~77383
substance or sub8 tance mixture in a ~uitable solvent or
solvent mixture ancl suhjecting the solution to spray
dryîn~ .
Other proce~ses, for example emlllsion processe~ or
precipitation processes, furthermore can al~o be employed
for the production of the particle~ according to the
invention (M. Bornschein et al~, ~ie Pharmazie, 44,
Volume 9, 1989, page 585 ff.; and Y. KawashLma et al,,
3. Pharm. Sciences, &1, Yolume 2, 1g92J page 135 f).
The spray drying is carried out, for e~ample/ in
accordance with the principle of ~et atomizativn in co-
current, i.e. the direction of the solution to be sprayed
and the dry ai.r run in the same direction.
The following adjustments of the essen~ial setting
parameters on a spray dryer (Buchi 190; Buchi GmbH,
Eislingen) have proved to be suita~le for production of
the echogenic particles according to the invention. The
int~ke temperature is 15C to 200C, in particular 20C
to 100C. HoweYer, the temperature can also vary within
wide lLmit~, and essentially depends on the solvent used
and the polymer.
The intake temperature is to be understood as meaning the
temperature of the dryin~ air, which is sucked through
the apparatu~ with the aid of the aspirator. Instead of
air, it is of course also possible to employ another gas,
if thP work to be carried out requires thi~.
During spray dryin~ of a solution, all or some of the
solvent is removed, and the liquid ~olution droplets are
thereby converted into particle~. So that the ~olvent
evaporates during the contact time available when the
goods to be dried are sprayed in a 6tream of air, the
temperature of the stream of air must be above or clo~e
to the boiling point of the solvent7
~77~83
-- 1$ --
That temperature of the stream of aix, which entrains the
solid particles, before entry into the cyclone is the
starting temperature. The starti.ng temperature results
from the combination of intake temperature, ~spirator
S setting, pump setting and concentration of the goods to
be sprayed, and last bu~ not least also dep~nds on the
heat o vaporization of the solvent.
The drying air is sucked through the apparatus with the
aspirator, and at the same time a reduced pressure is
generated. By regulating the aspirator output, on the one
hand the amount of heated drying air i8 increased or
reduced, and on the other hand the reduced pressure
prevailing in the apparatus is regulated. A pressure
which is in the range from 10 to 100 mbar below the
pressure outside the apparatus, in particular from 30 to
70 mbar, is preferred.
The object of the delivery pump is to feed the spray
solu~ion into the ~pparatus, and pump output~ of 100-
500 ml/hour have proved appropria~eO
The spray flow is understood as meaning the amount o
compressed air needed for atomization of the solution,
emulsion or di~persion. Instead of compressed air; it i9
of course also possible to employ another gas.
The spray flow can be ~et on th~ appaxatu6 in ~he range
~5 from 100 to 800 Nl/hour (Nl = normal liter), in parti~
cular 600 to 800 Nl~hour.
Suitable solvents or solYent mixtures arer for example,
water, tetrahydrofuran, methylene chloride, furan, DMSO
(dimethyl sulfo~ide), dioxane or acetone.
If the spray product doe~ not have a smooth ~urface after
a trial spraying, smooth surfaces can be produced by
addition of polymer pla~ticizers, such as DMSO or
2077383
~ 16
glycerol, to the solvent.
If the spray product shows smooth surfaces and circular
particles after a trial spr~ying, concave surface curves
inwards can be produced by addition of precipitating
agents and/or temperature changes.
The shape of the particles according to the invention
essentially results from the production pxocess and the
choice of tha parameters, The particles according to the
invention having concave surface segments are derived,
for example, from circular particles having A constant
diameter or hollow bodies. Shapes which look like cups,
hats, cigars or cylinders also occur. The particles can
be monolithic or in the form of hollow bodies, for
example as hollow hemispheres curved inwar~s. Monolithic
particles are tho~e which are essen~ially filled by the
shaping substance; hollow bodies have a ~reater or
smaller gas-filled interior. Hollow hemispherical par
ticles curved inwards can be formed by greater or lesser
invaginations of the hollow spheres. Spindle-shaped forms
with two invagination~ of a circular particle furthermore
have proved favorable. All the shape~ mentioned have
proved to be suitable echogenic particles.
The distribution of the shapes in a batch produced by
spray drying can vary within wide limits. Batches in
which at least 5%, preferably 5 to 90~, in particular 10
to 60%, particularly preferably 30 to 40~ of the par-
ticles have at least one concave surface segment have
proved to bP favorable. Echogenic particles of which at
least 5% have only one concave surface segment are parti-
cularly preferred. The distribution of the shapes can be
determined by computer a~sisted image analysis of the
scanning electron microscope Lmage~ of tha particles, or
with the aid of a cytophotometer.
- 17 - 2~773~3
The echogenic particles according to the invention can
also have a smooth ~urface. This feature c~n be deter-
mined, for example, by electron microscopy ~tudies, in
particular by scanning electron microscopy (SEM3. For
this, the particles are prepared and contrasted by the
standard methods of electron microscopy. Vapor-deposited
metals/ for example gold, have proved to be suitable
contrast media for SEM image3. Smooth surface of par-
ticles appear on the SEM image as homogeneous areas with
a largely constant gray shade. Disturbances in the
surface are recognized by different gray shades. Di tur-
bances have a distanc~ from the average ~urface of the
particles of more than 100 nm.
The particles according to the invention having a smooth
surface are not completely ~ree from disturbances of the
surface. The number of disturbances per ~m2 ranges from 0
to 4, preferably 0.5 to 3, in particular from 1 to 3. The
quality of the ~mooth ~urface can be determined, for
example, by selecting s~uare areas having an edge length
of 0.5 ~m on the SEM Lmages of the particles according to
the invention having a smooth surface, and determining
the number of disturbances of the surface. The charac-
teristic feature of a smooth surface is that, in a random
selection of 10 ~quare areas, 0 to 10 disturbances of the
surface are found, preferably on average 1 ~o 7.5~ in
particular 2. 5 to 7 . 5 .
The distribution of the particles aecording to the
i~ven~ion having a smooth surface in a batch produced by
spray drying can vary within wide limits. Batches in
which at least lO~, preferably 10 to 90%, in particular
20 to 60~, particularly preferably 30 ~o 40~ o~ the
particles have a smooth surface have proved to be
favorable.
The shaping substances can be employed by themselve or
as a mixture in the proce~s described. These shaping
:
2~77383
substances can al~o be employed as a mixture wi~h o~her
biologically degradable and/or biologically compatible
polymers, ~or example dex~ran~, polyethylene glycol~,
hydroxyethyl-starch and other degradable or excretable
polysaccharide3, or ph~siologically acceptable auxili-
aries (for example D~SO, glycerol or other polymer
plasticizers).
The invention furthe~more relates ~o diagnostic ag0nt~
and therapeutic agents, which can compri~e the ~chogenic
particles according to the invention, in addition to
pharmaceutically suitable and physiologically tolerated
excipients, additives and/or auxiliaries.
Before administration, the echogenic particles according
to the invention are conver~ed into a suitable diagnostic
or therapeutic pres~nta~ion form by addition of one or
more pharmaceutically suitable and physiologically
acceptable excipients and if appropriate other additives
and/or au~iliaries. For example, before administration,
the echogenic particles are suspended by addition of
water and mixi~g.
The physiological isotonicity of the particle suspen~ion
can be established by addition of substances having an
osmotic action, for example sodium chloride, galactose,
glucose or fructose. Suspension auxiliaries, such as
dextrans, or additives, such as colloidal infusion
solutions or plasma substitution furthermore can be
admixed to the particle suspensions.
In the processes described for the production of the
echogenic particles according to the i~vention, particle
sizes can be achieved in which 90% of the particles have
: a diameter of 0.1 ~m to 50 ~m, and particle size distri-
butions in which 90~ of the particles are less than 3 ~m
are achieved in particular. Larger particles are siaved
out with a sieve fabric which hae a mesh width of 1 ~m
19 2~7738~
to 50 ~m, in parkicular 3 ~m to 15 ~m. Particle ~izes o~
0.1 ~m to 7 ~m have pro~ed to be suitable for use of
these echogenic particles as ultrasonic contrast media
for diagnosis of cardiovascular diseases, and particle
sizes of 0.1 ~m to 3 ~m are advantageously employed. The
echogenic par~icles are injected, for example, into the
bloodstream. 0.1 mg to 1000 mg of echogenic particles,
preferably 1 mg to 100 mg, are employed per in~ection.
However, the echogenic particles can also be administered
orally, in order to render the gastrointestinal tract
accessible to ultrasonic examination.
The echogenic particles according to the invention can be
used both for diagnostic and for therapeutic methods. The
use of the echogenic particles according to the inYention
is not limited merely to visualization of the blood-
stream in the right ventricular portion of the blood
circulation following venous administration. The echo-
genic particles can be used with excellent success for
ex~mination of the left side of the heart and of the
myocardium. Furthermore, it i~ also possible to visualize
other organs supplied with blood, such as the liver,
spleen, kidneys or brain, using these echogenic
particles.
However, the echogenic particles according to the inven-
tion are also suitable for visualization of hollow
cavities in humans, anLmal~ or plants, for example the
urinary bladder, ureter, womb, vagina, ~ylem or phloem~
The invention is described in d~tail in the following
e~amples. Percentage data relate to the weight, unless
~tated o~herwise.
.,
s~77~83
- 20 -
Example 1
Determination of the shape and lung accessibility of the
echo~enic particles
The average size, the shape and the na~ure of the ~urface
are characteri~ed by means of scanning electron micro-
scope images.
For this, the echogenic particles are ~prinkled onto
conductive double-sided adhesive ~ape, and gold is vapor-
deposited on in a sputtering unit - coating thickness
about 1.8 nm, pressure ~bout 1 x 10-2 n~ar (sputtering
unit type 07 120B, Balzerz Union). ~he SEM Lmages are
produced with a Camscan ~canning electron microscope,
type series 4; tilt angle 15; high voltage 20 kV,
working distance 24 mm; secondary electrons, worXing
lS vacuum abou~ 2 10-6 mbar.
In each case 30 mg of particles are dispersed in 2 ml of
suspension auxiliary comprising 200 mg of dextran 40
(Roth, Germany), 10 mg of polysorbate and 16 mg of NaCl
in distilled wa$er, and are then filtered with sieve
fabrics (15 ~m and 3 ~m mesh width) and lyophili~ed.
The lung accessibility of the particles is tested in
vivo. 30 mg of the filtered particles are resuspended in
1. 5 ml of distilled water with the ai-l of a gla~s rod.
This suspension is injected into a peripheral veLn with
an injection syringe. An ultrasonic head of an ultrasonic
unit (Toshiba, FSH 160a, Japan) is held against the
thorax of the test animal, 50 that a typical cross-
section through the right and left ventricle i8 obtained.
As soon as the ultrasonic contra~t medium re~ches the
30 right ventricle, it can be ~een on the monitor of the
ultrasonic unit how the blood labeled by the contrast
medium reaches the right auricle, and then leave~ the
right Jentricle and sub~equently the heart via the
- 21 - 2~7~3
pulmonary artery. ~fter passage through the lung, the
contrast in th~ left ventricle is dis~inctly incrPa~ed by
the contrast medium.
Example 2
S PreparationofpolysuccinLmide-co~ (hydroxyethyl)-D,L-
aspartamide t70:30)
10 g (103 mmol) o polyanhydroaspartic acid are dissolved
in about 40 ml of N,N-dLmethylformamide (DMF), if approp-
riate with cautiou~ ~arming. 1.83 g (30 mmol) of freshly
distilled 2-aminoethanol are added dropwise to this
solution, and the mixture is stirred overnight at room
temperature. The reaction mixture is precipitated in
butanol and the product is washed several times with
dried acetone. Drying is carried out in vacuo at elevated
temperature. The white, wa~er-soluble product is formed
to the extent of approximately 100~, and is tested for
residues of DNF and butanol by NMR spectxoscopy. The
molar ratio of polyanhydroaspartic acid to aminoethanol
~mployed approximately corresponds to th~ copolymer
composition.
Example 3
Preparationofpolysuccinimide-co~ (nonyl carbonyloxy-
ethyl)-D,L-aspartamide (50:50)
6 g of a polysuccinimide-co-~ (hydroxyethyl)-D,~-
aspartamide (50:50) (= 24 mmol of hydro~yethyl groups),
which was prepared analogously to Example 4 from poly-
anhydroaspartic acid tmolecular weight = 14,000~ and
2-aminoethanol (molar ratio 2:1), are dissolved in 100 ml
of dry DNF, 8 g (100 mmol) of dry pyridi~e are added and
the mixture is cooled to 0C. 9.6 g of distilled decanoyl
chloride are slowly added dropwise.
22 2~7~
The mixture i~ ~tirred overn.igh~ and precipitated in
0.5 l o ether. The product which has precipita~ed is
filtered off with suction and washed with ethex, acetone,
water t acetone and ether.
About 8 g of a white, completely substituted polymer ~NNR
control), which is soluble, for example, in methylene
chloride and tetrahydrofuran, in each ca~e with a trace
of DMSO, or in methanol/methylene chloride mixtures, are
obtained.
Example 4
PreparationofpolysuccinLmide-co-~fl (nonyl-carbo~yloxy-
ethyl)-D,L-aspaxtamide
Various poly~uccinLmide-co~ -(hydro~yethyl)-D,L-aspart-
amides, inter alia having the composition 70:30, 50:50
and 30:70, are prepared from polyanhydroaspartic acids of
different molecular weight (molecular weight - 7000;
about 13,000, 30,000) analogously to ~xa~ple 2 and are
reacted with decanoyl chloride, as described in
Example 5, to give the corresponding poly~uccinLmide-co-
~,~-(nonylcarbo~yloxyethyl~-D,L-aspartamides.
a) - PolysuccinLmide-co~ (nonylcarbonyloxy-ethyl)-
D,L-aspartamide (70:30) ~rom polyanhydroaspartic
acid (molecular weight = 7000); characterized by
NMR.
b) - PolysuccinLmide-co-~ t ~- (nonylcarbonyloxy-ethyl)~
D,L-aspartamide (70:30) from polyanhydroaspaxtic
acid (molecular weight = 14,000); characteri~ed
by NMR.
c) - PolysuccinLmide-co~ -(nonylcarbonyloxy-ethyl)-
D,L-aspartamide (70:30) from polyanhydroaspartic
acid (molecular weigh~ = 30,000), characterized
~77.~3
- 2.~ -
by NMR.
Ex~mple 5
20 g of polyanhydroaspartic acid-co~ (nonylcarbonyl-
oxyethyl)-D,L-a~partamide 70:30 (prepared as in Example
6b) are dissolved in 400 ml of methylene chloride and the
solution is sprayed to paxticles under the following
conditions in a spray dryer (~ini Spray Dryer Buchi 190,
Buchi GmbH, EislingPn):
Spraying conditions
10 Intake temperature: 60C
Outlet temperature: 40C
Scale divisions heating: 1.5
Pump settingl ~
Aspirator: 12
15 Spray flow jet air: 700 Nl/h
Pressure in the filter: 55 mbar
Cooling jet: 20C
Yield: about 8 g
After spray drying, the methylene chloride concentration
in the particles is less ~han 0~05%. The average size and
shape and the nature of the surface are characterixed by
means of scanning electxon mi~roscopy image~; the lung
accessibility of the particles is determined as in
Example 1.
~5 100 mg of the particles produced are suspended in a 1%
strength ~Haemaccel solution (Behring Werke ~&, ~arburg,
Germany) and filtered throuyh sieve fabric (10 ym)~ The
filtrate is then lyophilized and used to determined the
lung accessibility.
2~77~3
- 24 -
Under the scanning electron microscope, the particles
produced have an average particle ~iæe of 2 ~m and a
smooth surface. Furthermore, the~e particle8 have access
to the lungs.
About 15~ of the particles are circular hollow spheres
and 50% of the particles are hollow spheres which have a
spheric~l invagination, so ~hat a hemispherical hollow
sphere is formed. The shell thickness of these hemi-
spherical hollow spheres at the maximum invagination can
be described by the equation rm - ri = 2 d (d = shell
thickness, r~ - radius of the outer hemisphere, r1 =
radius of the inner concave hemisphere. If the hemi-
sph~rical shape is not ideal, the radius is to be deter-
mined from the relative central point of the invaginated
hollow sphere.
About 25~ of the particles are hollow spheres having 2 ~o
5 invaginations of different size.
Example 6
Particles are produced from polyhydro~ybutyric acid type
159PHB (ICI Biological Products Ltd) in accordance with
instructions by J.R. Embleton, B.J. ~ighe (J.
Microencapsulation, 1992, ~olume 9, ~o. 1, 73-87) by the
so-called "double emulsion solvent evaporation proce~s".
The evaporation is carried out at room temperature. The
particles are fractionated by centrifugation and filtra-
tion through a 10 ~m sieve fabric. A particle content of
35~ having concave surface segments i6 then determined in
thè filtrate by scanning electron microscopy.
The evaporation is also carried out at 40C and the
particles are then fractionated. For comparison,
fraction with a particle content of only 5~ having
concave surface 8e~ments is determined in thi~ manner ~y
scanning electron microscopy.
~77383
-- 25 --
EY.amP1 e 7 ( LMP G 5 0 / 5 0 )
Preparation of poly(L-lysine methyl ester-fumaric acid/-
L-lysine methyl ester-glutaramide )
Copolymer 5 0 : 5 0 ~ LMFG 5 0: 5 0 )
0.7 g of glutaric acid dichloride and 0.84 g of fumaric
acid dichloride are dissolved in 170 ml of CH2Cl2. ~his
solution is added ~o a solution of 2.91 g of L-ly~ine
methyl ester ~nd 3.0 g of Na2C03 in 120 ml of H20 at room
temperature, while tirring vigoxously. The polyconden-
sation, which starts suddenly, is interrupted after15 minutes by addition of 150 ml of lN agueou~ HCl. The
me~hylene chloride is then driven off by passing in
steam. The hot aqueous mixture is filtered and the solid
product is washed ~everal tLmes with water~
Yield: 1.3 g (60% of theory) after vacuum drying
(20 hours)
Exampls 8
2 g of polymer from Example 7 are dissolved in 100 ml of
DNS0 and the solution is ~prayed to particles at an
intake temperature of about 200C in a Mini Spray Dryer
Buchi 190. The average size and the nature of the surface
are characteriz~d by means of scanning electron micro-
scopy images tsee Table 1). In each case 50 mg of par-
ticles ar~ suspended in 2 ml of suspension auxiliary l1%
of ~Haemaccel in isotonic water) and subsequently filtered
with sieve fabric (15 ~m and 3 ~m mesh width) and
lyophilized.
Example 9
2 g of polyhydroxybutyric acid type 159 PHB (ICI
Biological ~roducts ~td) are dissolved in 100 ml of a
- 26 - 2~773~3
furan/methylene chloride (9:1~ mixture, the solution is
sprayed to particles at about 45C in a Bushi Mini Spxay
Dryer, and the particles are further processed
analogously to Example 8.
Example 10
A solution of 2 g of polyhydroxybutyric acid type 159 PHB
(ICI Biological Products Ltd) in 100 ml of methylene
chloride/MeOH (1:2) is sprayed to particles at about 45C
in a Buchi Mini Spray Dryer, and the particles are
further processed analogously to Example 8.
Example 11
2 g of poly_e-caprolactone (Polysciences 19561~ are
dissolved in methylene chloride to give a 2% strength
solution, the solution is sprayed to particles at room
temperature in a BUchi Mini Spray Dryer, and the par-
ticles are further processed analogously to Example 8.
The lung accessibility of the particles from Examples 8
to 11 is tested in vivo and determined as in Example 1.
The following table shows the resultsO
- 27 2~7~
Table 1
PolymerSolvent ~verage Surf ace Lung
according particle access-
to size ibility
. Example ~ _
8 DMSO 3 ~m smooth yes
9 furan/ 3 ~m smooth yes
methylene
. ~ l~lori~ _ _
t0 CH2Cl2/ 2 ~m rough no
MeOH 1:2
.
11 CH2Cl2 3 ~m smooth yes
