NO305685B2 - Echogenic particles, processes for the preparation thereof, applications thereof, diagnostics or therapeutics containing the particles and preparation thereof. - Google Patents

Abstract

The present invention describes echogenic particles with a smooth surface and/or echogenic particles with 1 to 10 concave surface segments, the preparation thereof by spray drying, and the use thereof as ultrasonic diagnostic aid.

Description

The invention relates to echogenic particles containing gas and a shaping substance, whereby the surface of the particles is essentially smooth and the shape contains no or 1 to 10 concave surface segments or the surface is not smooth and contains 1 to 10 concave surface segments and which can pass the lung in the bloodstream and application thereof, especially as diagnostics and therapeutics. Furthermore, the present invention relates to a method for the production of said echogenic particles.

Ultrasound diagnostics has found very wide application in medicine due to the uncomplicated, simple handling. Ultrasound waves are reflected at the interfaces between different types of tissue. The resulting echo signals are amplified electronically and made visible.

The visualization of blood vessels and internal organs by means of ultrasound generally does not allow the visualization of the blood flow present therein. Liquids, especially blood, only provide ultrasound contrast when there are density differences to the surroundings. As a contrast agent used in medical ultrasound diagnostics, for example, substances that contain gases or produce gases, as the impedance difference between gas and surrounding blood is significantly greater than that between liquids or solid bodies and blood (LevineR.A., J. Am. Coll. Cardiol 3: 28, 1989; Machi, i.J. CU 11:3, 1983).

Several methods are known in the literature for the production and stabilization of gas bubbles. US patent 4,276,885 describes the production of gas bladders of defined size, which are surrounded by a gelatin envelope that protects the gas bladders against coalescence. The storage of the finished gas bladders can only take place in a frozen state, whereby the gas bladders must be brought back to body temperature for use.

EP-A2-0 123 235 and 0 122 624 describe gases containing ultrasound contrast agent which consist of mixtures of surfactants with a solid substance in a liquid carrier. The ultrasound contrast medium is produced by a laborious grinding process with an air jet mill. The particles thus obtained only have a limited useful life because they quickly lose the trapped gases.

EP-A2-0 224 934 describes ultrasound contrast agent in the form of gas-filled gelatin or albumin hollow cells. A disadvantage, however, is the use of foreign or denatured body proteins, due to the associated allergenic risk. The present invention is based on the task of finding echogenic particles that reflect, absorb, diffract, respectively scatter, ultrasound waves and consequently provide a clear contrast to surrounding tissue, which are so small and stable that they without significant gas loss and essentially quantitatively reach the left ventricle after intravenous administration and which can be produced quickly and easily.

It has been found that the quality of echogenic particles particularly depends on the shape and surfaces of these particles. It turned out that the echogenic particles with concave surface segments can significantly better pass the lung in the bloodstream and cause a higher contrast in ultrasound. These echogenic particles have an air-water interface whose scattering cross-section in ultrasound examinations is very high. Echogenic particles with an essentially smooth surface can pass through the lungs of vortex types in the bloodstream without the particles being filtered out to a large extent.

The invention thus relates to echogenic particles, containing a gas and a shaping substance or a mixture of substances which is biodegradable and/or separable and which can pass through the lung in the bloodstream of vertebrates, characterized by (a) the surface of the echogenic particles is generally smooth and the shape of the echogenic particles contains no or 1 to 10 concave surface segments or the surface of the echogenic particles is non-smooth and the shape of the echogenic particles contains 1 to 10 concave surface segments, and that (b) the maximum deflection of the concave surface segment constitutes from 30 to 90% of the smallest particle cross-section and that 10 to 60% of the echogenic particles exhibit at least one concave surface segment per particle.

Preferred features of the echogenic particles according to the invention appear from the accompanying claims 2-11 and as detailed below in the description.

Echogenic particles are particles with a diameter which in 90% of the particles reaches from 0.1 µm to 20 µm, preferably from 0.2 µm to 7 µm, especially from 0.5 µm to 3 µm. These particles are also able to reflect, absorb, diffract or scatter sound waves.

Concave surface segments of an echogenic particle mean a particle that at least exhibits an inwardly curved surface. The particles according to the invention include particles with a maximum bending of the concave surface segment of from 30 to 90%, especially 50 to 90%, of the smallest particle cross-section. A further characteristic is that 10 to 60%, especially 30 to 40%, of the echogenic particles exhibit at least one concave surface segment per particle.

The number of concave surface segments reaches from 1 to 10, preferably 1 to 5, especially 1 to 3.

The particles can also have a smooth surface. By an essentially smooth surface of an echogenic particle is meant surfaces which, at a magnification of 10,000 times, do not allow any structural differences to be recognized on the surface, i.e. disturbances on the surface of this particle such as elevations or depressions which are essentially less than 100 nm away from it mean the surface of the particle, especially 90 to 50 nm, preferably 60 to 80 nm. The average particle surface appears from the average of the elevations and depressions on the surface. Disturbances on the surface include changes to the homogeneous surface which in the electron microscopic image look like elevations, bridges, faults, stair, step, shell or layer-like structure of the surface as well as holes, depressions or pores in the surfaces.

The property of the particle according to the invention that it can pass through the lungs of vertebrates in the bloodstream is established, for example, by injecting the echogenic particle under sterile conditions into a peripheral vein of an experimental animal. An ultrasound head of an ultrasound device is held on the thorax of the experimental animal so that a typical cross-section through the right and left heart chambers is obtained. As soon as the ultrasound contrast medium reaches the right heart chamber, one can determine on the monitor of the ultrasound device how the blood marked by the contrast medium reaches the right atrium, then the right ventricle and finally leaves the heart again via the pulmonary artery. After the lung passage, a significant increase in contrast is observed in the left heart chamber with the help of the contrast agent.

Where the echogenic particle contains gas, for example air, nitrogen, noble gas such as helium, neon, argon or krypton, hydrogen, carbon dioxide, oxygen or mixtures thereof. The echogenic particle is filled with gas, for example by the particle being stored in a corresponding gas atmosphere or by spray drying directly during production, it is extracted in a corresponding gas atmosphere.

Forming substances or substance mixtures are all compounds that can be used to produce particles that contain at least one concave surface segment. These include, for example, naturally occurring polymers or polymers that can be produced synthetically, lipids that in aqueous solutions form micelles or low-molecular amino acids, fats

or carbohydrates.

Biodegradable and/or separable forming substance or substance mixture means substances which are converted into non-toxic decomposition products or excreted in the body of animals, humans or plants. The breakdown can take place enzymatically, hydrolytically or by partial or complete dissolution of the substances or decomposition of the particle into smaller subunits in water. The excretion of the breakdown products takes place via the kidney, skin, lung or intestine. When the substances completely or partially dissolve in water or do not break down, for example in the case of low molecular weight carbohydrates, excretion takes place without breakdown via the kidneys.

Forming substances or substance mixtures, which are biodegradable and/or separable, are selected from the following group: polyester, of a-, 13-, y- or s-hydroxycarboxylic acid, for example poly-s- caprolactone, polyhydroxybutyric acid, polylactic acid or

polyhydroxyvaleric acid,

copolymers of the above-mentioned polyesters with each other or with

polyglycolic acid,

polycondensates containing 2,3-O-alkylidene tartaric acid, 2,3-O-alkylidene-L-threitol, furo-[2,5] groups or terephthalates,

polyaminodicarboc-

syllic acid coimides such as polysuccinimide-co-α,B-(butyloxycarbonyl-propionyloxyethyl)-D,L-aspartamide,

polyamino acids such as polyglutamic acid, polyaspartic acid or polylysine, which react basic or acidic in aqueous solution, as well as their

derivatives,

polyamides, for example polylysine methyl ester fumaric acid-co-glutaramide or

polyhydroxyethylaspartamide,

polyorthoester,

polyanhydrides for example those composed of sebacic acid and bis(p-carboxyphenoxy)propane,

gelatin for example hydrophobically modified,

albumins such as bovine serum albumin,

polysaccharides such as dextrans, starch or basic or acidic derivatives thereof,

polymethylene malonate,

sugars such as glucose, galactose, lactose or sucrose, fatty acids with 8 to 20 carbon atoms,

amino acids such as proteinogenic amino acids and their hydrophobic derivatives, alcohols with 12 to 20 carbon atoms or

lipids such as phospholipids.

Particularly preferably, the echogenic particles according to the invention contain biodegradable and/or separable polymers from the following group: polyamides, polycondensates, polyhydroxybutyric acid or polyaminodicarboxylic acid co-imides.

The above-mentioned formative substances or substance mixtures can be produced by methods known from the literature (OEP-0.327.490; EP-0.398.935; EP-0.458.079; EP-0.123.235; EP-0.224.934, EP-0.245.840 , M. Bomstein et al., Die Pharmazie, 49, issue 9, 1989, pp. 585 ff, J. W. Freytag, J. Microencapsulation, 2, issue 1, 1985, pp. 31-38, Y. Kawashima et al. , J. Pharmaceutical Sciences, 81, Issue 2, 1992, pp. 135 et seq., EP-0,077,752, EP-0,131,540).

The above-mentioned polycondensates essentially contain at least 95 mol% of repeating structural units of formula I

where Ri stands for

a) compound of formula II

where R<5> and R<6> mean inert residues,

b) straight or branched chain alkyl, alkenyl, cycloalkyl or cycloalkenyl, which may be

substituted with one or more inert residues,

c) aryl, mono- or polynuclear, mono- or poly-substituted with inert residues,

d) compounds of formula V

e) heterocyclic residue, which may be mono- or polycyclic and may be single or

polysubstituted with inert residues,

where X stands for

a) -O-,

b) - NH - or

c) - S -,

where R<2> stands for

a) compound of formula III

where R<5> and R<6> have the meaning given above,

b) straight-chain or branched alkyl, alkenyl, cycloalkyl or cycloalkenyl, which may be substituted with one or more inert residues,

c) mono- or polycyclic aryl, mono- or multi-substituted with inert residues,

d) heterocyclic residue which may be mono- or polycyclic and mono- or poly-substituted with inert residues, or

d) compound of formula VI

where R.<5> and R<5> have the meaning given above,

on the condition that more than 50 mol-% of the residues R^ and/or R^ are present in the polycondensate of formula I, where Ri stands for a compound of formula II and/or R^ for a compound of formula III, or that more than 5 mol-% of the residues Ri and R^ are present in the polycondensate of formula I, where a) Ri stands for a compound of formula V and R^ for a compound of formula VI, or

b) R<1> stands for phenyl[1,2], phenyl[1,3] or phenyl[1,4] and

R<2> stands for a compound of formula VI.

The term "inert residues" refers to substituents which do not react with each other under the manufacturing and processing conditions for the polycondensates according to the invention or which are prevented from reacting with each other by means of protective groups. Inert residues can, for example, be inorganic residues such as halogen, or they can be organic residues such as alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkoxy or dialkylaminoalkyl.

Functional groups that are prevented from participating in the reaction by means of protective groups are, for example, amino or hydroxy.

Known protecting groups are, for example, the benzyloxy or phenylsulfonyl group.

Heterocyclic residues can be mononuclear or polynuclear and in particular have one or two oxygen, nitrogen or sulfur atoms in the nucleus.

Aryl stands for an aromatic carbon ring. Polynuclear aryl radicals can be condensed with each other, or linearly connected to each other via C-C bonds or via bridging groups such as -O-, -COO-, -CH2-, -CO-NH-, -S-, -CO-or a -S02 group.

Examples of polynuclear aromatic aryl radicals are 4,4'-biphenyl, naphthalene-1,5- or -2,6-groups or naphthalene-1,8-groups.

To produce the above-mentioned polycondensates, a dicarboxylic acid chloride of formula X is reacted

with one or more of the diols, diamines, dithio compounds or diazines of formulas XI, XII, XIII or XTV,

where R<1>, R<2>, r<7> and R^ have the meaning given above.

The dicarboxylic acid chloride with formula X and the individual diol, diamine, dithio or diazine types with formulas XI, XII, XIII or XIV can also be used in the form of mixtures with each other.

It is self-evident to the person skilled in the art that the sum of all the structural units derived from the dicarboxylic acids (A) and the sum of all the structural units derived from the dithiols, dithio compounds, diamines and diazines (B) are essentially the same, i.e. they differ by a maximum of approx. 1%, preferably approx. a maximum of 0.2%, especially that they are equally within the framework of the practical measurement and dosing possibilities.

The molecular weight of the polyamides formed can be controlled, among other things, by the selection of the ratio between A and B. These selection criteria are known to the person skilled in the field of polycondensation.

Examples of suitable dicarboxylic acids, from which the dicarboxylic acid chlorides of formula X are derived are 2,3-O-isopropylidene-L-tartaric acid, 2-chloroterephthalic acid, furandicarboxylic acid, 2-bromoterephthalic acid, 2-methylterephthalic acid and especially 2,3-O-isopropylidene-L-tartaric acid .

Examples of suitable diamines of formula XII are lysine methyl ester, naphthalene-1,4-diamine, naphthalene-1,5-diamine, naphthalene-2,6-diamine and especially lysine methyl ester or diazines.

Examples of suitable diols of formula XI are tartaric acid diethyl ester, 2,3-O-isopropylidene-L-threitol and tartaric acid diisopropyl ester.

The condensation of the above-mentioned components is generally carried out in solution.

For this purpose, the monomeric compounds to be reacted with each other are usually dissolved in an organic solvent. The organic solvent thereby preferably contains at least one solvent such as N-methyl-2-pyrrolidine, N,N-dimethylacetamide, pyridine, tetramethylurea, N-methyl-2-piperidone, dichloromethane, N,N'-dimethylethylene urea, N-methylcaprolactam , N-acetylpyrrolidine, N,N-dimethylpropyleneurea. For the method according to the invention, the preferred organic solvent is pyridine, dichloromethane, furan or a mixture of these compounds.

In a preferred form of carrying out the solvent polymerization, the monomers 2,3-O-isopropylidene-L-tartaric acid dichloride and tartaric acid diethyl ester are mixed in a mixture of pyridine and dichloromethane with vigorous stirring. The polycondensation temperatures in the solution polymerization usually reach from -20°C to +120°C, preferably from +10°C to +100°C. Particularly good results are achieved at reaction temperatures of +10°C to +80°C.

The sum of the concentrations of monomeric compounds i

the polymerization mixture solution can be adjusted taking into account the desired degree of polymerization, the desired viscosity of the polymerization mixture, the type of monomeric compounds used, the type of solvent used and the desired polymerization temperature. The most favorable sum of the concentrations can thereby be determined on the basis of a number of preliminary tests for the course of the polymerisation.

Polycondensation reactions are preferably carried out so that after completion of the reaction there are 2 to 15, preferably 5 to 15% by weight of polycondensate in the solution. Particularly good results are achieved at concentrations of 5.0 to 10% by weight.

During the course of the polycondensation, the molecular weight of the polymer increases and thus also the viscosity of the reaction mixture. Molecular weight from 500 to 1,000,000, preferably 3,000 to 100,000, is obtained. As a rule, viscosities of more than 0.1 dl-g"<l> (Staudinger index, dimethylformamide, 25°C) are achieved.

When the polymer solution has reached the viscosity required for further processing, the polycondensation can be stopped in the usual way by adding mono-functional compounds, such as acetyl chloride. The hydrogen chloride formed and dissolved in the amide solvent can then be neutralized by the addition of basic substances. Suitable for this purpose are, for example, pyridine, triethylamine, morpholine, but especially pyridine.

The 2,3-O-alkylidene-L-tartaric acid dichloride which can be used as a starting material is obtained, for example, by reacting tartaric acid alkyl esters with 2,2-dimethoxypropane to the corresponding tartaric acid dialkyl ester acetone ketals analogously to the reactions described by M. Carmack et al. (J. Org., Chem. 33, 1968, pp. 2171-2173). These tartaric acid dialkyl ester acetone ketals are transferred with alkali hydroxides to the corresponding salts and then transferred by reaction with phosphorus trichloride, phosphorus pentachloride, oxalyl chloride or thionyl chloride to the corresponding L-tartaric acid dichloride derivatives.

The preparation of 2,3-O-alkylidene-L-threitol takes place, for example, by reacting tartaric acid dialkyl ester acetone ketal with LiAltfy to the corresponding 2,3-O-alkylidene-2-trecol (P.W. Feit, J. Med. Chem. 7, 1964, p 14-17).

Furandicarboxylic acid dichlorides are, for example, obtainable by the method described by Moore and Kelley (American Chemical Society, 11, no. 3, 1978, pp. 568-573).

The invention further relates to a method for producing the echogenic particle according to the invention, which is characterized in that the shaping substance or substance mixture is dissolved in a suitable solvent or a

solvent mixture and the solution is then subjected to spray drying.

Furthermore, other methods can also be used, for example emulsification methods or precipitation methods for producing the particles according to the invention (M. Bornschein et al, Die Pharmazie, 44, issue 9, 1989, p. 585 ff; Y-Kawashima et al., J. Pharm. Sciences, 81, Issue 2, 1992, pp. 135 ff).

The spray drying takes place, for example, using the principle of nozzle atomization in co-flow, i.e. the direction of the solution to be atomized and the dry air proceed in the same direction.

For the production of the echogenic particle according to the invention, the following settings of the essential setting parameters on a spray dryer (Biichi 190; Biichi GmbH, Eislingen) have proven suitable. The inlet temperature is 15°C to 200°C, especially 20°C to 100°C. However, the temperature can also vary within wide limits and depends essentially on the solvent and polymer used.

Under inlet temperature is understood the temperature of the drying air that is sucked in through the device with the help of an aspirator. Instead of air, another gas can of course also be used, if the work to be carried out requires this.

When spray drying a solution, the solvent is completely or partially removed, thereby converting the liquid solution droplets into particles. In order for the solvent to evaporate during the atomization of the material to be dried in an air stream during the available contact time, the temperature of the air stream must be above or close to the boiling point of the solvent.

The exit temperature is the temperature that the air flow, which carries the solid particles with it, exhibits before entering the cyclone. The output temperature results from the combination of input temperature, aspirator setting, pump setting and concentration of the spray material and, not least, also dependent

of the heat of vaporization of the solvent.

With the aspirator, the drying air is sucked through the device and thus creates a negative pressure at the same time. By regulating the aspirator performance, on the one hand, the quantity of the heated drying air is increased or decreased, and on the other hand, the negative pressure prevailing in the apparatus is regulated. A pressure that lies in the range from 10 to 100 mbar below the pressure outside the apparatus, especially of 30 to 70 mbar, is preferred.

The task of the transport pump is to spray the spray solution into the device, a pump performance of 100-500 ml/h has proven appropriate.

Spray flow is understood as the amount of compressed air required to atomize the solution, emulsion or dispersion. Instead of compressed air, you can of course also use another gas.

The spray flow can be set on the device in the range from 100 to 800 Nl/h (NI = standard litre), especially 600 to 800 Nl/h.

Suitable solvents or solvent mixtures are, for example, water, tetrahydrofuran, dichloromethane, furan, DMSO (dimethylsulfoxide), dioxane or acetone.

If the spray product does not show a smooth surface after a trial spraying, then smooth surfaces can be produced by adding polymer softeners such as DMSO or glycerol to the solvent.

If the spray product shows smooth surfaces and round particles after a trial spraying, concave surface indentations can be achieved by adding precipitating agents and/or temperature changes.

The shape of the particles according to the invention essentially results from the production method and the choice of parameters. The particle according to the invention with concave surface segments is derived, for example, from round particles with a constant diameter or hollow bodies. There are also shapes that look like goblets, bowls, cigars or cylinders. The particles can be monolithic or exist as hollow bodies, for example as hollow inwardly curved hemispheres. Monolithic particles are those which are essentially filled with the shaping substance; hollow bodies have a more or less large gas-filled inner space. Hollow inwardly curved hemispherical particles can arise from more or less strong compression of hollow spheres. Furthermore, spindle-shaped forms with two indentations of a round particle have proven beneficial. All of the aforementioned forms have proven to be suitable echogenic particles.

The distribution of the shapes in a charge produced by spray drying can vary within wide limits. Advantageously, fillers have been found to be in which at least 5%, preferably 5 to 90%, especially 10 to 60%, especially preferably 30 to 40% of the particles exhibit at least one concave surface segment. The distribution of the shapes can be determined by computer-assisted image analysis by raster electronic recordings of the particle or can take place with the help of a cytophotometer.

The echogenic particles according to the invention can also have a smooth surface. This feature can, for example, be determined by electron microscopic examinations, especially by means of scanning electron microscopy (REM). Thereby, the particle is prepared by standard procedures for electron microscopy and treated with contrast. As a contrast agent for REM recordings, metal depositions, for example with gold, have proven beneficial. Smooth surfaces of particles appear on the REM recordings as homogeneous surfaces with an essentially constant gray tone. Disturbances of the surface can be recognized by different shades of grey. Disturbances have a distance from the mean surface of the particle of more than 100 nm.

Particles according to the invention with smooth surfaces are not completely free from disturbances on the surfaces. The number of disturbances per um<2> ranges from 0 to 4, preferably 0.5 to 3, especially 1 to 3. The quality of the smooth surface can be determined, for example, by selecting square areas of an edge length of 0.5 µm and determines the number of disturbances on the surfaces. A smooth surface is characterized by the fact that in a random selection of 10 square areas there are 0 to 10 disturbances on the surface, preferably on average from 1 to 7.5, especially 2.5 to 7.5.

The distribution of the particles according to the invention with a smooth surface in a charge produced by spray drying can vary within wide limits. Advantageously, fillers have been found to be in which at least 10%, preferably 10 to 90%, especially 20 to 60%, especially preferably 30 to 40% of the particles exhibit a smooth surface.

In the described method, the shaping substances can be used alone or as a mixture. These shaping substances can also be used in admixture with other biodegradable and/or biologically acceptable polymers, for example dextrans, polyethylene glycols, hydroxyethyl starch and other degradable or separable polysaccharides or physiologically acceptable excipients (for example DMSO, glycerol or other polymer softeners).

The invention further relates to diagnostics and therapeutics which are characterized in that they contain echogenic particles according to the invention alongside pharmaceutically suitable and physiologically acceptable carriers, and possibly further additives and/or auxiliary substances.

The echogenic particles according to the invention are transferred before use by adding one or more pharmaceutically suitable and physiologically acceptable carriers and possibly further additives and/or auxiliaries to a suitable diagnostic or therapeutic form of use. The echogenic particles are, for example, suspended before use by adding water and mixing.

Thus, the invention also provides a method for the production of a diagnostic or therapeutic, characterized by bringing echogenic particles according to the invention into a suitable diagnostic or therapeutic administration form by means of a pharmaceutically suitable and physiologically acceptable carrier and possibly further additional and/or auxiliary substances.

By adding osmotically active substances, for example table salt, galactose, glucose, fructose, the physiological isotonicity of the particle suspension can be produced. Furthermore, suspension aids such as dextrans or additives such as colloidal infusion solutions for plasma substitution can be added to the particle suspensions.

In the described method for producing echogenic particles according to

invention, particle sizes can be achieved whereby 90% of the particles have a diameter of 0.1 µm to 15 µm, in particular particle size distributions are achieved whereby 90% of the particles are smaller than 3 µm. Larger particles are screened out using a screening fabric having a mesh size of 1 µm to 50 µm, especially 3 µm to 15 µm. When using these echogenic particles as an ultrasound contrast agent for the diagnosis of cardiovascular diseases, particle sizes of 0.1 µm to 7 µm have proven advantageous, preferably using particle sizes of 0.1 µm to 3 µm. The echogenic particles are injected, for example, into the bloodstream. 0.1 mg to 1000 mg of them are used per injection

the echogenic particles, preferably 1 mg to 100 mg.

However, the echogenic particles can also be used orally, to make the gastrointestinal tract accessible for an ultrasound examination.

The invention also provides a use of the echogenic particles according to the invention for the production of diagnostics for visualizing the blood circulation or organs that are oxygenated with blood.

Furthermore, the invention provides the use of the echogenic particles according to the invention for the production of a diagnosticum for concentrating cavities in humans, animals and plants.

The echogenic particles according to the invention can be used for diagnostic as well as therapeutic procedures. The application of the echogenic particles according to the origin is not only limited to the visualization of the blood flow in the right ventricular part of the blood circulation after venous supply. The echogenic particles can be used with excellent results for examination of the left side of the heart and of the myocardium. Furthermore, it is also possible to visualize other organs supplied with blood, such as the liver, spleen, kidney or brain, with these echogenic particles.

As mentioned earlier, however, the echogenic particles according to the invention are also suitable for making visible cavities in humans, animals or plants, for example urinary bladder, ureter, uterus, vagina, xylem or phloem. In the following examples, the invention is described in more detail. Percentages refer to weight, unless otherwise stated.

Example 1

Determination of the shape and pulmonary accessibility of the echogenic particles.

The average size, shape and surface condition are characterized using raster electronic recordings.

For this purpose, the echogenic particles are sprinkled on a conductive double-sided adhesive tape and gold is vaporized in a cathode sputtering plant - layer thickness approx. 1.8 nm, pressure approx. 1x10" <2> mbar (atomization plant type 07 120B, Balzerz Union). The preparation of the REM images takes place with a "Camscan" scanning electron microscope, type series 4; tilt angle 15°; high voltage 20 kV, working distance 24 mm; secondary electrons, working vacuum approx. . 2 • 10"^ mbar.

In any case, 30 mg of particles are dispersed in 2 ml of suspension aid, consisting of 200 mg of "Dextran 40" (Roth company, Germany), 10 mg of polysorbate and 16 mg of NaCl in distilled water and then filtered with sieve tissues (15 µm and 3 µm mesh size) and lyophilized.

The lung accessibility of the particles is investigated in vivo. 30 mg of the filtered particles are resuspended in 1.5 ml of distilled water using a glass rod. With an injection syringe, this suspension is injected into a peripheral vein. An ultrasound head of an ultrasound device (Toshiba, FSH 160a, Japan) is held on the thorax of the experimental animal so that a typical cross-section through the right and left heart chambers is obtained. As soon as the ultrasound contrast agent reaches the right heart chamber, one can record on the monitor of the ultrasound device how the blood labeled with contrast agent reaches the right atrium, then the right ventricle and then again leaves the heart via the pulmonary artery. After the lung passage, the contrast in the left heart chamber is clearly enhanced by the contrast medium.

Example 2

Preparation of polysuccinimide-co-α,B-(hydroxyethyl)-D,L-aspartamide (70:30)

10 g (103 mmol) of polyanhydroaspartic acid are dissolved in approx. 40 ml of N,N-dimethylformamide (DMF) possibly under gentle heating. 1.83 g (30 mmol) of freshly distilled 2-aminoethanol is added dropwise to this solution and it is stirred at room temperature overnight. The reaction mixture is precipitated in butanol and washed several times with dry acetone. The drying takes place in a vacuum at an elevated temperature. The white, water-soluble product occurs in a yield of approximately 100% and is examined NMR spectroscopically for residues of DMF and butanol. The molar ratio used between polyanhydroaspartic acid and aminoethanol corresponds approximately to the copolymer composition.

Example 3

Preparation of polysuccinimide-co-α,B-(nonyl-carbonyloxyethyl)-D,L-aspartamide

(50:50)

6 g of a polysuccinimide-k<q->a,B-(hydroxyethyl)-D,L-aspartamide (50:50) (= 24 mmol hydroxyethyl groups), which is prepared analogously to example 4 from

polyanhydroaspartic acid (MG = 14000) and 2-aminoethanol (molar ratio 2:1), dissolve in 100 ml of dry DMF, mix with 8 g (100 mmol) of dry pyridine and cool to 0°C. 9.6 g of distilled decanoic acid chloride are added slowly, dropwise.

The mixture is stirred overnight and precipitated in 0.5 1 ether. The precipitated product is suctioned off, washed with ether, acetone, water, acetone and ether.

You achieve approx. 8 g of a white, fully substituted polymer (NMR check), which is, for example, soluble in dichloromethane and THF, in any case with a trace of DMSO or in methanol/dichloromethane mixtures.

Example 4

Preparation of polysuccinimide-co-α,B-(nonyl-carbonyloxyethyl)-D,L-aspartamide

Analogously to example 2, different polysuccinimide-co-α,B-(nonyl-carbonyloxyethyl)-D,L-aspartamides are prepared, among other things, with a composition of 70:30, 50:50 and 30:70 from polyanhydroaspartic acids of different molecular weight (MG = 7000; approx. .13000; 30000) and reacted with decanoic acid chloride, as described in example 5, to the corresponding polysuccinimide-co-α,B-(nonyl-carbonyloxyethyl)-D,L-aspartamides.

a) polysuccinimide-co-α,B-(nonylcarbonyloxyethyl)-D,L-aspartamide (70:30) from polyanhydroaspartic acid (MG = 7000); characterized by means of

NMR.

b) polysuccinimide-co-α,B-(nonylcarbonyloxyethyl)-D,L-aspartamide (70:30) from polyanhydroaspartic acid (MG = 14000); characterized by means of

NMR.

c) polysuccinimide-co-α,B-(nonylcarbonyloxyethyl)-D,L-aspartamide (70:30) from polyanhydroaspartic acid (MG = 30000); characterized by means of

NMR.

Example 5

20 g of polyanhydroaspartic acid-co-α,B-(nonylcarbonyloxyethyl)-D,L-aspartamide 70:30 (prepared as in example 6b) are dissolved in 400 ml of dichloromethane and sprayed into particles under the following conditions in a spray dryer ("Mini Spray Dryer Biichi 190 ", Biichi GmbH, Eislingen):

Application conditions

Yield: approx. 8 g

After the spray drying, the dichloromethane concentration in the particles amounts to less than 0.05%. The average size, shape and surface condition are characterized using raster electronic recordings; the lung accessibility of the particles is determined as in example 1.

100 mg of the prepared particles are suspended in a 1% "Haemaccell solution"

(Behring Werke AG, Marburg, Germany) and filtered through a sieve (10 µm). The filtrate is then lyophilized and used to determine lung availability.

In the scanning electron microscope, the produced particles have an average particle size of 2 µm and a smooth surface. Furthermore, these particles are lung accessible.

About. 15% of the particles are round hollow spheres, 50% of the particles are hollow spheres that exhibit a spherical entrapment, so that a hemispherical hollow sphere occurs. The shell thickness for these hemispherical hollow spheres can be described by the equation rm - rj = 2 d (d = shell thickness, rm = radius of the outer hemisphere), rj = radius of the inner, concave hemisphere) at maximum compression. When there is a non-ideal hemispherical shape, the radius is determined from the relative center point of the pinched one

the hollow ball.

About. 25% of the particles are hollow spheres with 2 to 5 differently sized inclusions.

Example 6

From polyhydroxybutyric acid "Type 159PHB" (ICI Biological Products Ltd.) is prepared according to a method by J.K. Embleton, B.J. Tighe (J. Microencapsulation, 1992, Vol. 9, No. 1, 73-87) particles by the so-called "double emulsion-solvent-evaporation process". The evaporation takes place at room temperature. The particles are fractionated by centrifugation and filtration through a 10 µm sieve. A particle proportion of 35% with concave surface segments in the filtrate is then recorded using scanning electron microscopy.

Likewise, the evaporation is carried out at 40°C and then it is fractionated. In this way, a fraction with a particle proportion of only 5% with concave surface segments is determined for comparison by means of scanning electron microscopy.

Example 7 (LMFG 50/ 50)

Preparation of poly(L-lysine methyl ester fumaric acid/L-lysine methyl ester glutaramide)

Copolymer 50:50 (LMFG 50:50)

0.7 g of glutaric acid dichloride and 0.84 g of fumaric acid dichloride are dissolved in 170 ml of CH 2 Cl 2 . This solution is added at room temperature with vigorous stirring to a solution of 2.91 g of L-lysine methyl ester and 3.0 g of Na 2 CO 3 in 120 ml of H 2 O. The immediately occurring polycondensation is interrupted after 15 minutes by the addition of 120 ml of 1 N aqueous HC1. When steam is introduced, the methylene chloride is then expelled. The hot, aqueous mixture is filtered and the solid product is washed several times with water.

Yield: 1.3 g (60% of theoretical) after vacuum drying (20 hours).

Example 8

2 g of polymer from example 7 are dissolved in 100 ml of DMSO and atomized at approx. 200°C inlet temperature in a "Mini Spray Dryer Biichi 190". The average size and surface condition are characterized using raster electronic recordings (see table 1). In any case, 50 mg of particles are suspended in 2 ml of suspension aid (1% "Haemaccel" in isotonic water) and then filtered with sieves (15 µm and 3 µm mesh size) and lyophilized.

Example 9

2 g of polyhydroxybutyric acid "Type 159 PHB" (ICI Biological Products Ldt.) are dissolved in 100 mL of furan-dichloromethane (9:1) and atomized at approx. 45°C in the "Biichi Mini Spray Dryer" into particles and further processed analogously to example 8.

Example 10

A solution of 2 g of polyhydroxybutyric acid "Type 159 PHB" (ICI Biological Products Ldt.) is dissolved in 100 ml of dichloromethane/MeOH (1:2), atomized at approx. 45°C in the "Biichi Mini Spray Dryer" into particles and further processed analogously to example 8.

Example 11

2 g of poly-e-caprolactone (Polysciences 19561) is dissolved to an extent of 2% in dichloromethane and atomized at room temperature in the "Biichi Mini Spray Dryer" into particles and further processed analogously to example 8.

The lung accessibility of the particles from Examples 8 to 11 is examined in vivo and determined as in Example 1.

The following table shows the results:

Claims (17)

1. Echogenic particles, containing a gas and a shaping substance or a substance mixture which is biodegradable and/or separable and which in the bloodstream of vertebrates can pass the lung, characterized in that (a) the surface of the echogenic particles is generally smooth and the shape of the echogenic particles contain none or 1 to 10 concave surface segments or the surface of the echogenic particles is non-smooth and the shape of the echogenic particles contains 1 to 10 concave surface segments, and that (b) the maximum deflection of the concave surface segment is from 30 to 90% of the smallest particle cross-section and that 10 to 60% of the echogenic particles exhibit at least one concave surface segment per particle. • 2. Echogenic particles according to claim 1, characterized in that the forming substance contains at least one polymer from the following group: polyesters of a-, B-, y- or e-hydroxycarboxylic acids; copolymers of the polyesters with each other or with polyglycolic acid; polyamides; polyamino acids; polyorthoesters; polycondensates containing 2,3-O-alkylidenetartaric acid, 2,3-O-alkylidene-L-threitol, furo-[2,5] groups or terephthalates; polyaminodicarboxylic acid co-imides; polyanhydrides; polymethylene malonate; gelatins; albumins or polysaccharides or at least one forming substance from the group of sugars; fatty acids; amino acids; alcohols with 12 to 20 carbon atoms or lipids. 3. Echogenic particles according to claim 1 or 2, characterized in that the forming substance contains at least one polymer from the following group: poly-s-caprolactone; polyhydroxybutyric acid; polylactic acid; polyhydroxyvaleric acid; polyglutamic acid; polyaspartic acid; polylysine; polysuccinimide-co-α,β-(butyloxycarbonyl-propionyloxyethyl)-D,L-aspartamide; polyanhydrides of sebacic acid and bis(p-carboxyphenoxy)propane; polylysine methyl ester fumaric acid co-glutaramide; dextrans; starch; basic or acidic derivatives of dextrans or starch; polyhydroxyethylaspartamide; bovine serum albumin; hydrophobically modified gelatins as well as at least one formative substance from the following group glucose; galactose; lactose; sucrose, fatty acids with 8 to 20 C atoms, proteinogenic amino acids, hydrophobic derivatives of proteinogenic amino acids, phospholipids or mixtures thereof. 4. Echogenic particles according to one or more of claims 1 to 3, characterized in that the shaping substance contains at least one polymer from the following group: polyamides; polycondensates; polyhydroxybutyric acid or polyaminodicarboxylic acid co-imides. 5. Echogenic particles according to one or more of claims 1 to 4, characterized in that the maximum deflection of the concave surface segment constitutes 50% to 90% of the smallest particle cross-section. 6. Echogenic particles according to one or more of claims 1 to 5, characterized in that they contain as gas air, nitrogen, a noble gas, hydrogen, carbon dioxide, oxygen or mixtures thereof. 7. Echogenic particles according to one or more of claims 1 to 6, characterized in that the particles are monolithic or exist as hollow bodies. 8. Echogenic particles according to claim 7, characterized in that the hollow body exhibits 2 to 10 concave surface segments. 9. Echogenic particles according to claim 7, characterized in that 30 to 40% of the echogenic particles exhibit at least one concave surface segment per particle. 10. Echogenic particles according to one or more of claims 1 to 5, characterized in that the surface exhibits 0 to 4, especially 1 to 3, disturbances per about<2>. 11. Echogenic particles according to claim 10, characterized in that at least 10%, especially 20 to 60%, of the echogenic particles exhibit 0 to 4, especially 1 to 3, disturbances per about<2>. 12. Method for producing echogenic particles according to one or more of claims 1 to 11, characterized in that the shaping substance or substance mixture is dissolved in a suitable solvent or a solvent mixture and the solution is subjected to spray drying. 13. Diagnostics or therapeutics, characterized in that it contains echogenic particles according to one or more of claims 1 to 11 or produced according to the method in claim 12, alongside pharmaceutically suitable and physiologically acceptable carriers and possibly further additional and/or auxiliary substances. 14. Process for producing a diagnostic or therapeutic according to claim 13, characterized by bringing echogenic particles according to one or more of claims 1 to 11 or produced according to claim 12 in a suitable diagnostic or therapeutic administration form using a pharmaceutically suitable and physiologically acceptable carrier and possibly further additives and/or auxiliaries. 15. Method according to claim 14, characterized in that the echogenic particles are filtered through a filter material with a mesh size of from 1 µm to 50 µm, in particular from 3 µm to 15 µm. 16. Use of the echogenic particles according to one or more of claims 1 to 11 or produced according to the method according to claim 12 for the production of a diagnosticum for visualizing the blood circulation or of organs that are supplied with blood. 17. Use of the echogenic particles according to one or more of claims 1 to 11 for the production of a diagnosticum for contrasting cavities in humans, animals or plants.