Ultrasonic contrast agent, its preparation and application as diagnostic or therapeutic.

0001The invention relates to ultrasound contrast media consisting of microparticles which contain a gas and polyaminodicarboxylic acid co-imide derivatives, processes for their production and their use as diagnostics and therapeutics.

0002Ultrasound diagnostics have found widespread use in medicine because of the simple, uncomplicated handling. Ultrasonic waves are reflected at interfaces from different types of tissue. The resulting echo signals are electronically amplified and made visible.

0003The visualization of blood vessels and internal organs using ultrasound generally does not allow the visualization of the blood flow contained therein. Liquids, especially blood, only provide ultrasound contrast if there are differences in density to the environment. As a contrast medium in medical ultrasound diagnostics, for example Gas-containing or gas-producing substances are used because the impedance difference between gas and surrounding blood is much greater than that between liquids or solids and blood (Levine RA, J Am Coll Cardiol 3: 28, 1989; Machi IJ CU 11: 3, 1983 ).

0004Several methods for producing and stabilizing gas bubbles are known in the literature. US Pat. No. 4,276,885 describes the production of gas bubbles of a defined size, which are surrounded by a gelatin shell which protects the gas bubbles from flowing together. The finished gas bubbles can only be stored in the frozen state, the gas bubbles having to be brought back to body temperature for use.

0005EP-A2-0 123 235 and 0 122 624 describe gases-containing ultrasound contrast media which consist of mixtures of surface-active substances with a solid in a liquid carrier. The ultrasound contrast media are produced by an elaborate grinding process using an air jet mill. The particles produced in this way only have a short service life because they quickly lose the enclosed gases.

0006EP-A2-0 224 934 describes ultrasound contrast media in the form of gas-filled gelatin or albumin hollow bodies. However, the use of foreign or denatured proteins from the body is disadvantageous because of the associated allergenic risk.

0007EP-A1-0 327 490 describes microparticles which consist of amyloses or synthetic, biodegradable polymers and a gas and / or a liquid with a boiling point of less than 60 ° C. Disadvantages of these polymers are their sticky consistency in water or blood, their poor biodegradability, their toxicity or the formation of toxic degradation products.

0008It has already been proposed (German patent application P 40 02 736.8) to use polyaminodicarboxylic acid co-imide derivatives as biodegradable depot preparations of medicaments with controlled release of active ingredients.

0009The object of the present invention was to develop ultrasound contrast agents based on microparticles which provide a clear contrast to the surrounding tissue, which are so small and stable that they reach the left half of the heart after intravenous application without substantial gas loss and essentially quantitatively, have good compatibility without allergenic potential, do not clump together in water or blood and can be produced quickly and easily.

0010Microparticles were produced from polyaminodicarboxylic acid co-imide derivatives (polydicarboxylic acid co-AHADS derivatives), which are surprisingly suitable as ultrasound contrast agents. Due to the incorporation of unopened imide rings (AHADS rings), the suspendability of the microparticles produced is particularly excellent in water. The microparticles do not have a sticky, greasy consistency in water-containing liquids and hardly collect together. The polymers form a pharmacologically inert matrix in which the gas is enclosed. In vivo these polymers are metabolized and excreted to non-toxic, non-allergenic and non-immunogenic compounds. Animal experiments have shown that the microparticles pass through the lungs essentially without significant gas loss and lead to an equally intense ultrasound contrast in both halves of the heart. The recorded echocardiograms show no wall movement disturbances during and up to 60 min after the application of the contrast medium. Furthermore, no changes were found in a six-channel ECG or in the contractility determined using a tip manometer.

0011The ultrasound contrast agents according to the invention bring about an improved increase in echogenicity in the myocardium and allow improved endocardium imaging. The following parameters can also be better assessed, for example: Chamber size, wall movement disorders, stroke volume, ejection fraction or intracavitary masses, eg thrombi or tumors. Furthermore, the ultrasound contrast agents according to the invention enable the evaluation of flow patterns in the case of valve insufficiency of the left and right half of the heart, intracardiac shunts, and the improved display of the large vessels in the case of congenital malformations. A massive amplification of the Doppler signal was also observed.

0012The invention thus relates to ultrasound contrast media, consisting of microparticles which contain a gas and a polyaminodicarboxylic acid co-imide derivative of the formula I,<chemistry id="chem0001" num="0001"><img file="EP0458079B1_D0001.tif" /></chemistry> in the<dl id="dl0001"><dt>n</dt><dd>1 or 2</dd><dt>x</dt><dd>1 up to 500</dd><dt>y</dt><dd>1 to 500, where</dd><dt>x + y</dt><dd>2nd is up to 1000 and</dd><dt>R</dt><dd>Means O-R¹ or NH-R², wherein R² H, (CH₂)_m-OR¹, (CH₂)_m-OC (O) -R¹ or (CH₂)_m-OC (O) -OR¹ means and m is 2 to 6 and R¹ is H, aryl, aralkyl, arylalkenyl, alkyl or C₃-C₈-cycloalkyl or a biologically inactive steroid alcohol or an amino acid, where aryl is unsubstituted or substituted by C₁-C₄-alkyl, C₂-C₄-alkenyl, C₁-C₄- Alkylcarbonyloxy, C₁-C₄-alkoxycarbonyl, C₁-C₄-alkoxy or hydroxy where the alkyl radicals mentioned for R¹ have 1 to 22 C atoms and the alkenyl radicals have 2 to 22 C atoms, which are not interrupted or interrupted by a carbonyloxy or oxycarbonyl group, where the repeat units in square brackets are randomly and / or distributed in blocks in the polymer and where both the repeat units labeled x and y are identical or different, and where the Amino acids are α- and / or β-linked contain.</dd></dl>

0013Aryl is understood to mean aromatic hydrocarbons such as phenyl and naphthyl, especially phenyl. In the substituted aryl radicals given, 1 to all replaceable hydrogen atoms have been replaced by identical or different substituents. The aryl radicals are preferably mono- or disubstituted. The alkyl and alkenyl radicals mentioned can be either straight-chain or branched. The biologically inactive steroid alcohols are preferably bound via their OH group. A preferred steroid alcohol is cholesterol. The amino acids mentioned for R 1 are preferably naturally occurring amino acids such as Tyr, Ala, Ser or Cys, particularly preferably Tyr and Ala. They can be bound via their NH₂ as well as their COOH function.

0014The invention also relates to processes for the production of gas-containing microparticles which consist of or contain the abovementioned polymers and their use also in admixture with other, biocompatible and / or biodegradable polymers or physiologically acceptable auxiliaries, for diagnostic or therapeutic processes.

0015The invention further relates to diagnostics or therapeutic agents, consisting of at least one of the contrast media mentioned above.

0016Furthermore, the invention also relates to methods for the production of diagnostic or therapeutic agents, which are characterized in that the above-mentioned ultrasound contrast agents are brought into a suitable dosage form with a physiological carrier and, if appropriate, further additives and / or auxiliary substances.

0017The invention is described in detail below.

0018Aspartic acid and / or glutamic acid are used as aminodicarboxylic acids, which react in a polycondensation reaction to give the corresponding polyimides (polyanhydroaminodicarboxylic acids, formula II). By partial reaction with one or more compounds of the formulas III and / or IV and / or NH₃         HO-R¹ (III)         H₂N- (CH₂)_m-OH (IV), wherein m and R 1 are as defined above for formula I, an α, β-poly-D, L-amino acid ester co-imide of the formula VIII is obtained<chemistry id="chem0002" num="0002"><img file="EP0458079B1_D0002.tif" /></chemistry>

0019It is essential in this reaction that the polyanhydroaminodicarboxylic acid (II) is only partially converted into the open-chain derivatives. The proportion of unopened anhydroaminodicarboxylic acid units is 0.1 to 99.9%, preferably 10 to 90% (the percentages relate to the total number of repeat units in the total polymer). Depending on which side the imide ring is opened in the reaction described above, α- or β-linked amino acids. Compounds of the formulas III and IV which are preferably used are: 2-aminoethanol, 3-aminopropanol, 2-aminopropanol, alcohols having 1 to 18 carbon atoms, in particular methanol, ethanol, isoamyl alcohol and isopropyl alcohol.

0020A process for the preparation of α, β-poly- (2-hydroxyethyl) -DL-aspartimide (PHEA) (formula I; y = O; R = NH-CH₂-CH₂-OH) is by P. Neri, G. Antoni , F. Benvenuti, F. Cocola, G. Gazzei, in J. Med. Chem. Vol. 16, 893 (1973). A general working procedure for the production of PHEA can be found in P. Neri, G. Antoni, Macromol. Synth. Vol. 8, 25. Reference is expressly made to this reference at this point. The conversion takes place in high yield to a product with a high degree of purity. In the same way, the substoichiometric use of NH₃ and / or compounds of the formulas III and / or IV, the analog poly-aspartic acid derivative-co-succinimide compounds of the formula VIII (n = 1) can be prepared.

0021A different, more elaborate process must be used to prepare pure poly (hydroxyalkyl) -L-glutamine, as described in US Pat. No. 4,356,166. The γ-standing COOH group of L-glutamic acid is initially protected by esterification with benzyl alcohol. This γ-benzyl glutamate is then reacted with phosgene to form an N-carboxy anhydride, which is then polymerized after the addition of triethylamine in an inert solvent, giving poly-γ- (benzyl) -L-glutamate. The protective group is split off either by adding an HCl / HBr mixture to the free poly-α-L-glutamic acid or in the presence of hydroxyalkylamines to the analogous poly-α- (hydroxyalkyl) -L-glutamines. A general procedure for the production of poly-α- (hydroxypropyl) -L-glutamine can be found in US Pat. No. 4,356,166, to which reference is expressly made here. In the same way, the analogous compounds of the formula VIII (n = 2) can also be prepared by using NH₃ and / or compounds of the formulas III and / or IV.

0022Compared to the complex presentation of pure polyglutamic acid and its derivatives, glutamic acid can be incorporated up to high proportions in the simple condensation of aspartic acid using phosphoric acid to polyanhydroaspartic acid-co-glutamic acid.

0023The polyamino acid amide co-imides of the formula VIII (R ′ = HN- (CH₂)_m-OH) can now, if necessary, in the following reaction step with one or more different, biologically inactive compounds of the formula V and / or VI and / or VII         X-R1 (V)<chemistry id="chem0003" num="0003"><img file="EP0458079B1_D0003.tif" /></chemistry> to be converted to further polyaminodicarboxylic acid-co-AHADS derivatives. Here X stands for a leaving group, which enables a gentle esterification of the polymer alcohol group. Chlorine, bromine, iodine, imidazolides, anhydrides or hydroxyl, in particular chlorine, are preferred.

0024The reaction with the compounds of the formula type V, VI or VII can take place either with a single such compound or with any combination of these compounds or also with compounds which have different radicals R 1, for example in the nature of their branching, in particular in their chain length, different .

0025The latter polymer-analogous alkylation or acylation is carried out according to known methods in organic chemistry. It runs selectively on the hydroxyl function (formula VIII, R '= HN- (CH₂)_m-OH) to ethers, esters or carbonates, without attacking further functions on the starting polymer. The unicorn variant of Schotten-Baumann acylation in the presence of pyridine is particularly suitable. Under mild conditions, very high degrees of derivatization (greater than 70%) are achieved.

0026The molecular weight of the polymers is 200 to 100,000, preferably 3,000 to 70,000.

0027Compounds of formula type V are commercially available or, if not, can be synthesized in a simple manner by methods known from the literature.

0028The chloroformic acid esters (formula VII) are obtained by reacting phosgene with the corresponding biologically inactive, physiologically acceptable, aromatic, araliphatic, aliphatic or cycloaliphatic, in particular unbranched alcohols. Alcohols which have an even number of carbon atoms are particularly preferably used. The chloroformylated steroids are also obtained in this way. In principle, all biologically inactive steroids with reactive hydroxyl groups are accessible. Examples include: cholesterol, cholestanol, coprostanol, ergosterol, sitosterol or stigmasterol.

0029The acid chlorides (formula VI) which can also be used are obtained, for example, from the corresponding carboxylic acids by reaction with phosphorus trichloride, phosphorus pentachloride, oxalyl chloride or thionyl chloride.

0030Compounds of formula type V, VI or VII, in which an alkyl chain is interrupted by an oxycarbonyl or carbonyloxy group, are prepared, for example, by reacting cyclic dicarboxylic anhydrides with alcohols. The dicarboxylic acid monoesters obtained in this way are then reacted analogously to the carboxylic acids described above, for example with oxalyl chloride, to give the corresponding acid chlorides.

0031An advantageous method for producing the ultrasound contrast agents is to dissolve one or more of the polyaminodicarboxylic acid co-imide derivatives of the formula I in a solvent or solvent mixture with a high melting point, or to dissolve these derivatives with one or more further polymers and / or physiologically acceptable Mix excipients and dissolve in a solvent or solvent mixture with a high melting point and in a condensed cold gas, for. B. liquid nitrogen to drip. The Leidenfrost phenomenon creates absolutely round particles. Examples of solvents which can be used are alcohols, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, methylene chloride, dioxane, acetonitrile or mixtures with alcohols. The high-melting and water-miscible solvent is, for. B. dissolved by transferring the microparticles into water and the polymer precipitated in the process, the spherical shape of the microparticles being retained.

0032If, in addition to a high melting point, the organic solvent used also has a low boiling point, this dropletization process can be further simplified by the solvent, for example tert. Butanol, can be gently removed directly using freeze drying.

0033Another method for producing the ultrasound contrast media is to dissolve one or more of the polyaminodicarboxylic acid co-imide derivatives of the formula I in a solvent or solvent mixture and, if appropriate, to precipitate them after adding another solvent and / or one or more other polymers or in To disperse water. Other suitable polymers are, for example, polyvinyl alcohol (®Mowiol 28-99) or polyoxyethylene polyoxypropylene (®Pluronic F 127). As a further solvent z. B. ether can be used. Microparticles with a diameter of 0.5 to 15 microns are obtained by vigorous stirring, for. B. with a mixer (25000 rpm). The solvents are then z. B. removed by lyophilization.

0034A particularly advantageous method is to obtain the microparticles by spray drying. For this purpose, one or more polyaminodicarboxylic acid co-imide derivatives of the formula I are dissolved or these derivatives are mixed with one or more other polymers and / or physiologically acceptable auxiliaries and brought into solution. Examples of suitable solvents or solvent mixtures are alcohol, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, methylene chloride, dioxane or acetonitrile. The solution is then sprayed into microparticles in a spray dryer.

0035In the process described, the polymers of the formula I can be used alone or as a mixture of different polymers of the formula I. These polymers can also be used in blends with other biodegradable and / or biocompatible polymers (e.g. ®Pluronic F68, PHEA, dextrans, polyethylene glycols, hydroxyethyl starch and other degradable or excretable polysaccharides) or physiologically acceptable additives (e.g. polymer plasticizers) .

0036The microparticles contain gas, for example air, nitrogen, noble gases such as helium, neon, argon or krypton, hydrogen, carbon dioxide, oxygen, or mixtures thereof. The microparticles are loaded with a gas, for example by storing the microparticles in an appropriate gas atmosphere after the lyophilization or by directly spraying them in a corresponding gas atmosphere during production.

0037Before application, the ultrasound contrast agents according to the invention are converted into a suitable diagnostic or therapeutic administration form by adding one or more physiologically acceptable carriers and, if appropriate, further additives and / or auxiliaries. The ultrasound contrast media are suspended, for example, by adding water and mixing before application.

0038The physiological isotonicity of the particle suspension can be produced by adding osmotically active substances, for example table salt, galactose, glucose, fructose.

0039In the described processes for producing the ultrasound contrast media according to the invention, particle sizes can be achieved in which 90% of the particles are between 0.1 μm and 15 μm. The spray drying process can be used to achieve particle size distributions in which 90% of the particles are smaller than<b>3</b> µm are. Larger particles are removed by sieving, for example using a 15 μm sieve cloth and / or 3 μm sieve cloth. When these microparticles are used as ultrasound contrast agents for the diagnosis of cardiovascular diseases, particle sizes from 0.1 µm to 7 µm have proven themselves, particle sizes from 0.1 µm to 3 µm are advantageously used. The ultrasound contrast agents are injected into the bloodstream, for example. 0.1 mg to 1000 mg of the microparticles, preferably 1 mg to 100 mg, are used per injection.

0040The ultrasound contrast agents described above can be used for both diagnostic and therapeutic procedures. The use of the ultrasound contrast agents according to the invention is not only limited to the visualization of the blood flow in the right ventricular part of the blood circulation after venous application. The ultrasound contrast agents can be used with excellent success for the examination of the left side of the heart and the myocardium. It is also possible to visualize other organs supplied with blood, such as the liver, spleen, kidney or brain, with these contrast media.

0041However, the ultrasound contrast media according to the invention are also suitable for making cavities in humans, animals or plants visible, for example urinary bladder, ureter, uterus or vagina.

0042The invention is described in detail in the following examples. Percentages relate to the weight, unless stated otherwise.

example 1

0043Preparation of polysuccinimide-co-α, β- (hydroxy-ethyl) -D, L-aspartamide (70:30)

004410th g (103 mmol) of polyanhydroaspartic acid are dissolved in about 40 ml of N, N-dimethylformamide (DMF), if necessary with gentle heating. 1.83 g (30 mmol) of freshly distilled 2-aminoethanol are added dropwise to this solution and the mixture is stirred at room temperature overnight. The reaction mixture is precipitated in butanol and washed several times with dried acetone. Drying takes place in a vacuum at an elevated temperature. Almost 100% of the white, water-soluble product is produced and is checked by NMR spectroscopy for residues of DMF and butanol. The molar ratio of polyanhydroaspartic acid to aminoethanol used corresponds approximately to the copolymer composition.

Example 2

0045Preparation of n-butyl 4-chloro-4-oxobutyrate

0046Excess thionyl chloride and a drop of DMF are added to succinic acid monobutyl ester. The reaction takes place with evolution of gas. The mixture is left to stir overnight with the exclusion of moisture and then the excess thionyl chloride is distilled off at normal pressure. The remaining crude product is fractionally distilled at 0.05 mbar and the pure product is recovered at approx. 70 ° C. In IR spectroscopic characterization, the product has bands of 1800 cm⁻¹ (acid chloride) and 1740 cm⁻¹ (ester) of the same intensity.

Example 3

0047Preparation of polysuccinimide-co-α, β- (butyloxycarbonylpropionyloxyethyl) -D, L-aspartamide (70:30)

00486 g of polysuccinimide-co-α, β- (hydroxyethyl) -D, L-aspartamide (= 16 mmol of hydroxyethyl groups), shown as described in Example 1, are dissolved in 100 ml of dry N, N-dimethylformamide (DMF). After adding 4 g (50 mmol) of pyridine, the mixture is cooled to 0 ° C. and 4.8 g (25 mmol) of n-butyl 4-chloro-4-oxobutyrate (see Example 2) are added over the course of 15 minutes. The mixture is stirred overnight and precipitated in 0.5 l of ether. The precipitated product is filtered off, washed with ether, acetone, water, acetone and ether. About 8 g of a white polymer with a degree of substitution of approximately 100% are obtained (can be checked by NMR spectroscopy). The resulting polymer is e.g. B. in acetonitrile with a trace of dimethyl sulfoxide (DMSO), soluble in DMSO or DMF.

Example 4

0049Preparation of polysuccinimide-co-α, β- (nonylcarbonyloxyethyl) -D, L-aspartamide (50:50)

00506 g of a polysuccinimide-co-α, β- (hydroxyethyl) -D, L-aspartamide (50:50) (≙ 24 mmol hydroxyethyl groups), which analogously to Example 1 from polyanhydroaspartic acid (MW = 14000) and 2-aminoethanol (molar ratio 2 : 1) was prepared, dissolved in 100 ml of dry DMF, mixed with 8 g (100 mmol) of dry pyridine and cooled to 0 ° C. 9.6 g of distilled decanoic acid chloride are slowly added dropwise and the procedure is continued as in Example 3. About 8 g of a white, fully substituted polymer (NMR control) is obtained, which, for. B. is soluble in dichloromethane and THF, each with a trace of DMSO or in methanol / dichloromethane mixtures.

Example 5

0051Production of polysuccinimide-co-α, β- (nonylcarbonyloxyethyl) -D, L-aspartamide of different copolymer compositions and different molecular weights

0052Analogously to Example 1, various polysuccinimide-co-α, β- (hydroxyethyl) -D, L-aspartamides, inter alia with the composition 70:30, 50:50 and 30:70, were obtained from polyanhydroaspartic acids of different molecular weights (MW = 7000; approx. 13000; 30000 ) prepared and reacted with decanoic acid chloride, as described in Example 4, to give the corresponding polysuccinimide-co-α, β- (nonylcarbonyloxyethyl) -D, L-aspartamides.<ul id="ul0001" list-style="none"><li>a) - polysuccinimide-co-α, β- (nonylcarbonyloxy-ethyl) -D, L-aspartamide (70:30) from polyanhydroaspartic acid (MW = 7000); characterized by NMR</li><li>b) - polysuccinimide-co-α, β- (nonylcarbonyloxy-ethyl) -D, L-aspartamide (70:30) from polyanhydroaspartic acid (MW = 14000); characterized by NMR</li><li>c) - polysuccinimide-co-α, β- (nonylcarbonyloxy-ethyl) -D, L-aspartamide (70:30) from polyanhydroaspartic acid (MW = 30000); characterized by NMR</li><li>d) - polysuccinimide-co-α, β- (nonylcarbonyloxy-ethyl) -D, L-aspartamide (30:70) from polyanhydroaspartic acid (MW = 12000); characterized by NMR</li></ul>

Example 6

0053Preparation of polysuccinimide-co-α, β- (octyloxycarbonyloxyethyl) -D, L-aspartamide (70:30)

00546 g Polysuccinimide-co-α, β- (hydroxyethyl) -D, L-aspartamide (70:30) (≙ 16 mmol hydroxyethyl groups), prepared as described in Example 1 from polyanhydroaspartic acid (MW = 37000) and aminoethanol, are analogous to Example 3 reacted with 4.8 g (25 mmol) of octyl chloroformate and worked up accordingly. About 8 g of a white, fully substituted polymer is obtained which is soluble in THF or methanol / dichloromethane mixtures.

Example 7

0055Preparation of polysuccinimide-co-α, β- (nonylcarbonyloxyethyl) -co-α, β- (hydroxyethyl) -D, L-aspartamide (60:20:20)

00566 g polysuccinimide-co-α, β- (hydroxyethyl) -D, L-aspartamide (60:40) (≙ 20 mmol hydroxyethyl groups), which was prepared analogously to Example 1 from polyanhydroaspartic acid and 2-aminoethanol (molar ratio 6: 4), are reacted analogously to Example 3 with 2.3 g of decanoic acid chloride (≙ 12 mmol). Due to the incomplete conversion (relatively small excess of acid chloride), only half of the free OH groups are esterified. About 7 g of a white polymer are formed. Microparticles of this substance show a firm consistency in water and are easily suspended.

Example 8

0057Preparation of polysuccinimide-co-α, β- (oleyl-oxyethyl) -D, L-aspartamide (10:90)

00586 g of polysuccinimide-co-α, β- (hydroxyethyl) -D, L-aspartamide (10:90) (≙ 40 mmol of hydroxyethyl groups), shown analogously to Example 1 with a molar ratio of polyanhydroaspartic acid to 2-aminoethanol such as 1: 9 reacted with 20 g of distilled oleic acid chloride as in Example 3. The heterogeneous reaction mixture becomes homogeneous by adding dichloromethane. It is precipitated twice in methanol, which is cooled to -20 ° C. The yellowish colored polymer is thermoplastic.

Example 9

0059a) Production of microparticles

006040 mg of polysuccinimide-co-α, β- (nonylcarbonyloxy-ethyl) -D, L-aspartamide (50:50) from Example 4 are dissolved in 1 ml of methylene chloride / methanol (50/1 by volume). The solution is introduced with stirring (800 rpm) into a beaker with 60 ml 0.1% by weight aqueous polyvinyl alcohol solution (®Mowiol 28-99), which is mixed with 0.3 ml methylene chloride / methanol (50/1 ) is saturated. At the same time, the solution is finely dispersed using a mixer (25000 rpm).

0061After 5 minutes, the contents are placed in a beaker with 200 ml of water and stirred for 30 minutes (200 rpm). The supernatant water is decanted off and the microparticles are lyophilized (diameter after lyophilization: 0.5 to 15 μm).

Example 10

0062Manufacture of microparticles

006380 mg of polysuccinimide-co-α, β- (octylcarbonyloxy-ethyl) -D, L-aspartamide (70:30) from Example 6 are dissolved in 1 ml of dimethyl sulfoxide at 50 ° C. and 20 mg of hydroxypropyl cellulose (®Klucel M.) are added . The solution of the two polymers is dripped into a reservoir of liquid nitrogen (100 ml) using a cannula (disposable syringe, cannula diameter outside 0.6 mm).

0064The resulting microparticles are transferred to 200 ml of water and extracted from residual solvent for 2 hours. Excess water is decanted off and the microparticles are lyophilized (diameter after lyophilization: 1-2 µm).

Example 11

0065Manufacture of microparticles

00664 g each of polysuccinimide-co-α, β- (octyloxycarbonyloxyethyl) -D, L-aspartamide (A) (Example 6) and polysuccinimide-co-α, β- (nonylcarbonyloxyethyl) -D, L-aspartamide (B) ( Example 5 d) are dissolved in 2% in the solvents listed in Table 1. The polymers are then sprayed into microparticles in a spray dryer (Mini Spray Dryer Büchi 190, Büchi, W-Germany).<tables id="tabl0001" num="0001"><img file="EP0458079B1_D0004.tif" /></tables>

0067The size distribution of the microparticles was determined in a Cilas 715 granulometer.

006830 mg portions of the microparticles prepared above are dispersed in 1.5 ml of suspension aid. The suspension aids consist of 150 mg of Dextran 40 (Roth, W.-Germany), 7.5 mg of polysorbate and 13.5 mg of NaCl in 1.5 ml of distilled water. The suspensions are filtered with sieve fabrics (15 µm and 3 µm mesh size) and then lyophilized. The microparticles are suspended with water before application.

Example 12

0069Echocardiographic examination in dogs

007030th mg microparticles (prepared according to Example 11, substance B, CH₂Cl₂ / methanol 2: 3 (vol.)) are resuspended in 1.5 ml of distilled water using a glass rod. This suspension is injected into a peripheral vein using an injection syringe. An ultrasound head of an ultrasound device (Toshiba, FSH 160a, Japan) is held against the thorax of the test animal, so that a typical cross section through the right and left heart is obtained. As soon as the ultrasound contrast medium reaches the right half of the heart, it can be seen on the monitor of the ultrasound device how the blood marked by the contrast medium reaches the right atrium, then the right ventricle and then the heart via the pulmonary artery. After passing through the lungs, the left half of the heart can be recognized by the contrast medium. The ultrasound contrast before and after the lung passage is of the same intensity, so that one can assume that the air remains essentially completely in the polymers and that the microparticles are transported to the left half of the heart essentially without loss.