CA2137910C

CA2137910C - Use of microcapsules as contrasting agents in colour doppler sonography

Info

Publication number: CA2137910C
Application number: CA002137910A
Authority: CA
Inventors: Michael Reinhardt; Thomas Fritzsch; Dieter Heldmann; Joachim Siegert
Original assignee: Schering AG
Current assignee: Acusphere Inc
Priority date: 1992-06-13
Filing date: 1993-04-24
Publication date: 2003-06-03
Anticipated expiration: 2013-04-24
Also published as: EP0644776A1; NO944806L; GR3029494T3; NO944806D0; DE4219724A1; DK0644776T3; ES2127277T3; NO308510B1; EP0644776B1; ATE173937T1; WO1993025241A1; CA2137910A1; JPH07507778A; DE59309189D1

Abstract

The invention concerns the use of gas-filled or liquid-filled microcapsules as ultrasonic contrasting agents to contrast regions of the body in which substantially no microcapsule movement is observed, the examination being carried out using colour Doppler sonography.

Description

The Use of Microcapsules as Contrasting Agents in Colour Doppler Sonography This patent application relates to the object described in the patent claims, i.e., the use of microcapsules to contrast the reticuloendothelial system, the myocardium and/or the intestinal tract and other body cavities by using colour Doppler sonography.
Various recording techniques, such as, for example, M-mode, B-mode, CW-Doppler, PW-Doppler and colour Doppler technology are used in the domain of ultrasound diagnostics, depending on the problem that is to be solved. M-mode and B-mode techniques are based on the principle the ultrasound waves in the Megahertz range (above 2 Megahertz with wavelengths between 1 and 0.2 nm) are reflected at the boundary surfaces of media that are of different acoustic densities. The resulting echoes are amplified and rendered visible. In principle, the method is suitable for showing different organs and for imaging moving structures, such as, for example, cardiac valves.
In contrast to the foregoing, Doppler techniques are used exclusively for examining moving structures, e.g., for determining the velocity of blood flow. The measurement principle underlying these techniques is based on the fact that in the ultrasound domain, the signals that are reflected on moving bodies (e.g., red blood cells) undergo a frequency shift (the so-called Doppler shift). In colour Doppler sonography, this frequency shift is converted into colour signals, so that structures that move are colour coded, whereas fixed structures are represented as grey-scale images [R. Omoto (1987) Real-time two-dimensional Doppler echocardiography, 2nd Ed., Lea & Febiger, Philadelphia] .
Common to both Doppler techniques and to the other known techniques, such as, for example, B-mode and M-mode, is the fact that the contrasting and thus the diagnostic pronouncement are enhanced if there are fine gas bubbles at the site that is to be examined (see Gramiak, Invest. Radiol 3 (1968) , 356/366) .
These gas bubbles can be generated by the most varied methods, e.g., by vibration or other agitation of physiologically compatible solutions (EP 0 077 752, Roelandt, J. Ultrasound. Med. Biol. 8: 471-492, 1982).
As described in EP 0 052 575, gas bubbles are generated in that a solid crystalline substance, for example, galactose, are suspended in a carrier fluid. When this is done, the gas bubbles are formed from gas that is enclosed in hollow cavities or adsorbed on the surface of the crystals.
EP 0 122 624 describes a similar contrasting agent based on a substance, such as galactose, that is not active on the boundary surfaces, which is added to a substance such as magnesium stearate that is active on the boundary surfaces; this results in stabilization of the gas bubbles.
This means that it is also possible to contrast the left-hand side of the heart, as well as different organs such as the liver, spleen, and kidneys in a 2D echo image or in an M-mode echo image.
DE 38 -3 972 discloses gas-filled or liquid-filled microcapsules that are based on biodegradable polymers, such 21~'~9~0 as, for example, polycyanacrylates or a-, b-, or g-hydroxycarboxylic acids.
Similar microcapsules are described in European Patent Application EP 0 441 468. In contrast to the particles disclosed in DE 38 03 972, in this case the particle shell is built up of polyaldehydes.
EP 0 224 934 and US 4,276,885 describe gas-filled microcapsules based on proteins or albumins (EP) or based on gelatine (US). Common to the particles described in DE 38 03 972 and in EP 0 441 468 is the fact that they are sufficiently stable and small enough (< lOmm) to pass through capillaries and for intracellular absorption in the reticulo-endothelial system (e. g., the liver, lymph nodes, and spleen). However, if B-mode or M-mode technique is used, contrasting is not satisfactory in all cases, e.g., it is often impossible to define healthy tissue (e.g., the liver, spleen, lymph nodes) as opposed to tumour tissue that contains only a few cells that belong to the reticulo-endothelial system (hereinafter: RES).
In addition to this, representation of the gastro-intestinal tract and perfusion of the myocardium presents some difficulties.
It is the task of the present invention to improve contrasting of the areas named above.
It has been found, most surprisingly, that if specific ultrasound contrasting agents are administered, even if conventional colour Doppler sonography is used, contrasting can be obtained even in areas of the body a.n which there is no movement of particles, which is to say in the RES

(spleen, liver, and lymph nodes), in the gastrointestinal tract, and in the myocardium, which is a contradiction of theoretical predictions.
The expression "no movement of particles" is to be understood to mean particle velocities that are null or significantly less than the flow velocities that occur on perfusion of the blood vessels. Such velocities are considered to be insufficient to generate a colour Doppler signal.
Consequently, the present invention relates to the use of contrast agents that consist of microcapsules in order to contrast areas of the body in which no movement of the micro-capsules can be distinguished, albeit recording is effected using colour Doppler technique.
Thus in one aspect, the invention provides use of gas-or liquid-filled microcapsules as an ultrasound contrasting agent for contrasting regions of the body by means of colour Doppler sonography in which there is substantially no observable movement of the microcapsules.
It was also surprising that the contrasting so achieved is better than that which can be achieved by using conventional methods (B-mode, M-mode). This leads to a significant improvement in the value of the diagnostic pronouncement.
As an example, during examination of the liver tissue using colour Doppler sonography, after intravenous injection of the contrasting agent, images of tumour areas (which, as is known, contain no or only a few cells capable of absorbing the particles) were formed in the usual grey values; in contrast to this, healthy tissue was, most surprisingly, shown in colour (see Illustration l, right-hand half of the image).
A comparison photograph in B-mode, which was taken under otherwise identical conditions (see I17_ustration 1, left-hand half of the image), confirms the clearly improved diagnostic value of the colour Doppler photograph. Tumour areas cannot be distinguished from the healthy areas.
After oral application, it is possible to mark the gastric canal, and this makes it possible to distinguish the intestinal structures from other abdominal organs.
Imaging a joint space is possible after administration of a contrasting agent and by using Doppler techniques, as is imaging of the tubes and the cavum uteri.
As a rule, suitable microcapsules consists of a thin-walled shell, e.g., a synthetic, biodegradable polymer material, and a nucleus that is of a gas or a low-boiling point liquid.
Suitable gases are, for example, air, nitrogen, oxygen, gaseous hydrocarbons, noble gases, and carbon dioxide.
Suitable low-boiling point liquids are 1,1-dichlorethylene, 2-methyl-2-butene, 2-methyl-1,3-butadiene, 2-butin, 2-methyl-1-butene, dibrom-difluormethane, furane, 3-methyl-1-butene, isopentane, diethylether, 3,3-dimethyl-1-butin, propylene oxide, bromethane, pentane, cyclopentane, 2,3-pentadiene, cyclopentane, and mixtures of these.
Suitable shell materials are, for example, biodegradable synthetic material, such as polyaldehydes, which, if desired, can contain additives and/or cross-linkers that are capable of copolymerization, optionally tensides or mixtures of tensides, coupling agents and, optionally, biomolecules or macromolecules that are bonded through these coupling agents, polycyanacrylate or polyester of a-, b-, or g-hydroxycarboxylic acids.

Suitable shell materials for gas-filled microcapsules are gelatins or proteins such as, for example, partially denatured albumins or human serum albumin.
The particles can be of any shape (e. g., spherical, ellipsoid, and the like); the wall thickness of the particles is best selected to be as thin as possible, and is preferably < 200 nm.
The particle, size is limited by the particular application that is intended; for examination of the RES and the myocardium, it should be between 1 and 10 um in order to ensure that they can pass through the capillaries, and a particle size of 100 um is suitable for examinations of body cavities and of the gastrointestinal tract.
Agents of this kind,,which are to be used according to the present invention, are described in European Patent Application 0 441 468, in European Patent 0 224 934, and in US Patent 4,276,885.
According to the present invention, gas-filled microcapsules based on polycyanacrylates are preferred.
A new, preferred, manufacturing process for these particles is such that monomer cyanacrylate is dispersed by a rotor-stator mixer in an acid aqueous solution saturated with a gas or gas mixtures, which optionally contains at least one surface active substance; after 5 minutes to 3 hours of dispersion, the particles that are obtained are separated off, optionally washed with water, and then taken up in a pharmaceutically acceptable suspension medium and freeze dried, when the suspension is advantageously moved energetically during the. freezing process. It is preferred that butylester be used as the cyanacrylate; and air, nitrogen, oxygen, noble gases, or carbon dioxide be used as the gas. Comparable apparatuses (such as a dissolver-stirrer, for example) that permit vigorous dispersal can be used in place of the rotor-stator mixer. It is preferred that (a) substances) from the polysorbate group, octylphenols or nonylphenols, macrogol-glycerolester or cetomacrogols or poloxamers~ or mixtures thereof be used as the surface active substance. The pH of the aqueous gas-saturated solution is preferably between 1.8 and 4.5;
hydrochloric acid and phosphoric acid are particularly well suited for adjusting the Ph. The particles are separated off by centrifuging or floatation. A suitable suspension medium is water as used for injection purposes, optionally with added cooking salt and/or glucose and/or mannitol and/or lactose, which can optionally also contain a surface active substance from the polysorbate group, octylphenols or nonylphenols, macrogol-glycerolester or cetomacrogols, or substances from the polox-amer° group, or mixtures thereof, and/or a polyvalent alcohol.
A new, preferred manufacturing process for particles based on polyesters is such that a polyester of an a-, b-, or g-hydroxycarboxylic acid, optionally together with a water-dispersible emulsifier, is dissolved in an hygienically harmless solvent, and this solution is added, during dispersal with a dissolver-stirrer or a sonic bar, to a liquid that contains a gas, and which, insofar as the emulsifier is not added with the polyester, contains a water-dispersible emulsifier; the particles that are obtained after 30 minutes to 2 hours dispersal are separated off, optionally washed with water, and then taken up in a pharmaceutically acceptable suspension medium and freeze dried. According to the present invention, polymers of lactic acid or glyoxal acid and mixed polymerisates of these are preferred. It is preferred that heated ethylalcohol be used as the harmless solvent. It is preferred that water or glycerol 87o be used as the liquid that contains gas, the preferred gases being air, nitrogen, oxygen, noble gases, or carbon dioxide. Phosphatidycholin or sucrose-palmitate-stearate 15, and mixtures of these, are suitable water-dispersible emulsifiers. The same media as in the case of particles based on polycyanacrylate are pharmaceutically acceptable suspension media.
The following particle concentrations have to be administered in order to arrive at the required concentration of 105 - 10' particles/cm3 of target organ:
10$ - 101° particles/kg body weight when examining the gastro-intestinal tract;
10' - 109 particles/kg body weight when examining the lymph system;
10' - 109 particles/kg body weight when examining the RE
system and the myocardium;
The concentration of contrasting agent that is administered can be in the range up to 2 x 1010 particles/ml.
There will have to be varying amounts of time between the application to the beginning of the examination in order to achieve the desired results:
seconds when examining the myocardium;
1 - 60 minutes when examining the RE system;
g _ - 10 minutes when examining the lymph system;
5 - 60 minutes when examining the stomach-intestine.
In the case of body cavities and the bladder, the examination can proceed immediately after application of the contrasting agent.
The present invention will be described below in the basis of the following examples:
Example 1 0.4 ml cyanacryl acid butylester are dispersed for five minutes with a rotor-stator mixer in 60 ml hydrochloric acid at pH 2.0 that contains 1% Poloxamer 407. The microparticles with an average size of 2mm are centrifuged off and taken up in 300 ml of an aqueous solution of 1%
poloxamer and 5% glucose. Determination of density resulted in a specific weight of 0.2 g/cm3.
example 2 The same procedure as in Example 1 was followed, although the hydrochloric acid has a pH of 2.5 and the Poloxamer 407 is replaced by octoxynol-9. The microparticles are of an average size of approximately 0.9mm and a specific weight of 0.2g/cm3. They are taken up in 300 ml 5-% mannitol solution that contains 0.1% polysorbate 20.

Example 3 The same procedure as in Example 1 was followed, although the hydrochloric acid has a pH of 3.0 and the Poloxamer 407 is replaced by cetomacrogol 1200. The microparticles are of an average size of approximately l.5mm and a specific weight of 0.3g/cm3. They are taken up in 300 ml 5-% mannitol solution that contains 0.1% cetomacrogol 1200 and 5%
povidone.
Example 4 The same procedure as in Example 1 was followed, although the Poloxamer 407 is replaced by 5% polysorbate 40. The microparticles are of an average size of approximately l.Omm and a specific weight of 0.4g/cm3. They are taken up in 300 ml 5-% mannitol solution that contains 1% macrogolglycerol hydroxy-stereate.
Example 5 The same procedure as in Example 1 was followed, although the Poloxamer 407 was replaced by 5% macrogolglycerol hydroxy-stereate. The microparticles are of an average size of approximately 0.9mm and a specific weight of 0.3g/cm3.
They are taken up in 300 ml 5-% mannitol solution that contains to macrogolglycerol hydroxy-stereate and 100 propyleneglycol.

Example 6 The same procedure as in Example 1 was followed, although the cyanacryl acid propylester is replaced by cyanacryl acid ethyl ester. The microparticles are of an average size of 1.5mm and a specific weight of 0.2g/cm3. They are taken up in 300 ml of an aqueous solution of 1% Poloxamer 407 and 5%
glucose.
Example 7 The same procedure as in Example 1 was followed, although the cyanacryl acid butylester is replaced by cyanacryl acid isopropyl ester. The microparticles are of an average size of approximately l.3mm and a specific weight of 0.2g/cm3.
They are taken up in 300 ml of an aqueous solution of to Poloxamer 407 and loo propyleneglycol.
Example 8 3 ml cyanacryl acid butylester are dissolved in 300 ml hydrochloric acid at pH 2.0 that contains 1o Poloxamer 407, and dispersed for 120 minutes with a dissolver-mixer. The microparticles, with an average size of 2mm and a specific weight of 0.1 g/cm3 are separated off by floatation, and taken up in 5 litres of a 5-% mannitol solution that contains to Poloxamer 407 and 10% propyleneglycol.
Example 9 The same procedure as in Example 8 is followed, although the Poloxamer 407 is replaced by octoxynol-9 and the pH is 213791fl adjusted to 2.5. The average size of the microparticles amounts to 0.8mm and their specific weight is 0.15g/cm3.
They are taken up in 5 litres of a 0.9-% solution of cooking salt that contains 0.1% cetomacrogol 1200.
Example 10 The same procedure as in Example 8 is followed, although the Poloxamer 407 is replaced by cetomacrogol 1200. The average size of the microparticles amounts to l.8mm and their specific weight is 0.4g/cm3. They are taken up in 5 litres of a glucose solution that contains cetomacrogol 1200.
Example 11 The same procedure as in Example 8 is followed, although the Poloxamer 407 is replaced by 5% polysorbate 60. The average size of the microparticles amounts to l.Omm and their specific weight is 0.4g/cm3. They are taken up in 5 litres of a 5-% mannite solution that contains 1% Poloxamer 407 and 10% propyleneglycol.
Example 12 The particles produced as in Examples 8, 9, 10, or 11 are . each taken up, in 15-ml portions, in 5-litre each of a 5-0 mannitol solution that contains 0.1% cetomacrogol 1200 and 5o povidone, in instead of the solutions described in the examples; they are then frozen during vigorous agitation, and freeze dried. Prior to use, the lyophilisate is resuspended using water for injection purposes and, if necessary, filtered.

Example 12a The particles produced as in Examples 8, 9, 10, or 11 are each taken up, in 15-ml portions, in 5-litre each of a 10-%
lactose solution that contains 0.1% cetomacrogol 1200, instead of the solutions described in the examples; they are then frozen during vigorous agitation, and freeze dried.
Prior to use, the lyophilisate is resuspended using water for injection purposes and, if necessary, filtered.
Example 13 1.0 g of hydrated Soya lecithin is dispersed in 200 ml glycerol with a dissolver-mixer. After 60 minutes, 2.0 g of poly-L-lactide (mean molecular weight 1100) dissolved in 10 ml boiling ethanol is added, drop by drop, to the dispersion. This is further dispersed for 60 minutes. The resulting microparticles are centrifuged at 1000 rpm, the result is taken up in 50 ml water, recentrifuged (1000 rpm), and the result is taken up in a 5-% solution of mannitol.
This suspension is divided into 10-ml portions and freeze dried. Prior to use, the lyophilisate is resuspended with water for injection purposes.
Example 14 1.0 g sucrose palmitate stearate (HLB 15) is dispersed in 200 ml glycerol with a dissolver-mixer. After 30 minutes, 1.0g of poly-L-lactide (mean molecular weight 1100) dissolved in 10 ml boiling ethanol is added, drop by drop, to the dispersion. The resulting microparticles have an average size of 2mm. They are centrifuged at 1000 rpm for 30 minutes, and the result is taken up in 50 ml of water, 21~'~91Q
recentrifuged (1000 rpm) and the result is taken up in 50 ml of a 5-% mannitol solution. This suspension is divided into ml portions and freeze dried. Prior to use, the lyophilisate is resuspended with water for injection purposes.
Example 15 A dog (11 kg, inhalation narcosis) is injected periphero-venously with microparticles (250mm/ml) at a dose of 300mm/kg body weight, as a rate of 0.1 ml/second. After 10 minutes, the liver was homogenously colour coded for sufficient time to effect a diagnosis during colour Doppler examination.
Example 16 Example 15 is repeated with the particles produced as in Examples 1 to 7 or 9 to 14. In these cases, too, the liver was homogeneously colour coded.
Example 17 A rabbit (3,5 kg, inhalation narcosis) is injected periphero-venously with microparticles produced as in Example 2, at a dose of 5mm/kg body weight. After 10 minutes, the liver was colour coded during colour Doppler examination, in contrast to which tumour areas appeared in the usual grey tones (See Illustration 1/right-hand half).
For purposes of comparison, a photograph was also made in B-mode under otherwise identical conditions (See Illustration 1, left-hand half). Even shading of the liver can be seen in this image, although it is impossible to define the tumour areas.
Example 18 4g of terephthalic acid dichloride is dissolved in 20 ml cyclohexane and emulsified by a vane stirrer in 500 ml 3-%
sodium carbonate solution that contains 1% Poloxamer 407.
After the addition of 600 mg L-lysine dissolved in 50 ml 1-%
Poloxamer 407 solution, the capsules, of an average size of 30mm, were centrifuged off and resuspended in a liquid suitable for injection, frozen, and freeze dried. After resuspension of the prepared product with water for injection purposes, a suspension of gas-filled capsules, approximately 30mm, was formed.
Example 19 6g human albumin is dissolved in 30 ml distilled water and processed for 3 minutes with a rotor-stator dispersal apparatus at 20,000 rpm. 150 ml of a solution of 1 part chloroform and 4 parts cyclohexane containing 2a Span~-85 is added during further stirring. After 1 minute, 2q terephthalic acid dichloride in 20 ml of the organic solvent is added, and stirring is then continued with a magnetic stirrer.
After 30 minutes the floating capsules are centrifuged off and then washed with isotonic cooking-salt solution.
This results in a suspension of approximately 30-mm gas-filled capsules.

213'910 Example 20 200 ml of a 5-s gelatin solution at 60°C and pH 4.5 is processed for 3 minutes at 20,000 rpm in a rotor-stator dispersal apparatus. 200 ml of a 5-% rubber-gum arabic solution is added during constant stirring, and the product is cooled to 5°C. After 2 hours, 50 ml of a 3-a glutaraldehyde solution is added and the pH is adjusted to 8.5. The floating capsules, with a size of 20-50mm are centrifuged off and washed several times with isotonic cooking-salt solution that contains 0.1% Poloxamer 188.
Example 21 A solution of 99.5 g water and 0.5g Poloxamer 407 is adjusted to pH 2.4 with 1 N hydrochloric acid. 1.0 g of cyanacrylic acid butylester is added to this solution while stirring (4000 rpm), and then stirring is continued for 60 minutes. The suspension so obtained is neutralized, the gas-filled capsules, of a size of approximately 30 to 100mm are separated off by centrifuging, and resuspended in a liquid that is appropriate for the intended application.
Example 22 30 ml of a suspension of gas-filled microparticles, produced as in Example 20, was administered to a 10-kg beagle, under heavy narcosis, by way of an oesophageal probe. The concentration is 1 mg\ml Immediately after administration, the stomach was examined with a colour Doppler ultrasound apparatus. The lumen was coloured (Illustration 2). The small intestine was similarly coloured 30 minutes later (Illustration 3).
Example 23 3m1 of microcapsules produced as in Example 19 were administered to the liquid-filled bladder of a beagle under narcosis by way of a bladder catheter; the microcapsules were at a concentration of 109 particles/ml. Illustration 4 shows the effect before and after the administration of the contrasting agent, as in colour Doppler mode.

Claims

The embodiments of the invention in which an exclusive-property or privilege is claimed are defined as follows:

1. Use of gas- or liquid-filled microcapsules as an ultrasound contrasting agent for contrasting regions of the body by means of colour Doppler sonography in which there is substantially no observable movement of the microcapsules.

2. Use of gas- or liquid-filled microcapsules for the manufacture of an ultrasound contrast agent for contrasting regions of the body by means of colour Doppler sonography in which there is substantially no observable movement of the microcapsules.

3. The use according to claim 1 or 2, wherein the gas- or liquid-filled microcapsules are synthesised from polycyanoacrylates or biologically degradable polyesters of .alpha.-, .beta.- or .gamma.-hydroxycarboxylic acids.

4. The use according to claim 1 or 2, wherein the gas- or liquid-filled microcapsules are synthesised from biologically degradable polymers synthesised from polymerisable aldehydes which, optionally contain one or more compounds selected from the group consisting of additives capable of copolymerisation, crosslinkers, surfactants, surfactant mixtures, coupling agents, and bio-or macro-molecules bonded by way of coupling agents.

5. The use according to claim 1 or 2, wherein the gas- or liquid-filled microcapsules are synthesised from gelatin.

6. The use according to claim 1 or 2, wherein the gas- or liquid-filled microcapsules are synthesised from partially denatured proteins.

7. The use according to claim 6, wherein the partially denatured proteins comprise denatured albumin or denatured human serum albumin.

8. The use according to any one of claims 1 to 7, for contrasting the reticuloendothelial system.

9. The use according to any one of claims 1 to 7, for contrasting the myocardium.

10. The use according to any one of claims 1 to 7, for contrasting the gastrointestinal tract.

11. The use according any one of claims 1 to 7, for contrasting the lymphatic system, the liver or the spleen.

12. The use according to any one of claims 1 to 11, wherein the agent is administerable in a quantity that results in 10 7 to 10 10 of microcapsules per kg of body weight.