NO161356B - CONTRACTOR FOR ULTRA SOUND DIAGNOSTICS CONTAINING MICROPARTICLES AND GAS BLAES. - Google Patents

Abstract

1. Contrast medium containing microparticles and gas bubbles for ultrasound diagnostics, characterised in that it contains microparticles of a solid surface-active substance, optionally in combination with microparticles of a non-surface-active solid, in a liquid carrier.

Description

The invention relates to the items indicated

in the characterizing parts of the subsequent patent claims.

Examination of organs with ultrasound (sonography) has been a well-established and practical diagnostic method for some years. Ultrasound waves in the mega-Hertz range (over 2 mega-Hertz with wavelengths between 1 and 0.2 mm) are reflected from the interfaces between different types of tissue. The resulting echoes are amplified and made visible. Of particular importance here is examination of the heart with this method, which is called echocardiography (Haft, J.I. et al.: Clinical echocardiography, Futura, Mount Kisco, New York 1978; Kohler,

E. Klinische Echokardiographie, Enke, Stuttgart 1979; Staffan,

F. et al.: Echokardiographie, Thime, Stuttgart-New York 1981; G. Biamino, L. Lange: Echocardiography, Hoechst AG. 1983.

As liquids - including the blood - only provide ultrasound contrast when there is a difference in density in relation to the surroundings, possibilities are sought to make blood and its flow visible for an ultrasound examination, which is also possible by adding of small gas bubbles.

Several methods for the production and stabilization of gas bubbles are known from the literature. They can, for example, be prepared before injection into the bloodstream by vigorous shaking or stirring of solutions such as salt solutions, dye solutions or in previously drawn blood.

Although one thereby achieves an ultrasound contrast, this method is accompanied by serious disadvantages which manifest themselves in poor reproducibility, greatly varying size of the gas bubbles and - due to a proportion of visible larger gas bubbles - a definite risk of embolism. These disadvantages were partially removed by other production methods, by e.g. the method according to US patent 3,640,271 whereby gas bubbles were produced with a reproducible size by filtering or by an electrode device that was under a direct current voltage. The possible advantage of being able to produce gas bladders of reproducible size is, however, associated with the disadvantage of using technically complicated equipment.

In US patent 4 2 76 885 the preparation is described

of gas bladders of defined size, which gas bladders are

surrounded by gelatin membranes that protect against coalescence. However, storage of the finished gas bladders can only take place in a "frozen" state, for example by storage at refrigerator temperature, whereby they must be brought back to body temperature for use.

US patent 4,265,251 describes the production and use of gas bladders with a solid membrane of saccharides, which can be filled with a pressurized gas. If they are under normal pressure, Jean they are used as an ultrasound contrast agent. When using elevated internal pressure, the gas bladders are used for blood pressure measurement. Although the storage of the solid gas bladders does not cause any problems, the use of complicated technical equipment is associated with a significant cost factor.

Risk with the contrast agents available according to the state of the art is caused by two factors: Size and number of both solid particles and gas bubbles.

The state of the technique discussed so far only allows the production of ultrasound contrast agents which always only have some of the required properties: 1) Elimination of embolism risk - gas bubbles (size and number)

- solid particles (size and number)

2) Reproducibility

3) Sufficiently long stability

4) Ability to pass lungs, e.g. to achieve ultrasound contrast rendering of the left half of the heart

5) Ability to pass capillary

6) Sterility and freedom from pyrogens during preparation

7) Easy manufacturability with reasonable costs

8) Hassle free storage.

In European patent application 52 5 75, the production of gas bladders which must be able to meet the required properties is described. When producing these, a solid, crystalline substance, such as galactose for example, is suspended in a carrier liquid, whereby the gas that is adsorbed on the particle surfaces is enclosed between the particles or in the intercrystalline cavities that form the gas bubbles.

The thus formed suspension of gas bubbles and microparticles is injected within 10 minutes. Although it is claimed in the European patent application 52 575 that the suspension produced according to the described method is suitable to be able to pass through both the right half of the heart and, after passing through the lungs, the left half of the heart after injection into a peripheral vein and there make the blood and its flow visible by ultrasound examination, these claims did not stand up to scrutiny. It was thus clarified that a contrast agent which was prepared according to the method described in European patent application 52 575 and injected into a peripheral vein, did not produce an ultrasound echo in the left half of the heart.

It was therefore the purpose of the present invention to produce a contrast medium for ultrasound diagnostics which is able, after intravenous injection, to make the blood and its flow paths visible not only on the right half of the heart but also after passage of the pulmonary capillaries on the left half of the heart. In addition, the agent must be able to make visible the flow paths of the blood through other organs such as the myocardium, liver, spleen and kidneys.

The agent produced according to the invention,

according to patent claims 1-3 has all the properties expected of such a contrast agent, and the properties indicated on page 2.

It was surprisingly found that by suspending microparticles of a solid surface-active substance, possibly in combination with microparticles of a non-surface-active solid substance in a carrier liquid of an ultra-contrast agent, which after injection into a peripheral vein also enables reproducible ultrasound recordings also of blood in the arterial left heart half. As the left half of the heart can be reached with ultrasound contrast medium according to the invention after intravenous injection, ultrasound contrasts of other organs supplied with blood from the aorta after venous injection are also possible, such as the myocardium, liver, spleen, kidneys. It goes without saying that the ultrasound contrast agent according to the invention is also suitable for obtaining contrasts of the right half of the heart and for all other applications as an ultrasound contrast agent.

Magnesium stearate, ascorbyl palmitate, sucrose monopalmitate, sucrose monostearate or sucrose distearate are preferably used as surface-active substance for the production of microparticles.

The surfactant is used in a concentration of 0.01 to 5% by weight, preferably 0.04 to 0.5% by weight.

If desired, the microparticles of the surface-active substance can be combined with microparticles of a physiological, crystalline solid, whereby galactose, lactose and α-cyclodextrin are preferred. These are present in a concentration of 5 to 50% by weight, preferably 9 to 40% by weight.

For the production of the microparticles, the substances used are recrystallized under sterile conditions. They are then divided under sterile conditions, e.g. by grinding in an air jet mill, until the desired particle size is achieved. A particle size is obtained from

<1 - 50 µm, preferably 1 - 10 µm. The particle size is determined in a suitable measuring instrument. The produced microparticles consist either of finely divided surface-active substances alone or of a mixture of microparticles of surface-active substances and non-surface-active solids. In this case, the weight ratio between solid surfactant and non-surfactant solid is 0.01 to 5:100.

Both the size of microparticles obtained by the division process and the size of the gas bubbles obtained in the contrast medium according to the invention ensure a safe passage in the capillary system and the lungs and exclude the formation of emboli.

The gas bubbles required for the contrast are partly transported by them. suspended microparticles, absorbed on the surface of the microparticles in the cavities between the microparticles or crystalline enclosed.

The gas volume transported by the microparticles in the form of gas bubbles is 0.02 to 0.6 ml per gram of microparticle.

In addition to the transport function, the carrier fluid has the task of stabilizing the suspension consisting of microparticles and gas bubbles, for example to prevent sedimentation of microparticles and coalescence of gas bubbles, respectively

delay dissolution of microparticles.

As a liquid carrier fluid, water, aqueous solutions of one or more inorganic salts such as physiological sodium chloride solution or buffer solution, aqueous solutions of mono- or disaccharides such as galactose, glucose or lactose are mentioned.

Water and physiological electrolyte solutions such as physiological saline solution and aqueous solutions of galactose and lactose are preferred. If solutions are used, the concentration of the dissolved substance is 0.1 to 30% by weight, preferably 0.5 to 25% by weight, in particular a 0.9% aqueous sodium chloride solution or a 20% aqueous galactose solution is used.

For the production of ready-to-use ultrasound contrast agents, the sterile, solid surface-active substance present in the form of microparticles, which is optionally combined with a sterile non-surface-active substance, is added to the sterile carrier liquid and this mixture is shaken until a homogeneous suspension has formed, why is it necessary approx. 5 to 10 seconds. The resulting suspension is injected immediately after its preparation, and no later than 5 minutes thereafter, as a bolus into a peripheral vein or an already existing catheter, whereby from 0.01 ml to 1 ml/kg body weight is used.

For expedient reasons, the necessary components such as carrier liquid (A) and microparticles of surface-active substance, possibly combined with microparticles of the non-surface-active solid (B) for the production of the agent according to the invention, are kept sterile in the quantity necessary for an investigation in two separate containers. Both containers have closure devices, which enable withdrawal and addition using injection syringes under sterile conditions (medicine vials).'' The size of container B must be such that the contents of container A can be transferred by means of syringes to B and that the combined components can be shaken.

The implementation of an echocardiographic examination on a 10 kg baboon shall demonstrate the use of the contrast agent according to the invention:

5 ml of carrier liquid (prepared according to example 1) is withdrawn from a medicinal glass with an injection syringe and added to 2 g of microparticles (produced according to example IB) which are in a second medicinal glass, shaken approx. 5-10 seconds until a homogeneous suspension has formed. One injects 2 ml of this suspension into a peripheral vein (V. jugularis, brachialis or saphena) via a 3-way stopcock with an infusion rate of at least 1 ml/sec., better with 2 - 3 ml/sec. After the contrast agent injection, 10 ml of physiological saline solution is immediately injected at the same speed so that the contrast agent bolus is maintained as completely as possible until it reaches the right half of the heart. Before, during and after the injection, a commercially available sound head for echocardiography is held to the thorax of the experimental animal so that a typical cross-section through the right and left halves of the heart is obtained. This experimental device corresponds to the state of the art and is known to the person skilled in the art.

If the ultrasound contrast agent reaches the right half of the heart, one can follow in the 2-D echo image or in the M-mode echo image how the blood marked by the contrast agent first reaches the height of the right atrium, then that of the right ventricle and the pulmonary artery, whereby a homogeneous filling occurs in approx. 10 seconds. While the cavities in the right half of the heart become empty again in the ultrasound image, blood marked with contrast is seen after passing through the lungs back into the pulmonary veins, filling the left atrium, the left ventricle and the aorta homogeneously, whereby the contrast lasts a total of 2 to 3 times longer than on the right half of the heart. In addition to the representation of the blood flow through the cavities of the left half of the heart, there is also a representation of the myocardium, which in turn provides the blood flow.

However, the use of the ultrasound contrast medium according to the invention is not limited to making visible the blood flow in the arterial part of the heart after venous application, but it is also used with excellent results when examining the right half of the heart and other organs as a contrast medium.

Example 1

A) Preparation of carrier liquid

80 g of galactose was dissolved in water for injection and made up to a volume of 400 ml, filtered through a 0.2 µm filter, 4 ml of this filtrate was filled into 5 ml vials and sterilized for 20 minutes at 120°C.

B) Preparation of microparticles:

Under sterile conditions, 198 g of galactose was intensively mixed with 2 g of magnesium stearate by homeopathic grinding, filtered through a 0.8 mm sieve, loosely mixed and ground with an air jet mill until the following particle size distribution was obtained:

The determination of the particle size and their distribution takes place in a particle measuring instrument after suspension in anhydrous isopropanol. The microparticles are filled in 5 ml vials, each with 2 g. C) To prepare 5 ml of ready-to-use ultrasound contrast agent, the contents of a vial with a carrier liquid (20% galactose solution in water, A) is added by means of an injection syringe to the vial with microparticles (B) and shaken until a homogeneous suspension is formed (5 - 10 seconds). Example 2

A) Preparation of carrier liquid:

Water is used for injection purposes, each

Once, 4 ml were filled into 5 ml medicine glasses and these were sterilized for 20 minutes at 120°C.

B) Preparation of microparticles:

Under sterile conditions, 198 g of galactose was intensively mixed with 2 g of ascorbyl palmitate by homeopathic shredding and processed further as described in example under B), whereby the following particle size distribution was obtained:

The particle size is determined as described in example 1 under B).

The microparticles are filled in 5 ml vials, each with 1200 mg. C) For the preparation of 4.5 ml of ready-to-use ultrasound contrast medium, the contents of a vial of carrier liquid (water, A) were added to a vial of microparticles (B) using a syringe and shaken until a homogeneous suspension was formed (5 - 10 seconds).

Example 3

A) Preparation of carrier liquid:

Dissolve 4.5 g of sodium chloride in water up to a volume of 500 ml, filter the solution through a 0.2 µm filter, fill 4 ral of this solution into a 5 ml medicine glass and sterilize for 20 minutes at 120°C.

B) Preparation of microparticles:

Under sterile conditions, 19 8 g became lactose anhydride (< 0.3 mm) intensively mixed with 2 g of ascorbyl palmitate by homeopathic shredding and the mixture was further processed as described in example 1 under B), whereby the following distribution of the particle size is obtained:

The particle sizes are determined as described in example 1 under B).

The microparticles are filled in 5 ral medicine glasses, each with 1.6 g.

C) For the preparation of 5 ral of ready-to-use ultrasound contrast agent, the contents of a vial (0.9% sodium chloride solution in water, A) were added to a vial of microparticles (B) using an injection syringe and shaken until a homogeneous suspension (5 -

10 seconds).

Example 4

A) Preparation of carrier liquid

As described in example 3 under A), a 0.9% aqueous sodium chloride solution was prepared, filled in 4 ml portions into 5 ml medicine glasses and sterilized for 20 minutes at 120°C.

B) Preparation of microparticles:

Under sterile conditions, 199 g of ct-cyclodextrin were intensively mixed with 1 g of ascorbyl palmitate by homeopathic grinding and the mixture was further processed as described in example 1 under B), whereby microparticles with the following size distribution were obtained:

The determination of the particle size takes place as described in the example under B).

The microparticles were filled in 5 ml vials each with 400 mg.

C) For the preparation of 4 ml of ready-to-use ultrasound contrast agent, the contents of a medicine glass (0.9% aqueous sodium chloride solution, A) were introduced into a medicine glass with microparticles (B) using an injection syringe and shaken until it formed a homogeneous suspension (5 -

10 seconds).

Example 5

A. Preparation of carrier liquid:

50 g of lactose should be dissolved in water for injection, made up to a volume of 500 ml, filtered through a 0.2 µm filter, filled into 5 ml vials, each containing 4 ml, and sterilized for 20 minutes at 120° C.

B. Preparation of microparticles:

Ascorbyl palmitate was dissolved in methanol, sterile filtered through a 0.2 µm filter, recrystallized under sterile conditions, dried and sieved through a 0.8 mm sieve. Then the sterile ascorbyl palmitate was ground under sterile conditions with an air jet mill, until the following size distribution of the particles is obtained:

The determination of the particle size and their distribution takes place in a particle measuring instrument after suspending 1 cold aqueous 0.1% "Pluronic"-F68 solution.

The microparticles were filled into sterile 5 ml vials, each containing 40 mg. C. For the production of 4 ml of ready-to-use ultrasound contrast agent, the contents of a medicine glass with a carrier liquid (10% lactose solution, A) were introduced into the medicine glass with the microparticles using an injection syringe and shaken until a homogeneous suspension was formed.

Example 6

A. Preparation of carrier liquid:

4.5 g of sodium chloride were dissolved in water to a volume of 500 ml, the solution was filtered through a 0.2 µm filter, this solution was filled into 5 ml vials, each with 4 ml, and sterilized for 20 minutes at 120° C>

B. Preparation of microparticles

Under sterile conditions, a sterile filtered solution of 0.5 g of ascorbyl palmitate in 40 g of isopropanol was coated on 199.5 g of sterile galactose particles, at 40° C. and 200 Torr the isopropanol was evaporated, and the particles were divided in an air jet mill until the following size distribution of the particles were obtained:

The determination of the particle size and their distribution took place in a particle measuring instrument, e.g. after suspension in isopropanol. The packing of the microparticles followed in 5 ml vials, each time with 2 g. C. For the preparation of 5 ml of ready-to-use ultrasound contrast agent, the contents of a vial with a carrier liquid (0.9% sodium chloride solution in water, A) were introduced using an injection syringe in the medicine glass with microparticles (B) and shaken until a homogeneous suspension was formed (5 - 10 seconds).

Example 7

A. Preparation of carrier liquid:

4.5 g of sodium chloride was dissolved in water, filled to a volume of 500 ml, the solution was filtered through a 0.2 µm filter, this solution was filled into 5 ml vials, each with 4 ml, and sterilized for 20 minutes at 120 °C.

B. Preparation of microparticles:

Under sterile conditions, 199.5 g of galactose was triturated with 0.5 g of ascorbyl palmitate, intensively mixed, sieved through a 0.8 ram sieve and divided with an air jet mill until the following size distribution of the particles was obtained:

The determination of the particle size and their distribution took place in a particle measuring instrument, e.g. after suspension in isopropanol. The microparticles were packaged in 5 ml vials, each with 2 g.

C. For the preparation of 5 ml of ready-to-use ultrasound contrast agent, the contents of a vial with a carrier liquid (0.9% sodium chloride solution in water, A) were introduced into the vial with microparticles (B) using an Eti injection syringe and shaken until a homogeneous suspension (5 - 10 seconds).

Example 8

A. Preparation of carrier liquid:

Water was used for injection purposes, 4 ml was • filled into 5 ml medicine glasses and these were sterilized for 20 minutes at 120°C.

B. Preparation of microparticles

Under sterile conditions, 0.5 g of sucrose monopalmitate was triturated with 199.5 g of galactose, mixed intensively, filtered through a 0.8 mm sieve and ground with an air jet mill until the following size distribution of the particles was obtained:

The determination of the particle size and their distribution took place in a particle measuring instrument, e.g. after suspension in isopropanol. The packing of the microparticles was carried out in 5 ml vials, each with 2 g. C. For the preparation of 5 ml of ready-to-use ultrasound contrast agent, the contents of a vial with a carrier liquid (sterile water for injection purposes, A) was introduced by means of an injection syringe into the vial with microparticles (B) and shaken homogeneously until a homogeneous suspension was formed (5 - 10 seconds).

Example 9

A. Preparation of carrier liquid:

4 ml of water for injection was filled into 5 ml vials and sterilized for 20 minutes at 120°C.

B. Preparation of microparticles:

Under sterile conditions, a sterile filtered solution of 0.5 g of sucrose monopalmitate in 40 g of isopropanol was placed on 199.5 g of sterile galactose particles, at 40°C and 200 Torr the isopropanol was evaporated and the particles were ground with an air jet mill until the following size distribution of the particles was obtained achieved:

The particle sizes and their distribution were carried out in a particle measuring instrument, e.g. after suspension in isopropanol. The packing of the microparticles was carried out in 15 ml vials, each with 2 g. C. To prepare 5 ml of ready-to-use ultrasound contrast agent, the contents of a vial with a carrier liquid (water for injection purposes, A) was introduced into the the vial with microparticles (B) and shaken until a homogeneous suspension was formed (5 - 10 seconds).

Example 10

A. Preparation of carrier fluid

4 ml of water for injection was filled into 5 ml vials and sterilized for 20 minutes at 120°C.

B. Preparation of microparticles

Under sterile conditions, a sterile filtered solution of 0.5 g of sucrose monostearate in 40 g of isopropanol was placed on 199.5 g of sterile galactose particles, at 40°C and 200 Torr the isopropanol was evaporated and the particles were ground with an air jet mill until the following size distribution of the particles was obtained achieved:

The determination of the particle size and their distribution was carried out in a particle measuring instrument, e.g. after suspension in isopropanol. The packing of the microparticles was carried out in 5 ml vials, each containing 2 g. C. To prepare 5 ml of ready-to-use ultrasound contrast agent, the contents of a vial with a carrier liquid (water for injection purposes, A) were introduced into the vial using an injection syringe. with microparticles (B) and shaken until a homogeneous suspension was formed (5 to 10 seconds).

Example 11

A. Preparation of carrier fluid

Water for injection purposes was used, 4 ml was filled into 5 ml vials and these were sterilized for 20 minutes at 120°C.

B. Preparation of microparticles

Under sterile conditions, 0.5 g of sucrose monostearate together with 199.5 g of galactose were mixed intensively, sieved through a 0.8 mm sieve and ground with an air jet mill until the following size distribution of the particles was obtained:

The determination of the particle size and their distribution takes place in a particle measuring instrument, e.g. after suspension in isopropanol. The packing of the microparticles was carried out in 5 ml vials, each containing 2 g. C. For the production of 5 ml of ready-to-use ultrasound contrast agent, the contents of a vial with a carrier liquid (sterile water for injection purposes, A) was introduced into the the vial with microparticles (B) and shaken until a homogeneous suspension was formed (5 - 10 seconds).

Example 12

A. Preparation of carrier fluid

4 ml of water for injection was filled into 5 ml vials and sterilized for 20 minutes at 120°C.

B. Preparation of microparticles

Under sterile conditions, a sterile filtered solution of 0.5 g of sucrose distearate in 40 g of isopropanol was coated on 199.5 g of sterile galactose particles, at 40°C and 200 Torr the isopropanol was evaporated and the particles were milled with an air jet mill until the following size distribution of the particles was obtained achieved:

The determination of the particle sizes and their distribution took place in a particle measuring instrument, e.g. after suspension in isopropanol. The packing of microparticles was carried out in 5 ml vials, each of 2 g. C. To prepare 5 ml of ready-to-use ultrasound contrast agent, the contents of a vial with a carrier liquid (water for injection purposes, A) was introduced into the vial using an injection syringe. with microparticles (B) and stirred until a homogeneous suspension was formed (5 - 10 seconds).

Example 13

A. Preparation of carrier fluid

Water for injection was used, 4 ml was filled into 5 ml vials and these were sterilized for 20 minutes at 120°C.

B. Preparation of microparticles

Under sterile conditions, 0.5 g of sucrose distearate was coagulated with 199.5 g of galactose, mixed intensively, sieved through a 0.8 mm sieve and ground with an air jet mill until the following size distribution of the particles was obtained:

The determination of the particle sizes and their distribution was carried out in a particle measuring instrument, e.g. after suspension in isopropanol. The packing of the microparticles was carried out in 5 ml vials, each containing 2 g. C. For the production of 5 ml of ready-to-use ultrasound contrast agent, the contents of a vial with a carrier liquid (sterile water for injection purposes, A) was introduced into the the vial with microparticles (B) and shaken until a homogeneous suspension was formed (5 - 10 seconds).

Claims (3)

1. Contrast agent for ultrasound diagnostics containing microparticles and gas bubbles, characterized in that it consists of microparticles of a solid, surface-active substance selected from sucrose esters or ascorbic acid esters of saturated (cio~C20^~ fatty acids or metal salts of said fatty acids, possibly in combination with microparticles of a non-surface-active solid such as cyclodextrins, monosaccharides or disaccharides, preferably galactose, lactose or cc-cyclodextrin as microparticles in a concentration of 5 to 50% by weight, preferably 9 to 40% by weight, in a liquid carrier containing water, physiological electrolyte solution or the aqueous solution of a mono- or disaccharide. 2. Means according to claim 1, characterized in that it contains magnesium stearate, ascorbyl palmitate, sucrose monopalmitate, sucrose monostearate or sucrose distearate as solid surfactant in the form of microparticles, in a concentration of 0.01 to 5% by weight, preferably 0.04 to 1% by weight. 3. Means according to claim 1, characterized in that the liquid carrier is water, physiological saline solution, 10% aqueous lactose solution or 20% aqueous galactose solution.