NZ207853A

NZ207853A - Ultrasound contrast agent containing microparticles and gas micro-bubbles

Info

Publication number: NZ207853A
Application number: NZ207853A
Authority: NZ
Inventors: J Hilmann; L Lange; I Zimmermann
Original assignee: Schering Ag
Priority date: 1983-04-15
Filing date: 1984-04-13
Publication date: 1988-01-08
Also published as: ZA842801B; FI81264C; DK78984A; EP0122624B1; NO841489L; NO161356C; ATE36958T1; NO161356B; DK165171C; IE57272B1; EP0122624A2; DK78984D0; JPS59205328A; EP0122624A3; DE3313946A1; DK165171B; AU566928B2; JPH0425934B2; IE840835L; DE3473828D1

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

New Zealand Paient Spedficaiion for Paient Number £07853 707853 Priori'!/ Dat5(s}: . "W o3 Complete Spacification F,led:^.~MSU Class: . B&Jft\ (>£} - Publication Date: .... JAN 1988' I P.O. Journal, No: ... 1303.

NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION t3AP*1964 "Ultrasound contrast agent containing microparticles and gas micro-bubbles." We, SCHERING AKTIENGESELLSCHAFT, a body corporate organized according to the laws of Germany, of 170-178 Mullerstrasse, D-1000 Berlin 65, Germany and Waldstrasse 14, 4619 Bergkamen, Germany, do hereby declare the invention, for which we pray that a Patent may be granted to us, and the method by which it is to be performed to be particularly described in and by tbe following statement:- - 1 - (Followed by 1A.) 207853 -l t\- Ultrasound contrast agent containing microparticles and gas micro-bubbles The invention relates to contrast agents for use in ultrasound diagnosis of the human or animal body.

The examination of organs using ultrasound (sonography) is a diagnostic method which has been well established and practised for some years. Ultrasound waves in the megahertz range (above 2 megahertz with wavelengths of between 1 and 0.2 mm) are reflected at the interfaces of various types of tissue. The resulting echoes are amplified and rendered visible. Of particular importance is the examination of the heart by this method which is known as echocardiography.

Since fluids, including blood, produce ultrasound image contrast only when there are differences in density with respect to the surroundings, possibilities were sought of rendering the blood and its circulation visible for ultrasound examination and this may be effected by injecting extremely fine gas bubbles into 207853 the bloodstream.

Several methods of producing and stabilising gas bubbles have been described in the literature. They can be produced, for example, before injection into the bloodstream, by vigorously shaking or stirring a liquid solution, such, for example, as sodium chloride solution, dye solution or previously removed blood.

Although ultrasound image contrast is achieved by these methods, they have serious disadvantages which are manifested in poor reproducibility, greatly fluctuating size of the gas bubbles and a certain risk of embolism due to a proportion of large visible bubbles. Some of these disadvantages have been eliminated by other production processes, such as, for example, by the process of U.S. Patent No. 3,640,271 in which bubbles of a reproducible size are produced by filtration or by the use of direct current electrode apparatus. Against the advantage of being able to produce gas bubbles of a reproducible size is the disadvantage of the considerable technical outlay involved.

U.S. Patent No. 4,276,885 describes the production of gas micro-bubbles of a specific size which are surrounded by a gelatine membrane which protects them from coalescence. The prepared bubbles can be stored only in the "frozen" state, for example by storing at refrigerator temperature, and they must 2 078 5 3 » be raised to body temperature again before they can be used.

U.S. Patent No. 4,265,251 describes the production and use of gas micro-bubbles with a solid saccharide covering, which bubbles may be filled with a pressurised gas. if they are under normal pressure, they can be used as ultrasound image contrast agents; when used at an elevated internal pressure, they can be used for measuring blood pressure. Although in this case the storage of the solid gas bubbles does not present any problem, the technical outlay involved in their production gives rise to high costs as a result of the complex techniques.

The risks involved with the hitherto known contrast agents for ultrasound diagnosis are caused by two factors: the size and number both of the particles of solid material and also of the gas bubbles.

The ultrasound contrast agents prepared by the previously described methods have, in all cases, possessed only some of the following properties that are required:- 1. Exclusion of the risk of embolism (dependent on size and number of gas bubbles and size and number of particles of solid material). 2. Reproducibility. 3. Sufficiently long stability.

? U78 53 4. Ability to pass through the lungs, for example in order to obtain ultrasound image contrast of the left-hand side of the heart.

. Ability to pass through the capillaries. 6. Sterility and freedom from pyrogens. 7. Easy production at reasonable cost. 8. Easy storage.

European Patent Application No. 52575 describes the production of ultrasound contrast agents containing gas bubbles that are supposed to possess these necessary properties. However, in order to produce them, microparticles of a solid crystalline substance, such as, for example, galactose, are suspended in a liquid carrier, and the gas, which is adsorbed at the particle surface and is enclosed in cavities between the particles or in intercrystalline cavities, forms the gas bubbles. The resulting suspension of gas bubbles and microparticles is injected over a period of 10 minutes. Although according to European Patent Specification 52575 the suspension prepared by the described method is capable, after injection into a peripheral vein, of appearing both on the right-hand side of the heart and also, after passing through the lungs, on the left-hand side of the heart and of rendering visible the blood there and its circulation during ultrasound examination, it was found when checked that the contrast medium prepared by the method 2078 5 3 described in European Application No. 52575 and injected into a peripheral vein did not in fact produce ultrasound echoes in the left-hand side of the heart.

An object of the present invention is to provide a contrast agent for ultrasound diagnosis which is capable, after being administered intravenously, of rendering visible for ultrasound the blood and its circulation conditions not only on the right-hand side of the heart but also, after passing through the capillary bed of the lungs, on the left-hand side of the heart. In addition, it should also permit the representation of the circulation of blood through other organs, such as the myocardium, the liver, the spleen and the kidneys.

The present invention provides a contrast agent for use in the ultrasound diagnosis of the human or animal body, which comprises microparticles of a solid surface-active substance and micro-bubbles of a gas in a liquid carrier. If desired, the liquid carrier also contains microparticles of a solid non-surface active agent.

It will be understood that the constituents of the contrast agents of the invention must be physiologically tolerable, and this, of course, equally applies to the liquid media and diagnositc kits described below.

The ultrasound contrast agents of the present ■■ V '• - • _ ' - • .. • + "07853 -6- * * invention possess all the above-mentioned properties that are expected of such a contrast agent.

The invention also provides a liquid medium for use in making up the ultrasound contrast agent, which 5 comprises a suspension of microparticles of a solid surface-active substance, and, if desired, microparticles of a solid non-surface active substance, in a liquid carrier.

Surprisingly, we have found that, by suspending 10 microparticles of a solid surface-active substance, optionally in conjunction with microparticles of a solid non-surface-active substance, in a liquid carrier, an ultrasound contrast agent is obtained which, after being injected into a peripheral vein, 15 permits reproducible ultrasound images even of blood in the arterial left-hand side of the heart. Since the left-hand side of the heart can be reached with the ultrasound contrast agent of the invention after intravenous administration, ultrasound contrasts of 20 other organs supplied with blood from the aorta, such as the myocardium, the liver, the spleen, the kidneys, inter aliar are therefore also possible after venous administration. The ultrasound contrast agent of the invention is, of course, also suitable for contrasts on 25 the right-hand side of the heart and for all other uses as an ultrasound image contrast medium.

All substances that are physiologically tolerable 2073 5 in the quantities used, that is, that have a low toxicity and/or are biologically degradable and the melting point of which is higher than room temperature, are suitable as the surface-active substance for the 5 production of microparticles. Especially suitable are lecithins, lecithin fractions and their conversion products, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerine polyethylene 10 glycol oxystearate, glycerine polyethylene glycol ricinoleate, ethoxylated soya sterols, ethoxylated castor oils and their hydrogenated derivatives, cholesterol, polyoxyethylene fatty acid stearates and polyoxyethylenepolyoxypropylene polymers 15 having a molecular weight of from 6800 to 8975, 13300 and 16250, saccharose esters, such, for example, as sugar esters, for example saccharose dipalmitate and saccharose monolaurate or saccharose glycerides and xyloglycerides, saturated or unsaturated (C^-Cjq)-20 fatty alcohols or (C4-C2Q)-fatty acids or their metal salts, mono-, di- and tri-glycerides, sorbitan fatty acid esters, fatty acid esters of saccharose or fatty acid esters such, for example, as butyl stearate and ascorbyl palmitate; calcium stearate, the saccharose 25 esters of lauric acid, stearic acid and palmitic acid, and ascorbyl palmitate are preferred. 2 07853 The rate at which the microparticles of the surface-active substance dissolve in the liquid carrier should be slower than the rate at which these microparticles dissolve in the blood. Advantageously, the solubility of the microparticles of the surface-active substance in the liquid carrier is such that when they are introduced into it they do not start to dissolve in it to a substantial extent for at least 10 minutes. It will be appreciated that upon administration of the contrast agent the microparticles of the surface-active substance will start to dissolve in the blood.

The solid surface-active substance of the contrast agent is used in a concentration of from 0.01 to 5 %, preferably from 0.04 to 0.5 %, by weight of the contrast agent.

If desired, the microparticles of the surface-active substance can be used in conjunction with microparticles of a physiologically tolerable non-surface-active crystalline solid. Organic and inorganic substances can be used as the crystalline solid, for example salts such, for example, as sodium chloride, sodium citrate, sodium acetate or sodium tartrate, monosaccharides such, for example, as glucose, fructose or galactose; disaccharides such, for example, as saccharose, lactose or maltose; pentoses such, for example, as arabinose, xylose or ribose; or *t'S" ,.'V i J © 2 07853 cyclodextrines such, for example, as a-, B- or y-cyclo-dextrine; galactose, lactose and a-cyclodextrine are preferred. They are contained in the contrast agent of the invention in a concentration of from 5 to 50 %, 5 preferably from 9 to 40 %, by weight.

The microparticles may be produced by recrystallising the surface-active substances and, if desired, non-surface-active substances under sterile conditions. They are then comminuted under sterile <i O 10 conditions, for example, by grinding in an air-jet mill, until the desired particle size is obtained. Preferably the microparticles should have a median particle size of less than 10 jjiti, advantageously less than 8 pin, more especially within the range of from 1 15 to 3 nm. The particle size is determined in a suitable measuring apparatus. The microparticles produced comprise either the comminuted surface-active substance alone or a mixture of the microparticles of the surface-active substance and a solid non-surface-active 20 substance. In the letter case the ratio by weight of solid surface-active substance to solid non-surface-active substance is preferably from 0.01 to 5:100.

Both the microparticle size achieved by the comminution process and also the size of the micro-25 bubbles containing a physiologically tolerable gas contained in the contrast agent of the invention ensure safe passage through the capillary system and the 2 07853 capillary bed of the lungs and preclude the occurrence of embolism.

Some of the micro-bubbles required to produce image contrast are transported by the suspended microparticles, adsorbed at the surface of the microparticles and enclosed in the cavities between the microparticles or enclosed in an intercrystalline manner.

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

Apart from its transporting function, the carrier liquid also has the function of stabilising the suspension comprising microparticles and gas micro-bubbles, for example of preventing the sedimentation of the microparticles and the coalescing of the gas micro-bubbles or of delaying the dissolving process of the microparticles.

There may be used as the liquid carrier, for example, water, aqueous solutions of one or more inorganic salts such, for example, as physiological sodium chloride solution and buffer solutions, aqueous solutions of mono- or di-saccharides such, for example, as galactose, glucose or lactose, monohydric or polyhydric alcohols, in so far as they are physiologically tolerable such, for example, as ethanol, propanol, isopropyl alcohol, polyethylene 2 07 - 5 3 glycol, ethylene glycol, glycerine, propylene glycol, propylene glycol methyl ester or their aqueous solutions.

Water and physiological electrolyte solutions, 5 such, for example, as physiological sodium chloride solution, and aqueous solutions of galactose and lactose, are preferred. If solutions are used, the concentration of the dissolved substance should be from .0.1 to 30 % by weight, preferably from 0.5 to 25 % by 10 weight, and, more especially there may be mentioned, 0.9 % aqueous sodium chloride solution or 20 % aqueous galactose.

The invention also provides a process for the preparation of the contrast agent of the invention, 15 wherein microparticles of a physiologically tolerable solid surface-active substance and, if desired, microparticles of a solid non-surface-active substance, are mixed with a liquid carrier and agitated until a homogeneous suspension is formed.

In order to prepare the ultrasound contrast agent in a form ready for use, the sterile liquid carrier may be added to the sterile solid surface-active substance, which is in the form of microparticles and which is optionally in conjunction with microparticles of a 25 sterile solid non-surface-active substance, and this mixture with the carrier liquid is shaken until a homogeneous suspension has formed, which takes 7 078 approximately from 5 to 10 seconds. Immediately after its preparation, and at the latest up to 5 minutes thereafter, the resulting suspension is injected in the form of a bolus into a peripheral vein or into a 5 catheter which is already present, from 0.01 ml to 1 ml/kg body weight being administered.

For reasons of expediency, the components necessary for the preparation of the contrast agent of the invention such, for example, as carrier liquid 10 and microparticles of the surface-active substance, optionally in conjunction with microparticles of the solid non-surface-active substance are stored under sterile conditions in two separate vessels (A) and (B) respectively in the quantity necessary to carry out an 15 examination. The size of vessel (B) should be such that the contents of vessel (A) can be transferred to (B) by means of an injection syringe and the resulting mixture can be shaken.

The present invention also provides a diagnostic 20 kit for use in the ultrasound diagnosis of the human or animal body, which comprises (A) a container which contains a liquid carrier, and (B) a second container which contains microparticles of a solid surface-active substance and, if desired, microparticles of a solid non-surface- active substance.

I J O 2 078 53 The contents of the containers are in a form ready for mixing together immediately before use.

Preferably container (A) is provided with a closure permitting the removal of the contents under 5 sterile conditions and container (B) is provided with a closure permitting, under sterile conditions, the addition of the contents of vessel (A) and the removal of the resulting contrast agent.

Advantageously the containers A and B both have a 10 volume of from 5 to 10 ml. When the non-surface active substance is present, the ratio by weight of the microparticles of the surface-active substance to the microparticles of the non-surface-active substance is •preferably from 0.01 to 5:100.

The use of the contrast agent of the invention is demonstrated by an echocardiographic examination of a baboon weighing 10 kg. which will now be described. ml of carrier liquid (prepared according to Example 1 A below) are removed from a phial using an 20 injection syringe a'nd are added to 2 g of microparticles (prepared according to Example 1 B below) which are in a second phial, and the mixture is shaken for approximately from 5 to 10 seconds until a homogeneous suspension has formed. 2 ml of this 25 suspension are injected into a peripheral vein (V. jugularis, brachialis or saphena) via a three-way tap having an infusion speed of at least 1 ml/sec.. 2 078 5^ preferably 2-3 ml/sec. Immediately after injecting the contrast agent, 10 ml of physiological sodium chloride solution are injected at the same speed so that the contrast agent bolus is maintained as complete as possible until the right-hand side of the heart is reached. Before, during and after injection, a commercially available transducer for echocardiography is held against the thorax of the experimental animal so that a typical cross-section is obtained through the right-hand side and the left-hand side of the heart.

This test procedure is understood and well known to a person skilled in the art.

If the ultrasound contrast agent reaches the right-hand side of the heart, it is possible to follow in a 2-D echo image or an M-mode echo image how the blood marked by the contrast agent first reaches the level of the right-hand atrium and then the level of the right-hand ventricle and the pulmonary artery, homogeneous filling occurring for approximately 10 seconds. While the cavities in the right-hand side of the heart in the ultrasound image empty again, the blood which is rendered visible with contrast agent, after passing through the lungs, appears again in the pulmonary veins and fills the left atrium, the left ventricle and the aorta homogeneously, the contrast image lasting from 2 to 3 times longer than on the right-hand side of the heart. In addition to the representation of the blood 2 078 53 flow through the cavities of the left-hand side of the heart, there is also a representation of the myocardium showing the circulation of the blood.

The use of the ultrasound contrast agent of the invention is, however, not limited to rendering visible the circulation of blood in the arterial part of the heart after venous administration but is also used with outstanding success as a contrast agent for examining the right-hand side of the heart and other organs.

The invention still further provides a method of ultrasound diagnosis of the human or animal body, wherein a contrast agent of the invention containing a dispersion of micro-bubbles is injected into a part of the human or animal body, preferably intravascularly, and an ultrasound image of the micro-bubbles at a site in the body which it is desired to investigate is obtained. 2078 5 The following Examples illustrate the invention, the parts and percentages being by weight unless otherwise indicated.

Example 1 A) Preparation of the carrier liquid 80 g of galactose are dissolved in water for injection purposes, made up to a volume of 400 ml and forced through a 0.2 |jm filter; 5 ml phials are each filled with 4 ml of this filtrate and sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles: Under sterile conditions, 198 g of galactose are mixed intensively with 2 g of magnesium stearate by homoeopathic trituration, passed through a 0.8 mm sieve, mixed loosely and ground in an air-jet mill until the following distribution of the particle size is obtained: Median: 1.9 |jm 99 % < 6 pm 90 % ^3 Mm.

The particle size and the distribution thereof are determined in a particle-measuring apparatus after suspension in anhydrous isopropanol. 5 ml phials are each filled with 2 g of the microparticles.

X ' : r: . 2 0785: C) For the preparation of 5 ml of the ultrasound contrast agent in a form ready for use, the contents of a phial containing carrier fluid (20 % galactose solution in water, A) are introduced by means of an 5 injection syringe into a phial containing microparticles (B) and shaken until a homogeneous suspension is formed (from 5 to 10 seconds).

Example 2 A) Preparation of the carrier liquid: Water for injection purposes is used and 5 ml phials are each filled with 4 ml of the water and sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles; Under sterile conditions, 198 g of galactose are 15 mixed intensively with 2 g of ascorbyl palmitate by homoeopathic trituration and further processed as described in Example 1 under B), the following distribution of the particle size being obtained: Median: 1.9 nm 20 100 % < 6 pm 90 % ^3 The particle size is determined as described in Example 1 under B). ml phials are each filled with 1200 mg of the 25 microparticles.

X '-■*>: 207853 1i C) For the preparation of 4.5 ml of the ultrasound i| contrast agent in a form ready for use, the contents of a phial containing carrier liquid (water, A) are introduced by means of an injection syringe into a 5 phial containing microparticles (B) and shaken until a homogeneous suspension is formed (from 5 to 10 seconds).

Example 3 A) Preparation of the carrier liquid: 10 4.5 g of sodium chloride are dissolved in water to a volume of 500 ml and the solution is forced through a 0.2 nm filter; 5 ml phials are each filled with 4 ml of this solution and sterilised for 20 minutes at 120°C. b) Preparation of the microparticles: Under sterile conditions, 198 g of anhydrous lactose ( 0.3 mm) are mixed intensively with 2 g of ascorbyl palmitate by homoeopathic trituration and the mixture is further processed as described in Example 1 under b), the following distribution of the particle 20 size being obtained: Median: 2.8 pim 100 % < 48 pm 99 % <12 pm.

The particle size is determined as described in 25 Example 1 under b). 207853 ml phials are each filled with 1.6 g of the microparticles.

C) For the preparation of 5 ml of the ultrasound contrast agent in a form ready for use, the contents 5 . of a phial (0.9 % sodium chloride solution in water. A) are introduced by means of an injection syringe into a phial containing microparticles (B) and shaken until a homogeneous suspension is formed (from 5 to 10 seconds), O Example 4 A) Preparation of the carrier liquid: In the same manner as described in Example 3 under A) , 0.9 % aqueous sodium chloride solution is prepared, introduced in 4 ml portions into 5 ml phials and sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles: Under sterile conditions, 199 g of tt-cyclodextrine are mixed intensively with 1 g of ascorbyl palmitate by homoeopathic trituration and the mixture is further processed as described in Example 1 under B), micro- particles having the following size distribution being obtained: Median: 2 jjm 99 % ^ 6 (jin 90 % 4 pm. i o 2 07853 € ■ . jj§ The particle size is determined as described in Example 1 under B). ml phials are each filled with 400 mg of the microparticles.

{Tn 5 . C) For the preparation of 4 ml of the ultrasound contrast agent in a form ready for use, the contents of a phial (0.9 % aqueous sodium chloride solution, A) are introduced by means of an injection syringe into a phial containing microparticles (B) and shaken until a 10 homogeneous suspension is formed (from 5 to 10 seconds).

Example 5 A) Preparation of the carrier liquid? 50 g of lactose are dissolved in water for 15 injection purposes, made up to a volume of 500 ml and forced through a 0.2 Mm filter; 5 ml phials are each filled with 4 ml of the solution and sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles: Ascorbyl palmitate is dissolved in methanol, filtered under sterile conditions through a 0.2 pm filter, recrystallised under sterile conditions, dried and passed through a 0.8 mm sieve. The sterile ascorbyl palmitate is then ground under sterile A 207853 conditions in an air-jet mill until the following size distribution of the particles is obtained: Median value: 1.9 |im 99 % <6 pm 90 % <3 pm.

The particle size and the distribution thereof are determined in a particle-measuring apparatus after suspension in cold aqueous 0.1 % Pluronic F68 solution.

Sterile 5 ml phials are each filled with 40 mg of the microparticles.

C) For the preparation of 4 ml of the ultrasound contrast agent in a form ready for use, the contents of a phial containing carrier liquid (10 % lactose solution. A) are introduced by means of an injection syringe into a phial containing the microparticles and shaken until a homogeneous suspension is formed.

Example 6 A) Preparation of'the carrier liquid: 4.5 g of sodium chloride are dissolved in water to a volume of 500 ml and the solution is forced through a 0.2 Mm filter; 5 ml phials are each filled with 4 ml of this solution and sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles: Under sterile conditions, a solution, filtered / 2 07853 under sterile conditions, of 0.5 g of ascorbyl palmitate in 40 g of isopropanol is absorbed on 199.5 g of sterile galactose particles, the isopropanol is removed by drying at 40° and 200 torr and comminution 5 is carried out in an air-jet mill until the following size distribution of the particles is obtained: Median value: 1.9 |im 99 % <6 nm 90 % <3 (jm.

The particle size and the distribution thereof are determined in a particle-measuring apparatus, for example after suspension in isopropanol. 2 g portions of the microparticles are each packed into 5 ml phials.

C) For the preparation of 5 ml of the ultrasound 15 contrast agent in a form ready for use, the contents of a phial containing carrier liquid (0.9 % sodium chloride solution in water. A) are introduced by means of an injection syringe into a phial containing microparticles (B) and shaken until a homogeneous suspension 20 is formed (from 5 to 10 seconds) .

Example 7 A) Preparation of the carrier liquid: 4.5 g of sodium chloride are dissolved in water and made up to a volume of 500 ml; the solution is 25 forced through a 0.2 nm filter and 5 ml phials are each } '• ■ • / o 2 07853 filled with A ml of this solution and sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles: Under sterile conditions, 199.5 g of galactose are 5 . triturated with 0.5 g of ascorbyl palmitate, mixed intensively,' passed through a 0.8 mm sieve and comminuted in an air-jet mill until the following size distribution of the particles is obtained: Median value: 1.9 nm 10 99 % ^ 6 pm 90 % <.3 |jm.

The particle size and the distribution thereof are determined in a particle-measuring apparatus, for example after suspension in isopropanol. 2 g portions 15 of the microparticles are each packed into 5 ml phials.

C) For the preparation of 5 ml of the ultrasound contrast agent in'a form ready for use, the contents of a phial containing carrier liquid (0.9 % sodium chloride solution in water, A) are introduced by means 20 of an injection syringe into a phial containing microparticles (B) and shaken until a homogeneous suspension is formed (from 5 to 10 seconds). ■ ■ c 7.. 078 5 3 Example 8 A) Preparation of the carrier liquid: Water for injection purposes is used and 5 ml phials are each filled with 4 ml of the water and 5 sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles: Under sterile conditions, 0.5 g of saccharose monopalmitate is triturated with 199.5 g of galactose, mixed intensively, passed through a 0.8 mm sieve and 10 ground in an air-jet mill until the following size distribution of the particles is obtained: Median value: 1.9 urn at least 99 % <6 nm at least 90 % <3 15 The particle size and the distribution thereof are determined in a particle-measuring apparatus, for example after suspension in isopropanol. 2 g portions of the microparticles are each packed into 5 ml phials.

C) For the preparation of 5 ml of the ultrasound 20 contrast agent in a form ready for use, the contents of a phial containing carrier liquid (sterile water for injection purposes. A) are introduced by means of an injection- syringe into a phial containing microparticles (6) and shaken until a homogeneous suspension 25 is formed (from 5 to 10 seconds). ) o vj 2 0/8 53 Example 9 A) Preparation of the carrier liquid: ml phials are each filled with 4 ml of water used for injection purposes and sterilised for 20 5 minutes at 120°C.

B) Preparation of the microparticles: Under sterile conditions, a solution, filtered under sterile conditions, of 0.5 g of saccharose o VSs-' monopalmitate in 40 g of isopropanol is absorbed on 199.5 g of sterile galactose particles, the isopropanol is removed by drying at 40°C and 200 torr and grinding is carried out in an air-jet mill until the • following size distribution of the particles is obtained: Median value: 1.9 Mn> at least 99 % <6 Mm at least 90 % <3 Mm.

The particle size and the distribution thereof are determined in a particle-measuring apparatus after 20 suspension in isopropanol. 2 g portions of the microparticles are each packed into 5 ml phials.

C) For the preparation of 5 ml of the ultrasound contrast agent in a form ready for use, the contents of a phial containing carrier liquid (water for 25 injection purposes. A) are introduced by means of an 2 07853 injection syringe into a phial containing microparticles (B) and shaken until a homogeneous suspension is formed (from 5 to 10 seconds).

Example 10 A) Preparation of the carrier liquid: ml phials are each filled with 4 ml of water used for injection purposes and sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles: Under sterile conditions, a solution, filtered under sterile conditions, of 0.5 g of saccharose monostearate in 40 g of isopropanol is absorbed on 199.5 g of sterile galactose particles, the isopropanol is removed by drying at 40°C and 200 torr and 15 grinding is carried out in an air-jet mill until the following size distribution of the particle is obtained: Median value: 1.9 pm at least 99 % <6 (am 20 at least 90 % <3 fim.

The particle size and the distribution thereof are determined in a particle-measuring apparatus after suspension in isopropanol. 2 g portions of the microparticles are each packed into 5 ml phials. 2078 5 a C) For the preparation of 5 ml of the ultrasound contrast agent in a form ready for use, the contents of a phial containing carrier liquid (water for injection purposes, A) are introduced by means of an 5 injection syringe into a phial containing micro- particles (B) and shaken until a homogeneous suspension is formed (from 5 to 10 seconds).

Example 11 A) Preparation of the carrier liquid: Water for injection purposes is used and 5 ml phials are each filled with 4 ml of the water and sterilised for 20 minutes at 120°C.

B) Preparation of the' microparticles: Under sterile conditions, 0.5 g of saccharose 15 monostearate is triturated with 199.5 g of galactose, mixed intensively, passed through a 0.8 mm sieve and ground in an air-jet mill until the following size distribution of ttte particles is obtained: Median value: 1.9 nm 20 at least 99 % < 6 ym at least .90 % <3 The particle size and the distribution thereof are determined in a particle-measuring apparatus after suspension in isopropanol. 2 g portions of the 25 microparticles are each packed into 5 ml phials.

-C v/8 53 C) For the preparation of 5 ml of the ultrasound contrast agent in a form ready for use, the contents of a phial containing carrier liquid (sterile water for injection purposes, A) are introduced by means of an injection syringe into a phial containing microparticles (B) and shaken until a homogeneous suspension is formed (from 5 to 10 seconds).

Example 12 A) Preparation of the carrier liquid: ml phials are each filled with 4 ml of water for injection purposes and sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles: Under sterile conditions, a solution, filtered under sterile conditions, of 0.5 g of saccharose distearate in 40 g of isopropanol is absorbed on 199.5 g of sterile galactose particles, the isopropanol is removed by drying at 40°C and 200 torr and grinding is carried out in an air-jet mill until the following size distribution of the particles is obtained: Median value: 1.9 (am at least 99 % < € p at least 90 % <3 nm.

The particle size and the distribution thereof are 2 078 ^ J determined in a particle-measuring apparatus after suspension in isopropanol. 2 g portions of the microparticles are each packed into 5 ml phials.

C) For the preparation of 5 ml of the ultrasound contrast agent in a form ready for use, the contents of a phial containing carrier liquid (water for injection purposes, A) are introduced by means of an injection syringe into a phial containing microparticles (B) and shaken until a homogeneous suspension is formed (from 5 to 10 seconds).

Example 13 A) Preparation of the carrier liquid: Water for injection purposes is used and 5 ml phials are each filled with 4 ml of the water and sterilised for 20 minutes at 120°C.

B) Preparation of the microparticles: Under sterile conditions, 0.5 g of saccharose distearate is triturated with 199.5 g of galactose, mixed intensively, passed through a 0.8 mm sieve and ground in an air-jet mill until the following size-distribution of the particles is obtained: Median value: 1.9 pan at least 99 % K. 6 pm at least 90 % ^3 jim. 2 078 53 The particle size and the distribution thereof are determined in a particle-measuring apparatus after suspension in isopropanol. 2 g portions of the microparticles are each packed into 5 ml phials.

C) For the preparation of 5 ml of the ultrasound contrast agent in a form ready for use, the contents of a phial containing carrier liquid (sterile water for injection purposes, A) are introduced by means of an injection syringe into a phial containing microparticles (B) and shaken until a homogeneous suspension is formed (from 5 to 10 seconds). *• 207853 A -§ NOV 1987 * ■ . 4 '»

Claims

WHAT WE CLAIM IS:

1. A contrast agent for use in the ultrasound diagnosis of the human or animal body, which comprises having a median particle size of less than 10 Lm microparticleslof a solid surface-active substance and micro-bubbles of a gas in a liquid carrier.

2. A contrast agent as claimed in claim 1, wherein the liquid carrier also contains microparticles of a solid non-surface-active substance.

3. A contrast agent as claimed in claim 1 or claim 2, wherein the solid surface-active substance is a lecithin, a polyoxyethylene fatty acid ester, glycerine polyethylene glycol ricinoleate, cholesterol, a polyoxyethylenepolyoxypropylene polymer, a saccharose ester, a xyloglyceride, a saturated or unsaturated (C4-C2Q)-fatty alcohol, a saturated or unsaturated (C4-C20)-fatty acid or a metal salt thereof, a mono-, di- or tri-glyceride or a fatty acid ester, or a mixture of any two or more of such substances.

4. A contrast ag'ent as claimed in claim 3, wherein the solid surface-active substance is magnesium stearate, ascorbyl palmitate, saccharose monopalmitate, saccharose monostearate or saccharose distearate, or a mixture of any two or more of such substances.

5. A contrast agent as claimed in any one of claims 2 to 4, wherein the solid non-surface active substance X v--" X -32- is a cyclodextrine, a mono-saccharide, a disaccharide, a trisaccharide, a polyol or an inorganic or organic salt, or a mixture of any two or more of such substances. 5

6. A contrast agent as claimed in any one of claims 2 to 5, wherein the sokd non-surface-active substance is galactose, lactose or ff-cyclodextrine, or a mixture of two or more of such substances.

7. A contrast agent as claimed in any one of claims 10 1 to 6, wherein the microparticles of the solid surface-active substance are present in a quantity of from 0.01 to 5 % by weight. 20

8. A contrast agent as claimed in claim 7, wherein the microparticles of the solid surface-active substance are present in a quantity of from 0.04 to 1 % by weight.

9. A contrast agent as claimed in any one of claims 2 to 3, wherein the microparticles of the solid non-surface-active substance are present in a quantity of from 5 to 50 % by weight.

10. A contrast agent as claimed in claim 9, wherein the microparticles of the solid non-surface-active iMI 207853 -33- substance are present in a quantity of from 9 to 40 % by weight. 1 ].

A contrast agent as claimed in any one of claims 2 to 9, wherein the ratio by weight of the microparticles of the surface-active substance to the microparticles of the non-surface-active substance is from 0.01 to 5:100.

12. A contrast agent as claimed in any one of claims 1 to 11, wherein the liquid carrier is water, a physiological electrolyte solution, an aqueous solution of a monohydric or polyhydric alcohol, or an aqueous solution of a mono- or di-saccharide.

13^ A contrast agent as claimed in claim *2 wherein the liquid carrier is an aqueous solution of glycerine, polyethylene glycol or propylene glycol.

14. A contrast agent is claimed in claim 12, wherein the liquid carrier is physiological sodium chloride solution, 10 % aqueous lactose solution or 20 % aqueous galactose solution.

15. A contrast agent as claimed in any one of claims 1, 3, 4 and 7 or 8, which comprises microparticles of magnesium stearate in a 20 % aqueous galactose solution. .

16. A contrast agent as claimed in any one of claims 2 to 11 > which comprises microparticles of ascorbyl palmitate and galactose in water. -34-

17. ft contrast agent as claimed in any one of claims 2 to H which comprises microparticles of ascorbyl palmitate and a-cyclodextrine in physiological sodium chloride solution.

A contrast agent as claimed in any one of claims 1, 3, 4 and 7 or 8, t which comprises microparticles of ascorbyl palmitate in a 10 % aqueous lactose solution.

19 A contrast agent as claimed in any one of claims 2 to 11, which comprises microparticles of saccharose monopalmitate and galactose in water.

20. A contrast agent as claimed in any one of claims 2 to 11', which comprises microparticles of saccharose monostearate and galactose in water.

21. A contrast agent as claimed in any one of claims 2 to 11. which comprises microparticles of saccharose distearate and galactose in water.

22. a contrast agent as claimed in any one of claims 1 to 21, wherein the microparticles have a median particle size of from 1 to 3 ^m. 207853 - 35 -

23. A contrast agent as claimed in claim 1, substantially as described in any one of the Examples herein.

24. A diagnostic kit for use in the ultrasound diagnosis of the human or animal body, which comprises 10 (A) a container which contains substantially 4 ml of a liquid carrier, and (B) a second container which contains between substantially 0.04g and 2.0g of microparticles having a median particle / ofa. solid Surfnct -size of less than'lU um*|substance and, if desired, microparticles of a solid non-surface-active substance. 15 20 25 CA '1987 A

25. A diagnostic kit as claimed in claim 24, wherein the containers A and B each has a volume of from 5 to 10 ml.

26 A diagnostic kit as claimed in claim 24 or r ^5 claim >6", wherein the ratio by weight of the microparticles of the surface-active substance to the microparticles of the non-surface active substance is from 0.01 to 5:100.

27. A diagnostic kit as claimed in any one of claims 24 to 26, wherein the surface-active substance is a substance (s) as mentioned in claim 3 or claim 4.

28*. A diagnostic kit as claimed in any one of claims 207853 - 36 - 24 to 27 wherein the optionally present non-surface active substance is a substance (s) as mentioned j.n claim 5 or claim 6.

29. A diagnostic kit as claimed in any one of 5 claims 24 to 28, wherein the liquid carrier is a liquid ■M as mentioned any Qf claims 13 to 15.

30- A diagnostic kit as claimed in any one of claims 24 to 29 r which also comprises an injection syringe for ^ transferring the contents of container (A) to container ^ 10 (B).

31. A diagnostic kit as claimed in any one of claims 24 to 30, wherein each of the containers (A) and (B) is a phial or ampoule.

32. A diagnostic kit as claimed in any one of claims 15 24 to 31, wherein the microparticles have a median particle size of from 1 to 3 pm.

33. a diagnostic kit as claimed in claim 24, wherein the contents of containers (a) and (b) are substantially as described in any one of the Examples herein. 20

34. An ampoule or phial for use in ultrasound diagnosis of the human or animal body, which contains a contrast agent as claimed in any one of claims 1 to ^ 23. 0 20785a - 37 -

35. A method of ultrasound diagnosis of the non-human animal body, wherein a contrast agent as claimed in any one of claims 1 to 23 is injected into a part of the animal body, and an ultrasound image of the micro-bubbles at a site in the body which it is desired to investigate is obtained.

36. A process for the preparation of a contrast agent as claimed in any one of claims 1 to 23, wherein microparticles of a solid surface-active substance and, if desired, microparticles of a solid non-surface- 15 active substance, are mixed with a liquid carrier and agitated until a homogeneous suspension is formed.

37. a process as claimed in claim 36, conducted substantially as described in any one of the Examples herein. SCHERING AXTIENGESELLSCHAFT By Their Attorneys HENRY HUGHES LIMITED By: