NO163654B - MICROBOBLE PROCEDURES IN STERILE, INJECTABLE UNIT DOSAGE FORM, PROCEDURE FOR THE PREPARATION OF AN Aqueous SUSPENSION MICROBOBLES AND USE OF THE MICROBOBLE PROCESSOR FOR INJECTION IN THE BLOOD FLOW. - Google Patents

Description

This invention relates to a microbubble precursor in sterile, injectable unit dosage form, adapted for injection into a fluid-containing area of a living being, for example the blood stream; furthermore, it relates to a method for producing an aqueous suspension of microbubbles suitable for such injection as mentioned, and use of the microbubble precursor for injection into the bloodstream of a living being.

It is known that the contrast in ultrasonic images is enhanced by the presence of microbubbles in a liquid, e.g. the blood flow in a test subject. The literature describes the formation of such microbubbles both outside the test individual with subsequent injection of the microbubbles into the individual's blood stream, and formation of microbubbles in situ in the blood stream.

Microbubbles have, for example, been formed in a simple way by vigorously stirring a liquid solution, such as normal saline solution, a dye solution or a temporarily withdrawn amount of blood, before it is injected into the blood stream. This can lead to significant enhancement of ultrasound image contrast, but these bubbles are usually of non-uniform size, often as large as 2000 µm in diameter, which are potentially dangerous as gas emboli. Furthermore, neither the size nor the concentration of the microbubbles can be regulated quantitatively for optimal contrast,

which limits their applicability. A method for obtaining microbubbles of a defined size by filtering bubbles produced by applying a direct current potential over a silver plate is described in US patent 3,640,271.

In the method according to US patent 4,276,885, microbubbles of very precise size are formed with a membrane that is resistant to coalescence, especially gelatin, and the precision microbubbles are then injected into the blood stream. This method leads to significant image enhancement compared to an air-filled saline solution. However, storage of the microbubbles produced in this way requires refrigeration or other techniques that preserve the microbubbles.

In the method according to US patent 4,265,251, a solid microbubble precursor, in particular a saccharide material with enclosed microbubbles of gas, usually a gas under pressure, is added to the blood stream. When the saccharide dissolves in the bloodstream, the individual microbubbles are released into the bloodstream. The microbubbles thus formed can be used to obtain an enhanced ultrasound echographic image of a liquid containing the microbubbles. When the pressure in the cavities is different from the pressure in the blood stream, ultrasonic signals are produced by the formation of the microbubbles, which can be used to measure local blood pressure. Although these microbubble precursors do not require the special storage treatment required for the precursors in US patent 4,276,885, the number of microbubbles per unit volume of precursor that can be formed, naturally limited due to technological factors.

While some of the microbubbles according to the state of the art, especially the gelatin microbubbles and the microbubbles formed from the solid "precursor", can be made relatively small, e.g. 10 pm average size or even smaller, it is relatively more difficult and time-consuming to make them in such a size range and the use of special equipment and carefully regulated parameters is required. This increases costs, and the very small gelatin microbubbles cause storage difficulties due to their relatively short lifetime. Furthermore, the number of microbubbles that can be formed in a liquid of microbubble-containing precursors naturally limited to the number of microbubbles present in the precursor Because the volume of precursor that can be added to a liquid is sometimes limited, e.g. in the blood for physiological reasons, and both the number of microbubbles that can be formed per unit volume of precursor and the average size of these is limited by technological factors, is the echogenic impenetrability of liquid r that can be achieved with such microbubble-containing precursors is less than optimal.

It would be desirable if a solid microbubble precursor could produce microbubbles in a liquid, and especially in blood, with a very small average diameter, e.g. about. 10 pm or less, in quantities greater than what is possible to produce in a gel or solid microbubble-containing precursor.

An object of this invention is to provide new solid microbubble precursors which have the previously mentioned microbubble-producing ability. Another object is to provide solid microbubble precursors that are cheaper to manufacture than the solid precursors of US Patent 4,265,251.

The invention thus relates to a microbubble precursor in sterile, injectable unit dosage form, adapted for injection into a fluid-containing area of a living being, for example the blood stream, characterized in that it comprises a particulate solid substance which is of non-toxic, physiologically acceptable material , soluble in the liquid/blood and mainly free of microbubbles, which particles are aggregates of microparticles with an average size in the range from 1 to 250 pm, which microparticles delimit gas-filled cavities between them and may contain intracrystalline gas pockets, and where the ratio between the mass of the microparticles and the volume of gas in the cavities between them is sufficiently large that the liquid/blood in which the microbubble precursor is dissolved will be supersaturated with respect to the gas in the area of the liquid/blood which surrounds the microbubbles when they are formed therein.

Furthermore, the invention relates to a method for producing an aqueous suspension of microbubbles suitable for injection into a fluid-containing area of a living being, for example the blood stream, characterized in that immediately before the injection a suspension of (a) a unit dosage amount of a sterile injectable material is produced in unit dosage form and suitable for injection into a liquid-containing area, for example the blood stream, of a living being and which will produce microbubbles in the liquid/blood stream, comprising a solid substance in particulate form which is of non-toxic, physiologically acceptable material, soluble in the liquid /blood and mainly free of microbubbles, whose particles are aggregates of smaller particles and contain gas-filled cavities, where the smaller particles have an average size in the range of 1-250 pm, and where the ratio between the mass of the smaller particles and the volume of gas in the cavities those in between is sufficiently large that liquid/blood in which matter ale is dissolved, will be supersaturated with respect to the gas in the region of the fluid/blood surrounding the microbubbles when they are formed therein, and (b) a unit dosage amount of a carrier fluid which is non-toxic and physiologically acceptable and in which the solid in particle form is at least temporarily stable so that a fluid injectable suspension can be formed.

The invention also relates to the use of the above-mentioned microbubble precursor for injection into the bloodstream of a living being. Reference is made to requirement 6.

With the help of the invention, a liquid's transmission characteristics towards an electromagnetic or elastic wave can be changed to form a detectable signal by dissolving a suitable substance in particle form or a suspension of it in the liquid/blood. this in a suitable carrier liquid which is effective when it comes to forming, in the liquid/blood, a quantity of microbubbles which act to significantly change such transmission characteristics in the liquid in the area of it containing the microbubbles.

In a preferred aspect, the impermeability of the liquid is measured

for ultrasound waves.

The drawing illustrates greatly enlarged a preferred solid microbubble precursor 1 suspended in a carrier liquid 3 and consisting of an aggregate 5 of microparticles 7 of the solid precursor with surfaces 9 and with adsorbed gas and intra-crystalline gas 15 enclosed in the microparticles. Between the microparticles, the aggregate has passage 11 which provides a connection to the interior of the aggregate, which contains trapped gas 13 in cavities between the microparticles 7.

The present invention will be described in particular

for ultrasonic imaging of the blood flow as a main illustration regarding the application. Similar applications for imaging other fluid-containing vessels and chambers in the body, changing the transmission characteristics of such other fluids to electromagnetic waves and other elastic waves sent through the fluids, will then be obvious to those skilled in the art.

By using the microbubble precursor and/or the method according to the present invention, a cloud of very small microbubbles of substantially uniform size, usually 10 µm or less in diameter, can be produced in the fluids, such as the blood stream, of a living individual, preferably a human. Furthermore, so many of these microbubbles are produced that by an ultrasonic imaging of the part of the blood flow that contains these microbubbles, the blood flow can be shown to be impermeable, if desired, i.e. that an ultrasonically impermeable cloud of the microbubbles can be formed which has a tendency to completely fill the blood vessel. If desired, a lower concentration of microbubbles can be formed by reducing the amount of microbubble precursor injected into the blood stream. That is, the density of the cloud of microbubbles can be regulated by regulating the amount of microbubble precursor that is added to the blood stream. This provides a much better contrast and contrast control than is normally achieved with microbubbles according to the state of the art. The ultrasonic images can be used for quantitative diagnostic purposes, for determining blood flow rate and the like, as described in US patents 3,640,271, 4,265,251 and 4,276,885.

Because the microbubbles formed according to the present invention can essentially completely fill the blood vessels through which they flow, wall effects can be observed in the blood vessel, i.e. the degree of turbulence near the vessel walls and the internal structure of the walls can be observed. In addition, the very small microbubbles that are formed can pass through capillaries, thus providing ultrasound contrast enhancement even in capillary systems and without the risk of gas embolism. tiny right-to-left shunts in the heart are visible on ultrasound images of the heart, since the tiny microbubbles can flow through such shunts. Small left-to-right shunts can also be detected, since the outflow sides of such shunts are free of microbubbles.

It has been found that under certain conditions a solid microbubble precursor does not need to have microbubbles, i.e. tiny balls or spheres of gas completely surrounded by solid, present in it for microbubbles to form in a liquid when the precursor dissolves in it. All that is needed is that the precursor provides an environment in the liquid in which it dissolves which allows for the formation of a number of microbubbles in the liquid and for their continued existence for a limited period of time after their formation.

In order for microbubbles to form at all in a liquid in which a microbubble precursor is dissolved, there must of course be a gas source that provides the gas volume needed for the formation of the microbubbles. Until now, this source has been the gas in the microbubbles which are present in the carrier itself. These microbubbles are only transferred from the precursor to the liquid in which it is dissolved. However, the gas may be supplied from one or more of (a) gas present in voids between the microparticles of the solid precursor aggregates; (b) gas adsorbed on the surface of the precursor particles; (c) gas which is an essential part of the internal structure of the precursor particles; (d) gas formed when the precursor reacts chemically with the liquid and (e) gas dissolved in the liquid and which is released from it when the precursor dissolves in it. It was initially assumed that the latter was the most important gas source. However, it is now believed that in many cases the gas comes

in the microbubbles produced according to this invention predominantly from voids between the microparticles in the aggregates of the precursor and from surface adsorbed gas rather than from the liquid in which the precursor is dissolved, which has been shown by experiments in which the solid microbubble precursor according to this invention has been carefully degassed (under vacuum >), is added to a liquid that is saturated with gas. In such experiments, the degassed precursor produced much fewer microbubbles in the liquid than the same precursor before degassing. Because the gas-saturated liquid was unchanged, it cannot be the sole source of the gas in the microbubbles. In another experiment, when the microbubble precursor (without degassing) is added to the same liquid that has been carefully degassed, microbubbles are formed therein, albeit again in significantly smaller amounts than the same liquid before degassing. The latter experiment confirms that the precursor itself is a significant, if not the predominant, source for the gas in the formed microbubbles. That a smaller quantity of microbubbles is formed can be explained either by the fact that the degassed liquid is no longer a supplementary source of the gas for the microbubbles or by the fact that the degassed liquid is

a poorer environment than a gas-saturated liquid for the maintenance of the microbubbles after they are formed, as explained below. Current signs point to the latter.

As stated above, a further condition for the formation of a number of microbubbles according to this invention is that the microbubble precursor provides an environment in the liquid in which it dissolves, which gives the microbubbles the opportunity to continue to exist in the liquid for a suitable period of time, e.g. one or more seconds, after they are formed. This condition is met if the amount of precursor dissolved in the liquid in the area immediately surrounding each microbubble at the time of its formation and thereafter during its useful life is sufficient to make this liquid area supersaturated with respect to the gas in the microbubble. The required degrees of supersaturation must be sufficient to compensate for the elevated pressure inside the microbubble that occurs due to its surface tension, which will be described. If this condition is not met, a microbubble with a diameter of 10 µm or less will collapse in the course of less than a second upon dissolution in the liquid together with the precursor. The microbubble precursor must thus have sufficient mass to achieve this localized supersaturation.

Finally, if the number of microbubbles produced is to significantly exceed the number of solid precursor particles added to the liquid, each particle must provide a number of nuclei from which microbubbles can form. As explained below, aggregates of microparticles of the solid precursor provide such a number of nuclei.

The aforementioned conditions can be met with many different types of solids, provided that these substances have the necessary physical structure. This necessary physical structure can be determined based on the criteria explained below.

Microbubble formation according to this invention is determined by the following factors: 1. The total volume of the microbubbles produced is equal to the sum of the volume of gas supplied by the solid precursor plus the gas that is released from the surrounding liquid upon dissolution of the solid precursor. 2. The total number of microbubbles produced is equal to the total number of microbubble cores supplied by the solids precursor. 3. The size of each microbubble is determined by the volume of gas available to each bubble core. 4. The lifetime of microbubbles is determined by the length of time in which the microbubble precursor can produce localized gas supersaturation in the liquid in the area surrounding each microbubble. This prevents the microbubble from dissolving in the liquid.

Each of the above-mentioned factors will be discussed in the following:

I. GAS SOURCE FOR MICROBUBBLE FORMATION

a. Total Gas Volume Available for Microbubble Formation The volume g* ass V g introduced when a solid microbubble precursor is dissolved in a liquid can be calculated as follows:

V = V + V + V. + V

g c a i r

where V c = g3 ash volume in the interparticle cavities between the microparticles in an aggregate precursor,

Va = volume of gas adsorbed on the surface of the microparticles,

V^ = volume of intracrystalline gas in the microparticles,

and

Vr = gas volume produced by chemical reaction between the precursor and the dissolving liquid.

The total final volume V^ of the gas available for the formation of microbubbles is this gas volume V^ introduced at the solid precursor plus the volume of dissolved gas Vd separated from the liquid upon dissolution of the solid precursor, i.e.

V, = V + V,

b g + d

In the preferred embodiment of this invention, Vc>Vd>Va>Vi and Vr = °-

Stated in another way, the total final volume of the microbubbles is V^ = -g- ND3 where D is the final average diameter of the microbubbles and N is the total number of bubble cores (and microbubbles).

The origin and relative importance of each of these sources involved in the gas supply will now be described.

b. Gas trapped between the particles Microparticles of a solid can join together to form aggregates by electrostatic, chemical or physical bonding. When the space between the microparticles is filled with gas, the gas will, in the right environment, be released as microbubbles when the aggregates dissolve in a liquid. The total amount of enclosed gas V depends on the size distribution, shape and degree of compression of the microparticles that make up the aggregates. The rate of dissolution of the aggregate, and therefore the rate of bubble production, also depends on these geometric properties as well as on the solubility of the microparticles present. The dependence of the size and number of intermediate cells on these geometric properties of the aggregates is well known due to its importance in the production of and properties of concrete. Under many conditions, this gas is the main source of gas for microbubble formation according to the present invention.

c. Intracrystal- trapped gas

Different methods for the production of crystalline solid types (single or polycrystalline) can cause a gas volume V\ to be enclosed in individual crystals (crystallites) or in the grain boundaries between the crystallites. Since pockets of this intra-crystal gas can be formed by nucleation of gas molecules enclosed (sorbed) in the lattice, the gas pressure in such pockets can be. much higher than the pressure at which the gas was introduced into the solid, and much higher than the pressure in the bubbles they form. The microbubbles that form when such a solid dissolves in a liquid close to atmospheric pressure can therefore become much larger than the size of the intracrystal gas pockets, they can have a much greater echogenicity, and the energy released by bubble formation can set the liquid in strong motion, including formation of ultrasound impulses. Also, the intracrystal-enclosed gas can be a gas different from the gas present in the cavities between the microparticles in the microbubble precursor aggregates. The latter gas volume is considerably larger than the volume of intracrystal-enclosed gas in the preferred precursors according to this invention.

d. Surface-adsorbed Gas

Gas can be adsorbed on the surface of the microparticles

in the aggregates of "a microbubble precursor. When the microparticles are very small, the total surface area becomes very large, and the volume V of adsorbed gas released can be significant compared to the volume V of the aggregate. It can be easily shown that V /V = 7rkT/ pd 2D , where k is the Boltzmann con-

a P P

the stent, T is temperature, p is the gas pressure in the microbubbles,

d is the thickness of the gas molecule coating adsorbed on. the surface of the particles in the aggregate, and D per the average diameter of the particles. At T = 300 oK, p = 1 atm and d = 3.6Å (the diameter of the oxygen molecule), this equation gives V /V —1.0 D , when D is measured in micrometers. Therefore can

a P P P

an aggregate of microparticles of diameter l^um, which has a complete monolayer of gas molecules adsorbed on its surface, will produce a volume of microbubbles approximately equal to the volume of the aggregate, and an aggregate of 0.1^,um particles will produce a ten times greater volume of microbubbles when dissolved.

Although a densely packed aggregate of very small microparticles can theoretically produce a large volume of adsorbed gas, it is preferred that the microbubble precursors according to this invention form loosely packed aggregates, the voids between the microparticles of the aggregates being the main source of the gas produced by the preferred precursors.

e. Gas produced by reaction with the liquid If the microbubble precursor undergoes a chemical reaction to form a gas when dissolved in a liquid, e.g. if microparticles of sodium bicarbonate are enclosed in the aggregate, the volume of gas V rwhich is thus formed can help to provide gas for the microbubbles. The preferred precursors according to this invention only dissolve in the liquid without undergoing a gas-forming chemical reaction. Therefore, <V>r is usually 0.

f. Gas dissolved in the liquid

When a solid soluble in a liquid saturated with dissolved gas dissolves in the liquid, molecules of the dissolved solid can combine with molecules of the solvent, the concentration of the solvent being reduced and causing the liquid to become supersaturated with gas. Such molecular association of a solute with its solvent is usually termed "solvation" ("hydratation" in aqueous solutions), and the resulting reduction in gas solubility is termed "salting out". If the dissolved liquid contains nuclei for the formation of gas bubbles, gas will be released from the solution of these nuclei during the formation of bubbles.

This phenomenon can easily be seen when salt or sugar is added to a carbonated drink. If the number of such nuclei and their distribution in the liquid are in the right proportions, the gas will form microbubbles of the required size in the liquid. The gas volume separated from such liquids V contributes significantly to the gas volume in the microbubbles that are formed according to the method according to this invention, either by the volume being increased by the supply of gas from the microbubble precursor, or by the amount of the latter gas that is dissolved in the liquid during microbubble formation, is reduced. If the microbubble precursor only provides the nuclei for the microbubbles and little or no gas, e.g. a degassed precursor containing insoluble bubble nuclei, the gas in the liquid can be the essential or even the only gas source for the microbubbles. Conversely, if the liquid is completely degassed, it cannot add any gas.

The fraction of the dissolved gas that is separated from a volume VT of a gas-saturated liquid by solvation (salting out) at a. mass M of solid precursor is

where a is the Bunsen absorption coefficient (cm Dissolved gas per cm 3 liquid), and K is the solvation proportionality factor. The volume of excreted gas is therefore V^ = V^Aa.

II. MICROBUBBLE CORE

It is shown below that in order to achieve the greatest echogenic ability for a given amount of gas, it is necessary to "achieve the smallest practical bubble diameter. Microbubbles less than 10 µm in diameter, i.e. close to capillary diameter, are also desirable physiologically since they should be harmless if they pass from the venous to the arterial system.Furthermore, an additional resonance enhancement of echogenicity (greater than 10-fold) can be achieved at typical MHz ultrasound imaging frequencies if bubbles in

this size range is used.

It can be shown from the above equations that to achieve a given desired microbubble diameter D, the number of bubble cores per mass unit. solid precursor n = N/M be

where Vg and d are the volume and density of the solid precursor. If it is assumed that there are e nuclei associated with each microparticle of mass M in the aggregate, the ratio of particle diameter to bubble diameter must be

III. REQUIREMENTS FOR BUBBLE STABILITY

The lifetime of microbubbles of the desired small size is usually very short in gas-saturated water or blood. For example, the lifetime of a nitrogen bubble of diameter 10 µm in nitrogen-saturated water or blood is only about 1 second, and for a bubble of diameter l^urn it is only a few milliseconds. The degree of gas supersaturation e in a liquid which is sufficient to prevent microbubbles of the required size D from dissolving in the liquid over a suitable period of time is given by

where t is the surface tension in the liquid-gas interface and p is the saturation pressure of the dissolved gas in the liquid. In the present invention, this degree of supersaturation is achieved by solvation of a. mass M of solid precursor in the liquid region with volume VT containing the microbubbles. The above equation plus the equation that defines the fraction of the dissolved gas that is separated from a volume of a gas-saturated liquid therefore requires for stability that

IV. ULTRASONIC PROPERTIES OF MICROBUBBLES

The ultrasonic scattering cross section of each microbubble is a = D<2>Fr, where Fr = {[ (Dr/D) 2-l ] <2> + 6 <2>j<-1> where Dr is the resonant bubble diameter and <5 is the damping constant. For . air bubbles in water is Dr = 650/f cm at frequency f and 6 - 0.2.. When D <>>>Dr, Fr s 1, and when D = Dr, Fr = 25.

The ultrasonic echogenicity of the microbubbles per mass-unit solid precursor, namely E = na, is therefore given by

For a given gas solubility a and a given mass M of solid f-f precursor with density d, more ultrasonic contrast can therefore be formed with a small bubble diameter D, a large fraction of trapped gas Vg/Vg, a high resolution factor K and operation near bubble resonance.

V. SUMMARY OF REQUIREMENTS FOR THE MICROBUBBLE PRECURSOR

AND QUANTITATIVE EXAMPLES OF THESE

The following conditions are essential for the formation and maintenance of microbubbles of a given required diameter useful for an appreciable time in a liquid with a given initial gas content: a) Sufficient gas must be available in the microbubble precursor and in the liquid volume VT to provide the overall

volume Vtø of microbubbles that provide the required ultrasonic

echogenicity in the volume VT.

b) A sufficient number of gas nuclei n must be provided by the microbubble precursor for the formation of the required diameter D of the microbubbles with the available gas volume V^. c) Sufficient mass M of solid must be present in the microbubble precursor to supersaturate the liquid volume VT with

gas by solvation so that bubbles with the required diameter D do not dissolve during the required application period.

Condition a) requires sufficiently high values for contained ga■ ssf raks . ion V g /V s and solvation factor K, which together are required physical and chemical properties of the precursor.

Condition b) requires a number of gas cores which are in accordance with Condition a) and the required microbubble diameter D, and which is primarily a required physical property of the precursor.

Condition c) requires a sufficient concentration

of solid precursor M/VT which is primarily dependent on the ratio t/K between surface tension t and solvation factor K, And both of these can be influenced by the chemical properties of the solid precursor and its carrier liquid.

The following "set of values is typical of the various physical and chemical properties of solid-for-runners suitable for microbubble ultrasound image intensification: In water, a = 0.029 for O2, 0.015 for N2, and 0.76 for CC>2 at 25°C and one atmosphere partial pressure for each of them.

In venous blood, in which the partial pressures for 02, N2 and CO., are respectively approx. 40, 760 and 45 mm Hg, a = 0.06. For ionic salt solutions, see P.S. Albright and J.W. Williams, Trans. Faraday Soc. 33/ 247 (1937), is K = 300 Z/W cm<3>/g, where Z and W are the valency and atomic weight of the salt. If V is equal to the void volume in an aggregate of spherical particles, V /V = 0.35, 0.9 and 1.9 for close-packed, respectively

g p

single-cubic and rhombic devices. For gas adsorbed on particles with diameter D ,um, V /V = l/D .

p/ g s p

As a typical example in terms of echogenicity, the above formulas give, when D = 10.um bubbles, and dextrose or galactose as solid precursors have d = 1.5 g/cm ,

W 180 g, Z = 1, Vg/Vs = 1 and a = 0.06, for non-resonant ultrasound echogenicity in venous blood

E. = 4 x IO<3> [1 + 0.15] cm<2>/g..

Under these conditions, it can therefore be seen that a very high echogenicity is achieved per grams of solid precursor, of which approx. 15% comes from excreted gas. In the case of a densely packed aggregate of effective divalent solid precursor, however, released gas will contribute approx. half or more of the total echogenicity.

In terms of the required cores, e is 0.75, 1.0 and 1.5 for center-centered, close-packed and single-cubic devices, respectively, if each interparticle cavity in a device of spherical particles acts as a bubble core. It follows from the above formulas that the required particle diameter is approximately equal to the desired bubble diameter, e.g. Dp/D = (1.5/1.15)<1/3> = 1.1. It has been found that efficient formation of microbubbles of diameter less than 10 µm from a solid precursor requires a precursor particle size in this range. It can be seen from the foregoing that non-wetting solid cores of this number and of this diameter can be used as microbubble cores, rather than gas contained in the solid precursor, provided that the solid precursor and the liquid in which it is dissolved together provide the volume of gas which is needed for the formation of a stable microbubble from the cores.

In terms of the required mass of solid precursor, t = 42 dyne/cm for blood, so the above formulas give p = 1 atmosphere and D = 10.um bubbles, is

It has been observed that an injection of galactose solid microbubble precursor with M = 1.5 is about the maximum for enhancing an ultrasound image of a dog heart for which the above calculated example gives a final volume of blood-galactose mixture of V = 15 cm<3>. The ventricular stroke volume for the dog is in the range 15-25 cm<3.> It therefore appears that the solid precursor injection is carried as a bolus into the ventricle where it is almost completely mixed with blood.

From the foregoing, it can be seen that bubble formation with a solid microbubble precursor can be improved in one or more of the following ways: a) Increasing the volume of gas separated from the liquid, e.g. by using a strongly solvating compound as a solid precursor, or more precursor; b) Increasing the amount of gas contained in a given mass of the precursor, e.g. by reducing the size and/or packing density of the microparticles in aggregates that form the precursor; c) Increasing the number of bubble-forming nuclei (in accordance with a and b), e.g. by reducing the size

of the microparticles or by adding a non-wetting nucleating agent to these;

d) Reduction of the quantity needed of precursor to achieve the required degree of localized gas supersaturation

in the liquid in which the precursor is dissolved; by using a fast-dissolving strongly solvating compound as a precursor, by adding the precursor as a mixture with another liquid or solid that reduces the solubility of the gas dissolved in the gas-saturated liquid, or by means of a. low bubble surface tension, e.g. by mixing a small amount of surfactant into the solid precursor or its carrier liquid;

All of the previously described processes are likely to be active to varying degrees in a good solid microbubble precursor. The formation of localized gas supersaturation in the liquid surrounding the microbubbles is essential.

Formation of Solid Microbubble Precursors and Their Observed Behavior

a. Commercially Available Salts and Saccharides Various salts and saccharides in their commercially available form have been evaluated as solid precursors for microbubble formation. Visual observations of microbubble formation are in good agreement with quantitative measurement

of the ultrasound contrast effect produced by any given metal. In the case of samples consisting of relatively large individual crystals, without any visual sign of entrapped gas, their behavior is consistent with the gas separation (salting out) condition for micro-bubble production.

It has been confirmed by quantitative experiments that the smaller the particles, the more effective a given material is as a solid microbubble precursor. This is in accordance with the gas separation mechanism because the greater dissolution rate for a finely divided material should result in a higher degree of local gas supersaturation and therefore in a greater formation rate and total number of microbubbles for a given amount of material. Materials that were an-

shown to have good solvation tendencies proved to have the best properties when it came to microbubble formation. Microbubble formation in degassed water using precursors that are highly active in gas-saturated water, and in gas-saturated water using degassed (in vacuum) precursors that were highly active in this before degassing, is significantly less, which confirms that the solid precursor and the test liquid together must add enough gas to provide stable bubbles. Since the test liquids and microbubble precursors are rarely degassed, this requirement can be easily met.

Physical Form of Solid Microbubble Precursors

a. Optimal Physical Form

The microbubble precursors according to this invention, unlike the microbubble precursors according to the U.S. patents 4,265,251 and 4,276,885, even substantially free of microbubbles, i.e. microspheres or balls of gas completely surrounded by the precursor material.

The microbubbles are formed by means of the solid precursors according to this invention by their formation in the liquid in which the precursor is dissolved. The present precursors are, like the precursors according to U.S. patent 4,265,251, stable solids at ambient temperatures and are non-toxic and physiologically satisfactory when injected into the bloodstream of a living subject, e.g. a human being.

The physical structure of the microbubble precursor is extremely important and determines the number and size of the microbubbles

.which is different from this one. A precursor in microcrystalline form whose crystals are separated, compact and relatively free of structural defects has few nuclei for the formation of microbubbles and therefore little bubble-forming capacity, i.e. so

as little as one bubble or less per particle, while aggregates of particles with many internal cavities that are in connection with the outer part of the aggregate are the most effective. Aggregates of microparticles, each microparticle having an average size approximately corresponding to the size of the desired microbubbles, are preferred.

The number of particles in each aggregate is preferably

greater than 10 to achieve a significant volume of internal structure in the aggregate, and is more preferably greater than 100 so that one approaches the advantages of a fully developed aggregate structure. The preferred size of microparticles in the aggregate is approximately equal to the microbubble size that is maximum for the intended use and falls in the range of 1-50 µm. Most preferably, however, the microparticle diameter falls in the range of 5-10 µm since this size range of bubbles is most useful for injection into the blood stream, as described earlier.

The preferred size of the microparticle aggregate is most often determined by the maximum dissolution time for the precursor for a given use. For example, if bubble formation is required immediately after injection at the injection site, an average aggregate size of approx. 20 µm preferred, while it has been found that for enhancing the ultrasonic image of the right half of the heart an average size of 40 µm is preferable, and when passing through the lungs to image the left heart half it has been found that an average size of 125 µm is preferable.

For normal use, aggregates in the size range 30-50 µm are most preferred.

• It is clear, however, that the preferred average microparticle and aggregate sizes are subject to certain reservations. For example, the microparticle size must be in accordance with it. preferred- number of particles for a given aggregate size, or vice versa, depending

of which claim, i.e. bubble size or illumination time, which is the most important in the particular application of the precursors. To achieve the desired average aggregate sizes, it may be necessary to pass the precursor through a sieve so that particles larger than approx. 250 µm is not present and the precursor can freely pass through an injection needle.

Because the solid precursor is usually the main source

for the gas for the microbubbles, the cavities in the aggregates usually and preferably contain a volume of gas roughly equivalent to the volume of gas in the microbubbles formed by the precursor, although, as explained above, this is not absolutely necessary if the liquid is gas-saturated.

In addition to the physical structure of the particles, which must provide the necessary number of microbubble nuclei, the mass of the precursor must be sufficient to cause the areas of the liquid in which it dissolves, immediately surrounding the place where a microbubble is formed and later used, to be supersaturated with respect to to the gases dissolved in it, or otherwise expressed, in apparent•equilibrium with. consideration of the gas in the microbubble. It is usually insufficient for the liquid to simply be saturated with gas at the ambient liquid pressure, ■ because the pressure of the gas in the microbubble usually mainly exceeds the ambient liquid pressure due to surface tension effects. Before the microbubble is to have a useful life after it is formed in the liquid in which the precursor dissolves, the liquid must therefore be supersaturated with respect to the surrounding, pressure to the same degree that the pressure of gas in the microbubble exceeds the surrounding pressure.

The excess pressure in the bubble due to surface tension is inversely proportional to the bubble diameter, and is about one atmosphere for a 3^um bubble in water. If this overpressure in the bubble is not compensated for by a similar overpressure in the dissolved gas, the large pressure difference and the large surface/volume ratio will therefore cause microbubbles of this order of magnitude to dissolve in the liquid immediately after they are formed, or even before the gas cores are released from the unit. The solid precursor according to this invention provides both the gas nuclei and the solid mass needed to stabilize the resulting microbubbles upon solvation-induced supersaturation of a liquid. The ratio mass/gas volume M/Vg. for the precursor which is needed•to form, stabilize and supplement microbubbles of a given diameter depends on the ratio t/K as explained in the preceding. The surface tension t and the solvation factor K both depend on the properties of the liquid and the solid, so that for a given liquid the required ratio between mass and gas depends on the properties of the solid. As discussed below, it has been found that the bubble-forming properties of different solid materials in the same aggregate form can vary to a large extent, but that the best bio-compatible solid materials have bubble-forming properties in the above-described optimal aggregate precursor form which are essentially similar and which are very effective for the production of ultrasonic contrast in the blood stream. The leading mass/gas volume ratio for these materials, not including gas outside the aggregates, is achieved when the density of the aggregate is in the range 1/3-2/3 of the specific gravity d of the solid. Most preferred is the density, of the aggregate approx. d/2, so that

the average size of the interparticle voids is approximately equal to the average size of the microparticles which

in turn is approximately equal to the desired size of the microbubbles formed by the precursor. For the saccharides, d is 1.5 g/cm<3>, and for NaCl, d =2.17 g/cm<3>.

Although it may seem difficult to provide

all the conditions necessary for the formation of microbubbles of the required size in suitable quantities and with a suitable lifetime, many different microbubble precursors according to this invention can easily provide these conditions if the above-mentioned criteria are met. The criteria for a satisfactory microbubble precursor can be determined theoretically, as discussed earlier.

It was found that the same solid material provided

from different commercial sources or powdered by different methods can have remarkably different microbubble formation characteristics because they differ in various ways from the optimal physical form described above.

Therefore, in order to achieve the high degree of reliability, reproducibility and efficiency required for clinical diagnosis, it was necessary to establish methods for the production of standard solid-state microbubble precursor materials that are independent of the shape of the raw starting material. and which results in optimum physical shape. It has been found that such standard materials can be prepared from commercially available starting materials by carefully controlled ball milling to a finely divided powder or by controlled recrystallization.

b. Ballpoint pen

Commercial batches of various saccharides selected for potential biocompatibility were ball milled for 10 minutes in a Pitchford Chemical Model 3300 ball mill with three 1.5 cm diameter alumina balls. The resulting powders are aggregates of microparticles, and the aggregates are in the size range 1-160µm with a predominant proportion of aggregates near 40µm, and the microparticles are in the range 1-20µm with a predominant proportion of microparticles close to 10µm. The lower limit of l^um is the resolution limit of the light microscope used. Although a large number of smaller microparticles and aggregates could also be present, they would not occupy any significant fraction of the total puiver volume. Relatively few of the largest aggregates occur and these also occupy an insignificant volume. They can be removed by sifting, if desired.

Among the saccharide precursors prepared by this preparation method, galactose was found to give the best ultrasonic contrast in all particle size ranges obtained by sieving. It was chosen as the standard material for a series of in vitro and in vivo experiments.

Since a large amount of the interparticle-trapped gas is found in loose aggregates of fine particles of these materials, it is possible that such gas may contribute significantly to their microbubble formation.

Heating and extensive plastic processing of the material occurs during the painting process. Diffusion of air into the hot material or entrapment of air during the plastic processing can occur in this material, so that

view \ p

is enabled that the intra-crystalline trapped gas can contribute to its microbubble-forming characteristics. Since a significant fraction of the material consists of particles with a diameter smaller than 10^,um, adsorbed gas could also contribute significantly to microbubble formation. Finally, the large degree of plastic processing, together with the possibility of including gas and solid matter associated with the painting process, should provide a high density of bubble-forming cores in this material.

Although gas release (salting out) is a significant effect of these materials, they also form some bubbles when dissolved in degassed water. However, the volume of microbubbles formed in this is much smaller, which suggests that the sum of the other effects, particularly of gas enclosed in the cavities between microparticles, is a significant, if not dominant, factor.

c. Water - Recrystallization

Galactose was found to be clearly superior to dextrose and sodium chloride as a solid microbubble precursor for obtaining ultrasonic contrast when processed by the ball-painting method. However, dextrose and NaCl are preferred as biological contrast agents because their suitability for injection in large quantities into the bloodstream is well established, while the universal suitability for. galactose is less safe. Methods have been found for the production of highly active dextrose and NaCl microbubble precursors which can be compared to or are superior to galactose processed by the ball milling method.

The first of these methods, which is most suitable

for dextrose and other materials which tend to decompose when heated to high temperatures, consists of recrystallization of dextrose from a saturated aqueous solution at low temperatures. Commercial USP dextrose is dissolved in boiling deionized water to obtain a saturated solution which is rapidly cooled by stirring in an ice water bath until recrystallization begins. The solution is then completely recrystallized for several hours in air at 6°C and ambient pressure. The resulting crystals are washed with USP ethanol

and dried in air with 2.5% moisture at ambient pressure.

The resulting precipitate consists of large, i.e.

from approx. 100 to 1000 µm in size, relatively hard and dense aggregates of microparticles of dextrose with an average size of 5-20 µm. The aggregates dissolve relatively slowly in gas-saturated water and continuously develop copious amounts of strongly expelled microbubbles in the diameter range 1-10 2 um until they are completely dissolved. When the aggregates are crushed, the resulting individual particles are in the 10-100 µm range and also develop microbubbles in the same way, but aggregates and particles much smaller than this dissolve without producing microbubbles. The rate of formation and the total number of microbubbles formed by the aggregates is much smaller in degassed water than in gas-saturated water, and the lifetime of the bubbles formed is shorter in the degassed water. The ultrasonic contrast obtained with this precursor is equal to or superior to ball milled galactose.

The second recrystallization method is faster and

is suitable for materials such as NaCl that can be heated to high temperatures. Commercial NaCl crystals are dissolved in a minimal amount of warm deionized water. The solution is then heated rapidly in a crucible which is kept at a temperature which causes all the water to boil off, preferably for approx. one minute. The NaCl crystals that remain are immediately cooled to room temperature. The visual appearance and microbubble formation properties of this activated NaCl material are the same as those of the activated dextrose obtained by low temperature recrystallization.

d. Solvent- Recrystallization

Another method for producing standard microbubble precursors from commercially available materials does not produce more microbubbles than the water-recrystallized materials, but has unique microbubble-producing properties that can be important in special applications that require these properties.

In this method, a solid precursor is dissolved

in a warm organic solvent, e.g. dissolve dextrose in boiling hot (80°C) pure ethanal and recrystallize

and dried in the same way as described for the method for low-temperature water recrystallization. Dextrose recrystallized by this method has a fine, dendritic structure and forms aggregates with a large, open structure similar to new snow. This material dissolves instantly in water, leaving behind a "shadow" of relatively

few microbubbles with a fairly large size range (10-100 µm) of the same shape as the aggregate.

e. Irradiation activation

Large crystals (1-0.1 mm) of the polysaccharide arabinose were observed to form negligible microbubbles even in water supersaturated with CC^- After irradiation with high-energy cobalt gamma rays, this material formed copious amounts of lO^um bubbles in CC^- supersaturated water as it dissolved. This result suggests that crystal defects caused by the irradiation can serve as nuclei for the formation of solvation-separated microbubbles. The other effects seem unlikely to be significant with this method.

Chemical Types of Solid Microbubble Precursors

High solubility of the solid precursors is desirable because the degree of supersaturation achievable depends on the concentration of the solvation solid precursor achievable in the liquid. A degree of supersaturation of 10% enables marginal stabilization of a 30 µm bubble, so that the preferred solubility is greater than the concentration of 0.3 mol/liter of an effective monovalent solid to which this degree corresponds. Solubilities equal to or greater than 1 mol/liter are preferred because such concentrations provide more than 30% supersaturation, which can stabilize bubbles smaller than 10 µm.

Since there is an optimum dissolution time, however, there is an associated optimum dissolution rate, which is proportional to the solubility, for the preferred aggregate sizes previously mentioned. For applications that benefit from a long exposure time, such as when imaging the left hemisphere of the heart, a low resolution is advantageous, but it must be consistent with that required for adequate supersaturation described above. The optimal solubility for such applications should therefore be close to 1 mol/litre. In reality, it has been found that galactose

is superior to the other saccharides for the formation of contrast for the left half of the heart and its solubility is approx. 1 mol/liter (0.20 g/cm 3 ).

Because the microbubble precursors are prepared prior to use, they should be stable at ambient temperatures for storage purposes. For economic reasons, they are also preferably stable in air. Although crystalline compounds are preferred, amorphous compounds which form aggregates of microparticles of the required size may also be used.

The microbubble precursors can thus be formed from many chemicals or mixtures thereof. For example, various salts such as sodium chloride, sodium citrate, sodium acetate and sodium tartrate can be used, provided that these have the necessary physical structure. Polyvalent electrolytes such as CaCl2 and AlCl^ are effective but are biologically less satisfactory. Many organic and inorganic compounds are also suitable as microbubble precursors, including those listed in the table below, of which glucose, galactose, maltose and sodium chloride are preferred. Other sugars and sugar-related compounds that may be used include arabinose, maltotriose, maltotetrose, sorbitol, mannitol, gluconic acid and saccharic acid. Particularly suitable organic compounds are those sugars which have the ability to form hydrogen bonds with water. Monosaccharides and disaccharides have been found to be excellent microbubble precursors. Raffinose,

a trisaccharide, is also satisfactory as a precursor. Table I shows sodium chloride and various sugars formed as microbubble precursors by the ball milling technique that has been tested.

It should be noted that although the maximum bubble size (Table I) was 15-25 µm, the average bubble size was approx. lO^um. Such very small microbubbles have the ability to enter capillaries and provide ultrasonic contrast in them. Furthermore, many relatively small microbubbles form a very fine and dense contrast agent with mainly complete ultrasonic opacification in the possible cavities. This gives rise, for example, to the discovery of small shunts and other small structures and flows in the heart. Furthermore, more contrast can be obtained for the same amount of contrast agent than with prior art microbubble contrast agents, because of it. particularly effective achieved opacification. The degree of opacity (density) can be regulated by regulating the amount of solid precursor used. Furthermore, the arrival time of microbubbles to any particular area of the blood stream can be regulated by choosing a particular saccharide or salt, since the different saccharides dissolve at different rates and different salts at yet other different rates. Thus, any one of many peripheral injection sites can be used for the production of microbubbles

on any particular area of the blood stream,

for example, in the heart, by selecting a saccharide or salt that dissolves at a particular rate. Different carrier fluids can also be used with a special solid precursor to select the site of microbubble production. Furthermore, because of the very small microbubble size achievable, the microbubbles can cross through the lungs when the contrast agent is injected on the venous side and can therefore provide contrast to the left side of the body. Due to the relatively small bubble size that is achievable,

the dangers of air embolism are essentially eliminated. This size is small enough for harmless passage through brain capillaries, for example, and enables amplification of ultrasonic images of brain blood vessels.

B carrier fluid

In a preferred embodiment of the invention, the solid microbubble precursor is added to the liquid in which the microbubbles are to be produced as a dispersion or suspension in a carrier liquid in which the solid precursor is at least transiently stable, i.e. it is chemically and physically stable therein and does not form all of its microbubbles therein within at least several seconds after being dispersed therein, preferably for at least several minutes or longer.

The carrier fluid has several important functions:

a) It acts as a dispersant, allowing the solid precursor powder to form and disperse uniformly

in a stable or quasi-stable suspension suitable for injection into the blood stream or other test fluid in which the microbubbles are to be formed.

b). It serves to hold the suspension of solids flowing in it together as a viscous bolus for transport in

the test liquid to the place where the microbubbles are to be used.

c) It serves as a surfactant to modify the surface properties of the test fluid to aid in the formation and

stabilization of the very small microbubbles formed by the impact of the solid precursor on the test liquid.

Regarding the carrier liquid's function as a dispersant, it must wet the solid precursor sufficiently so that a homogeneous slurry can be formed, but it must preserve and not significantly dissolve the aggregates of microparticles and the gas nuclei contained therein. It has been found that many gas-saturated non-aqueous carrier liquids, especially those low-polarity liquids which do not solvate the solid precursor and in which the solid precursor is insoluble, meet these criteria. Relatively diluted aqueous solutions can also be used as carrier liquids, but only if sufficient carrier liquid is used to form a concentrated slurry of solids from the precursor. Although a small amount of precursor dissolves

in the carrier liquid of such a slurry, the carrier liquid becomes saturated with the solid so that no further significant dissolution occurs. Furthermore, the high concentration of solid matter in the carrier liquid causes it to become heavily supersaturated with gas. Therefore, the gas nuclei in the solid precursor, or even microbubble nuclei partially released therefrom, are stable against dissolution. Furthermore, the viscosity of the slurry should be sufficiently high so that the suspended solids flow and the microbubble cores do not separate from the suspension for a sufficiently long time between their formation and their injection. For in vivo use, the viscosity of the slurry must be sufficiently low to facilitate injection. The fact that water can be used as the main component of the carrier fluid is important for use in the bloodstream to reduce potential adverse physiological effects of other substances. Such other substances should only be added so that it is sufficient to achieve the required wetting and surface-active properties of the carrier liquid, e.g. 5% propylene glycol in water is found to be a satisfactory carrier liquid. When used with e.g. ball milled galactose precursor, this carrier fluid forms a slurry sufficiently stable for injection up to one hour after it.

is formed without appreciable loss of bubble-forming efficiency.

Regarding the function of the carrier fluid as a transport facilitating medium, the carrier fluid mixed with the solid precursor in the prescribed proportions forms a slurry sufficiently viscous to prevent it from mixing to any significant extent with the test fluid during its transport in the test fluid to the location where the bubbles are to be formed and are used. It has been found that the maximum viscosity of the slurry depends on the distance from the injection site to the site of application of the microbubbles. The viscosity of the slurry can be adjusted by varying the amount of carrier liquid in relation to the amount of solid precursor or by varying the amount of non-aqueous dispersant is varied in a given amount of carrier. For example, it has been found that a volume of 5% propylene glycol in aqueous solution as a carrier fluid, which provides maximal ultrasonic imaging of the right hemisphere with easy injectability,

is also easily injected when the same volume of more viscous 10% propylene glycol in aqueous solution is used, which is excellent for imaging the left half of the heart.

Regarding the function of the carrier liquid as a surfactant, the importance of low surface tension on the formation of very small microbubbles and their stabilization against dissolution has already been highlighted. Another important feature is

a surfactant role in preventing the coalescence of the small microbubbles so that they form physiologically undesirable large bubbles. An important criterion when choosing a carrier liquid is therefore the extent to which it reduces the surface tension and suppresses bubble coalescence.

In order to achieve the above-described functions at once, the preferred viscosity of the slurry formed by the carrier liquid and the solid precursor is in the range of 5-30 centipoise, 15 centipoise being the most preferred for ordinary purposes. For in vivo use, a viscosity no greater than 1,000 eps is usually required. Bubbles formed by a surfactant carrier liquid maintain a coating of surfactant molecules on their surface and preserve low surface tension and anti-coalescence even after the slurry is dispersed in the test liquid at the test site.

Table II lists a number of liquids that have been tested as carrier liquids in that they have first been mixed with a special microbubble precursor (ball milled galactose) to form a dispersion, and then water has been added and possible microbubble formation have been observed. The ratings are from zero to ten, and higher numbers indicate increased microbubble formation for equal amounts of galactose. The set-up in Table II is not exhaustive for all possible carrier liquids, but is rather made as an example of the fact that a large number of different liquids can serve as a carrier liquid. Such liquids as dimethylacetamide, glycerol formalin, glycofurol, benzyl benzoate, various oils and various dioxolanes, for example, are suitable as carrier liquids, at least for some solid precursors. Although methanol and ethanol are usable, they inactivate the galactose within only a few minutes. Consequently, only very fresh dispersions of galactose in these carrier liquids are suitable for microbubble formation.

Carrier liquids are useful in situations where the liquid is used in vitro, to regulate the concentration of microbubbles formed per unit volume of test liquid by increasing the volume of test liquid in which the solid precursor is dispersed before it dissolves therein. Because the microbubbles formed according to this invention are so effective when it comes to changing a liquid's transmission characteristics towards elastic and electromagnetic waves, significantly less solid precursor is needed to change these characteristics than microbubble sources..'according to the state of the art.

It is not necessary that the carrier liquid be soluble in blood. Although sesame oil, corn oil, peanut oil, and other triglyceride esters or fatty acids are not soluble in water (or blood) to any significant extent, they are excellent carrier fluids. Basically, although these triglyceride esters moisten and coat the precursor aggregates, apparently the shear stresses in the blood flow and the dynamics of the heart serve to physically erode them from the microbubble precursor, which then dissolves in the blood and provides the desired number of microbubbles. This is particularly desirable because the triglyceride ester coating can prevent the microbubble precursor from producing most of the microbubbles before it has reached e.g. the heart/where the erosion is greatest.

Some of the carrier liquids according to Table II were also tested using recrystallized glucose and either recrystallized or ball milled sodium chloride as microbubble precursor. While ball milled sodium chloride was inferior to ball milled galactose as a microbubble precursor, recrystallized glucose and sodium chloride were as good as, or better than, galactose.

For use in the body, both the carrier liquid and the microbubble precursor must be bio-compatible, i.e. non-toxic and non-allergenic and otherwise physiologically acceptable in the quantity in which they are injected. As can be seen, only some of the liquids (or liquid mixtures) in Table II satisfy this criterion.

Aqueous solutions are suitable as carrier fluids for injection into the blood stream, provided that the solution is viscous enough to prevent instantaneous formation of all the microbubbles upon mixing of the microbubble precursor with the carrier fluid. Such an aqueous solution preferably has a viscosity which is substantially greater than that of water, preferably at least 5 and not more than 1,000 centipoise. Suitable agents for increasing the viscosity of aqueous carriers include glycerol, propylene glycol, polyethylene glycol 300 and 400, polyvinyl pyrrolidone, and other water-dispersible polymers which are biocompatible as explained above.

It is also desirable that the microbubble precursor has a density approximately equal to the density of the carrier liquid so that a stable dispersion is maintained. This can be achieved by using a mixture of liquids having one component that has a higher density and another component that has a lower density than the microbubble precursor.

Articles of manufacture

Where applicable, articles of manufacture are provided

the preferred non-toxic, physiologically acceptable solid f-f precursors of this invention in sterile dosage unit form, i.e. about. 0.25-10 and preferably approx. 0.5-3.5 g, sealed in a container with aids to extract the contents, e.g. an ordinary glass ampoule with a threaded part that can be separated, a bottle with a cap, a flexible plastic bag or an ordinary ampoule with an opening that is sealed with a membrane of rubber or other elastomer through which a cannula can be inserted for mixing carrier liquid with solid the precursor in the ampoule and removing the mixture therefrom after a homogeneous dispersion of the latter has been formed in the former.

In another article of manufacture aspect, the aforementioned solid precursors are provided as a kit together with a separate sterile packaged dosage unit quantity, e.g. sealed in a glass ampoule or in the cylindrical chamber of a syringe, i.e. celebrate approx. 0.25 to 10 ml, preferably approx. 1 - 7.5 ml, of a physiologically acceptable sterile carrier liquid for this, as explained above, in which the solid precursor is at least transiently stable, i.e. that after mixing with this, microbubbles do not form in significant quantities or they continue to form for at least several minutes.

In a further article of manufacture aspect, sterile dosage unit amounts of the solid precursor and the carrier liquid are provided in separate chambers in a single sealed two-chamber ampoule with means for mixing the ingredients within the ampoule. Such ampoules are common and are described in US patents 2,694,614, 2,908,274, 3,073,471, 3,081,899,

3,464,414, 3,940,003 and 4,089,432. Preferably, the two-chamber ampoule has an elastomeric stopper between the upper and lower chambers which can be pierced with a needle or which can be pressed down into the lower chamber by increasing the gas pressure in the upper chamber. The ampoule has an elastomeric capsule that can be pierced with a needle and is covered by a removable dust cover. The solid precursor is stored in the lower chamber and the carrier liquid in the upper chamber. To form a suspension of the solid precursor in the carrier liquid, the dust cap is removed, and if the plug can be forced down into the lower chamber, this is done by injecting air into the upper chamber with a cannula or by the pressure in the upper chamber on other way is increased, e.g. in that the elastomeric capsule is pressed into the upper chamber. If the stopper is adapted to be pierced by a needle, the cannula is inserted through the elastomeric capsule, the carrier fluid is drawn into the cannula, the needle is inserted through the stopper, and the carrier fluid is injected into the lower chamber. After a uniform dispersion or suspension of the solid precursor in the carrier liquid has been formed, the mixture is drawn into the cannula and injected into the test liquid to form microbubbles therein.

Test fluids

The relatively highly polar liquid in which the microbubble precursors according to this invention can be added for the formation and stabilization of microbubbles therein by solvation, whereby one can predict what results can be expected when blood is used, generally has a very high dielectric constant (above 40) and will generally be an aqueous liquid with a dielectric constant of approx. 80. It is essential that the carrier liquid does not have any significant effect on the solvation (polar or hydrogen bond coordination) of the test liquid with the dissolved substance formed by dissolving the solid microbubble precursor in it. This ensures that the water concentration in the test liquid will reduce locally, whereby gas dissolved in the test liquid will be excreted or the solubility in the test liquid for gas supplied from the solid precursor will be reduced. A strongly polar carrier liquid can be used if, as previously described, its properties are such that the solid precursor is prevented from completely dissolving therein before the mixture is added to the test liquid.

Application methods

The microbubble precursor according to the invention can be used for

to change transmission characteristics, i.e. either reflectivity or absorption for electromagnetic and sound (elastic wave) radiation that is transmitted through a liquid to form a detectable signal, by providing the liquid with a quantity of microbubbles that are effective for significantly to change the transmission characteristics of such radiation through the liquid in the area of it that contains the microbubbles.

Because microbubbles generally change the physical properties of liquids that affect the transmissibility and reflectivity of electromagnetic and acoustic (elastic wave) radiation incident on a region of the liquid containing the bubbles, the microbubble precursor of this invention is useful in various analytical, diagnostic and operational processes, which will be clear to those who have knowledge in imaging, detection, grading and testing when using such radiation. Examples of such electromagnetic properties of a liquid that can be changed by microbubbles are: resistivity, electric and magnetic susceptibility, dielectric constant and absorption, nuclear and electron paramagnetic resonance response. ' Examples of elastic-wave properties that can be changed are: compressibility, density, acoustic impedance and absorption. Examples of such radiation and the associated applications that could potentially benefit from the use of microbubble-forming materials are: medical ultrasonic imaging (both transmission and echographic), X-ray imaging (e.g. CT scan), NMR imaging and microwave - image.

After the microbubble precursor is injected into the blood stream, an ultrasonic image of the stream is obtained on the opposite side of the place where the carrier liquid is dissolved or dispersed, and the solid precursor has been dissolved and has dispersed microbubble nuclei, thereby supplying the blood with the required number of microbubbles. Blood flow velocity can be determined by simultaneous measurement of the positions and velocities of the microbubbles, or of the cloud of microbubbles, from such an ultrasonic image. Blood flow velocity can also be determined by measuring the intensities of two ultrasonic images, one from a proximal wall and the other from a distal wall of a blood vessel, at a location in the blood flow and applying conventional dye dilution equations, or by measuring the intensity from a distal interface before and during flow of the microbubble-containing liquid. In addition, the boundary between the flowing blood and the blood vessel can be observed with a view to any turbulence that may be caused by plaque formation on the blood vessel walls.

According to another embodiment of the invention, the microbubble precursor described above can be dissolved in a strongly polar liquid, e.g. temporarily withdrawn blood, normal saline or water, preferably in a syringe or the like, to form a cloud of microbubbles, and the polar liquid with the microbubble cloud can be injected into the blood stream. The microbubble precursor can also be added directly to the liquid without a carrier liquid.

The aforementioned improvement has been shown to be particularly useful

by amplifying ultrasonic images of the blood flow, i.e.

of the cardiovascular system. Very high ultrasonic contrast has been achieved compared to any microbubble contrast agent according to the state of the art. The microbubbles that are formed are usually much smaller than microbubbles with contrast agents according to the state of the art, and a much higher concentration of such microbubbles can be formed for better contrast, with greater ease and better control, and at a lower cost.

All these advantages can also be achieved when it comes to enhancing ultrasound images of other fluid-filled parts of the body, such as the lymphatic system, digestive system, urinary, reproductive, biliary and other systems, as well as intra-peritoneal, intracranial, intrathoracic and other cavities and spaces in the body.

Although at least some of the microbubbles are usually formed in situ in the liquid through which a signal is sent, the microbubbles can first, as will be seen, be formed in another liquid, e.g. a carrier liquid as explained herein, and the carrier liquid containing the microbubbles can then be added to the liquid in which the wave energy is transmitted, e.g. the flow of blood in a living individual. However, the lifetime of such microbubbles therein is less than when the microbubbles are formed in situ.

MATERIAL COMPOSITION

Example 1 Ball milled galactose

About. 5 g (10 ml) commercial d(+)-galactose (anhydrous

powder') is placed in an aluminum oxide chamber with aluminum oxide ends containing three aluminum oxide balls with a diameter of approx. 13 mm, under a low humidity atmosphere (20% relative humidity). The above device is placed in a Pitchford ball mill, model 3800. The galactose is pulverized in the ball mill for 10 minutes, then taken out of the chamber and stored in an environment with low humidity (20% relative humidity) and room temperature (19-29°) until use.

Unlike the starting powder, which actually doesn't

has any ability to form microbubbles, the ball-milled galactose has an average particle size of 45 µm, which particles are aggregates of microcrystals with an average size of 10 µm with cavities between the microcrystals of approximately the same size, which are highly active microbubble cores.

Example 2 Recrystallized Dextrose

15 g of commercial d-dextrose (monohydrate powder USP.) is placed in a 100 ml beaker and moisten with 4.8 ml of warm (100°C) deionized water. The beaker is heated in a water bath (100°C) while its contents are stirred until all the dextrose crystals are dissolved. The beaker is cooled in an ice bath, and the contents are stirred until crystals begin to form. Cover the beaker with plastic foil and cool down (6°C) for 12 hours.

The contents of the beaker are taken out, placed on filter paper and washed with 100% ethanol. The crystals on the filter paper are placed in an atmosphere of low humidity (20% relative humidity) until they are dry (12 hours). The dry crystals are crushed using a mortar and pestle, and then sieved through a sieve with 180 µm openings. The sieved material is stored in an environment with low humidity (20% relative humidity) and room temperature (19-29°C) until use.

Unlike the starting powder, the sieved material has aggregates with an average size of 75 µm, of microcrystals with an average size of 10 µm, with voids between the microcrystals of approximately the same size, which are highly active microbubble cores.

E xample 3 Recrystallized Sodium Chloride

10 ml of food-grade NaCl (separate crystals of 295 µm) are dissolved in 20 ml of deionized water in a crucible. The pot is heated quickly until all the water has boiled off. The remaining crystals are then sieved through a sieve with openings of 180 µm. The sieved material is stored at room temperature (19-29°C) until use.

The recrystallized NaCl has aggregates (58 µm average size) of microcrystals (26 µm average size) with voids between the microcrystals of approximately the same size that are highly active microbubble cores.

Example 4 Suspension of Solid Precursor in Carrier Liquid

10 ml of propylene glycol (PG) sterilized by dry heat (120°C, 4 hours) is mixed with 90 ml of sterile, commercial 5% dextrose in water (D5W).

Add 1.5 g of the ball-milled galactose according to Example 1 to 3 ml of the thus prepared PG/D5W carrier liquid in a 50 ml polyethylene plastic beaker. Stir for approx. 15 seconds until the milky (with microbubbles with an average of about 10 µm) mixture has become homogeneous. Draw the mixture into a sterile needle and remove any trapped gas pockets. Use the mixture within approx. 10 minutes after mixing for maximum microbubble delivery.

Follow the procedure in Example 4, using either the same amount of the recrystallized dextrose according to Example 2 or the recrystallized NaCl according to Example 3 to produce liquid mixtures with corresponding microbubble-forming capabilities. Use the mixture within approx. 10 minutes after mixing for maximum microbubble formation.

ARTICLE OF MANUFACTURE

Example 5 Equipment set

Draw into a 10 ml syringe approx. 5.4 ml of the PG/D5W carrier liquid according to Example 4 and cover the constricted part of the syringe where the needle is connected with a removable cover. Sterilize the closed device, e.g. in that it is postponed

for approx. 1.5 M-rad cobalt-60 gamma rays. Fill, under a dry nitrogen atmosphere, a 10 ml plastic bag formed from flexible polyethylene film with approx. 2.7 g of the ball milled galactose according to Example 1, of the recrystallized dextrose according to Example 2 or the recrystallized NaCl according to Example 3. Seal the opening of the bag with a thin rubber membrane and cover the membrane with a metal lid and a corrugated cover to keep the lid in place . Sterilize the sealed bag, e.g. in that it is exposed to approx. 1.5 M-rad cobalt-60 gamma rays. Pack the syringe bag as a kit, possibly together with a sterile No. 16 needle.

In another version, the plastic bag is replaced with an ordinary ampoule with a rubber stopper as described above. Solids f-f the precursor and the carrier liquid are mixed by transferring the mixture back and forth between the ampoule and the syringe, several times.

Example 6 Procedure for Formation of Microbubbles

For the production of a suspension of solid microbubble precursor in a carrier liquid according to this invention using the equipment set according to Example 5, the cover is removed from the syringe and the cannula is sterilely mounted on it.

Remove the corrugated cover and lid from the opening of the plastic bag, poke through the rubber membrane and inject the contents of the syringe into the bag. Mix the solid microbubble precursor with the carrier liquid manually in the bag until a viscous milky (due to microbubble formation) homogeneous mixture is formed. Pull the mixture out of the bag into the syringe and use it within approx. 10 minutes.

Example 7 Procedure for Application of Microbubbles

Within a few minutes of forming the suspension

of galactose in the PG/D5W carrier liquid as described in Example 6, a bolus injection of 3 ml of the mixture containing 1.5 g of galactose precursor is carried out through a plastic catheter into the femoral vein of a dog of 25-40 kg . Immediately after the injection, a 10 ml bolus saline rinse is injected through the same injection site. During the contrast injection and the saline rinse, a normal ultrasonic transducer, connected to acoustic gel, is held with the hands against the dog's right chest side wall at a rib-between-

room while the dog lies on its right side. Record the signals. M-mode and/or 2D echo-contrast-enhanced ultrasound images are obtained of the right hemisphere, lasting for = 10 seconds.

The above procedure can also be followed using 2.7 g of solid precursor in 5.4 ml of carrier fluid and a rinse with 18 ml of saline, to obtain enhanced ultrasound images of the right hemisphere of the heart in humans, with the transducer held by the hands against the patient's left chest wall at a rib-interspace or in the subxiphoid (under the sternum) position while the patient is supine.

By using the above method, the following ultrasonic image intensifications are achieved:

Claims (6)

1. Microbubble precursor in sterile, injectable unit dosage form, adapted for injection into a liquid-containing area of a living being, for example the blood stream, characterized in that it comprises a particulate solid which is of non-toxic, physiologically acceptable material, soluble in the liquid /blood and essentially free of microbubbles, which particles are aggregates (5) of microparticles (7) with an average size in the range from 1 to 250 pm, which microparticles (7) delimit gas-filled cavities (11, 13) between them and can contain intracrystalline gas pockets (15), and where the ratio between the mass of the microparticles (7) and the volume of gas in the cavities (11, 13) between them is sufficiently large that the liquid/blood in which the microbubble precursor is dissolved will be supersaturated with consideration of the gas in the area of the liquid/blood which surrounds the microbubbles when they are formed therein. 2. Microbubble precursor according to claim 1, characterized in that the particulate solid is dextrose, galactose, maltose or sodium chloride. 3. Microbubble precursor according to claim 1 or 2, characterized in that the solid particulate substance is aggregates (5) of at least 10 microparticles (7) whose average size is from 1 to 50 pm. 4. Microbubble precursor according to any one of claims 1-3, characterized in that it consists of a liquid injectable suspension in a carrier liquid (3) which is non-toxic and physiologically acceptable and in which the particulate solid is at least transiently stable . 5. Method for producing an aqueous suspension of microbubbles suitable for injection into a fluid-containing area of a living being, for example the blood stream, characterized in that, immediately before the injection, a suspension is prepared of (a) a unit dosage amount of a sterile injectable material in unit dosage form and suitable for injection into a liquid-containing area, for example the blood stream of a living being and which will produce microbubbles in the liquid/blood stream, comprising a solid substance in particulate form which is of non-toxic, physiologically acceptable material, soluble in the liquid/blood and essentially free of microbubbles, whose particles are aggregates (5) of smaller particles (7) and contain gas-filled cavities, where the smaller particles ( 7) has an average size in the range of 1-250 pm, and where the ratio between the mass of the smaller particles (7) and the volume of gas in the cavities (11, 13) between them is sufficiently large that the liquid/blood in which the material becomes dissolved, will be supersaturated with respect to the gas in the region of the fluid/blood surrounding the microbubbles when they are formed therein, and (b) a unit dosage amount of a carrier fluid which is non-toxic and physiologically acceptable and in which the particulate solid is at least temporarily stable so that a fluid injectable suspension can be formed. 6. Use of a microbubble precursor in a sterile, injectable unit dosage form comprising a particulate solid which is of non-toxic, physiologically acceptable material, soluble in blood and substantially free of microbubbles, which particles are aggregates (5) of microparticles (7) with a average size in the range from 1 to 250 pm, which microparticles (7) delimit gas-filled cavities (11, 13) in between and may contain intracrystalline gas pockets (15), and where the ratio between the mass of the microparticles (7) and the volume of gas in the cavities (11, 13) between them are sufficiently large that blood in which the microbubble precursor is dissolved will be supersaturated with respect to the gas in the region of the blood surrounding the microbubbles when they are formed therein, for injection into the bloodstream of a living being.