CA2273140C - Gaseous ultrasound contrast media and method for selecting gases for use as ultrasound contrast media - Google Patents

Abstract

Disclosed herein are agents for enhancing the contrast in an ultrasound image. These agents are extremely small bubbles, or "microbubbles," comprised of specially selected gases. The microbubbles described herein exhibit long life spans in solution and may be produced at a size small enough to traverse the lungs, thus enabling improved ultrasound imaging of the cardiovascular system and other vital organs. Also disclosed herein is a method for selecting gases from which contrast agents may be produced. The method is based on calculations using inherent physical properties of gases and describes a means to associate the properties of a gas with the time for dissolution of microbubbles comprised of the gas.

Description

CA 02273140 1999-06-07 , GASEOUS ULTRASOUND CONTRAST MEDIA AND
METHOD FOR SELECTING GASES FOR USE AS
ULTRASOUND CONTRAST MEDIA
PATENT
DESCRIPTION
This invention relates to agents that enhance the contrast in an ultrasound image generated for use in medical diagnosis. The contrast-enhancing media disclosed herein are comprised of extremely small gas bubbles which are present in a solution that is infused into the body during or just before an ultrasound image is generatE~d. This invention is also directed to a method for enhancing such images by selecting gases from which a collection of free gas microbubbles can be prepared that have novel and superior properties. These microbubbles, composed of the gases whose selection is enabled by the process of this invention, may be extremely small in size and yet survive in the bloodstrE:am long enough to allow contrast-enhanced imaging of: parts of the cardiovascular system, peripheral vascular system, and vital organs previously believed to be inaccessible to free gas mic:robubbles.
BACKGROUND
When using ultrasound t:o obtain an image of the internal organs and structures of a human or animal, ultrasound waves -- waves of sound energy at a frequency above that discernable by the human ear --CA 02273140 1999-06-07 , GASEOUS ULTRASOUND CONTRAST MEDIA AND
METHOD FOR SELECTING GASES FOR USE AS
ULTRASOUND CONTRAST MEDIA

PATENT

This invention relates to agents that enhance the contrast in an ultrasound image generated for use in medical diagnosis. The contrast-enhancing media disclosed herein are comprised of extremely small gas 15 bubbles which are present in a solution that is infused into the body during or just before an ultrasound image is generatE~d. This invention is also directed to a method for enhancing such images by selecting gases from which a collection of free gas 20 microbubbles can be prepared that have novel and superior properties. These microbubbles, composed of the gases whose selection is enabled by the process of this invention, may be extremely small in size and yet survive in the bloodstrE:am long enough to allow 25 contrast-enhanced imaging of: parts of the cardiovascular system, peripheral vascular system, and vital organs previously believed to be inaccessible to free gas mic:robubbles.
BACKGROUND
30 When using ultrasound t:o obtain an image of the internal organs and structures of a human or animal, ultrasound waves -- waves of sound energy at a frequency above that discernable by the human ear --PATENT
are reflected as they pass through the body.
Different types of body tissue reflect the ultrasound waves differently and the :reflections, often aptly described as "echoes," that are produced by the ultrasound waves reflecting off different internal structures are detected and converted electronically into a visual display. This disp-lay may prove invaluable to a physician or other diagnostician in several ways, including evaluating the progression of cardiovascular disease or the existence or nature of a tumor.
For some medical conditions, obtaining a useful image of the organ or structure of interest is especially difficult because the details of the structure may not be adequately discernible from the surrounding tissue in an ultrasound image produced by the reflection of ultrasound waves absent a contrast-enhancing agent. Additionally, traditional ultrasound images are notoriously poor in quality and resolution. For these reasons, detection and observation of certain physiological conditions may be substantially improved by enhancing the contrast in an ultrasound image by infusing an agent into an organ or other structure of interest. In other cases, detection of the movement of the contrast-enhancing agent itself is particularly important.
For example, a distinct blood flow pattern that is known to result from particular cardiovascular abnormalities may only be discernible by infusing a contrasting agent into the bloodstream and observing the dynamics of the blood flow.
Medical researchers have made extensive investigation into the use of solids, gases and liquids in an attempt to discover ultrasound contrast-enhancing agents suitable for particular -:3-PATENT
diagnostic purposes. Composite substances such as gelatin encapsulated microbubbles, gas-incorporated liposomes, sonicated partially denatured proteins and emulsions containing highl~r fluorinated organic compounds have also been studied in an attempt to develop an agent that has certain ideal qualities, primarily, stability in thE~ body-and the ability to provide significantly enhanced contrast in an ultrasound image.
Small bubbles of a gars, termed "microbubbles,"
are readily detected in an image produced using standard ultrasound imagine techniques. When infused into the bloodstream or a particular site in the body, microbubbles enhance the contrast between the region containing the microbubbles and the surrounding tissue.
A substantial amount of the research effort directed at contrast-enhancing agents has focused on the use of extremely small gas bubbles.
Investigators have long known that free gas bubbles provide a highly effective contrast agent because a gas bubble has unique physical characteristics that affect ultrasound energy as it is directed through the body. The advantages offered by free gas bubbles as opposed to liquid or solid agents that exhibit contrast enhancement is described in detail below in the context of the discussion of ultrasound diagnostic techniques.
Despite the known advantages, however, the rapid dissolution of free gas bubbles in solutions such as blood or many aqueous intravenous solutions, severely limits their use as an ultrasound contrast-enhancing agent. The most important limitations are the size of the microbubble and the length of time that a PATENT
microbubble will exist before dissolving into the solution.
Examining the size requirements for microbubbles more closely, the gas bubbles must, of course, be sufficiently small that a suspension of the bubbles does not carry the risk of embolism to the organism in which they are infused. At the same time, extremely small free gas bubbles composed of the gases generally used in uli~rasound contrast imaging dissolve into solution so rapidly that their image-enhancing capability exists only immediately proximate to the infusion site. An additional obstacle exists for ultrasound imaging of the cardiovascular system. Medical researchers have studied the time required for microbubbles composed of ordinary air, pure nitrogen, pure oxygen, or carbon dioxide, to dissolvE~ into solution.
Microbubbles of these gases that are sufficiently small to be able to pass through the lungs and reach the left heart, less than about 8 microns in diameter, have a life span of less than approximately 0.25 seconds. Meltzer, R.S., Tickner, E.G., Popp, R.L., "Why Do the Lungs Clear Ultrasonic Contrast?"
Ultrasound in Medicine and Bioloav, Vol. 6, p.263, 267 (1980). Since it takes over 2 seconds for blood to pass through the lungs, microbubbles of these gases would be fully dissolved during passage through the lungs and would never reach the left heart.
Ibid. Primarily because oi= this tradeoff between bubble size and life span, many researchers concluded that free gas microbubbles were not useful as a contrast-enhancing agent for ultrasound diagnosis of certain parts of the cardiovascular system.
However, the ultrasound contrast-enhancing media described herein comprises microbubbles, composed of PATENT
the biocompatible gases whose selection is also provided by this invention, that are sufficiently small that they pass through the pulmonary capillary diameter of approximately F1 microns and thereby allow contrast-enhanced ultrasound diagnosis of the left chambers of the heart. The: free gas microbubbles survive in the bloodstream long enough that they may be peripherally intravenou:~ly infused, travel through the right heart, through the lungs, and into the left cardiac chambers without dissolving into solution.
Additionally, certain of these media have extremely long persistence in solution and will enable contrast-enhancement of many other organs and structures.
This invention overcomes many of the inherent limitations thought to exi~~t with the use of free gas microbubbles by providing, in part, a method for selecting special gases based on particular physical criteria such that microbubbles composed of these gases do not suffer from tree same limitations as the microbubbles previously investigated. Therefore, it has been discovered that the ultrasound contrast-enhancing media described herein comprising a composition of microbubble:~ produced using a biocompatible gas or combination of gases selected by the physical and chemical parameters disclosed herein can exist for a sufficient length of time and be of sufficiently small size that their stability in the bloodstream allows enhanced ultrasound contrast imaging of particular structures in the body previously thought inaccessible to free gas microbubbles.
By using the term "biocompatible gas" I mean a chemical entity which is capable of performing its functions within or upon a living organism in an acceptable manner, without undue toxicity or PATENT
physiological or pharmacological effects, and which is, at the temperature of t:he living organism, in a state of matter distinguished from the solid or liquid states by very low density and viscosity, relatively great expansion and contraction with changes in pressure and temperature, and the spontaneous tendency to become distributed uniformly throughout any container. The following Table contains the assumed body temperatures for various living organisms:
Organism Rectal Temperature (degree Fahrenheit) Swine (Sus Scrofa) 101.5-102.5 Sheep (Ovis sp.) 101-103 Rabbit (Oryctolaqus cunicu7_us) 102-103.5 Rat (Tattus morvegicus) 99.5-100.6 Monkey (Macaca mulatta) 101-102 Mouse (Mus Musculus) 98-101 Goat (Capra hircus) 101-103 Guinea pig (Cavia porcellus) 102-104 Hamster (Mesocricetus sp.) 101-103 Man (Homo sapiens) 98.6-100.4 Horse (Equus sp.) 101-102.5 Dog (Canin familiaris) 101-102 Baboon (Papio) 98-100 Cat (Felis catus) 101-102 Cattle (Bos taurus) 101.5-102.5 Chimpanzee (Pan) 96-100 Techniques For Measuring Ultrasound Contrast-Enhancement Phenomena To more fully appreciate the subject matter of the present invention, it is useful to describe what is presently known about the technology of ultrasound imaging and to review the search for improved ultrasound contrast-enhancing agents in that light.
Materials that are useful as ultrasound contrast agents operate by having an effect on ultrasound waves as they pass through the body and are reflected PATENT
to create the image from which a medical diagnosis is made. In an attempt to develop an efficient image-contrast agent, one skilled in the art recognizes that different types of substances affect ultrasound waves in different ways and to varying degrees.
Moreover, certain of the ei°fects caused by contrast-enhancing agents are more readily-measured and observed than others. Thus, in selecting an ideal composition for a contrast--enhancing agent, one would prefer the substance that has the most dramatic effect on the ultrasound wave as it passes through the body. Also, the effect. on the ultrasound wave should be easily measured. There are three main contrast-enhancing effects which can be seen in an ultrasound image: backscatt~er, beam attenuation, and speed of sound differentia:~L. Each of these effects will be described in turn.
A. BACKSCATTER
When an ultrasound wave that is passing through the body encounters a structure, such as an organ or other body tissue, the structure reflects a portion of the ultrasound wave. Different structures within the body reflect ultrasound energy in different ways and in varying strengths. This reflected energy is detected and used to generate an image of the structures through which the ultrasound wave has passed. The term "backscat:ter" refers to the phenomena in which ultrasound energy is scattered back towards the source by a substance with certain physical properties.
It has long been recognized that the contrast observed in an ultrasound image may be enhanced by the presence of substances known to cause a large amount of backscatter. When such a substance is _g_ PATENT
administered to a distinct part of the body, the contrast between the ultrasound image of this part of the body and the surrounding tissues not containing the substance is enhanced. It is well understood that, due to their physical properties, different substances cause backscatter in varying degrees.
Accordingly, the search for contr-ast-enhancing agents has focused on substances that are stable and non-toxic and that exhibit the maximum amount of backscatter.
Making certain assumptions about the way a substance reflects ultrasound energy, mathematical formulae have been developed that describe the backscatter phenomenon. Working with these formulae, a skilled researcher can estimate the ability of gas, liquid, and solid contrast-enhancing agents to cause backscatter and the degree to which a particular substance causes measurablE~ backscatter can be compared with other substances based on the physical characteristics known to cause the backscatter phenomenon. As a simple e:cample, the ability of substance A to cause backscatter will be greater than substance B, if, all other factors being equal, substance A is larger than substance B. Thus, when both substances are encountered by an ultrasound wave, the larger~substance scatters a greater amount of the ultrasound wave.
The capability of a substance to cause backscatter of ultrasound energy also depends on other characteristics of the substance such as its ability to be compressed. Of particular importance is the dramatic increase in backscatter caused by gas bubbles due to the bubble resonance phenomenon which is described below. When examining different substances, it is useful to compare one particular _g_ PATENT
measure of the ability of .a substance to cause backscatter known as the "scattering cross-section."
The scattering cross-section of a particular substance is proportional to the radius of the scatterer, and also depends on the wavelength of the ultrasound energy and on other physical properties of the substance, J. Ophir and K. J.- Parker, Contrast Agents in Diagnostic Ultrasound. Ultrasound in Medicine & Biology, vol. IS, n. 4, p. 319, 323 (1989) .
The scattering cross-section of a small scatterer, a, can be determined by a known equation:
4 rcs - ~c Z 1 3 (Ps ' P) Z
Q = - ~ra2 (ka) 4 + -9 ~c 3 2ps - P
where x - 2~r/a, where a is the wavelength; a = the radius of the scatterer; acs = adiabatic compressibility of the scatterer; ~c - adiabatic compressibility of the medium in which the scatterer exists, ps = density of thE~ scatterers and p - the density of the medium in which the scatterer exists.
P. M. Morse and K. U. Ingard, Theoretical Acoustics, p. 427, McGraw Hill, New York (1968).
In evaluating the utility of different substances as image contrasting agents, one can use this equation to determine which agents will have the higher scattering cross-section and, accordingly, which agents will provide the greatest contrast in an ultrasound image.
Referring to the above equation, the first bracketed quantity in the above equation can be assumed to be constant for the purpose of comparing solid, liquid and gaseous scatterers. It can be assumed that the compressibility of a solid particle PATENT
is much less than that of t:he surrounding medium and that the density of the particle is much greater.
Using this assumption, the scattering cross section of a solid particle contrast-enhancing agent has been estimated as 1.75. Ophir and Parker, supra, at 325.
For a pure liquid scat:terer, the adiabatic compressibility and density of the scatterer rcs and the surrounding medium ~c ai:e likely to be approximately equal which would, from the above equation, yield the result that liquids would have a scattering cross-section of: zero. However, liquids may exhibit some backscattE:r if large volumes of a liquid agent are present pi_-esumably because the term a in the first bracketed quantity in the above equation may become sufficiently large. For example, if a liquid agent passes from a very small vessel to a very large one such that the liquid occupies substantially all of the vessel the liquid may exhibit measurable backscat:ter. Nevertheless, in light of the above equation and the following, it is appreciated by those skilled in the art that pure liquids are relatively inei=ficient scatterers compared to free gas microbubbles.
It is known that chances in the acoustic properties of a substance are pronounced at the interface between two phases, i.e., liquid/gas, because the reflection characteristics of an ultrasound wave change mar~;edly at this interface.
Additionally, the scatter cross-section of a gas is substantially different than a liquid or solid, in part, because a gas bubble can be compressed to a much greater degree than a liquid or solid. The physical characteristics of gas bubbles in solution are known and standard values for compressibility and density figures for ordina~_y air can be used in the PATENT
above equation. Using there standard values, the result for the second bracl~:eted term alone in the above equation is approximately 1014, Ophir and Parker su ra, at 325, with the total scattering cross section varying as the radius a of the bubble varies.
Moreover, free gas bubbles in a liquid exhibit oscillatory motion such that, at-certain frequencies, gas bubbles will resonate at a frequency near that of the ultrasound waves commonly used in medical imaging. As a result, the scattering cross-section of a gas bubble can be over a thousand times larger than its physical size.
Therefore, it is recognized that gas micro-bubbles are superior scattE~rers of ultrasound energy and would be an ideal contrast-enhancing agent if the obstacle of their rapid dissolution into solution could be overcome.
B. BEAM ATTENUATION
Another effect which can be observed from the presence of certain solid contrast-enhancing agents, is the attenuation of the ultrasound wave. Image contrast has been observed in conventional imaging due to localized attenuation differences between certain tissue types. K. J. Parker and R. C. Wang, "Measurement of Ultrasonic Attenuation Within Regions selected from B-Scan Images," IEEE Trans. Biomed.
Enar. BME 30(8), p. 431-37 (1983); K. J. Parker, R.
C. Wang, and R. M. Lerner, "Attenuation of Ultrasound Magnitude and Frequency Dependence for Tissue Characterization," Radiology, 153(3), p. 785-88 (1984). It has been hypothesized that measurements of the attenuation of a region of tissue taken before and after infusion of an agent may yield an enhanced image. However, techniques based on attenuation contrast as a means to measure the contrast PATENT
enhancement of a liquid agent are not well-developed and, even if fully developed, may suffer from limitations as to the internal organs or structures with which this technique can be used. For example, it is unlikely that a loss of attenuation due to liquid contrast agents could be observed in the image of the cardiovascular system because of the high volume of liquid contrast agent that would need to be present in a given vessel before a substantial difference in attenuation could be measured.
Measurement of the attenuation contrast caused by microspheres of Albunex*(Molecular Biosystems, San Diego, CA) in vitro has been accomplished and it. has been suggested that in vivo attenuation contrast measurement could be achieved. H. Bleeker, K. Shung, J. Burnhart, "On the Application of Ultrasonic Contrast Agents for Blood Flowometry and Assessment of Cardiac Perfusion," J. Ultrasound Med. 9:461-471 (1990). Albunex*is a suspension of 2-4 micron encapsulated air-filled microspheres that have been observed to have acceptable stability in vivo and are sufficiently small in size that contrast enhancement r can occur in the left atrium or ventricle. Also, attenuation contrast resulting from iodipamide ethyl ester (IDE) particles accumulated in the liver has been observed. Under such circumstances, the contrast enhancement is believed to result from attenuation of the ultrasound wave resulting from the presence of dense particles in a soft medium. The absorption of energy by the particles occurs by a mechanism referred to as "relative motion." The change in attenuation caused by relative motion can be shown to increase linear:Ly with particle concentration and as the square of the density difference between the particles and the surrounding *Trade-mark PATENT

medium. K. J. Parker, et al., "A Particulate Contrast Agent with Potential for Ultrasound Imaging of Liver," Ultrasound in Medicine & Bioloc~v, Vol. 13, No. 9, p. 555, 561 (1987). Therefore, where substantial accumulation o:E solid particles occurs, attenuation contrast may be a viable mechanism for observing image contrast e:ahancem8nt although the effect is of much smaller magnitude than the backscatter phenomenon and would appear to be of little use in cardiovascular diagnoses.
C. SPEED OF SOUND DIFFERENTIAL
An additional possible technique to enhance contrast in an ultrasound image has been proposed based on the fact that the speed of sound varies depending on the media through which it travels.
Therefore, if a large enough volume of an agent, through which the speed of sound is different than the surrounding tissue, can be infused into a target area, the difference in the speed of sound through the target area may be measurable. Presently, this technique is only experimental.
Therefore, considering the three techniques described above for contrast enhancement in an ultrasound image,, the marked increase in backscatter caused by free gas microbubbles is the most dramatic effect and contrast-enhancing agents that take advantage of this phenomenon would be the most desirable if the obstacle of their limited stability in solution could be overcome.
The Materials Presently Used as Contrast-Enhancing A ents In light of what is known about the various techniques described above, attempts to develop a contrast-enhancing agent whose presence generates PATENT
substantial contrast in an ultrasound image, and whose survival in vivo is sufficiently long to allow contrast-enhanced imaging of the cardiovascular system, has led to the investigation of a broad variety of substances -- gases, liquids, solids, and combinations of these -- as potential contrast-enhancing agents. -A. SOLID PARTICLES
Typically, the solid substances that have been studied as potential contrast-enhancing agents are extremely small particles that are manufactured in uniform size. Large numbers of these particles can be infused and circulate freely in the bloodstream or they may be injected into a particular structure or region in the body.
IDE particles are solid particles that can be produced in large quantities with a relatively narrow size distribution of appro:~imately 0.5-2.0 microns.
Sterile saline injections of these particles may be injected and will tend to <~ccumulate in the liver.
Once a substantial accumulation occurs, contrast enhancement may be exhibited by either attenuation contrast or backscatter mechanisms. Although suspensions comprising these solid particles dispersed in a liquid may exhibit acceptable stability, the backscatter or attenuation effects are relatively minor compared 'to free gas bubbles and a substantial accumulation of the particles must occur before appreciable contrast is observed in an ultrasound image. Thus, use of these suspensions has been limited to certain cell types in which the particles have the tendency to coagulate because unless the suspension becomes highly concentrated in particular tissue, the contrast enhancement will be minor.

_1~5_ PATENT
SHU-454*(Schering, A. G., West Berlin, Germany) is an experimental contrast-enhancing agent in powder form that, when mixed with a saccharide diluent, forms a suspension of crystals of various rhomboid and polyhedral shapes ranging in size from 5 to 10 microns. Although the precise mechanism by which these crystals enhance ultrasound contrast is not completely understood, it is suspected that the crystals may trap microbubbles in their structure or that the crystals themselves may backscatter ultrasound energy by an as-yet undetermined mechanism.
B. LIQUIDS AND EMULSIONS
In another attempt to achieve a satisfactory agent, emulsions are prepared by combining a chemical species compatible with body tissue and a species that provides high ultrasound contrast enhancement.
European Patent Application 0231091 discloses emulsions of oil in water containing highly fluorinated organic compounds that have been studied in connection with their possible use as a blood substitute and are also capable of providing enhanced contrast in an ultrasound image.
Emulsions containing perfluorooctyl bromide (PFOB) have also been examined. Perfluorooctyl bromide emulsions are liquid compounds known to have the ability to transport oxygen. PF08 emulsions have exhibited a limited utility as ultrasound contrast agents because of a tendency to accumulate in certain types of cells. Although the mechanism is not completely understood, PFOB emulsions may provide ultrasound contrast because of their high density and relatively large compressibility constant.
United States Patent nfo. 4,900,540 describes the use of phospholipid-based l.iposomes containing a gas *Trade-mark -lE~-PATENT
or gas precursor as a contrast-enhancing agent. A
liposome is a microscopic, spherical vesicle, containing a bilayer of phospholipids and other amphipathic molecules and an inner aqueous cavity, all of which is compatible with the cells of the body. In most applications, liposomes are used as a means to encapsulate biologically-active materials.
The above reference discloses the use of a gas or gas precursors incorporated into the liposome core to provide a longer life span for the gas when infused into the body. Production of stable liposomes is an expensive and time consuming process requiring specialized raw materials a.nd equipment.
C. MICROBUBBLES
As noted above, a critical parameter that must be satisfied by a microbubble used as a contrast-enhancing agent is size. Free gas microbubbles larger than approximately 8 microns may still be small enough to avoid impeding blood flow or occluding vascular beds. However, microbubbles larger than 8 microns are z-emoved from the bloodstream when blood flows through the lungs. As noted above, medical researchers have reported in the medical literature that microbubbles small enough to pass through the lungs will dissolve so quickly that contrast enhancement of lei=t heart images is not possible with a free gas microbubble. Meltzer, R.S., Tickner, E.G., Popp, R.L., "Why Do the Lungs Clear Ultrasonic Contrast?" Ultrasound in Medicine and Bioloav, vol. 6, p.263, 26'7 (1980).
However, cognizant of the advantages to be gained by use of microbubb:Les as contrast-enhancing agents by virtue of their :Large scattering cross-section, considerable attention has been focused on developing mixtures containing microbubbles that are PATENT
rendered stable in solution. Enhancing the stability of gas microbubbles may be .accomplished by a number of techniques.
Each of the following techniques essentially involves suspending a collection of microbubbles in a substrate in which a bubble of ordinary gas is more stable than in the bloodstream.
In one approach, microbubbles are created in viscous liquids that are injected or infused into the body while the ultrasound diagnosis is in progress.
The theory behind the use of viscous fluids involves an attempt to reduce the rate at which the gas dissolves into the liquid and, in so doing, provide a more stable chemical environment for the bubbles so that their lifetime is extended.
Several variations on this general approach have been described. EPO Application No. 0324938 describes a viscous solution of a biocompatible material, such as a human protein, in which microbubbles are contained. By submitting a viscous protein solution to sonication, microbubbles are formed in the solution. Partial denaturation of the protein by chemical treatment or heat provides additional stability to mic:robubbles in the solution by decreasing the surface tension between bubble and solution.
Therefore, the above approaches may be classified as an attempt to enhance the stability of microbubbles by use of a stabilizing medium in which the microbubbles are contained. However, none of these approaches have addressed the primary physical and chemical properties of gases which have seriously limited the use of free gas. microbubbles in ultrasound diagnosis, particularly with respect to the cardiovascular system. None of these approaches PATENT
-la_ suggest that selection of the gases, by precise criteria, would yield the ability to produce stable microbubbles at a size that would allow transpulmonary contrast-enhanced ultrasound imaging.
The behavior of microbubbles in solution can be described mathematically based on certain parameters and characteristics of the <~as of-which the bubble is formed and the solution in which the bubble is present. Depending on the degree to which a solution is saturated with the gas o:E which the microbubbles are formed, the survival time of the microbubbles can be calculated. P.S. Epstein, M.S. Plesset, "On the Stability of Gas Bubbles in Liquid-Gas Solutions,"
The Journal of Chemical Phv~sics, Vol. 18, n. 11, 1505 (1950). Based on calculations, it is apparent that as the size of the bubble decreases, the surface tension between bubble and surrounding solution increases. As the surface tension increases, the rate at which the bubble dissolves into the solution increases rapidly and, therefore, the size of the bubble decreases more and more rapidly. Thus, the rate at which the bubble shrinks increases as the size of the bubble decreases. The ultimate effect of this is that a population of small free gas microbubbles composed of ordinary air dissolves so rapidly that the contrast-enhancing effect is extremely short lived. Using known mathematical formula, one can calculate that a microbubble of air that is 8 microns in diameter, which is small enough to pass through the lungs, will dissolve in between 190 and 550 milliseconds depending on the degree of saturation of the surrounding solution. Based on these calculations, medical investigators studying the way in which the lungs remove ultrasound contrast agent have calculated the dissolution times of oxygen PATENT
and nitrogen gas microbubbl.es in human and canine blood and have concluded treat free gas microbubble contrast agents will not a7.low contrast-enhanced imaging of the left ventricle because of the extremely brief life of thE: microbubbles.
The physical properties of the systems that feature gas bubbles or gases dissolved in liquid solutions have been investigated in detail including the diffusion of air bubbles formed in the cavitating flow of a liquid and the scatter of light and sound in water by gas bubbles.
The stability of gas bubbles in liquid-gas solution has been investigated both theoretically, Epstein P.S. and Plesset M.S., On the Stability of Gas Bubbles in Liquid-Gas Solutions, J. Chem. Phys.
18:1505-1509 (1950) and experimentally, Yang WJ, Dynamics of Gas Bubbles in Whole Blood and Plasma, J.
Biomech 4:119-125 (1971); 'Yang WJ, Echigo R., Wotton DR, and Hwang JB, Experimental Studies of the Dissolution of Gas Bubbles in Whole Blood and Plasma-I. Stationary Bubbles. J. Biomech 3:275-281 (1971);
Yang WJ, Echigo R., Wotton DR, Hwang JB, Experimental Studies of the Dissolution of Gas Bubbles in Whole Blood and Plasma-II. Moving Bubbles or Liquids. J.
Biomech 4:283-288 (1971). The physical and chemical properties of the liquid a:nd the gas determine the kinetic and thermodynamic :behavior of the system.
The chemical properties of the system which influence the stability of a bubble, and accordingly the life time, are the rate and extent of reactions which either consume, transform, or generate gas molecules.
For example, a well known reaction that is observed between a gas and a liquid takes place when carbon dioxide gas is present in water. As the gas dissolves into the aqueous solution, carbonic acid is PATENT
created by hydration of the carbon dioxide gas.
Because carbon dioxide gas is highly soluble in water, the gas diffuses rapidly into the solution and the bubble size diminishes rapidly. The presence of the carbonic acid in the solution alters the acid-base chemistry of the aquec>us solution and, as the chemical properties of the solution are changed by dissolution of the gas, the' stability of the carbon dioxide gas bubbles changer as the solution becomes saturated. In this system, the rate of dissolution of a gas bubble depends in part~on the concentration of carbon dioxide gas that is already dissolved in solution.
However, depending on the particular gas or liquid present in the system, the gas may be substantially insoluble in the liquid and dissolution of a gas bubble will be slower. In this situation, it has been discovered than it is possible to calculate bubble stability in a gas-liquid system by examining certain physical parameters of the gas.
BRIEF DESCRIPTIOrd OF THE INVENTION
It has been discovered that it is possible to identify chemical systems where extremely small gas bubbles are not reactive in an aqueous solution.
Relying on the method disc:Losed herein one skilled in the art may specially select particular gases based on their physical and chemical properties for use in ultrasound imaging. These gases can be used to produce the contrast-enhancing media that is also the subject matter of this invention. The microbubbles can be produced using certain existing techniques that use ordinary air, and can be infused as in a conventional ultrasound diagnosis.

-2:1-PATENT
The method that is the subject matter of this invention requires that calculations be made, consistent with the equations provided herein, based on the intrinsic physical properties of a gas and a liquid. Particularly, the density of a gas, the solubility of a gas in solution, and the diffusivity of a gas in solution, which in turn is dependent on the molar volume of the gas and the viscosity of the solution, are used in the equations disclosed below.
Thus, by the method disclosed herein, the physical properties of a given gas-liquid system can be evaluated, the rate and extent of bubble collapse can be estimated, and gases that would constitute effective contrast-enhancing agents can be selected based on these calculations.. Using existing techniques, substantially improved contrast-enhancing media may then be produced and used to improve the quality and usefulness of ultrasound imaging.
DETAILED DESCRIPTION OF THE INVENTION
To understand the metriod of this invention, it is useful to derive the mathematical relationships that describe the parametex-s of a gas-liquid system and the effect on bubble stability that occurs when a value for one or more of these parameters is altered.
It is assumed that, at an initial time, To, a spherical gas bubble of gas X, with a radius of Ro, is placed in a solution in which the initial concentration of gas X dissolved in the solution is equal to zero. Over some period of time, the bubble of gas X will dissolve into the solvent at which time its radius R will equal zero. Assume further that the solution is at constants temperature and pressure and that the dissolved gas concentration for a solution saturated with thE~ particular gas is PATENT
_ 2 ;~ _ designated Cs. Thus, at To, the concentration of the gas in the solution is zero, meaning that none of the gas has yet dissolved and all of the gas that is present is still contained within the bubble of radius Ro .
As time progresses, due to the difference in the concentration of the gas in the bubble and the gas in solution, the,bubble will tend to shrink as gas in the bubble is dissolved into the liquid by the process of diffusion. The change in bubble radius from its original radius of Ro to, after the passage of a particular amount of time, a smaller radius R is expressed by Equation (1), R 2 DC$ 1!z Ro _ P Ro where R is the bubble radius at time T, D is the coefficient of diffusivity of the particular gas in the liquid, and p is the dE:nsity of the particular gas of which the bubble is composed.
It follows that the time T required for a bubble to dissolve completely may be determined from Equation (1) by setting R/Ro = 0, and solving for T:
z RoP
Equation (2) T =

This result qualitatively indicates that bubble stability, and hence life span, is enhanced by increasing the initial bubble size Ro or by selecting a gas of higher density P, lower solubility CS in the liquid phase, or a lower coefficient of diffusivity D.
The diffusivity D of a gas in a liquid is dependent on the molar volume of the gas (Vm), and PATENT
-2:3-the viscosity of the solution (n) as expressed by a known Equation;
(Equation 3) D = 13.26 x 10 5 . n-1.14 . V~o.sa9 By substituting the expression for D given in Equation (3) into Equation (2) it=is revealed that bubble stability is enhanced by using gases of larger molar volume Vm, which tend to have a higher molecular weight, and liquids of higher viscosity.
By way of example, a comparison of the stability of air microbubbles and microbubbles composed of gases specially selected by the method disclosed herein may be made. Taking' the value of D for air in water at 22°C as 2 x 10 5 cm2sec 1 and the ratio CS/p - 0.02 (Epstein and Pl~~sset, Ibid.), one obtains the following data for the time t for complete solution of air bubbles in water (unsaturated with air) TABLE I
INITIAL BUBBLE DIAMETER, mi.croas TIME, milliseconds If the blood transit tame from the pulmonary capillaries to the left ventricle is two seconds or more (Hamilton, W.F. editor, Handbook of Physiology, Vol. 2, section 2, CIRCULATION. American Physiology Society, Washington, D.C., p. 709, (1963)), and recognizing that only microbubbles of approximately 8 microns or less will be small enough to pass through the lungs, it is clear that: none of these bubbles PATENT
have a life span in solution long enough to be useful contrast agents for ultrasound contrast-enhanced imaging of the left ventric:le.
The method of the pre~;ent invention allows identification of potentially useful gases by comparing the properties of: any particular gas, denoted gas X in the following description, to air.
Taking Equations (2) and (3) above, a coefficient Q
may be formulated for a pai:ticular gas X that will describe the stability of microbubbles composed of gas X in a given liquid. The value of the Q
coefficient determined by this method for a particular gas X also can be used to determine the utility of gas X as an ultrasound contrast-enhancing agent as compared to ordinary air.
From Equation (2) above, an equation that describes the time for complete dissolution of a bubble of gas X compared to the same size bubble of ordinary air under identic<~1 conditions of solution temperature and solution viscosity may be written based on the physical props~rties of gas X and air:
Equation (4) Px ( Cs ) air Dair 2 5 Z'x - Tair _ Pair (Cs)x 13.26 X 10 5 . n-1.14 . (vm)X.589 or, if D is known for gas :K, Equation (5) P x ( Cs ) air Dair Tx - Z'air P air ( Cs ) x Dx PATENT
To formulate this equation so that the value Q may be obtained to enable comparison of gas X with air, the above equation may be rewritten:
Equation ( 6 ) TX = QTair where Px (Cs) air pair Q =
P air ( Cs ) x Dx Assuming for comparison, a solution of water at 22 degrees C, the density, dif:fusivity, and solubility of air in the solution are known quantities which may be substituted into the above equation yielding:
_ Px Equation (7) Q = 4.0 x 10~
( Cx ) ~X
Substituting Equation (3) into the above for gases whose diffusivity DX is not readily known, and assuming that the viscosity term n below for water at 22 degrees C is approximately equal to 1.0 cP, Equation (8) Q = 3.0 x 10 3 PX
(CS)x(Ym)x~589 Thus, knowing the density, solubility and molar volume of a gas, this method allows the calculation of the value of the Q coefficient.
If Q is less than one,, microbubbles of gas X
will be less stable in a given solvent than microbubbles of air. If Q :is greater than one, microbubbles formed of gas X are more stable than microbubbles of air and wi:Ll survive in solution longer than air bubbles. All other properties being the same for a given microbubble size, the time for complete dissolution of a microbubble of gas X is equal to the time for comp:Lete dissolution of a microbubble of ordinary ai:r multiplied by the Q

PATENT
-2E;-coefficient. For example, if the Q coefficient for gas X is 10,000, a microbub:ble of gas X will survive 10,000 times as long in solution compared to a microbubble of air. A Q value can be determined for any gas in any solution assuming the quantities identified herein are known or can be estimated.
Different methods for determining or estimating values for the individual parameters of density, diffusivity, and solubility may be needed depending on the chemical structure of the gas. Values for these parameters may or may not be available from known scientific literature: sources such as the Gas Encyclopedia or the tabulations published by the American Chemical Society. Values for the density of most gases are readily available from sources such as the Handbook of Chemistry and Physics. CRC Press, 72d Ed. (1991-92). Additional7.y, the solubility in water and molar volume of some gases has been measured with accuracy. In many cases however, calculations for the numerical values for molar volume and solubility may need to be calculated or estimated to provide the data used to determine the value of the Q coefficient for an individual gas by the method described above.
An example of the calculation of Q values for a preferred selection of gases illustrates how the method of this invention c<~n be applied to individual gases.
Generally, many fluorine-containing gases exhibit extremely low solubility in water, and have relatively high molecular weights, high molar volumes, and high densities. To determine the Q
value for several gases, the solubility, molar volume and density of the individual gases are determined and the values are substituted into Equations (7) or (8) above.

_2~7_ PATENT
Determination of Gas Solubility for Fluorocarbons This method for estimating the gas solubility of fluorocarbons uses extrapolation of the experimental data of Kabalnov AS, Makarov KN, and Scherbakova OV.
"Solubility of Fluorocarbons in Water as a Key Parameter Determining Fluorocarbon Emulsion Stability," J. Fluor. Chem. 50, 271-284, (1990). The gas solubility of these fluorocarbons is determined relative to perfluoro-n-pentane which has a water solubility of 4.0 x 106 moles per liter. For a homologous series of non-branched fluorocarbons, the gas solubility may be estimated by increasing or reducing this value by a factor of about 8.0 for each increase or reduction in the number of additional -CFz- groups present in the molecule.
Determination of Molar Volume The molar volume (Vm) is estimated from the data of Bondi A., "Van der Waals Volumes and Radii," J.
Phys. Chem. 68, 441-451 (1964). The molar volume of a gas can be estimated by identifying the number and type of atoms that make up the molecule of gas in question. By determining the number and type of atoms present in the molecule and how the individual atoms are bound to each other, known values may be applied for the molecular volume of the individual atoms. By considering the contribution of each individual atom and its frequency of occurrence, one may calculate the total molar volume for a particular gas molecule. This calculation is best demonstrated with an example.
It is known that a carbon molecule in an alkane carbon-carbon bond has a molar volume of 3.3 cubic centimeters per mole, a carbon atom in an alkene PATENT
-2f3-carbon-carbon bond has a molar volume of 10.0 cubic centimeters per mole, and when multiple fluorine atoms are bound to an alkane carbon, a fluorine atom has a molar volume of 6.0 cubic centimeters per mole.
Examining octafluoropropane, this molecule contains three carbon atoms in alkane carbon-carbon bonds (3 atoms at 3.3 cubic centimeters per mole) and 6 fluorine atoms bound to alkane carbons (6 atoms at 6.0 cubic centimeters per mole) hence, octafluoro-propane has a molar density of 58 cubic centimeters per mole.
Once density, molar volume, and solubility are determined, the Q value is calculated using Equation 8 above.
The following Table lists the Q value for a number of gases based on th.e calculations detailed above.
TABLE II
MOLAR

kg/m3 micromoles/ cm3/mole liter Argon 1.78 1500 17.9 20 n-Butane 2.05 6696 116 5 Carbon Dioxide 1.98 33000 19.7 1 Decafluorobutane 11.21 32 73 13,154 Dodecafluoropentane 12.86 4 183 207,437 Ethane 1.05 2900 67 13 Ethyl ether 2.55 977,058 103 0.1 3 Helium 0.18 388 8 5 Hexafluorobuta-1,3-diene 9 (*) 2000 56 145 Hexafluoro-2-butyne 9 (*) 2000 58 148 Hexafluoroethane 8.86 2100 43 116 Hexafluoropropene 10.3 260 58 1299 3 Krypton 3.8 2067 35 44 Neon 0.90 434 17 33 Nitrogen ## ## ## 1 Octafluoro-2-butene 10 (*) 220 65 1594 Octafluorocyclobutane 9.97 220 61 1531 4 Octafluoropropane 10.3 260 58 1299 Pentane 2 1674 113 58 PATENT
Propane 2.02 2902 90 30 Sulfur Hexafluoride 5.48 220 47 722 Xenon 5.90 3448 18 28 * These density values are estimated from the known density of homologous fluorocarbons.
## The solubility/density ratio value of 0.02 (supra) and the diffusivity of 2 x 10 5 cmZSec-1 given above were used in equation 7 for this Q-value determination.
Once the Q value has been determined the utility of an individual gas as an ultrasound contrast-enhancing agent can be analyzed by determining the life span of a collection of microbubbles composed of the gas in question at different sizes, as was done for air in Table I above. Taking the value of Q for decafluorobutane and examining the time necessary for various sized bubbles to dissolve in water, one obtains the values in Table: III below by multiplying each of the time values in Table I by the Q value for decafluorobutane:
TABLE III
INITIAL BUBBLE DIAMETER, macrons TIME, minutes $ 44 3 6.1 2 2.9 1 0.7 Notice that the time acale in Table III is minutes rather than milliseconds as was the case for air. All bubbles of decaf:luorobutane, even as small as 1 micron, can be injected peripherally and will not dissolve into solution during the approximately 10 seconds needed to reach the left ventricle.
Similar calculations can b~~ perforned for a gas with any Q coefficient. Slightly larger bubbles will be PATENT
able to pass through the lungs and yet survive long enough to permit both examination of myocardial perfusion and dynamic abdominal organ imaging.
Moreover, as with many of the gases identified by this method, decafluorobuta:ne features low toxicity at small dosages and would, therefore, offer substantial advantages as a contrast-enhancing agent in conventional ultrasound diagnosis.
Manual creation of a microbubble suspension may be accomplished by several methods. United States Patent 4,832,941 refers to a method for producing a suspension of microbubbles with a diameter less than seven mi~~rons created by spraying a liquid through a quantity of gas using a three-way tap. Although techniques could vary in practice, the three-way tap is a preferred method to manually suspend a quantity of high Q~coefficient gas to produce the contrast-enhancing media described herein.
The general techniques for use of a three-way tap device are well known in connection with preparation of the common Freund~s adjuvant for immunizing research animals. Typically, a three-way tap is comprised of a pair of syringes, both of which are connected to a chamber. The chamber has outlet from which the suspension may be collected or infused directly.
Techniques for use of t:he three-way tap may differ from that described in U.S. Patent No.
4,832,941 because different gases are being used in this procedure. For example, use of one of the high Q coefficient gases disclosed herein may be more efficient if the system is purged of ordinary air or -31.-PATENT
flushed with another gas before the microbubble suspension is produced.
In a preferred embodimE:nt of the present invention, a 40-50% Sorbito7_ (D-glucitol) solution is mixed with approximately 1-10% by volume of a high Q-coefficient gas with approximately 5o gas being an optimal value. Sorbitol is a commercially available compound that when mixed in an aqueous solution substantially increases the viscosity of the solution. Higher viscosity solutions, as seen in equation 3 above, extend thE~ life of a microbubble in solution. A 40-50% Sorbitol solution is preferred to maintain as a bolus upon injection; that is as intact as possible without exceeding a tolerable injection pressure. To produce the suspension of microbubbles, 'a quantity of the chosen gas is collected in a syringe. In the same syringe, a volume of the Sorbitol solution may be contained. A quantity of Sorbitol solution is drawn :into the other syringe so that the sum of the two volumes yields the proper percentage of gas based on i~he volume percentage of microbubbles desired. Using the two syringes, each featuring a very small aperi~ure, the liquid is sprayed into the gas atmosphere approximately 25 times or as many times as is necessary to create a suspension of microbubbles whose size distribution is acceptable for the purposes described herein. This technique may be varied slightly, of course, in any manner that achieves the resulting suspension of microbubbles of the desired size in a desired concentration. Microbubble size may be checked either visually or electron:i.cally using a Coulter Counter*(Coulter Electronics) by a known method, Butler, B.D., "Production oi° Microbubble for Use as *Trade-mark _3~>_ PATENT
Echo Contrast Agents," J. C:Lin. Ultrasound. V.14 408 (1986) .
EXAMPLES
EXAMPLE 1. An ultrasound contrast agent was prepared using decafluorobutane as the microbubble-forming gas. A solution was prepare=d containing:
sorbitol 20.0 g NaCl 0,9 g soy bean oil 6.0 mL
Tween 20* 0.5 mL
water q.s. 100.0 mL
A soapy, clear, yellow solution was afforded with stirring. A 10 mL aliquot of this solution was taken up in a 10 mL glass syringe. The syringe was then attached to a three-way stopcock. A second 10 mL syringe was attached to t:he stopcock and 1.0 cc of decafluorobutane (PCR, Inc., Gainesville, FL) was delivered to the empty syringe. The stopcock valve was opened to the solution-containing syringe and the liquid and gas phases mixed rapidly 20-30 times. A
resulting milky-white, slightly viscous solution was obtained.
EXAMPLE 2. The gas emulsion. obtained in Example 1 was diluted with water (1:10 to 1:1000), placed in a hemocytometer, and examined under the microscope using an oil immersion lens. The emulsion consisted of predominately 2-5 micron bubbles. The density was 50-100 million microbubbles per mL of original undiluted formulation.
EXAMPLE 3. The formulation of Example 1 was prepared and echocardiography performed in a canine model. A
17.5 kg mongrel dog was anesthetized with isoflurane and monitors established to measure ECG, blood *Trade-mark -3:3-pressure, heart rate, and arterial blood gases PATENT
according to the methods described by Keller, MW, Feinstein, SB, Watson, DD: Successful left ventricular opacification following peripheral venous injection of sonicated contrast agent: An experimental evaluation. A.m Heart J 114:570d (1987).
The results of the safety evaluation are as follows MAXIMUM PERCENTAGE CHANGE IN MEASURED

DOSE AORTIC PRESSURE BLOOD GASES HEART RATE
SYSTOLIC DIASTOLIC MEAN Pa02 PaC02 pH
mm Hg 0.5 mL +6, -14 +9, 0 +8, -6 329 58.1 7.26 +10 -19 1.0 mL +9, -2 +5, -1 +4, -4 +1, -4 2.0 mL +5, -3 +5, -1 +5, -1 0, -1 3.0 mL +6,_ -2 +7, 0 +4, -3 0, -3 4.0 mL +5, -1 +3, -3 +5, -3 0, -3 5.0 mI~ 0, -10 +1, -3 0, -4 +1, -1 2 0 7.0 mL 0, -13 0, -8 0, -9 313 28.6 7.36 0, -1 All changes were tran:~ient and returned to baseline values typically within 3-6 minutes. The above safety data demonstrate minimal changes in the measured hemodynamic parameter. All doses provided both right and left ventricular chamber opacification.
The intensity increased with the increasing dose.
Example 4. The above specific determinations of the suitability of a particular gas for use as an ultrasound agent can be approximated if the molecular weight of a particular gas is known, can be calculated, or can be measured. This <~pproximation is based on the determination that there is a linear relationship PATENT
_3~6_ between the logarithm of the Q-value and the molecular weight for a gas, as shown :in the Figure below.

t ", d y a 1a , 250 Molaculpr Weight Based on this Figure, the following guidelines can be used to estimate a Q-value::
Molecular Weight Estimated Q-Value < 35 < 5 171-220 1001-10,000 221-270 10,001-100,000 >270 >100,000 The following Table contains a series of gases with the relevant data on molecular weight and estimated Q-value. The higher the Q-value the more PATENT
promising is the particular gas. Especially promising are gases with Q-values greater than five. Additional issues, including, but not .Limited to, cost and toxicity, should be considered in addition to longevity of the derived microbubbles (as estimated by the Q-value) in determining the suitability of any particular gas as an ultrasound contrast agent.
TASi,E =v Chemical Name Molecular Estimated O

Wei ht Value Acetone, hexafluoro 166.02 81-1000 Acetylene, isopropyl 68 5-20 ~r 28.4 < 5 Allene 40.06 5-20 Allene, tetrafluoro 112.03 81-1000 Argon 39.:B 5-20 Borne, dimethyl, methoxy 71.19 21-80 Borne, trimethyl 55.91 5-20 Boron fluoride dihydrate 103.84 81-1000 1,2-Butadiene 54.09 5-20 1,3-Butadiene 54.09 5-20 1,3-Butadiene, 1,2,3-trichloro157.43 81-1000 1,3-Butadiene, 2-fluoro 72.08 21-80 1,3-Butadiene, 2-methyl 68.12 5-20 1,3-Butadiene, hexafluoro 162.03 81-1000 Butadiyne 50.06 5-20 n-Butane 58.12 5-20 Butane, 1-fluoro 76.11 21-80 Butane, 2-methyl 72.15 21-80 3 Butane, decafluoro 238.03 10,001-100,000 1-Butene 56.11 5-20 2-Butene {cis} 56.11 5-20 2-Butene {traps} 56.11 5-20 1-Butene, 2-methyl 70.13 5-20 3 1-Butene, 3-methyl 70.13 5-20 2-Butene, 3-methyl 68 5-20 1-Butene, perfluoro 200.03 1001-10,000 2-Butene, perfluoro 200.03 1001-10,000 3-Butene-2-one, 4-phenyl {traps}146.19 81-1000 4 1-Butene-3-yne, 2-methyl 66.1 S-20 Butyl nitrite 103.12 81-100 1-Butyne 54.09 5-20 2-Butyne 54.09 5-20 Butyne, 2-chloro-1,1,1,4,4,4-hexafluoro199 1001-10,000 45 1-Butyne, 3-methyl 68.12 5-20 2-Butyne) perfluoro 162.03 81-1000 Butyraldehyde, 2-bromo 151 81-1000 Carbon dioxide 44.01 5-20 Carbonyl sulfide 60.08 5-20 5 Crotononitrile 67.09 5-20 Cyclobutane 56.11 5-20 Cyclobutane, methyl 70.13 5-20 Cyclobutane, octaffuoro 200.03 1001-100,000 Cyclobutene, perffuoro 162.03 81-1000 Cyclopentene, 3-chloro 102.56 81-1000 Cyclopropane 42.08 5-20 Cyclopropane, 1,2-dimethyl 70.13 5-20 {traps, dl}

Cyclopropane, 1,1-dimethyl 70.13 5-20 Cyclopropane, 1,2-dimethyl 70.13 5-20 {cis}

Cyclopropane, 1,2-dimethyl 70.13 5-20 {traps, 1}

Cyclopropane) ethyl 70.13 5-20 Cyclopropane, methyl 56.11 5-20 Deuterium 4.02 < 5 Diacetylene 50.08 5-20 Diaziridine, 3-ethyl-3-methyl 86.14 21-80 Diazoethane, 1,1,1-trifluoro 110.04 81-1000 Dimethyl amine 45.08 5-20 Dimethyl amine, hezafluoro 153.03 81-1000 Dimethyl disulfide, heaafluoro202.13 1001-10,000 2 Dimethylethylamine 73.14 21-80 bis-(Dimethyl phosphino) amine137.1 81-1000 2,3-Dimethyl-2-norbornano 140.23 81-1000 Dimethylamine, perffuoro 171.02 1001-10,000 Dimethyloxonium chloride 82'.53 21-80 1,3-Dioxolane-2-one, 4-methyl 102.09 81-1000 Ethane 30.07 < 5 Ethane, 1,1,1,2-tetrafluoro 102.03 81-1000 Ethane, 1,1,1-triffuoro 84.04 21-80 Ethane, 1,1,2,2-tetraffuoro 102.03 81-1000 Ethane, 1,1,2-trichloro-1,2,2-triffuoro187.38 1001-10,000 Ethane, 1,1-dichloro 98 21-80 Ethane, 1,1-dichloro-1,2,2,2-tetrafluoro170.92 1001-10,000 Ethane, 1,1-dichloro-1-fluoro 116.95 81-1000 Ethane, 1,1-diffuoro 66.05 5-20 Ethane, 1,2-dichloro-1,1,2,2-tetrafluoro170.92 1001-10,000 Ethane, 1,2-diffuoro 66.05 5-20 Ethane, 1-chloro-1,1,2,2,2-pentafluoro154.47 81-1000 Ethane, 1-chloro-1,1,2,2-tetraffuoro136.48 81-1000 Ethane, 2-chloro, 1,1-diffuoro100 21-80 4 Ethane, 2-chloro-1 ,1 ,1-trifluoro118.49 81-1000 Ethane, Chloro 64.51 5-20 Ethane, chloro pentafluoro 154.47 81-1000 Ethane, dichlorotriffuoro 152 81-1000 Ethane, fluoro 48.06 5-20 4 Ethane,hexaffuoro 138.01 81-1000 Ethane, vitro-pentaffuoro 165.02 81-1000 Ethane, nitroso-pentafluoro 149.02 81-1000 Ethane, perfluoro 138.01 81-1000 Ethyl amine, perfluoro 171.02 1001-100,000 5 Ethyl ether 74.12 21-80 Ethyl methyl ether 60.1 5-20 Ethyl vinyl ether 72.11 21-80 Ethylene 28.05 < 5 Ethylene, 1,1-dichloro 96.94 21-80 55 Ethylene) 1,1-dichloro-2-fluoro114.93 81-1000 Ethylene, 1 ,2-dichloro-1 ,2-difluoro132.92 81-1000 Ethylene, 1 ,2-diffuoro 64 5-20 PATENT

Ethylene, 1-chloro-1 ,2,2-trifluoro116.47 81-1000 Ethylene, chloro triffuoro 116.47 81-1000 Ethylene) dichloro difluoro 132.92 81-1000 Ethylene, tetrafluoro 100.02 21-80 Fulvene 78.11 21-80 Helium . 4 < 5 1,5-Heptadiyne 92.14 21-80 Hydrogen (H2) 2.02 < S

Isobutane 58.12 5-20 1 Isobutane, 1,2-epoxy-3-chloro 106.55 81-1000 Isobutylene 56.1 1 5-20 Isoprene 68.12 5-20 Krypton 83.8 21-80 Methane 16.04 < 5 Methane sulfonyl chloride, 168.52 81-1000 triffuoro Methane sulfonyl fluoride, 152.06 81-1000 triffuoro Methane, (pentaffuorothio)triffuoro196.06 1001-10,000 Methane, bromo diffuoro nitroso159.92 81-1000 Methane, bromo ffuoro 112.93 81-1000 2 Methane, bromo-chloro-fluoro 147.37 81-1000 Methane, bromo-triffuoro 148.91 81-1000 Methane, chloro difluoro vitro131.47 81-1000 Methane, chloro dinitro 140.48 81-1000 Methane, chloro fluoro 68.48 5-20 2 Methane, chloro trifluoro 104.46 81-1000 Methane, chloro-diffuoro 86.47 21-80 Methane, dibromo difluoro 209.82 1001-10,000 Methane, dichloro diffuoro 120.91 81-1000 Methane, dichloro-fluoro 102.92 81-1000 3 Methane, diffuoro 52.02 5-20 Methane, diffuoro-iodo 177.92 1001-10,000 Methane, disilano 76.25 21-80 Methane, ffuoro 34.03 < 5 Methane, iodo- 141.94 81-1000 3 Methane, iodo-triffuoro 195.91 1001-10,000 Methane, vitro-triffuoro 115.01 81-1000 Methane, nitroso-trifluoro 99.01 21-80 Methane, tetrafluoro ~ 88 21-80 Methane, trichlorofluoro 137.37 81-1000 4 Methane, triffuoro 70.01 5-20 Methanesulfenylchloride, triffuoro136.52 81-1000 2-Methyl butane 72.15 21-80 Methyl ether 46.07 5-20 Methyl isopropyl ether 74.12 21-80 4 Methyl nitrite 61.04 5-20 Methyl sulfide 62.13 5-20 Methyl vinyl ether 58.08 5-20 Neon 20.18 < 5 Neopentane 72.15 21-80 5 Nitrogen (N2) 28.01 < 5 Nitrous oxide 44.01 S-20 1,2,3-Nonadecane tricarboxylic acid, 2-.....hydroxy~rimethylester 500.72 > 100,000 1-Nonene-3-yne 122.21 81-1000 5 Oxygen (02) 32 < S

1,4-Pentadiene 68.12 5-20 n-Pentane 72.15 21-80 PATENT

PATENT
-3~3-Pentane, perfluoro 288.04 > 100,000 2-Pentanone, 4-amino-4-methyl 115.18 81-1000 1-Pentene 70.13 5-20 2-Pentene {cis} 70.13 5-20 2-Pentene {traps} 70.13 5-20 1-Pentene, 3-bromo 149.03 81-1000 erfluoro 250.04 10,001-100,000 1-Pentene, p Phthalic acid, tetrachloro 303.91 > 100,000 Piperidine, 2,3,6-trimethyl 127.23 81-1000 1 0 Propane 44.1 5-20 Propane, 1,1,1,2,2,3-hexafluoro 152.04 81-1000 Propane, 1,2-epoxy 58.08 5-20 Propane, 2,2-difluoro 80.08 21-80 Propane, 2-amino 59.11 5-20 1 5 Propane, 2-chloro 78.54 21-80 Propane, heptafluoro-1 -vitro 215.03 1001-10,000 Propane) heptafluoro-1 -nitroso 199.03 1001-10,000 Propane, perfluoro 188.02 1001-10,000 Propene 42.08 5-20 2 0 Propyl, 1 ,1 ,1 ,2,3,3-hexaffuoro-2,3-dichloro221 10,001-100,000 Propylene, 1 -chloro 76.53 21-80 Propylene, 1-chloro-{traps} 76.53 5-20 Propylene, 2-chloro 76.53 5-20 Propylene, 3-fluoro 60.07 5-20 2 5 Propylene, perfluoro 150.02 81-1000 Propyne 40.06 5-20 Propyne, 3,3,3-trifluoro 94.04 21-80 Styrene, 3-fluoro 122.14 81-1000 Sulfur hexafluoride 146.05 81-1000 30 Sulfiu (di),decafluoro(S2F10) 298 >100,000 Toluene, 2,4-diamino 122.17 81-1000 Trifluoroacetonitrile 95.02 21-80 Trifluoromethyl peroxide 170.01 81-1000 Trifluoromethyl sulfide 170.07 81-1000 3 5 Tungsten hexafluoride 298 > 100,000 Vinyl acetylene 52.08 5-20 Vinyl ether 70 5-20 Xenon 131.29 81-1000 Example 5. The relationship b etween a.

40 calculated Q-value for a given and the gas persistence of microbubbles of thatgas was studied to determine what Q-value would a lower limit be for utility as an ultrasound contrast agent. For these experiments, a 190 x 100 mm PyrexTT'(No. 3140) 45 evaporation dish was filled with approximately mL of water at 37C. Five mL of 20% sorbitol a solution was taken up in a 10 mL syringe connected to a three way stopcock. A 10 mL syringe, containing 2 cubic centimeters of the subject gas (or low boiling PATENT
_3c~_ liquid, as pertinent) was attached to the syringe containing the sorbitol solution. The sorbitol and gas or liquid are rapidly mixed 25-times to create a suspension of microbubbles or dispersed liquid and then rapidly added to the water. The microbubbles of this method are generally albout 100 microns in size, and if composed of air would have ~ calculated persistence of 31 sec (0.5 'min). Ultrasound scans before, during and after the addition were made with a Hewlett-Packard Model Sonos 500 ultrasound scanner operating at 5 MHz. The time during which the microbubbles could be observed was recorded. The results are contained in Table V below. The experimental Q-values were obtained by dividing the measured persistence of a given gas by the measured persistence for air.
TABLE V
RELATIONSHIP BETWEEN Q-VALUE FOR A GAS
AND THE PERSISTENCE OF MICROBUBBLES

(Calculated) (Experimental Q-Value) . Diethyl ether 0.1 0.1 min (0.2) Air 1 0.6 min (1.0) Butane 5 1.5 min (2.6) Helium 5 2.0 min (3.5) Propane 30 3.2 min (6.0) Pentane 58 20.6 min (36) Dodecafluoropentane 207,437 >5760 min (>10,105) These experiments indicate an excellent agreement between the calculated Q-value and the experimentally determined ,values. Based on these data, gases with Q-values calculated to be greater than five should be potentially useful as contrast agents for ultrasound imaging.
Example 6. The relationship of the state of matter of a given chemical entity with a high Q-_~, ~ f!' ~ . , PATENT

coefficient and its utility as an ultrasound contrast agent was tested by comparing the efficiency of perfluoropentane and perfluorohexane to act as an ultrasound contrast agent. Perfluoropentane (dodecafluoropentane) has a calculated Q-coefficient of 207,437 and a boiling point under standard pressure conditions of 29.5 degrees Centigrade.
Perfluorohexane (PCR, INc., Gainsville, FL) has a calculated Q-coefficient of 1,659,496 and a boiling point under standard pressure conditions of 59-60 degrees C. Therefore, at 3'7 degrees C, the body temperature of man, perfluo:ropentane is a gas while perfluorohexane is a liquid.
Aqueous dispersions of perfluoropentane and perfluorohexane (2% w/v) were formed at 4 degrees C
by vigorous homogenization. A plastic beaker, containing approximately 1000 mL of water at 37 degrees C, was prepared to simulate human blood and was ultrasonically-scanned, as indicated in Example 5 above, before and after the addition of samples of each of the above dispersions.
Less than 1.0 mL of the perfluoropentane dispersion, when mixed with the simulated blood, produced an extremely bright ultrasound signal which persisted for at least 30 minutes. A 1:10,000 dilution was still detectable.
In contrast, a 1.0 mL sample of the perfluorohexane dispersion was undetectable by ultrasound scanning under the same conditions, as was even a 10 mL sample (1:100 dilution).
The conclusion to be drawn is that both a high Q-coefficient and a gaseous. state at the body temperature of the organism being scanned is necessary for a substance t:o be effective as an -41.-PATENT
ultrasound contrast agent according to the method that is the subject matter of this invention.
Although the invention has been described in some respects with referencE~ to specified preferred embodiments thereof, variat:Lons and modifications will become apparent to those skilled in the art. It is, therefore, the intention that~the following claims not be given a restrictive interpretation but should be viewed to encompass variations and modifications that are derived from the inventive subject matter disclosed. ~Cn this regard the invention allows those skil7_ed in the art to determine the suitability oi' various chemicals as ultrasound contrast agents where density, solubility and molar volume data are available.

Claims (44)

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

1. A contrast media for ultrasound image-enhancement comprising liposomes filled with microbubbles of gas as enhancing agent, wherein said gas is, at least in part, selected from the group consisting of dodecafluoropentane, decafluorobutane and octafluoropropane wherein said microbubbles are less than 8µm in size.

2. A contrast media according to claim 1 wherein said gas is dodecafluoropentane.

3. A contrast media according to claim 1 wherein said gas is decafluorobutane.

4. A contrast media according to claim 1 wherein said gas is octafluoropropane.

5. A contrast media for ultrasound image-enhancement comprising encapsulated gas-filled microspheres, wherein said gas is, at least in part, selected from the group consisting of dodecafluoropentane, decafluorobutane and octafluoropropane wherein said microspheres are less than 8µm in size.

6. A contrast media according to claim 5 wherein said gas is dodecafluoropentane.

7. A contrast media according to claim 5 wherein said gas is decafluorobutane.

8. A contrast media according to claim 5 wherein said gas is octafluoropropane.

9. A contrast media for ultrasound image-enhancement comprising a suspension of crystals in a saccharide diluent, wherein said suspension further includes microbubbles of a fluorine containing gas in said suspension wherein said fluorine-containing gas is, at least in part, selected from the group consisting of dodecafluoropentane, decafluorobutane and octafluoropropane wherein said microbubbles are less than 8µm in size.

10. A contrast media according to claim 9 wherein said gas is dodecafluoropentane.

11. A contrast media according to claim 9 wherein said gas is decafluorobutane.

12. A contrast media according to claim 9 wherein said gas is octaflouoropropane.

13. A contrast media for ultrasound image enhancement comprising gaseous perfluorohexane.

14. A method of performing ultrasound imaging, comprising administering biacompatible contrast media into a mammal by sequentially conducting a scan on the required organ of said mammal, characterized in that the biocompatible contrast media used is represented by a chemical which comprises free gas microbubbles of a gas selected from decafluorobutane, dodecafluoropentane, and octafluoropropane wherein said microbubbles are less than 8µm in size.

15. The use of a collection of free gas microbubbles of a chemical selected from octafluoropropane, decafluorobutane and dodecafluoropentane for ultrasound imaging and performing ultrasound scan on an animal.

16. Use of a collection of microbubbles of a chemical which is compatible with an animal, where said chemical is selected from the group consisting of octafluoropropane, decafluorobutane and dodecafluoropentane for ultrasound imaging and for performing an ultrasound scan on said animal.

17. Use of a biocompatible ultrasound contrast agent comprising dodecafluoropentane for subjecting an organism to an ultrasound scan.

18. Use of a biocompatible ultrasound contrast agent comprising dodecafluoropentane for subjecting a human to an ultrasound scan.

19. Use of a biocompatible ultrasound contrast agent comprising a member of the group consisting of octafluoropropane, decafluorobutane and dodecafluoropentane for performing an ultrasound scan on an animal.

20. Use of a biocompatible contrast medium for ultrasound image-enhancement, said biocompatible contrast medium comprising a gas emulsion, the gas emulsion including microbubbles of a gas selected from decafluorobutane, dodecafluoropentane, sulfur hexafluoride and octafluoropropane.

21. Use of a biocompatible contrast medium according to claim 20 comprising microbubbles of decafluorobutane.

22. Use of a biocompatible contrast medium according to claim 20 comprising microbubbles of sulfur hexafluoride.

23. Use of a biocompatible contrast medium according to claim 20 comprising microbubbles of octafluoropropane.

24. Use of a biocompatible contrast medium according to claim 20 further comprising a viscosity enhancing agent.

25. Use of a biocompatible contrast medium according to claim 20 comprising microbubbles less than 8µm.

26. Use of a biocompatible contrast medium according to claim 20 wherein said microbubbles are suspended in an aqueous solution.

27. Use of a biocompatible ultrasound contrast medium according to claim 23 further comprising a surfactant for stabilising the gas emulsion.

28. Use of a contrast medium according to claim 27 further comprising a viscosity enhancing agent.

29. Use of a contrast medium according to claim 27 wherein said microbubbles have a diameter less than 8µm.

30. Use of a contrast medium comprising gaseous dodecafluoropentane as enhancing agent, for ultrasound image-enhancement.

31. Use of a contrast medium comprising gaseous decafluorobutane as enhancing agent, for ultrasound image-enhancement.

32. Use of a contrast medium comprising gaseous octafluoropropane as enhancing agent, for ultrasound image-enhancement.

33. Use of a contrast medium according to any of claims 30 to 32 wherein said enhancing agent is dispersed in aqueous solution.

34. Use of a contrast medium according to any of claims 30 to 33 wherein said enhancing agent is in the form of a microbubble having a diameter less than 8µm.

35. Use of a contrast medium comprising liposomes filled with gas as enhancing agent, wherein said gas is, at least in part, a fluorine or sulfure hexafluoride containing gas, for ultrasound image-enhancement.

36. Use of a contrast medium according to claim 35 wherein said fluorine containing gas is one of sulfur hexafluoride, dodecafluoropentane, decafluorobutane or octafluoropropane.

37. Use of a contrast medium comprising encapsulated gas-filled microspheres, wherein said gas is, at least in part, a fluorine or sulfure hexafluoride containing gas, for ultrasound image-enhancement.

38. Use of a contrast medium according to claim 37 wherein said fluorine containing gas is dodecafluoropentane.

39. Use of a contrast medium according to claim 37 wherein said fluorine containing gas is decafluorobutane.

40. Use of a contrast medium according to claim 37 wherein said fluorine containing gas is octafluoropropane.

41. Use of a contrast medium according to claim 37 wherein said fluorine containing gas is sulfur hexafluoride.

42. Use of a contrast medium comprising a suspension of crystals in a saccharide diluent comprising microbubbles of a fluorine containing gas in said suspension, for ultrasound image-enhancement.

43. Use of a contrast medium according to claim 42 wherein said fluorine containing gas is one of dodecafluoropentane, decafluorobutane, or octafluoropropane.

44. Use of a contrast medium comprising gaseous perfluorohexane, for ultrasound image-enhancement.