CA2164410C

CA2164410C - Emulsions as contrast agents and method of use

Info

Publication number: CA2164410C
Application number: CA002164410A
Authority: CA
Inventors: Rolf Lohrmann; Ashwin M. Krishnan; Dung K. Hong; Jialun Meng; Kenneth J. Widder
Original assignee: Molecular Biosystems Inc
Current assignee: Molecular Biosystems Inc
Priority date: 1993-06-04
Filing date: 1994-05-26
Publication date: 2000-02-22
Anticipated expiration: 2014-05-26
Also published as: US5536489A; WO1994028939A1; EP0701451B1; EP0852145A2; DE69412610D1; DK0701451T3; US5716597A; JP3016592B2; JPH08509984A; DE69412610T2; EP0701451A1; GR3027727T3; US6030603A; EP0852145A3; ES2120053T3; JPH11193247A; ATE169828T1

Abstract

This invention relates to an oil-in-water emulsion that is of a water-isoluble gas-forming chemical and a stabilizer, the emulsion being capable of forming microbubbles of gas upon application of ultrasonic energy. This composition allows for site-specific imaging as the image-enhancing bubbles can be released upon the application of ultrasonic energy at the specific location where the image is desired.

Description

.a~ s44 ~ o EMULSIONS AS CONTRAST AGENTS AND METHOD OF USE
Background of the Invention 1. Field of the Invention:
This invention relates to diagnostic ultrasonic imaging and contrast agents for use thereof. More particularly, it relates to ultrasonic contrast agents comprising emulsions capable of forming gas microbubbles upon the application of ultrasonic energy and methods for their use in diagnostic imaging.

2. Brief Description of the Hackcrround Art:
Diagnostic ultrasonic imaging is based on the principal that waves of sound energy can be focused upon an area of interest and reflected in such a way as to produce an image thereof. The ultrasonic scanner utilized is placed on a body surface overlying the area to be imaged, and sound waves are directed toward that area. The scanner detects reflected sound waves and translates the data into images. When ultrasonic energy is transmitted through a substance, the amount of energy reflected depends upon the velocity of the transmission and the acoustic properties of the substance. Changes in the substance's acoustic properties (e.g. variations in acoustic impedance) are most prominent at the interfaces of different substances, such as liquid-solid or liquid-gas. Consequently, when ultrasonic energy is directed through media, changes in acoustic properties will result c~~~~~~~

in more intense sound reflection signals for detection by the ultrasonic scanner.
Ultrasonic imaging agents of particular importance are composed of gas-containing substances which, when injected into the circulatory system, provide improved sound reflection and image clarity. One class of gas-containing imaging agents consists of microspheres of gas surrounded by a shell made of a biocompatible substance. These are best typified by ALBUNEX°
(Molecular Biosystems, San Diego, California: U.S. Patent Nos. 4,572,203; 4,718,433; 4,744,958; 4,844,882 and 4,957,656) which consists of microspheres of air surrounded by albumin shells. Another such microspheric imaging agent is described by Holmes et al. These microspheres consist of either non-proteinaceous crosslinked or polymerized amphipathic moieties forming micelles (PCT WO 92/17212) or crosslinked proteins (PCT

3), both of which encapsulate gasses such as nitrogen, SF6 and CF4.
Another class of ultrasonic imaging agents can be described as microparticles of a solid or semi-solid substance containing gas which is entrapped in the microparticle matrix during production. Glajich et al.
(U.S. Patent No. 5,147,631) describe the formation of porous particles of an inorganic material containing entrapped gas or liquid such as 02, CF4, perfluoroethane and argon. Erbel et al. (U. S. Patent No. 5,137,928) describe polyamino-dicarboxylic acid-co-imide derivatives capable of entrapping gasses such as air, argon and krypton. Albayrak et al. (European Patent Specification 0 357 163) describe crystalline complexes entrapping gasses such as nitrogen, krypton, SF6, cyclopropane and pentane which are dissolved in an aqueous vehicle such as protein or glycerol causing the release of gas bubbles.
The aqueous vehicle, now containing a plurality of T _.

WO 94128939 216 4 ~~ 1 0 p~IVS~4I05965 microbubbles of gas in solution,_is then ready for use as an-injectable ultrasonic imaging agent. stein et al.
!European Patent Specification 327 490) describe microparticlea containing amyloses or synthetic S biodegradable polymers entrapping gasses or liquids with a boiling point Less than 60°C.
Another class of gas-containing imaging agents are lipid vesicles Br liposomes. Unger (U. S. Patent Nos.
5,088,499 and 5,123,414) describes the encapsulation of ga$aes yr gaseous precursors in liposvmes, more particularly liposomee which contain ionophores for aCtivatlon of gaseous precursors by way of a pH gradient.
Render~on et al. !PCT WO 92/15824) describe lipid vesicles with gas-filled center cores.
iS still another class of imaging agents is -compoaed of microbubblee of ga$ in solution. For example. Tickner et al. (U.9. Patent No. 4,276,88S) describe microbubblee dispersed in liquitied gelatin_ More recently, Quay (pCT WO 93/05819) describes ultrasound imaging agents comprising microbubbles of selected gasses in solution. In a specific embodiment Quay describes the formation og a gas-liquid emulsion of decaEluorobutane_ Also disclosed therein are imaging agents comprising aqueous dispersion of biocompatible 2S gasses, some of which are gaseous at ambient temperature and others of which beevme gaseous at the body temperature .og the subject being imaged.
The efficiency of gas as an ultrasound imaging agent is described by J_ Ophir and K.J. Parker, Contrast Ac~entB in Dipqnpsti~ Ultraq~, Ultrasound in M~diCine and Biology (1989), Vol. iS(4) p. 319-333. However, the disadvantages of using gas as an ultrasound imaging agent have been and continue to be lacking of sufficient persistence of the gas in-vivo and in-vitro, and toxicity 35 due to the introduction of gas into the venous system.

WO 94/28939 2 16 4 410 PCT/US94/05965 .

The present invention relates to site specific oit-in-water emulsions and is based on the unexpected observation that emulsions of gas-forming chemicals can be stabilized in the liquid state and will produce microbubbles when subject to ultrasonic energy. The advantages are that such emulsions are more stable than most of the gas-containing imaging agents heretofore described, and their ability to form microbubbles when subject to ultrasonic energy makes them site-specific and inherently less toxic due to less overall gas being introduced into the venous system.
~ummarv of the Invention This invention provides an emulsion which can be used as an ultrasonic imaging agent. The emulsion is made of at least one water-insoluble gas-forming chemical and at least one stabilizer. This emulsion is capable of forming microbubbles of gas upon application of ultrasonic energy. The stabilizer is either a hydrophobic or amphipathic compound having a boiling point higher than that of the gas-forming chemical and, when present in the emulsion with the gas-forming chemical, acts as a stabilizer (maintains the gas-forming chemical in the liquid state) until the application of ultrasonic energy. The stabilizer causes the effective boiling point of the gas-forming chemical to be raised thereby preventing the volatilization of the gas-forming chemical until it reaches a temperature above its boiling point at atmospheric pressure (760 nm). In this way, upon application of ultrasonic energy, the emulsified chemical becomes volatilized and produces gas microbubbles. In a specific embodiment the water-insoluble gas-forming chemical is perfluoropentane and the stabilizer is lecithin. This invention also provides additional means to stabilize the emulsion for delivery ~~. ~4~ ~. D

to a patient. These means include delivery vehicles such as a natural polymer matrix, a synthetic polymer matrix or a liposome. More specifically, it is provided that the natural polymer matrix is an albumin matrix. This albumin matrix can be derivatized to contain polyethylene glycol.
This invention also provides a method to enhance the contrast of tissues and organs in an ultrasonic image comprising: (a) injecting at least one stabilized water-insoluble gas-forming chemical into a patient; (b) applying a sufficient amount of ultrasonic energy to volatilize said chemicals to release microbubbles; and (c) detecting an ultrasonic image. The water-insoluble gas-forming chemical is stabilized with a hydrophobic or amphipathic stabilizer.
Brief Description of the Drawing's.
Fig. lA shows the effects of increasing ultrasonic energy transmit power on reflectivity of the ultrasonic signal (expressed as video brightness) in the presence of an ALHUNEX~ (Molecular Biosystems, San Diego, California) sample.
Fig. 1H shows the effects as descried in Fig.
lA in the presence of the perfluoropentane emulsion of Example 1.
Fig-2 shows the difference in video brightness observed when the emulsion of Example 5 is either continually exposed to ultrasonic energy, or exposed only during 30 second intervals every 5 minutes.
Fig-3 shows the difference in video brightness observed when Emulsion C of Example 7 is either continually exposed to ultrasonic energy, or exposed only during 30 second intervals every 5 minutes.
Fig. 4 shows the 1H NMit spectrum of a CDC13 solution of nonafluoro-t-butylmethane (C4F9CH3).

Fiq. 5 shows the 19F NNfft spectrum of a CDC13 solution of nonafluoro-t-butylmethane (C4F9CH3).
Detailed Description of the Invention We have now found that particularly effective site-specific ultrasonic contrast agents may be obtained by preparing emulsions of water-insoluble gas-forming chemicals. These gas-forming chemicals are stabilized by emulsification with a stabilizer. Additionally, the emulsification of the gas-forming chemicals, which are for the most part insoluble in water, serves to make the contrast agent more soluble and thus administrable to a patient. The water-insoluble gas-forming chemical must be capable of forming gas at the body temperature of the animal being imaged and will generally have a boiling point below body temperature. As discussed herein, boiling point will refer to the temperature at which the thermal energy of the molecules of a chemical are great enough to overcome the cohesive forces that hold them together in a liquid state (or solid state for chemicals which sublime and thus have no liquid state) at atmospheric pressure (760 nm). A stabilizer having a boiling point higher than that of the gas-forming chemical is necessary to stabilize the gas-forming chemical in the liquid state until the application of ultrasonic energy. The stabilizer causes the temperature at which the gas-forming chemical volatilizes to a gas to be raised to a temperature above its boiling point. In this way, the gas-forming chemical is actually both stabilized (maintained in a liquid state above its boiling point) and destabilized (capable of being volatilized upon exposure to ultrasonic energy) simultaneously. When the emulsion of the present invention is volatilized by exposure to ultrasonic energy, such as 50% transmit power at 5.0 Ngiz, gas _7_ microbubbles are formed and released from the emulsion thereby increasing the ultrasonic reflectivity in the area being imaged.
The water-insoluble gas-forming chemicals useful in the present invention can be further characterized as being non-toxic, physiologically compatible and generally having a boiling point below 37°C, and preferably between 26°C and 34°C. Some of the gas-forming chemicals which would be useful in the present invention and their boiling points at atmospheric pressure are:

Gas-forming Chemical Roiling Point C

pentane 1-pentene 30 perfluoropentane 29.5 2-methyl butane (isopentane) 27.8 tetramethylsilane 26 2-bromo-1,1,1-trifluoroethane 26 dibromodifluoromethane 25 fluorotrichloromethane 24 2 H-perfluoro-t-butane 13 cyclobutane 12 heptafluoropropylbromide 12 1-chloro-1,1,2,2-tetrafluoroethane 10.2 neopentane 9,5 teflurane 2-chloro-1,1,1-trifluoroethane 6.g decafluorobutane 4 butane -0.5 2-chloro-1,1,1,2-tetrafluoroethane -12 2 H-heptafluoropropane -15 iodotrifluoromethane -22.5 cyclopropane -33 wv 94128939 216 4 410 ~'T~S94~as9~s perfluoroethyla~nine . - 3 5 octafluoropropane -36 SF6 (sulrur hexafluoride) -&4 The stabilizer of the present invention may be a hydrophobic or amphipathic (containing both hydrophobic and hydrophilic entities) compound. Hydrophobic compounds include di- and triglycerides; saturated and unsaturated hydrocarbons; perfluorocarbons such as perfluorohexane or perfluorodecalin; gate and fatty oils such as trivlein.
Amphipathic compounds include phospholipids such ae phvaphatidic acid, phosphatidylglycerol, and phoephatidylinoaitol; alkali salts of fatty acids; ionic aurractants such as sodium dodecyl sulfate; non-ionic surfactants Such ae PLURONIC~ F-68 (trade name for pvloxamer 188, a block copolymer ot.polyoxyethylene and polyoxypropylene (CAS-9003-11-6) HD (CH2CHa0) n ('CHCH20) p (CH2CH20) nH
~3 whorein the average value of n~~5 and the average value of p-3o such that the average molecular weight of said compound is 8350 daltons) and polysorbate 80; zwitterionic surfactants such ae phoaphatidylcholine tlecithin), phosphatidylethanolamine and phosphatidylserine; amino acid polymers or proteins with hydrophilic and hydrophobic moieties such ag albumin.
Amphipathic compounds which are particularly useful a.s stabilizers of fluorinated gas-forming compounds are themselves rluorinated. TheBe compounds act ae both stabilizers and solubilizerg of fluorinated gas-forming compounds, due to the fluorine-fluorine interactions between the two compounds. Such fluvrirated stabilizers generally have a hydrvphobiC fluorocarbon B

,:,;
_g_ chain connected to a hydrophilic moiety, such as a pol.yether, sugar, carbvxylate, sulfonate or a quaternary ammonium group. Examples of fluorinated stabilizers can be found in U.S. Patent Nos. 5,077,036, 5,080,855 and 4,987,154.
When the boiling point of the gas-forming chemical is below the temperature at which the emulsion is prepared and stored, such as less than 24°C, it is still possible to form a liquid-liquid oiI-in-water emulsion of the present invention by using a stabilizer which is capable of strong hydrophobic interactions with the gas-forming chemical which will maintain the gas-gvrming chemical in a liquid state above its boiling point. particularly useful stabilizers for this purpose are C5 to C20 pertluorocarbons or hydrocarbons and can be either hydrophobic or amphipathic.
The stabilizer may be used singly or in various combinations in the emul8ions of the present invention.
However, when the stabilizer is a hydrophobic compound, it will be necessary to also have present a surface active agent either within the emulsion or in association with the emul8ion in order for the emulsion to be soluble and thus physiologically tolerated. Surface active agents, or surfactants, are characterized as being substances that lower the surface tension between two liquids. A surface active agent will generally be an amphipathic Compound as described above or may also be a cationic or anionic compound. Additionally, a surfactant and a co-surfactant combination, such as phvephatidyl choline and PLURONIC~ F-68 is also contemplated.
When the stabilizer is amphipathic, the presence of an additional hydrophobic compound is generally not necessary. zn particular, the chemical 3S PLURONIC~ F-68 has been found tv surticiently aolubilize WO 94/28939 216 4 410 PCT~S94/05965 and stabilize the gas-forming chemical in the absence of an additional hydrophobic compound.
The amount of stabilizer present in the emulsion of the present invention will vary over a wide range of concentrations depending on the concentration and properties of the other components of the emulsion and will be principally dependent on the amount and characteristics of the,gas-forming chemical. This is exemplified in the example section.
Optionally present in the emulsion are viscosifiers which are generally polyalcohols or carbohydrates such as glycerol, sorbitol, lactose, sucrose and dextrans, and preferably glycerol at a concentration between 5-15% (w/v). Other optional constituents are anti-oxidants such as a-tocopherol, preferably at a concentration of 0.1 to 0.25% (w/v).
Still another class of optional components are compounds which impart organ or tissue target specificity to the emulsion. These compounds may include steroids such as cholesterol, proteins, lipoproteins and antibodies.
The emulsion of the present invention may be useful as an ultrasonic imaging agent either by itself or in combination with a delivery vehicle which may be used to impart greater stability, both in-vivo and in-vitro, or tissue or organ target specificity. One such delivery vehicle can be made from a natural polymer which forms a matrix, such as an albumin matrix, with multiple chambers which contain the emulsion of a gas-forming chemical.
The surface of the albumin matrix so described may also be modified to contain a polymer such as polyethylene glycol to reduce reticular endothelial system uptake in vivo.
Further examples of delivery vehicles comprise the use of synthetic polymers, such as the polyamino dicarboxylic acid-co-imide derivatives disclosed in U.S.

WO 94/28939 2 16 4 4 l 0 ~~US94~05965 Patent No. 5,190,982 or the crosslinkable synthetic polymers such ag pvlyphoaphazinea de9cribes in U.8_ Patent No. S,I49,543, Another delivery vehicle may comprise a liposome. In addition to the delivery vehicles described, it is understood that any delivery vehicle designed to make hydrophobic compounds, whether they are therapeutic or diagnostic compounds, administrable to a patient is also cvntemplated_ The emulsions of the present invention, whether or not they are incorporated into a delivery vehicle, will generally have a size below 8.0 ~C, and preferably below 5.0 ~,c. It is additionally anticipated that microemulsions can be prepared according tv the pre9ent IS invention with a size below I.0 ~.
~rtu~u~rtrt~ t An emulsion useful for stabilizing the gas-forming chemical wag made by mixing the following components together by rotating under vacuum_ Glycerol,Trioleate (triolein) 1.25 g 1.2-dioleoyl-glycero-3-phosphvcholine 15 (20 mg/ml in chlvrofozm) cholesterol 0.05 g a-tocopherol 0.012 g Any remaining solvent was removed by drying under high vacuum at room temperature (20-2S°C)_ After 16 hours, 1.58 g of glycerol (1.26 g/ml) and 0.2 g perfluoropentane were added. Then, 9.6 m1 of water were added slowly while mixing at 10,000 rpm in a POLXTRON~
PT3000 (Hrin.k~nan, We9tbury, New York) for 2 minutes at 0°C. The resultant emu181on was further homogeni2ed for 3 minutes at 30,000 rpm.
t,t~., WO 94128939 216 4 410 PCTIUS94l05965 The ultrasonic imaging characteristics of the emulsion of Example 1 where studied using an HP SONOS 100 Ultrasound Imaging system (Hewlett-Packard, Palo Alto, California) with a 5 IrBiz transducer (focal zone = 3.5 cm) in sector mode to detect the scattering capability of the sample solution_ The compression was adjusted to obtain the greatest dynamic range possible, i.e. 60 d8. The time gain compensation control of the ultrasound system to was adjusted until the image sector being imaged ie judged visually to be optimal.
The imaging sequence was started by optimizing the instrument as described in 1.0 L of water at 37°C at transmit power. A 1.0 ml sample was then injected into the Water. Thereafter, every 2 minutes the transmit power was adjusted upwardB to 10, 20, 30, ~0, 50, 60, 70, 80, 90 and 99ic. The entire sequence of images was recorded on videotape (attached to the ultrasound system) for storage and analysis.
To prepare quantitative results of this experiment, videodensitometry analysis was performed.
Selected video frames stored on the videotape were digitized using an Apple Macintosh zh~computer equipped with a Data Translation QuickCapture~frame grabber board.
These frames were analyzed using CineProbe~ version 2.0 (Molecular Hioeyatems, San Diego, California) image processing software. A Region of Interest (ROI) within the beaker was selected and the mean pixel intensity (video brigritneee) within the region was determined.
Each frame was then analyzed as to its mean videodenaicy within the region of interest. The videodensity of a water blank is subtracted and the resultant videodensity is expressed as Video Hrightness yr Normalized Video Brightness when the initial value is set to 100 for comparison.
*Trade-mark WO 94!7,.8939 216 4 410 PCT/US9~110596s An ALHUNEX~ (MoleCUlar_Biasyatema, Ban Diego, California) (micrebubbles surrounded by a protein shell prepared as described in U.S. Patent Noa. 4,572,203;
4,718,433; 4,744,958; 4,844,882 and 4,957,656) control was also prepared and analyzed as described by injecting a 1.0 mL sample of ALHUNEXm (Molecular Hioeystems, San Diego, California) into 1.0 liter of 37°C water_ The results of this experiment are depicted in Figure lA and 1H. Due to the unchanging number of l0 microbubbles present in the ALBUNEX~ (Molecular Hiosystems, San. Diego, California) sample, there would be expected to be a linear relationship between transmit power and video brightness. This linear relationship is depicted in Pigure lA. In.comparison, using the emulsion of Example 1, there would be the expectation of a bilinear or step Lunction between video brightness and transmit power which would be due to some threshold energy of cavitation for micrvbubblea to be formed upon exposure to ultrasonic energy Such a relationship was observed, and these results are depicted in Figure IH.
The following components were added together and homogenized in the POLYTROtHm (Hrinkman, Westbury, New York) at 0°C for 3 minutes at 10,000 rpm while slowly adding 10 ml ultrapure water:
Trialein 0.6 g Glycerol 1.57 g Lecithin 0.6 g Pertluoropentane 1.5 g These components were further homogenized for an additional 2 minutes at 30,000 rpm to produce a milky white emulei.an. This emulsion was filtered successively thxough a 5 P. and 1.2 ~ filter. The particle size was determined in a Nicomp 770*(Particle Sizing Systems, *Trade-mark WO 94/28939 216 4 410 pCT/LTS94/05965 Santa Barbara, California) to be 95% less than 3.8 ~,. It was stable (no appreciable phase separation or particle size increase) for several days at 4°C. When imaged as described in Example 2, this emulsion demonstrated microbubble formation above 40% transmit power as observed in the ultrasonic image.

The following components were added together and homogenized in the POLYTRON~ (Brinkman, Westbury, New York) at 0°C for 3 minutes at 10,000 while slowly adding ml water:
Triolein 1.0 g Glycerol 1.0 g 15 a-Tocopherol 0.02 g PLURONIC~ F-68 0.2 g Gas-forming Chemical 1.5 g of one of the following:
Emulsion A: FCC13 (Fluorotrichloromethane) 20 Emulsion H: Br2F2C (Dibromodifluoromethane) Emulsion C: TMS (Tetramethylsilane) Emulsion D: 2-Methyl butane (Isopentane) The above emulsions were filtered through a 1.2 ~, filter and the particle sizes were determined as described in Example 4 to be:
A 95% less than 2.97 H 95% less than 4.02 C 95% less than 2.18 D 95% less than 2.99 ~C

The following components were added together and homogenized in the POLYTRON~ (Brinkman, Westbury, New York) at 0°C for 5 minutes at 10,000 rpm while slowly adding 20 ml water:

WO 94/28939 216 4 410 pCT/US94/05965 Triolein 1.0 g Glycerol 3.0 g a-Tocopherol 0.02 g Lecithin 1.0 g Perfluoropentane 1.0 g The emulsions were further homogenized for 3 minutes at 20,000 rpm and filtered successively through a 5 ~, and 1.2 ~, filter. The ultrasonic imaging characteristics of the emulsion were studied as described in Example 2 and exhibited microbubble formation above 40% transmit power as observed in the ultrasonic image.

To further study the effects of ultrasonic energy on the production of microbubbles, the emulsion of Example 5 (perfluoropentane) was imaged in two separate experiments either continually or in 30 second intervals.
For each experiment, a 1.0 ml sample of the emulsion was added to 1.0 liter of water at 37°C. In the first experiment, ultrasonic imaging as described in Example 2 was carried out at 99% transmit power continuously for 30 minutes. In the second experiment, the ultrasonic imaging was carried out for 30 second durations once every 5 minutes (intermittent imaging). Image brightness was quantified as described in Example 2 and the results are depicted in Figure 2. These results demonstrate that with continuous ultrasonic energy, due to the constant production of microbubbles and depletion of the bubble-forming capability of the emulsion, image brightness was significantly diminished at the end of 30 minutes. In comparison, with intermittent imaging which exposed the emulsions to only one-tenth the energy as compared to constant imaging (30 seconds every 5 minutes), the microbubble-forming capability of the emulsion persisted WO 94/28~'" 2 ~ 6 4 410 PCT/US94/05965 and a substantial amount of microbubbles continued to be produced even after 30 minutes.

An alternative emulsion formulation comprises a viscosifier, a stabilizer which is amphipathic and a gas-forming chemical formed by mixing together the following components in a final volume of 50 mL water:
Viscosifier: Stabilizer:
Emulsion A PLURONIC° F-68 Sucrose (8.6 g) (0.5 g) Emulsion B Sodium dodecyl- Sucrose (8.6 g) sulfate (1.44 g) Emulsion C PLURONIC° F-68 Lactose (9.0 g) (0.5 g) Emulsion D Sodium dodecyl- Lactose (9.0 g) sulfate (1.44 g) The solutions from above were filtered through a 0.2 ~, filter. A 10 mL aliquot of each of the above were mixed with 0.168 mL of perfluoropentane in the POLYTRON° (Brinkman, Westbury, New York) at 0°C for 1 to 3 minutes at 10,000 to 20,000 rpm and then for an additional 5 minutes at 20,000 rpm. Each of these four emulsions demonstrated microbubble formation as observed in the ultrasonic image above 40% transmit power when studied as described in Example 2.
To study the effects on these emulsions of continuous versus intermittent exposure to ultrasonic energy, a 1.0 mL sample of Emulsion C was placed in 1.0 liter of degassed water at 37°C. This solution was ultrasonically imaged either continuously or in intervals as described in Example 6. The results are depicted in Figure 3.

SYNTHESIS OF NONAFLUORO-t-BUTYL METHANE C4F9CH3 Starting materials (methyl iodide and cesium fluoride) were obtained from Aldrich Chemical Company and perfluoroisobutylene gas was obtained from Flura Corporation. Nuclear magnetic resonance spectra were obtained using a 200 MHz instrument tuned for determination of proton (1H) or fluorine (19F) resonances.
In a flask equipped with a gas inlet, mechanical stirrer and a dry ice condenser was placed a suspension of dry cesium fluoride (42.5 g, 0.279 mol) in diglyme (200 mL). Perfluoroisobutylene gas (55.5 g, 0.278 mol) was bubbled in. The gas reacted rapidly with cesium fluoride and a yellow solution resulted. The mixture was stirred for 30 minutes and then methyl iodide (38.5 g, 0.271 mol) was added dropwise. The reaction was slightly exothermic and the cesium iodide separated out.
The mixture was stirred for 3 hours and was allowed to stand overnight. A cold solution (2M, sodium chloride, 500 mL) was added to the mixture with cooling (5°C) for minutes. Sodium iodide and most of the diglyme solvent dissolved in the aqueous phase which was then decanted off from the solid giving a crude yield of 45 g 25 ( 40%). Distillation of the compound sublimed at head temperature 35-39°C and bath temperature not exceeding 50-55°C. The product was collected in a receiver cooled to -30°C with dry ice and ethanol. The proton 1H NMR
spectrum of its CDC13 solution showed a single resonance 30 relative to TMS; 1.65 (s, 3H, CH3) ppm (see Figure 4) and the 19F spectrum, in the same solvent, showed also one single resonance at -69.99 (s, 9F) ppm relative to CDC13 (see Figure 5).
NONAFLUORO-t-BUTYL METHANE C4F9CH3 is shown according to either of the following chemical formulas:

F
I
F-C - F
CFs F F
I I
F-C C C -F
FsC - C - CFs I I
F ~ F
CHs H-C.-H
I
H

The following components were mixed together and homogenized in the POLYTRON~ (Hrinkman, Westbury, New York) at 0°C for 3 minutes at 10,000 rpm while slowly adding 10 mL ultrapure water.
Triolein 1.01 g Glycerol 1.05 g cx-Tocopherol 0.02 g PLURONIC~ F-68 0.099 g C4F9CH3 0.780 g The resultant emulsion was filtered through a 5 ~. filter. The particle size was determined as described in Example 4 to be less than 4.30 microns.
The ultrasonic imaging characteristics of the 2'S emulsion were studied as described in Example 2. The formation of gas bubbles was observed even at low transmit power (<25%) settings which became brighter as the transrnit power was slowly increased to 99%.
Also for comparison a control experiment without C4F9CH3 was conducted by mixing the following components:
Triolein 1.01 g Glycerol 1.05 g a-Tocopherol 0.021 g PLURONIC° F-68 0.204 g I . ..._. . __ v VYO 94IZ8939 216 4 41 Q ~CT~US9di05965 The emulsion was prepared as described above.
In contrast to the pxevioua ultrasound imaging experiment, micropubble formation was not observed even at 99~ transmit power.
F~BM~E .10 The Following components were added together and homogenized in the P4LYTRON~ (Brinlanan, Westbury, New York) at 0°C for 2 minutes at 10,000 rpm while slowly adding 10 ml water:
Triolein 1_0 g Glycerol I.0 g a-Tocvpherol 0.03 g PLURONIC~ F-68 0_1 g 1$ Isvpentane 0.15 g n-Pentane 0_85 g The emulsion was further homogenized for 6 minutes at 30,000 rpm and filtered through a 1.2 filter. The ultrasonic imaging characteristics of the emulsion were studied ae described in Example 6 and a higher level v~ video brightness was observed with intermittent imaging than with continuous imaging.
EMULSION-CONTAININC3 ALBUMIN MICRaPARTZCLE
The emulsion of the present invention can be encapsulated into a delivery vehicle comprising a multi-chamber albumin matrix as follows:
A primary emulsion is prepared by first dissolving 2.0 g human aenim albumin in 20.0 ml buffer (0.45 N Na2C03, pH 9.8) and then adding I.0 g perfluoropentane. This mixture ie emulsified in an osteriaer*at high speed for 10 rninutee.
*Trade-mark tV0 94/x.8939 216 4 4 i 0 ~r/US94/DSg65 A double emulsion is then prepared by adding 100 ml Chloroform:Cyclohexane (1:4 v/v) with L0~ (v/v) sorbitan trioleate with continued mixing for 10 minutes.
The albumin is croeslinked by adding, while continuing to mix, an additional 100 m3 Chlorofozm:
Cyclohexane (1:4 v/v) containing 2.5 g terephthaloyl chloride and continuing to mix for an additional 30 minutes. The reaction is quenched with X00 mL of cyclohexane containing Sg polysorbate and 10~
(v/v) ethanolamine. The micrvcapsules are washed 3 times with cyclohexane:ethanol (i:i v/v), followed by 2 wa$hea in S~ polyavrbate-95~ ethanol, 2 washes in 95~ ethanol and a washes in water. The microparticles are then resuapended in normal saline and comprise multi-chambered vesicles containing an inner emulsiLied matrix of perfluoropentane.
Although the invention has been described primarily in connection with special and preferred embodiments, it will be u~derstvod that it is capable of modiLication without departing from the scope of the invention. The following claims are intended to cover all variations, uses or adaptations of the invention, following, in general, the principles thereof and including such departures from the present disclosure as come within known or customary practice in the field to which the invention pertains, or as are obvious to persons skilled in the art.

B

Claims

-21

1. An emulsion for use as an ultrasonic imaging agent comprising at least one water-insoluble gas-forming chemical and at least one stabilizer, said emulsion being capable of forming microbubbles of gas upon application of ultrasonic energy.

2. The emulsion of claim 1 wherein said stabilizer is a hydrophobic compound.

3. The emulsion of claim 2 wherein said emulsion includes a surface active agent.

4. The emulsion of claim 1 wherein said stabilizer is amphipathic.

5. The emulsion of claim 4 wherein said stabilizer is wherein the average value of n=75 and the average value of p=30 such that the average molecular weight of said stabilizer is 8350 daltons.

6. The emulsion of claim 1 wherein said emulsion includes a viscosifier.

7. The emulsion of claim 1 wherein said emulsion includes antioxidants.

8. The emulsion of claim 1 wherein said emulsion includes a component that imparts tissue specificity to said emulsion.

9. The emulsion of claim 1 wherein said emulsion includes a component that imparts organ specificity to said emulsion.

10. An ultrasound imaging agent comprising the emulsion of claim 1 and a delivery vehicle.

11. The ultrasound imaging agent of claim 10 wherein said delivery vehicle is selected from the group consisting of:
a natural polymer matrix, a synthetic polymer matrix and a liposome.

12. The ultrasound imaging agent of claim 11 wherein said natural polymer matrix is an albumin matrix.

13. The emulsion of claim 1 wherein said water-insoluble gas-forming chemical is nonafluoro-t-butylmethane.

14. The emulsion of claim 1 wherein said water-insoluble gas-forming chemical is perfluoropentane and said stabilizer is lecithin.

15. A method to enhance the contrast of tissue and organs in an ultrasonic image comprising. (a) injecting the emulsion or imaging agent of claims 1-14 into a patient; (b) applying a sufficient amount of ultrasonic energy to volatilize said chemicals to release microbubbles; and (c) detecting an ultrasonic image.

16. The use of an emulsion or imaging agent according to any of claims 1 to 14 to enhance the contrast of tissues and organs in an ultrasonic image, which forms microbubbles of gas on application of sufficient ultrasonic energy.