WO1999042138A1

WO1999042138A1 - The use of bjerknes forces to manipulate contrast agents

Info

Publication number: WO1999042138A1
Application number: PCT/US1999/003358
Authority: WO
Inventors: Katherine W. Ferrara; Paul Dayton; Allen Goode
Original assignee: University Of Virginia Patent Foundation
Priority date: 1998-02-19
Filing date: 1999-02-17
Publication date: 1999-08-26
Also published as: AU2683899A

Abstract

Microbubbles used for contrast-assisted imaging can be manipulated by taking advantage of both primary and secondary Bjerknes forces. The primary Bjerknes force allows microbubbles to be pushed away from the inducing pressure of the ultrasound. This can be used to push microbubbles against a desired target such as a tissue wall to which they can bind. Advantage is made of the secondary Bjerknes forces which results in microbubbles forming aggregates. The positioning of these aggregates can be manipulated and can result in an improved ultrasound image. Manipulation of drug-filled microbubbles using the primary and secondary forces, especially in combination with a pulse of ultrasound to destroy the microbubbles, allows for targeted drug delivery.

Description

TITLE OF THE INVENTION THE USE OF BJERKNES FORCES TO MANIPULATE CONTRAST AGENTS

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to Serial No. 607075,147, filed 19 February 1998, to which priority is claimed and which is incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to the field of ultrasound contrast agents.

DESCRIPTION OF THE BACKGROUND ART

Physicians and physiologists have long recognized the significance of local perfusion in the assessment of wound healing, diabetes, viability of transplanted organs and reattached limbs, the diagnosis of cancerous lesions and the assessment of myocardial function, however to date there has been no method that can provide a direct assessment. It has been clear that fundamental changes in tissue perfusion are involved in disease progression, or in some cases in the onset of a disease state. Many imaging techniques have been developed to indirectly detect ischemia, for example ultrasonic and magnetic resonance schemes that evaluate the extent and quality of the regional motion of the beating heart. An opportunity is presented by contrast-assisted ultrasonic imaging with second and third generation agents and new imaging strategies, since the echoes from blood are now far stronger, and local bubble destruction may allow us to map microvascular transit time on a far smaller scale. In addition, we may have the ability to remotely manipulate agents within the body using radiation force. Contrast-assisted imaging is also less expensive than alternative vascular imaging techniques.

Contrast-assisted imaging methods have failed to produce the expected results for reasons that include the lack of fundamental understanding of the interaction between ultrasound and the microbubbles, and difficulties associated with some of the artifacts that can arise. In cardiology, the use of the returned signal amplitude has been particularly difficult since the contrast agent can also attenuate the signal and therefore make interpretation of amplitude fluctuations difficult.

Cancer currently accounts for 15.8% of the deaths within the United States. Although malignant breast masses can often be differentiated using ultrasound due to an increase in the 2 attenuation and decrease in the backscattered echo, these changes are absent in approximately 10% of malignant masses.

More than 60 million people in the United States have some form of cardiovascular disease (CVD), with CVD playing a major role in more than 954,000 deaths annually. This is more than 42 percent of the deaths in the United States each year. Ultrasound remains the premier imaging modality for the detection and valuation of CVD, however the current inability of ultrasound to map flow within the coronary arteries, or to map myocardial perfusion is a significant limitation. Ultrasound contrast imaging shows the potential to address these current limitations but needs improved signal processing with definitive detection of bubbles to improve clinical acceptance.

While ultrasonic contrast agents were first considered in 1968, the development of agents with extended persistence provides exciting new opportunities to image the micro vasculature. In the past few years, microbubbles have been developed that can survive within the circulation for extended periods. New agents include high molecular weight gases that have a low diffusion constant and have been incorporated into lipid or albumin shells. Agents under development include substances that are intended to adhere to particular tissues and improve the detection of plaque or tumor vasculature.

Also, while contrast agents have been used to increase the amplitude of scatter since 1970, techniques to differentiate bubble and tissue echoes have been proposed very recently. The scattering and attenuation properties of certain contrast agents have been studied. Experiments have shown the nonlinear increase in scattered echoes and the change in harmonic signal content with transmitted pressure. Use of harmonic imaging in vitro has been demonstrated and has provided evidence that cross correlation can be used to track microbubbles. Use of 5 MHz transmission and 2.5 MHz subharmonic reception has been evaluated, demonstrating that subharmonic peaks from the contrast agent Albunex^® can be detected in vitro. Studies of attenuation and transmission have shown the high attenuation of acoustic contrast agents, a significant factor in cardiac imaging. New agents have been presented in many recent conference abstracts.

Clinical evaluation of current contrast-assisted imaging techniques has shown that artifacts can reduce the effectiveness of estimations of myocardial perfusion. Specifically, attenuation and rib artifacts are problematic for operating modes which base the perfusion 3 estimate on the video intensity of the returned signal to map perfusion. Concerns about safety have been reported, particularly for high intensities and low frequencies (<2 MHz). There remains the need in the art for improved ultrasound techniques.

The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method of using ultrasound to direct a contrast agent to a particular site of interest is disclosed. The method causes the contrast agent to form aggregates and to adhere to the targeted tissue. The method uses a high pulse repetition frequency and a low instantaneous intensity.

Both primary and secondary radiation force can be shown to have an effect on the flow of microbubbles through a small vessel during insonation, depending on the operating parameters. Primary radiation force can displace a streamline of microbubbles across the entire radius of a tube. At 2-5 MHz, a low-power, high pulse repetition frequency (prf) ultrasound system pushes the bubbles most effectively. At high frequencies, the microbubbles are more resistant to breaking and can be pushed with either high prf or high power. Secondary radiation force produces a reversible attraction and aggregation of microspheres with a significant attraction. The microspheres accelerate towards each other as suggested by the theory of the secondary Bjerknes force, which predicts an inverse square relationship between force and separation distance.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram of an experimental setup used to study primary and secondary

Bjerknes forces on microbubbles.

Figure 2 shows the orientation of a transducer and vessel as well as the forces on the contrast agent.

Figure 3 is an optical image of contrast agent displacement. Figure 4 shows the displacement of contrast streamline as a function of pulse repetition frequency at 120 kPa (solid line) and at 150 kPa (dashed line) 4

Figure 5 illustrates the contrast displacement for 38 MHz transducer center frequency. Figure 6 illustrates the predicted velocity variation.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for controlling microbubbles in vivo and in vitro by use of primary and secondary Bjerknes forces associated with ultrasound. These forces can be used to direct microbubbles to a desired target location. It has been discovered that the secondary Bjerknes forces cause the microbubbles to form aggregates at a desired target location. In accordance with one embodiment of the invention, microbubbles are manipulated into forming aggregates at a desired target and these can be used for contrast imaging directly. The aggregates can be forced against a tissue wall of interest and will result in a good contrast- assisted image being obtained.

In accordance with a second embodiment of the invention, microbubbles are filled with a drug or drugs or other desired compound such a vector for gene therapy, the microbubbles are manipulated to bind to a specific location or to form aggregates binding to specified location.

The drugs or other enclosed compounds are then released by use of a high intensity pulse thereby resulting in targeted drug delivery.

In accordance with a third embodiment of the invention, two or more chemicals or drugs are encapsulated individually in microbubbles and with ultrasound the microbubbles are targeted to a desired location then destroyed with a pulse of ultrasound thereby releasing the chemicals or drugs which react to form a third compound or drug which has a desired activity.

Primary and secondary Bjerknes forces result from pressure gradients in the incident and scattered ultrasonic fields. These forces and their dependence on experimental parameters are described. Both primary and secondary Bjerknes forces are shown to have a significant effect on the flow of microbubbles through a small vessel during insonation. The primary Bjerlcnes force produces displacement of microspheres across the entire vessel radius for a small transmitted acoustic power. The secondary Bjerknes force produces a reversible attraction and aggregation of microspheres with a significant attraction over a distance of approximately 100 microns, and with the force proportional to the square of the inverse separation distance. The relative velocity of approach thus depends on the inverse of the separation distance with the magnitude of the velocity accurately predicted by the theory of the secondary Bjerknes force. 5

We show that this force is sufficient to produce aggregates that remain intact for a physiologically appropriate shear rate. Brief interruption of the acoustic power allows an immediate disruption of the aggregate.

We show that a system with a high pulse repetition frequency and a low instantaneous intensity (particularly at a frequency far above the resonant frequency of the bubbles) allows a stream of contrast material to be delivered to the vessel wall. Specifically, a pulse repetition rate above 1 kHz, and an instantaneous intensity below 100 kPa are required for this application.

An ultrasound system is described herein which combined with a contrast agent can direct the contrast agent to a particular site of interest and will cause the contrast agent to aggregate and to adhere to the targeted tissue site. A diagram of such a system is shown in Figure 1. The system comprises transmission of a high prf, low intensity ultrasound signal, directed along a single line of sight. A frequency near the resonance frequency of the bubbles is chosen in order to produce an attractive secondary Bjerknes force that locates bubbles in a particular site. Transmission away from the resonance frequency can be interleaved to improve the effectiveness of the primary radiation force (a smaller percentage will be broken). The line-of-sight is specifically chosen to allow primary radiation force to direct the contrast agent to the targeted tissue. The system further comprises use of a contrast agent with a high compressibility (a gas filled agent is currently used). The capacity for adhesion to a tissue component such as plaque is also desirable. Alternatively, the system comprises the use of contrast agents containing two drugs or chemicals that are mixed while aggregated producing a third substance used in treatment. The system further comprises a final application of a high intensity, low prf pulse to deliver the drug or chemical contained in the microsphere or attached to its shell. High intensity ultrasound will also cause the enclosed gas to leave the shell. The transmitted ultrasonic field and pulsing strategy are optimized for control and destruction at a known time. Ultrasound contrast agents are used to enhance the intensity of scattered echoes from the blood and are currently useful in the assessment of tissue perfusion. Such agents consist of gas encapsulated by an albumin or a phospholipid shell. Ultrasonic interrogation is performed after a small quantity of microspheres is injected into an artery or vein. The nonlinear properties of wave motion in continuous media produce a primary acoustic radiation force experienced by objects placed within the field. It has been shown that this primary radiation force can levitate bubbles placed within a stationary field. Acoustic manipulation of gas filled microspheres using 6 primary radiation force has been proposed as a mechanism for improved image contrast or for localized drug delivery. We provide evidence to support the suggestion of Fowlkes (Fowlkes et al, 1993) and others who have recently postulated that primary radiation force can be used to manipulate these agents. In addition, we show that secondary radiation force (also known as the secondary

Bjerlcnes force) can play a role in the acoustic manipulation of such agents. Bjerknes postulated a secondary force between bubbles exposed to a stationary sound field (Bjerknes, 1906). The secondary Bjerknes force is an attractive force that produces a reversible aggregation of microbubbles, which immediately disperse in a flowing stream when the ultrasonic beam is removed. This aggregation is important since we observe that a high percentage of bubbles which persist at a moderate acoustic pressure are within an aggregate.

These primary and secondary radiation forces take on new importance when evaluating the behavior of acoustic contrast agents. Through an evaluation of the microscopic behavior of contrast agents, we demonstrate that primary radiation force does not adequately predict the motion or concentration of these agents. As predicted by primary radiation force, they can be concentrated at a predictable distance form the transducer with the agents moving from a higher to a lower pressure. As predicted by the secondary Bjerknes force, their velocity increases as they approach one another.

The following sets out details of our experimental system and also presents evidence of primary and secondary radiation force as shown by several experiments. First, the effect of primary radiation force through estimation of the displacement of a streamline of microspheres is demonstrated. Second, the velocity of two aggregates as they approach one another in a direction perpendicular to the transducer axis is evaluated.

Theory of Primary Radiation Force

Primary force results from a pressure gradient created by nonlinear wave propagation; secondary radiation force results from a pressure minimum produced by energy dissipation near each resonant bubble. In either case, the magnitude of the radiation force experienced by a compressible sphere exposed to a pressure gradient can be evaluated from equation 1. The effect of primary radiation force on bubbles and compressible spheres has been considered by Leighton

(1994), Zheng and Apfel (1995), Lee and Wang (1993), and Crum (1975). Letting F indicate the 7 force, V(t) indicate the volume as a function of time and P_a(z,t) indicate the acoustic pressure as a function of space and time,

F=(V(t)VP_a(z,t))(l) where () indicates time average and V indicates spatial gradient.

Secondary Radiation Force: Attraction between Two Spheres Exposed to an Acoustic Field

The pulsation of bubbles exposed to an ultrasonic field produces a secondary scattered field with local pressure minima associated with the location of each bubble. The secondary Bjerknes force then predicts an attraction between bubbles as they are drawn to the local pressure inhomogeneity. The conditions required for the derivation of this force specify that each sphere and their separation must be much smaller than an acoustic wavelength, conditions that can be satisfied for contrast agents interrogated at medical ultrasound frequencies.

The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.

Example 1 Experimental Conditions In order to evaluate the microscopic behavior of this agent, we have developed a unique interface between ultrasound pulser/receivers, a flow phantom and a microscope. The flow phantom contains an optically transparent small (200 micron diameter) cellulose vessel suspended within a water bath. Flow through the small vessel is controlled by a syringe pump. The ultrasound transducer is coupled to the phantom through the water bath. A variable concentration of contrast agents is then pumped through the vessel. The output of the ultrasound scanner and the optical images observed through the microscope were recorded and analyzed. Transducer center frequencies of 2-38 MHz were used in the following experiments, with acoustic energy applied in a pulsed mode. Experiments in the 2-5 MHz transducer center frequency involved a Panametrics 5900 pulser/receiver. Energy was directed to a single line-of- sight at pulse repetition frequencies of 10 Hz to 10 kHz. The vessel was located at the focal 8 depth of the transducer. A diagram which shows the orientation of the transducer and vessel, as well as the forces on the contrast agent, is shown in Figure 2.

The contrast agents used in these studies were Albunex® and MP1950. Albunex® is a commercial product consisting of 5% sonicated human albumin. MP1950 is an experimental phospholipid agent and is prepared as follows: Phosphatidyl choline and surfactant were dispersed in physiological saline solution at 5 mg/mL each and then sonicated using an XL2020 probe-type sonicator. The microspheres were then created by higher intensity sonication while decafluorobutane gas (FC31-10) was bubbled through the aqueous lipid/surfactant dispersion. The mean microsphere size for Albunex® was 3-5 microns and 1.5 microns for MP1950.

Example 2 Primary and Secondary Radiation Forces Effects were observed that are accurately predicted by the Bjerknes model in several experiments. The primary radiation force offsets the streamline and the secondary radiation force produces a reversible aggregation of microbubbles.

A) Primary Radiation Force

Even with a rapidly flowing stream, a radiation force directed at ninety degrees will push a fraction of bubbles toward the far vessel wall. The effect of this force is shown in Figure 3. The transducer is located above the top edge of this figure, which corresponds to the side of the vessel. Flow is entering from the right side of the figure. The microscope objective is above the vessel (corresponding to above the page for this figure) with the focus adjusted to view the top of the vessel. Using a transmitted pressure of 130 kPa at a center frequency of 4 MHz and a prf of 1250 Hz with Albunex®, the contrast streamline is displaced across the vessel. The field of view in Figure 2 is approximately 200 by 800 microns. This image was taken with the beam center oriented at the left hand side of the vessel to show the increasing displacement corresponding with the beam focus. The transmitted pressure of 130 kPa corresponds to the minimum pressure increment available on the commercial ultrasound scanner used in these experiments. The displacement was then evaluated for transducer center frequencies between 2 and 38

MHz at different pulse repetition rates. Between 2 and 5 MHz, we observed that a low power 9 level with a high pulse repetition rate was able to displace the microsphere streamline to the edge of the tube. Using a higher power and low pulse repetition rate tended to break the bubbles before the streamline could be pushed.

Figure 4 shows the displacement of the streamline as a function of pulse repetition rates at 2 MHz and 120 kPa-150 kPa. At 200 kPa and higher, the bubbles were broken before they could be displaced even at the lowest prf of 100 Hz. The displacement does not increase at the higher transmitted power due to a change in the bubble distribution since some bubbles are broken at the higher power. Also, the displacement distance begins to taper off at higher displacements due to the buoyancy of the bubbles in the rounded tube. Figure 5 shows the displacement of an Albunex® streamline at a transducer center frequency of 38 MHz and different pulse repetition frequencies. At this frequency, the bubbles were not destroyed by the ultrasound and it was possible to measure displacements for a 1-20 kHz prf and 1-16 μJ of transducer excitation. In this system, a 1 μJ transducer excitation corresponds to 600 kPa. Thus, the agent can be displaced to the far wall of this small tube with 2 μJ excitation and 20 kHz prf, corresponding to a 13 mW/cm² spatial -peak temporal-averaged intensity.

B. Secondary Radiation Force

We show the effect of secondary radiation force through quantification of the velocity of approach of a pair of bubbles. We demonstrate that bubbles accelerate rapidly as their separation distance decreases below 40 microns. The transducer center frequency was 2.3 MHz, transmitted along a single line-of-sight.

Figure 6 shows three plots of velocity of approach vs. separation distance for pairs of microsphere aggregates. In each case, a small amount of MP 1950 was injected into a vessel and subjected to an ultrasound beam which caused the bubbles to attract each other. Successive image frames of two microsphere aggregates as they approached one another in a direction perpendicular to the beam axis were captured and analyzed. This experiment involved an acoustic pressure of 1.89 MPa, a flow of 3.53 mm/sec, and a concentration which provided fewer than 10 bubbles in the final aggregate. The resulting plots show the inverse square relationship trend of the velocity vs. separation distance between the aggregates. The solid line represents theoretical inverse square data. 10

Example 3 In Vivo Uses The primary and secondary Bjerknes forces are very useful with practical in vivo as well as in vitro uses. The primary Bjerknes force allows one to direct microbubbles to a specific site. This force can push microbubbles against a vessel or tissue wall. This can be combined with the secondary Bjerlcnes force to form aggregates which can be localized and pushed against vessel or tissue walls and result in a layer of aggregated microbubbles at specific targeted points. One advantage of the aggregates is that they attract other aggregates and they "sound" different from simple microbubbles and are therefore easy to monitor. The Bjerknes forces can be used to generate aggregates of microbubbles at a specific location and then the microbubbles can be destroyed at that location by changing the ultrasound to a high intensity. This is desirable for freeing drugs that were enclosed in the microbubbles at the specified location. One variation is to use a combination of two or more different sets of microbubbles which each contain at least one chemical. The chemicals from each set will be released and can combine with chemicals from the other set or sets of microbubbles to form a new chemical compound which is useful. Thus drugs can be kept in an essentially inert form while in the microbubbles and be released at the desired site of action by use of ultrasound. This allows very high local concentrations of drugs without high systemic concentrations, thereby improving drug efficacy without added adverse effects. The microbubbles can be preloaded with other desired compounds such as a vector for gene delivery. Destruction of microbubble aggregates at a specific location directs the vector to the desired target at a higher concentration thereby increasing the chance that the gene therapy will be successful.

While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims. 11

LIST OF REFERENCES Bjerknes VFK (1906). Fields of Force (Columbia University Press, New York). Crum L. (1975). J. Acoustical soc. Am. 57:1363-1370. Fowlkes JB, et al. (1993). J. Acoustical Soc. Am. 93:2348. Lee CP and Wang TG (1993). J. Acoustical Soc. Am. 93:1637-16540. Leighton TG (1994). The Acoustic Bubble (Academic Press). Zheng X and Apfel R (1995). J. Acoustical Soc. Am. 97:2218-2226.

Claims

12WHAT IS CLAIMED IS:

1. A method for producing in a liquid stream aggregates of microbubbles that remain intact wherein said method comprises subjecting said microbubbles to a high pulse repetition rate and a low intensity of ultrasound transmission.

2. The method of claim 1 wherein said high pulse repetition rate is above 1 kHz and wherein said low intensity is below 100 kPa.

3. The method of claim 1 wherein said method is performed with microbubbles which are in vivo.

4. The method of claim 1 wherein said ultrasound is directed along a single line of sight to form aggregates at a desired location.

5. The method of claim 1 wherein said microbubbles have a capacity for adhesion to a tissue component.

6. The method of claim 5 wherein said tissue component is plaque.

7. The method of claim 1 wherein said microbubbles comprise a chemical.

8. The method of claim 7 wherein said chemical is a drug or vector comprising a nucleic acid for gene therapy.

9. The method of claim 7 further comprising an application of a high intensity, low pulse repetition pulse of ultrasound.

10. The method of claim 9 wherein said application of a high intensity, low pulse repetition pulse of ultrasound causes release of said chemical from said microbubbles. 13

11. The method of claim 1 wherein two or more sets of microbubbles are present wherein each set of microbubbles comprises a chemical and wherein said chemical is different in each set of microbubbles.

12. The method of claim 11 wherein said chemical is a drug or vector comprising a nucleic acid for gene therapy.

13. The method of claim 12 further comprising an application of a high intensity, low pulse repetition pulse of ultrasound.

14. The method of claim 13 wherein said application of a high intensity, low pulse repetition pulse of ultrasound causes release of said chemical from said microbubbles.

15. The method of claim 1 wherein said microbubbles comprise a contrast agent with a high compressibility.

16. The method of claim 15 wherein said contrast agent is a gas filled agent.

17. A method of performing contrast-assisted imaging wherein said method comprises forming aggregates of microbubbles according to the method of claim 1.

18. A method of delivering a chemical to a targeted site in vivo by the method of claim 7.

19. The method of claim 1 wherein said ultrasound transmission is interleaved with a second ultrasound transmission away from a resonance frequency of said microbubbles.

20. A method of forcing microbubbles to adhere to a tissue component at a desired location in an organism said method comprising injecting microbubbles into said organism and subjecting said microbubbles to a high pulse repetition rate and a low intensity of ultrasound along a line of sight which forces said microbubbles to contact said tissue component. 14

21. The method of claim 20 wherein said method results in said microbubbles forming aggregates.

22. The method of claim 20 wherein said high pulse repetition rate is above 1 kHz and wherein said intensity is below 100 kPa.

23. The method of claim 20 wherein said ultrasound transmission is interleaved with a second ultrasound transmission away from a resonance frequency of said microbubbles.