U.S. patent application number 12/540338 was filed with the patent office on 2009-12-03 for method for ultrasound triggered drug delivery using hollow microbubbles with controlled fragility.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. Invention is credited to Stanley R. Conston, Thomas B. Ottoboni, E. Glenn Tickner, Ronald Yamamoto.
Application Number | 20090297567 12/540338 |
Document ID | / |
Family ID | 21845142 |
Filed Date | 2009-12-03 |
United States Patent
Application |
20090297567 |
Kind Code |
A1 |
Conston; Stanley R. ; et
al. |
December 3, 2009 |
Method For Ultrasound Triggered Drug Delivery Using Hollow
Microbubbles With Controlled Fragility
Abstract
A method is provided for site specific delivering therapeutic or
diagnostic agents to a region in a fluid-filled cavity, vessel or
tissue using an agent-loaded microbubble population. The population
has controlled fragility characterized by a uniform wall thickness
to diameter ratio which defines the discrete threshold intensity
value of ultrasonic power where microbubble rupture occurs in the
population. The location of the microbubble population may be
monitored by ultrasound to determine its presence at the region
prior to application of the ultrasonic power to rupture to
microbubbles.
Inventors: |
Conston; Stanley R.; (San
Carlos, CA) ; Yamamoto; Ronald; (San Francisco,
CA) ; Ottoboni; Thomas B.; (Belmont, CA) ;
Tickner; E. Glenn; (Watsonville, CA) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
Suite 350, 101 Howard Street
San Francisco
CA
94105
US
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
|
Family ID: |
21845142 |
Appl. No.: |
12/540338 |
Filed: |
August 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11108158 |
Apr 14, 2005 |
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12540338 |
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10028738 |
Oct 22, 2001 |
6896659 |
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11108158 |
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09638167 |
Aug 11, 2000 |
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10028738 |
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09389868 |
Sep 2, 1999 |
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09638167 |
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09245781 |
Feb 5, 1999 |
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09389868 |
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09020046 |
Feb 6, 1998 |
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09245781 |
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Current U.S.
Class: |
424/400 ; 514/46;
514/47 |
Current CPC
Class: |
A61K 9/5073 20130101;
A61M 37/0092 20130101; A61B 5/4839 20130101; A61K 9/0009 20130101;
A61K 49/223 20130101; A61K 47/6925 20170801; A61B 8/481 20130101;
A61K 41/0028 20130101 |
Class at
Publication: |
424/400 ; 514/46;
514/47 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 31/7076 20060101 A61K031/7076; A61P 9/00 20060101
A61P009/00 |
Claims
1. A composition comprising a microbubble population, wherein said
microbubble population comprises a plurality of microbubbles,
wherein each of said microbubbles comprise the same wall thickness
to diameter ratio.
2. The composition of claim 1, wherein said ratio ranges between
approximately 0.016-0.049.
3. The composition of claim 1, wherein said wall thickness ranges
between approximately 25 nm to 1000 nm.
4. The composition of claim 1, wherein said diameter ranges between
approximately 1 to 10 microns.
5. The composition of claim 1, wherein each of said microbubbles
comprise a single layer shell.
6. The composition of claim 1, wherein each of said microbubbles
comprise a bi-layer shell.
7. The composition of claim 1, wherein each of said microbubbles
comprise a biodegradable polymer.
8. The composition of claim 7, wherein said biodegradable polymer
is biocompatible.
9. The composition of claim 6, wherein said bi-layer shell
comprises an outer layer of an amphiphilic material.
10. The composition of claim 9, wherein said amphiphilic material
comprises a protein.
11. The composition of claim 10, wherein said protein is selected
from the group consisting of collagen, gelatin, albumin, and
globulin.
12. The composition of claim 7, wherein said biodegradable polymer
is selected from the group consisting of polycaprolactone,
polylactide, polyglycolide, polyhydroxyvalerate,
polyhydroxybutyrate, or copolymers thereof.
13. The composition of claim 1, wherein each of said microbubbles
further comprise at least one therapeutic agent.
14. The composition of claim 13, wherein said therapeutic agent
comprises a cardiovascular drug.
15. The composition of claim 13, wherein said therapeutic agent
comprises an anti-restenosis drug.
16. The composition of claim 14, wherein said cardiovascular drug
is selected from the group consisting of a fibrinolytic agent,
vasodilator, calcium channel blocker, angiogenesis agent,
antiplatelet agent, anti-white cell agent, endocardium acting
agent, free radical scavenging agent, or anti-restenosis agent.
17. The composition of claim 13, wherein said therapeutic agent is
selected from the group consisting of adeno sine, adeno sine
monophosphate, adeno sine diphosphate, adenosine triphosphate or
chemical derivatives of adenosine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of application Ser. No. 10/028,738,
filed Oct. 22, 2001, which is a continuation-in-part of application
Ser. No. 09/638,167, filed Aug. 11, 2000, of application Ser. No.
09/389,868, filed Sep. 2, 1999, and of application Ser. No.
09/245,781, filed Feb. 5, 1999, which in turn is a
continuation-in-part of application Ser. No. 09/020,046, filed Feb.
6, 1998, now abandoned, the disclosures of which are all
incorporated by reference herein in their entirety.
FIELD OF INVENTION
[0002] The current invention relates to a method of delivering a
therapeutic agent to a localized region within a subject using
ultrasound to trigger the release of the agent from hollow
microbubbles having a specified set of mechanical properties.
BACKGROUND OF THE INVENTION
[0003] Ultrasound imaging has a wide application in the field of
medical practice. Ultrasonic diagnostics refers to the imaging of a
region of the human or animal patient using an ultrasound
transducer to generate and receive ultrasound waves. Typically, the
transducer is placed on the patient's body over the region to be
imaged and high frequency sound waves are generated by the
transducer and directed at the region. The transducer receives
reflected ultrasonic waves from the region and converts the
received waves into electrical signals from which an image is
generated. Due to the extremely high acoustic reflectivity of
gases, contrast agents comprised of gas bubbles with and without
encapsulating shells are used to improve the quality of ultrasound
images by highlighting the blood pool and the vascular perfusion of
organs within the body.
[0004] The use of ultrasound contrast agents serving also as drug
carriers has been described for gas-filled liposomes in U.S. Pat.
No. 5,580,575. A quantity of liposomes containing drug is
administered into the circulatory system of a patient and monitored
using ultrasonic energy at diagnostic levels until the presence of
the liposomes are detected in the region of interest. Ultrasonic
energy is then applied to the region that is sufficient to rupture
the liposomes to release drugs locally for therapeutic purposes.
The ultrasonic energy is described in U.S. Pat. No. 5,558,082 to be
applied by a transducer that simultaneously applies diagnostic and
therapeutic ultrasonic waves from therapeutic transducer elements
located centrally to the diagnostic transducer elements.
[0005] The use of gas-filled microcapsules to control the delivery
of drugs to a region of the body has also been described in U.S.
Pat. No. 5,190,766 in which the acoustic resonance frequency of the
drug carrier is measured in the region in which the drug is to be
released and then the region is irradiated with the appropriate
sound wave to control the release of drug. Separate ultrasound
transducers are described for the imaging and triggering of drug
release in the target region.
[0006] Exemplary contrast agents include, for example, stabilized
microbubbles, sonicated albumin, gas-fulled microspheres,
gas-filled liposomes, and gas-forming emulsions. A variety of
methods have been developed for their manufacture. These methods
usually involve spray drying, emulsion, or interfacial
polymerization techniques. Typically, the result is a microbubble
population having a range of diameters with either a fixed or an
arbitrarily variable wall thickness. An ultrasonic contrast agent
produced by one methodology, for example, may contain microbubbles
where each has a shell wall of the same thickness regardless of its
diameter. Alternatively, a different method of production may
result in a microbubble population with wall thickness varying even
between those microbubbles having the same diameter.
[0007] Conceptually, for an ultrasound contrast agent to be used as
a carrier for therapeutics, the agent would typically be, through
processing, internally loaded with a drug. The treated microbubbles
are then injected intravenously and allowed to circulate
systemically. An ultrasound signal of sufficient energy to rupture
the drug-containing microbubbles is applied to a region where the
delivery of the drug is desired. The insonating beam destroys the
microbubbles and thus releases its payload.
[0008] An ultrasound contrast agent having a fuxed or an
arbitrarily variable wall thickness may not by optimal as a carrier
of therapeutic agent. A microbubble population having an arbitrary
wall thickness could result in the drug being released prematurely
or not at all. Those with thinner more fragile walls may rupture
from hydrostatic pressure before reaching the site. Those with
thicker more durable walls may not rupture at all. A microbubble
population with a fixed wall thickness would similarly be
unsuitable. While the strength of an encapsulated microbubble is a
function of the thickness of its wall, it is also a function of its
diameter. Thus, a relatively smaller microbubble would show more
resistance to hydrostatic and acoustic pressures than would a
relatively larger bubble having the same wall thickness.
[0009] A drug-containing ultrasound contrast agent having a
controlled fragility would therefore represent an improvement to
the state of the art. For purposes herein, the term "controlled
fragility" is taken to describe a microbubble population having the
characteristic of being rupturable only when exposed to acoustic
energy equal to or greater than a predetermined intensity. That is,
below this acoustic intensity threshold, substantially all the
microbubbles remain intact while above the acoustic intensity
threshold the microbubbles rupture. While in the unruptured state,
bubble agents can still be seen ultrasonically in the larger blood
pool so that the sonographer can position and focus the scanner
transducer on the region of interest prior to increasing ultrasound
intensity to initiate agent rupture and concomitant delivery of
drug. Thus, the agent can be turned-on or turned-off by controlling
the intensity of the insonating signal.
SUMMARY OF INVENTION
[0010] The present invention provides a method of delivering
therapeutic or diagnostic agents to a region of interest within a
subject comprising the steps of introducing an agentloaded
microbubble population having a controlled fragility into the
bloodstream of the subject, directing at the region an insonating
beam of ultrasound energy at a power intensity sufficient to induce
rupture of the microbubbles and subsequent release of the active
agent into the region to achieve therapeutic or diagnostic effect,
and maintaining the power intensity until at least a substantial
number of microbubbles are ruptured. The microbubble population has
a controlled fragility characterized by a uniform wall thickness to
diameter ratio that defines a discrete threshold power intensity
value of ultrasonic energy where microbubble rupture in the
population occurs. An advantage of the invention is that the
microbubbles have specifuc and predetermined acoustic properties
such that the specific ultrasound power intensity required to
rupture the microbubbles can be predetermined as a release
threshold prior to injection into the subject. In addition,
microbubbles can be tailored for specifuc rupture characteristics
to allow use of ultrasound conditions which will not cause rupture
except in the desired body region.
[0011] The method may also include the step of monitoring the
location of the microbubbles by ultrasound or other suitable
detection technique to detect their presence at the region of
interest.
[0012] Particularly preferred microbubbles will have a bi-layered
shell having an outer layer of a biologically compatible
amphiphilic material and an inner layer of a biodegradable polymer.
Preferred threshold conditions for rupture are those at power,
frequency, and waveform sufficient to provide a mechanical index
from about 0.1 to about 1.9.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a plot of acoustic densitometry measured along the
length of the flow phantom described in Example 1.
[0014] FIG. 2 is a plot of fragility slope verses signal intensity
described in the same experiment.
[0015] FIG. 3 is a plot of acoustic densitometry decay curve
measured according to Example 3.
[0016] FIG. 4 is a plot of fragility slope v. mechanical index as
described in Example 3.
[0017] FIG. 5 is a plot of fragility slope v. intensity described
in Example 7.
DETAILED DESCRIPTION OF INVENTION
[0018] The method for ultrasound triggered drug delivery according
to the present invention relies upon an ultrasonic contrast agent
consisting of a population of drug-carrying microbubbles having a
controlled fragility which is derived from a specific and constant
relationship between the microbubble shell thickness and its
diameter. This relationship requires that, irrespective of
diameter, the microbubbles exhibit an equivalent resistance to
acoustic and hydrostatic stresses associated with an echographic
imaging environment.
[0019] Controlled fragility of a microbubble in drug release is an
important consideration in localizing drugs with ultrasound.
Microbubbles containing drugs should be resistant to rupture and
inadvertent drug release by normal physiological pressures or by
ultrasound conditions of the beam passing through tissues not at
the target region. By normal physiological pressures, it is meant
those pressures encountered in vivo including pressures within the
heart and arteries, as well as compressive pressures of passing
through constrictions such as capillaries. At minimum, in the use
of microbubbles within the circulatory system, the microbubbles
should be resistant to normal intracardiac pressures. For example,
albumin microbubbles filled with air have been reported to
"disappear" in significant amounts in the left ventricle (Gottlieb,
et al., 1984), potentially causing problems in use as a drug
delivery system to regions other than the left ventricle or with
drugs with significant cardiotoxicity.
[0020] The controlled fragility characteristic of the drug
containing microbubble is derived from the provision that the wall
thickness of the microbubble is linearly related to its diameter.
That is, for a given microbubble population the ratio of wall
thickness to diameter for each microbubble within the population is
a constant. An ultrasound contrast agent so produced would exhibit
essentially an equivalent resistance to the stresses imparted by
the hydrostatic and acoustic forces present in the ultrasound
imaging environment.
[0021] It can be shown mathematically that the strength, that is
resistance to a hoop stress, of a thin-walled hollow sphere is a
function of the diameter of the sphere and the thickness of its
wall. Further, this relationship is linearly proportional with
diameter and inversely proportional with thickness. Thus, for a
given applied pressure, if the ratio of thickness to diameter is
constant, then the hoop stress on the sphere wall remains constant
irrespective of diameter.
[0022] An ultrasonic contrast agent including a spectrum of
microbubbles all having the same wall thickness to diameter ratio
would therefore exhibit essentially an equivalent resistance to the
stresses imparted by the hydrostatic and acoustic forces present in
the ultrasonic imaging environment.
[0023] The maximum wall stress in a pressurized sphere has been
solved by Timoshenko and Goodier. (Timoshenko, S. and J. N.
Goodier; "Theory of Elasticity"; McGraw-Hill Book Company, New
York, N.Y.; (1951): 356-359.) The authors fund that the hoop
stress, ae, for an elastic homogeneous material is given by:
.sigma..theta.=(P/2)(2a.sup.3+b.sup.3)/(b.sup.3-a.sup.3) (1)
where
[0024] a is the inner radius,
[0025] b is the outer radius, and
[0026] P is the pressure difference across the wall.
If we assume that wall thickness, h, is very thin, then equation 1
simplifies to:
.sigma..theta.=(P/2)(R+h)/h (2)
where R is the mean radius. If the wall thickness, h, is thin
compared to mean radius, R, that is R>>h, equation 2 can be
further simplified:
.sigma..theta.=(P/2)(R/h) (3)
Rewriting this expression in terms of the diameter, d, we
obtain
.sigma..theta.=(P/4)(d/h) (4)
The hoop stress for all elastic materials has a limit above which
the material will fail. It is a physical constant. Letting this
value be identified as sq-max and the corresponding pressure
causing the failure identified as Pmax and rewriting equation 4, we
obtain:
Pmax=46e.sub.--ma(h/d) (5)
[0027] Thus, it can be noted from equation 5 that the maximum
allowable pressure, Pmax, is also a constant so long as the wall
thickness to diameter ratio (h/d) remains constant. It becomes a
property of the microspheres. If there were a spectral distribution
of bubble diameters but all possessed the same h/d ratio, all
microspheres would begin to fail at the same pressure. In contrast,
if the hid ratio varies, then there is a plethora of pressures
where the agent begins to fail.
[0028] The mechanical index (MI) identified on all modem ultrasound
scanners is a measure of the maximal rarefactional (negative)
pressure in the propagated ultrasound field. If the MI were
increased systematically from a transducer focused on a plethora of
microbubbles possessing a constant h/d ratio, there would be no
breakage until the rarefactional pressure of P.sub.max was reached.
The value of MI when this event is achieved is referred to as the
critical MI (MI.sub.crit). Microbubbles continue to break for all
values above this value of MI.sub.crit. However, the rate of
destruction increases as the power level rises. If the h/d ratio is
not constant, then there is not a clean initiation of agent
destruction. Failure begins at near zero MI and the rate of
destruction increases thereafter. Thus, there is no critical MI,
there is no controlled fragility.
[0029] In a preferred method of manufacture, a microbubble
population having a constant diameter to wall thickness ratio can
be produced by an emulsion solvent evaporation process. First, two
solutions are prepared. One is an aqueous solution containing an
appropriate surfactant material which may be an amphiphilic
biopolymer such as gelatin, collagen, albumin, or globulins.
Viscosity enhancers may additionally be included. This becomes the
outer continuous phase of the emulsion system. The second is made
from the dissolution of a wall-forming polymer in a mixture of two
water immiscible organic liquids. One of the organic liquids is a
relatively volatile solvent for the polymer and the other is a
relatively non-volatile non-solvent for the polymer. The relatively
non-volatile non-solvent is typically a C6-C20 hydrocarbon such as
decane, undecane, cyclohexane, cyclooctane and the like. The
relatively volatile solvent is typically a C5-C7 ester such as
isopropyl acetate. Other polymer solvents, methylene chloride for
example, may be used so long as they are miscible with the
accompanying non-solvent. Typically about three parts of the
organic polymer solution having a concentration of about 0.5 to 10
percent of the polymer is added to one part of the aqueous
biomaterial solution having a concentration of about 1 to 20
percent of the biomaterial.
[0030] The wall forming polymer may be selected for its modulus of
elasticity and elongation which define the mechanical properties of
the polymer. Preferred polymers useful in the fabrication of
drug-carrying microbubble ultrasound contrast agent would be
biodegradable polymers such as polycaprolactone, polylactic acid,
polylactic-polyglycolic acid copolymers, co-polymers of lactides
and lactones such as epsilon-caprolactone, delta valerolactone,
polyamides, polyhydroxybutryrates, polydioxanones,
poly-beta-aminoketones, polyanhydrides, poly-(ortho)esters, and
polyamino acids, such as polyglutamic and polyaspartic acids or
esters of same. References on many biodegradable polymers are cited
in Langer, et al. (1083) Macromol. Chem. Phys. C23, 61-125.
[0031] The polymer solution (inner organic phase) is added to the
aqueous solution (outer phase) with agitation to form an emulsion.
A variety of devices can be used to produce the emulsion, e.g.
colloid mills, rotor/stator homogenizers, high pressure
homogenizers, and sonicating homogenizers. The emulsifucation step
is carried out until the inner phase droplets are in the desired
size spectrum. It is this droplet size that will determine the size
of the microbubble.
[0032] The emulsion thus produced may optionally be further diluted
into a water bath with moderate mixing. Mixing is continued while
maintaining a bath temperature appropriate for the relatively
volatile solvent to evaporate while the relatively non-volatile
non-solvent remains. A typical temperature is in the range of
30.degree.-35.degree. C. As the solvent volatilizes, polymer
concentration in the droplet increases to a point where it
precipitates in the presence of the less volatile non-solvent. This
process forms a film of polymer at the surface of the emulsion
droplet. As the process continues, an outer shell wall is formed
which encapsulates an inner core of the non-solvent liquid. Once
complete, the resulting microcapsules can then be retrieved,
washed, and formulated in a buffer system. Subsequent drying,
preferably by freeze-drying, removes both the non-solvent organic
liquid core and the water to yield airfilled hollow
microbubbles.
[0033] Use of an amphiphilic biopolymer as a surfactant in the
outer continuous phase as described above provides for the option
of creating a microbubble having a bi-layered shell. If during
processing the biopolymer is rendered insoluble, by chemical
crosslinking using an aldehyde, glutaraldehyde for example, the
material forms a permanent contiguous outer layer enveloping and
adhering to the inner polymer layer. The advantage to this
construct is to allow separate tailoring of the inner and outer
layers to serve different functions. While the inner layer can be
modified to provide the mechanical and acoustic properties
optimized to a specific drug delivery application, the outer layer
can be independently altered chemically, for example to enhance
biocompatibility or to increase circulation half-life. Such
chemical modifications may include pegylation, succinylation, or
amidation as well as attaching to the surface a targeting moiety
for binding to selected tissues.
[0034] The free circulation of the microbubbles is important to the
effective delivery of drugs to a local region targeted with
ultrasound. As very few regions of the body receive 100% of the
cardiac output of the heart, only a fraction of the total number of
microbubbles injected into the circulatory system will reach target
regions such as the liver, a tumor, etc., on the first circulating
pass. In order to effectively dose the target region, the
microbubbles need to recirculate with sufficient half-life to
eventually reach the target after a number of cardiac passes. The
smaller the fraction of total cardiac output received by the target
tissues, the greater the need for extended half-life of the
microbubbles to achieve significant delivery of drug to the target.
In the case where the microbubbles are targeted in the tissues with
biological agents such as antibodies or by mechanical trapping,
half-life of the agent becomes less important as the microbubble
will preferentially accumulate at the target tissues.
[0035] In the bilayer microbubble, the inner layer permits the
modification of the mechanical properties of the shell of the
microbubble which are not provided by the outer layer. The
mechanical properties of the inner layer may be adjusted to provide
varying threshold levels of microbubble rupture with ultrasound
conditions such as output power. Moreover, the inner layer may
provide a drug carrier and/or drug delivery capacity. For dual
purpose use as an ultrasonic contrast agent, the inner layer will
typically have a thickness which is no larger than is necessary to
meet the minimum mechanical or drug carrying/delivering properties
in order to maximize the interior gas volume of the microbubble.
Generally, the greater the gas volume within the microbubble the
better the echogenic properties.
[0036] For use as an ultrasonically triggered drug delivery system,
the inner layer wall thickness to diameter ratio can be varied to
provide varying thresholds to rupture, allowing a threshold release
characteristic to be utilized. In addition, the mechanical
properties of the inner layer such as ultimate elongation, modulus,
stress at failure and fatigue properties can be tailored by
material selection.
[0037] Selection of the appropriate mechanical strength of the
inner layer allows imaging at conditions which do not necessarily
trigger drug release, but drug release may be triggered during
imaging by altering the ultrasound characteristics. These
characteristics are important for controlling and localizing
release, especially as matched to the localization of the threshold
ultrasound conditions for drug release.
[0038] Any of a variety of therapeutics may be encapsulated in the
microbubbles. By therapeutic, as used herein, it is meant an agent
having a pharmacological or diagnostic effect on the patient. As
used herein, the term therapeutic agent is synonymous with the term
drug.
[0039] The therapeutic agent could be incorporated into the
microbubble agent by a number of techniques. In one method, for
example, the drug is dissolved or otherwise incorporated into the
organic polymer solution during microbubble fabrication.
Alternatively, the drug may be incorporated into the microbubbles
through a series of secondary steps where the finished product is
reconstituted with a solution containing the drug, the suspended
microbubbles are made to flood with the drug containing solution,
and the result dried, typically by lyophilization. Finally, the
drug may be affixed by chemical means to the surface of the
microbubble.
[0040] Preferred methods of incorporation produce a drug-carrying
microbubble that would, upon rupture with insonation, allow ready
desolution of the active agent into the blood or other body fluids
as required. Those methods which incorporate the drug into the wall
structure of the microbubble or provide attachment to the surface
may also be useful. In this case it is envisioned that the
mechanical properties of the wall would be such that microbubble
rupture would result in ultra-small wall fragments which would then
carry drug to the local site.
[0041] Microbubbles produced by the general procedures outlined
above will be of a size approximate to the emulsion droplets from
which they were derived. In such a case, a relationship between the
concentration of the wall forming material in the polymer solution,
the microbubble diameter, and the wall thickness can be
derived.
[0042] Defining vw=volume of the microbubble wall,
v.sub.w=VC/.rho. (6)
where,
[0043] V=volume of the emulsion droplet,
[0044] C=mass concentration of wall forming material, and
[0045] .rho.=dry density of the wall forming material
[0046] For d>>h,
h=v.sub.w/S (7)
[0047] where S=surface area of the emulsion droplet.
[0048] Substituting,
h=VC/Sr (8)
[0049] Since for a sphere, V=.pi.d.sup.3/6 and S=.pi.d.sup.2,
h=C(.pi.d.sup.3/6)/p(.pi.d.sup.2) (9)
[0050] Simplifying,
h=Cd/6.rho. (10)
[0051] rearranging,
h/d=C/6.rho. (11)
[0052] Since C and .rho. are constants for a given set of process
conditions, it follows that h/d is also constant.
[0053] Upon review of equation 5 and equation 11, it is evident
that a methodology exists that can be used to fashion a microbubble
population having a constant wall thickness to diameter ratio and
that a microbubble population so constructed possesses a controlled
fragility useful as a drug-carrying ultrasonic contrast agent.
[0054] Adjustment of the controlled fragility property of a
microbubble population produced in a manner described above can be
achieved in several ways. By manipulating the initial concentration
of the wall-forming material in the inner phase solution of the
emulsion system, for example, the ratio of h/d can be adjusted to
increase or decrease the acoustic intensity threshold at which the
microbubble ruptures. Wall material selection can also be used to
modify the ultimate wall strength of the microbubble. Those
materials having a higher yield stress property would provide for a
less fragile microbubble. Average molecular weight of the material
may also be manipulated to modify the controlled fragility
characteristic as a lower molecular weight material would generally
produce a more fragile wall. Use of additives such as plasticizers
may also be considered since such additives typically affect the
mechanical properties of the material including yield strength.
[0055] The thickness of the microbubble shell will depend in part
on the mechanical properties required for the particular drug
delivery application and within a given lot would vary in
accordance with the ratio h/d. Typically, for populations of drug
carrying microbubbles in the size range suitable for vascular
applications shell thickness will be in the range from about 25 nm
to about 100 nm.
[0056] In many drug delivery applications, it is important that the
microbubbles circulate through the capillary network unimpeded. For
such instances, microbubble diameter should be in the range of 1 to
10 microns.
[0057] The controlled fragility characteristics of a microbubble
agent may be predetermined by a number of ways. A simple approach
is to place a test agent in a beaker after first examining the
suspension for optical density and then insonating the suspension
at progressively higher MI settings until a degradation in optical
density is observed. This point will identify the critical MI.
[0058] Acoustic densitometric (AD) methods can also be used. One
technique utilizes a Doppler Flow Phantom. The transducer of a
commercial scanner is placed in the water well atop the phantom,
oriented downwards, and focused on the flow tube. The suspended
microbubble agent is pumped through the phantom at constant flow
rate and viewed on the scanner. AD measurements are then made from
the images. If there is no agent destruction, the AD reading is
constant along the length of the flow-tube, as backscatter remains
constant therein. The scanner power level is increased and the
process is repeated. At some point, the microbubbles will fail and
there will be a decrease in AD readings axially along the phantom.
This decrease provides two useful pieces of information. The rate
of acoustic degradation axially along the tube identifies the
fragility rate (measured in AD/cm) and is a function of the local
MI. The intercept value with the y-axis, essentially the AD
measurement at the phantom entrance, provides a measure of the peak
backscatter of the test agent. The process can be repeated for
several power settings and the data collected. Following the tests,
the measured fragility slopes can then be plotted as a function of
MI. The intercept of the x-axis (MI) provides the critical MI for
the test agent.
[0059] Another AD method utilizes a clear chamber placed under
water. Microbubble agent is contained within the chamber without
flow. An ultrasound transducer is submerged and oriented toward the
center of the chamber containing the test agent. As the scanner
emits pulses of ultrasound, the backscattered data is collected
over time. If there is no agent destruction, the backscattered
signal remains constant over time. The power level is increased and
the process is repeated. Again at some point, the agent will begin
to fail. When this occurs, the backscatter will decrease over time
as more and more of the agent is destroyed. The decay time constant
is noted and the process continued at a higher power level. When
sufficient data are collected, the decay time constants are plotted
against MI. Again, the intercept of the regression fit to the data
with the x-axis identifies the critical MI, that is the value of MI
required to initiate microbubble destruction.
[0060] Analytical means may also be employed to establish the
fragility characteristics of the agent. Microbubble wall thickness
and diameter can be determined directly by Scanning Electron or
Transmission Electron Microscopy or indirectly from knowledge of
the wall material mass density, the microbubble count, and the size
histogram. These results may then be incorporated into the
appropriate equations along with the wall material constants to
estimate the acoustic and/or hydrostatic pressures necessary to
rupture the microbubbles. Finally, in-vivo procedures using an
appropriate animal model could be established that would be useful
in collaborating the in-vitro and analytical results.
[0061] These approaches permit the determination of the critical MI
which is the primary manifestation of controlled fragility. If the
agent does not exhibit a unique characteristic as identified here,
the critical MI will be essentially zero. With such an agent, there
is no turnon and turn-off capability. There is no controlled
fragility.
[0062] The microbubbles may contain a pharmacological agent or
agent-carrying reservoir in the shell or more preferably in the
central core. The cores of the microbubbles may contain a
physiologically compatible gas such as air or nitrogen. If a gas
with low solubility is preferred, a perfluorocarbon may be used.
Alternatively, the microbubbles may be fulled with carbon dioxide
if an application calls for rapid dissolution of the gas upon
microbubble rupture.
[0063] Typically, the drug-carrying microbubbles will be introduced
intravenously by injection, but they also may be injected
intra-arterially. The microbubbles may also be injected
interstitially or directly into any body cavity.
[0064] A useful dosage of the therapeutic agent will be
administered and the mode of administration will vary depending
upon the age and weight of the subject and upon the particular
therapeutic application intended. Typically, the dosage is
initiated at a lower level and increased until the desired
therapeutic affect is achieved.
[0065] Typically, the rupture of drug-carrying microbubbles would
be achieved using ultrasound scanning devices and employing
transducers commonly utilized in diagnostic contrast imaging. In
such instances a single ultrasound transducer would be employed for
both imaging and triggering of the microbubbles by focusing the
beam upon the target site and alternately operating at low and high
power levels as required by the application.
[0066] Another option is to utilize a plurality of transducers
focused at the region so that the additive wave superposition at
the point of convergence creates a local intensity suffucient to
rupture the microbubbles. A separate imaging transducer would be
used to image the region for treatment.
[0067] A specially designed transducer or multiple transducer set
may be incorporated into a wearable object to treat a selected
region or organ to alleviate the need for manual placement of the
transducer and to facilitate concentration of the ultrasound at the
target site.
[0068] The transducer may also be incorporated into the distal
section of a cannula; or be implanted within the body near the
target site. In the first case, an intra-vascular ultrasound
catheter is used to provide the specific ultrasound energy required
to disrupt the microbubbles as they pass the catheter. The use of
such a system provides for the treatment of target sites downstream
from the catheter and in places that standard ultrasound imaging
would be impaired, in the lungs for example.
[0069] In the case of an implant, the ultrasound transducer is
surgically implanted within the body at or near the target site for
treatment. The transducer may be treated by induction means through
the body wall such that it is inert at all times except during use.
The drug-containing microbubbles are injected into the body
intra-vascularly and the transducer energized to rupture the
microbubbles at the target site. The method is useful for longer
term or chronic treatment.
[0070] To monitor the location of the drug-carrying microbubbles,
one or several pulses of sound may be used and the machine may be
paused between pulses to receive the reflected sonic signals. In
the rupturing of microbubbles, a distinct ultrasound pulse is
received which can be used to calculate the number of microbubbles
releasing drug and the cumulative microbubbles triggered.
[0071] The drug-containing microbubbles can be imaged with
ultrasound under clinically accepted diagnostic power levels for
patient safety. While not required, it is preferred that the
microbubbles be rupturable for drug release at threshold power
levels below the clinically accepted power levels for diagnosis.
Specifuc matching of ultrasound conditions and microbubble response
to such conditions are important factors in achieving such
controlled release conditions. Preferred acoustic threshold
conditions for rupture are those at a power, frequency, and
waveform sufficient to provide a mechanical index from about 0.1 to
about 1.9.
[0072] The sound energy may be pulsed, but continuous wave
ultrasound energy is preferred for maximal triggering of drug
release from the microbubbles. If pulsing is employed, sound will
preferably be pulsed in echo trained links of at least about 3 wave
periods and preferably be pulsed in echo trained links of at least
about 5 wave periods at a time.
[0073] Either fixed frequency or modulated frequency sound may be
used. For example, a high to low pulse with an initial frequency of
10 MHZ of sonic energy may be swept with increasing power from one
to five watts/cm.sup.2. Focused, frequency modulated, high energy
ultrasound may increase the rate of local gas expansion within the
microbubbles, rupturing them to provide local delivery of
therapeutics.
[0074] The types of agents to be released from the microbubble
agent may, for example, be cardiovascular drugs (endocardium
agents) with short circulatory half-lives that affect the cardiac
tissues, vasculature and endothelium to protect and treat the heart
from ischemic or reperfusion injury or coronary artery from
restenosis (anti-restenosis agent). Drugs which target platelets
(anti-platelet agent) and white cells (anti-white cell agent) which
may plug the microvasculature of the heart after a heart attack are
also useful for local cardiac delivery. Another type of drug useful
for local delivery is one for which a local effect is required but
where systemic effects of the drug would be detrimental. These are
typically drugs with high toxicity, for example, locally
administered potent vasodilators which would increase blood flow to
hypoxic tissue but if delivered systemically would cause a
dangerous drop in blood pressure. Suitable drugs include
fibronolytic agents such as tissue plasminogen activator,
streptokinase, urokinase, and their derivatives, vasodilators such
as verapamil, multifunctional agents such as adenosine, adenosine
agonists, adenosine monophosphate, adenosine diphosphate, adenosine
triphosphate, and their derivatives, white cell or platelet acting
agents such as GPIIb/IIIa antagonists, energy conserving agents
such as calcium channel blockers, magnesium and beta blockers,
endothelium acting agents such as nitric oxide, nitric oxide
donors, nitrates, and their derivatives, free-radical scavenging
agents, agents which affect ventricular remodeling such as ACE
inhibitors and angiogenic agents, and agents that limit restenosis
of coronary arteries after balloon angioplasty or stenting.
[0075] In addition to therapeutic agents delivered locally to the
heart, the use of vasodilators in the microbubbles will have
enhanced diagnostic application. Vasodilators are used in
cardiology to assess the coronary blood flow reserve by comparing
blood flow in the heart with and without the maximal vasodilation
by the pharmacological agent. Coronary blood flow reserve
correlates well with patient prognosis since the reserve capacity
enables the myocardium to remain viable during a heart attack.
Adenosine and other vasodilators are used during interventional
cardiology and nuclear imaging to determine coronary reserve. A
microbubble agent which contains a vasodilator will be useful in
echocardiography to examine the myocardium under normal conditions,
and then upon release of the vasodilator by the ultrasound beam
conditions to stimulate local vasodilation. The coronary blood flow
reserve may be estimated non-invasively using ultrasound imaging by
the extent of hyperemia of the myocardium, Doppler regional flow,
or by other well known methods of characterizing the ultrasound
imaging data.
[0076] The following examples are provided by way of illustration
but are not intended to limit the invention in any way.
Example 1
Preparation of Polylactide Microbubbles Having a Constant Wall
Thickness to Diameter Ratio
[0077] A 6% aqueous solution was prepared from a 25% solution of
USP grade human serum albumin by dilution with deionized water. The
pH of the solution was adjusted to 4 using 1M HCI. Separately, 0.41
gm poly d,l-lactide and 5.63 gm cyclooctane were dissolved in 37.5
gm isopropyl acetate. The organic solution was then slowly
incorporated into 25 gm of the prepared albumin solution with mild
stirring while the mixture was maintained at 30.degree. C. The
resulting coarse o-w emulsion was then circulated through a
stainless steel sintered metal filter element having a nominal pore
size of 7 microns. Recirculation of the emulsion was continued for
8 minutes. The emulsion was added with stirring to 350 ml deionized
water maintained at 30.degree. C. and containing 1.0 ml of 25%
glutaradehyde. During the addition, the pH of the bath was
monitored and adjusted as necessary with 1N NaOH to insure that it
remained between 7 and 8. Low sheer mixing was continued for
approximately 21/2 hours until the isopropyl acetate had
substantially volatilized. Poloxamer 188 in the amount of 0.75 gm
was dissolved into the bath. The resulting microbubbles were
retrieved by centrifugation and washed two times in an aqueous
solution of 0.25% poloxamer 188. The washed microbubble suspension
was then formulated in a glycine/polyethylene glycol solution and
lyophilized. The resulting dry white cake was reconstituted with
deionized water and examined under the microscope to reveal
spherical discrete microbubbles.
Example 2
In-Vitro Method for Establishing Acoustic Fragility Threshold
[0078] A Hewlett Packard 5500 ultrasound scanner was used for this
study in conjunction with an ATS Laboratories, Model 524 Doppler
Flow Phantom. The S4 transducer was positioned vertically downward
and oriented along the centerline of the 6 mm diameter flow tube
within the phantom. The tip of the transducer was placed in the
water well above the flow tube. The flow tube appeared as a
constant diameter tube (dark interior) in the sector of the scan.
The scanner was set in the harmonic mode (1.8/3.6 MHz) with a beam
width of approximately 4 cm at the 4 cm depth of the tube
centerline below the transducer. A peristaltic pump delivered
liquid containing test agent from a 500 ml beaker placed on a
magnetic mixer through the phantom and into a discharge container.
This fluid was not recirculated. The fluid was stirred constantly
in the beaker throughout the test.
[0079] Microbubble agent prepared in accordance with the procedures
in Example 1 was placed in the beaker, thoroughly mixed with
degassed water and pumped through the phantom with a mean velocity
of almost 1 cm per second. Flow velocity was maintained constant
throughout the test. Axial positions were marked on the scanner
monitor and measured from the proximal end of the flow tube along
the centerline using the caliper function of the system. A circular
(11.times.11) region of interest (ROI) was selected for the study
and used exclusively throughout. The interval was set at 200 ms for
any acoustic densitometric (AD) measurement made. Power levels
could be varied and local AD measurement made for each power
setting and each location. The highest mechanical index (MI) that
the system was capable of producing with this setup was 1.6. The
system provides 60 reading at 200 ms intervals. The average reading
was used for each local reading.
[0080] Using the experimental set-up with the HP 5500 and Doppler
Flow Phantom as detailed above, an experiment was performed to
determine the fragility threshold of the test agent. If the agent
does not rupture, the AD reading is constant along the flow
phantom. If the agent does rupture, there is a decrease in AD
reading along the flow phantom, and there is a slope associated
with this decease. This is shown in FIG. 1 for measurements made at
an MI of 1.6. A linear regression fit to the data permits the
determination of the fragility slope. The fragility slope can then
be determined at various MIs when the data is plotted against the
square of MI as seen in FIG. 2. The resulting graph permits a
determination of where agent begins to fail, i.e., the slope
becomes zero. The value of the MI at this point is the critical MI
(MI.sub.crit). Examination of these results reveals that when the
mechanical index exceeds 0.64 (MI.sub.crit=0.64), the agent
ruptures. A value below MI.sub.crit results in the agent remaining
intact.
Example 3
Method for Establishing Acoustic Fragility Threshold Using the
Decay Method
[0081] A Hewlett Packard 5500 ultrasonic scanner was utilized for
this study in conjunction with an ATS Laboratories, Model 524
Doppler Flow Phantom. An S4 ultrasound transducer was positioned
vertically downward and orthogonal to the center-line of a 12 mm
diameter flow tube within the phantom. The tip of the transducer
was placed in the water well of the flow phantom. The flow tube
appeared with a dark, circular cross-section in the sector of the
scan when bubbles were not present and a bright white, solid circle
when bubbles were present in the sector scan. The scanner was set
in the 2D Harmonic mode (1.8/3.6 MHz) with a beam width of
approximately 3 cm at an 8 cm depth from the top of the flow tube.
A peristaltic pump delivered water containing test agent from a 1
liter beaker placed on a magnetic mixer through the flow phantom
and into a discharge container. This fluid was not recirculated and
stirred constantly throughout the test to maintain the suspension
uniform.
[0082] Microbubble agent prepared in accordance with the procedures
in Example 1 was placed in the beaker with thoroughly degassed
water and pumped through the phantom. The scanner was placed in the
Freeze mode (does not emit excitation signals) and a new batch of
test agent pumped into the test section. An 11.times.11 circular
region of interest (RO) was established at the top center of the
flow as seen on the sector scan and the interval was set at 200 ms
(0.2 sec between scans). When ready, the scanner was unfrozen and
samples collected. Between 12 and 15 scans were collected and the
average of these utilized in the data analysis for each power level
investigated. An example of one such decay curve in shown in FIG.
3.
[0083] For very low power levels, the backscatter did not decay,
but would continue at the same level over all evaluation frames.
This means that the agent was not breaking. Therefore, backscatter
remained constant over time. The power level was step-wise
increased until a decay curve was measured up to a maximum MI of
1.6. The first three points of the decay curve were fit with a
linear regression line, which established both (1) the peak
backscatter and 2) the decay slope. The peak backscatter is a
measure of the backscatter potential of the agent and is dosage
dependent. The slope, called fragility slope herein, is a measure
of the agent fragility. This slope was found to be proportional to
MI. The fragility slope can be plotted against MI. Extrapolation of
the data to a zero fragility slope identifies the acoustic
fragility threshold or the critical MI. A graph of the fragility
slope as a function of MI for the agent is shown in FIG. 4. From
the plot the critical MI is determined to be 0.22.
Example 4
Comparison of the Fragility Threshold of Microbubble Agent Having
Different Wall Thickness to Diameter Ratios
[0084] A family of fuve agents was prepared in accordance with the
procedures described in Example 1. Each agent was fabricated in
identical fashion except that the concentration of the polylactide
polymer in the organic solution was varied for each run. Each agent
would thus be provided with a different wall thickness to diameter
ratio. The agents were then tested according to the procedures
described in Example 3. From the data, the critical MI for each was
determined. The results are presented in Table 1.
[0085] The results show that the thicker walled microbubbles
require more power to rupture than the thinner walled agent. Thus,
it is clearly demonstrated that the fragility threshold, or
critical MI of the microbubble can be controlled by the
construction of the capsule. Further, the fragility of the drug
carrying ultrasound contrast agent may be tailored to rupture
within the range of diagnostic ultrasound power levels.
TABLE-US-00001 TABLE 1 Comparison of Critical MI with
Wall-to-Diameter Ratio Estimated Wall to Diameter Ratio Critical MI
.016 .22 .024 .29 .032 .34 .041 .41 .049 .52
Example 5
Dye Loading of Microbubbles Having Controlled Fragility
[0086] To test an agent-carrying ultrasound microbubble population
having controlled fragility, a population with a polylactide wall
was furst prepared in a manner described in Example 1. The
resulting lyophilized cake which was prepared in a 10 ml serum vial
was removed and then placed into a 50 ml centrifuge tube. Enough
isopropyl alcohol was added to cover the cake. The microbubbles
quickly became flooded with the isopropyl alcohol. After 30
seconds, a 0.25% w/w aqueous poloxamer 188 solution was added to
fill the tube. After centrifuging, supernatant was removed and
another rinse performed. A saturated, filtered solution of
rhodamine B dye was added to the microparticles and allowed to soak
overnight. Under the microscope, the microparticles appeared filled
with dye solution. Four ml of a dye saturated poloxamer 188
solution was combined with the approximately 2 ml of the
microcapsule suspension. The resulting mixture was divided equally
into two 10 ml serum vials and then lyophilized. Both vials were
purged with perfluorobutane gas by five pump-down purge cycles with
a vacuum pump. Microscopic inspection revealed that roughly half
the microbubbles contained gas. The microbubbles were then rinsed 4
times with 20 ml portions of 0.25% poloxamer solution on a vacuum
filter. The microbubbles were placed in a cuvette, centrifuged, and
an initial spectra was taken. The cuvette was sonicated in an
ultrasonic bath, centrifuged, and another spectra taken.
TABLE-US-00002 Initial Absorbance (553-800 nm): 1.164 Absorbance
after sonication (553-800 nm): 1.86
The higher absorption after sonication indicates that encapsulated
dye was released upon insonation of the microbubbles.
Example 6
Dye Loading of Polycaprolactone Microbubbles Having a Controlled
Fragility
[0087] Microbubbles were prepared in accordance with the
methodology described in Example 1 except that polycaprolactone
polymer was used in lieu of polylactide and paraffin in the amount
equal to 20% of the polymer was added to the organic phase. The
lyophilized cake from one of the prepared vials was placed into a
50 ml centrifuge tube, covered with methanol and allowed to soak
for 30 seconds. The tube was then filled with an aqueous solution
of 0.25% (w/w) poloxamer 188, gently mixed, and centrifuged in
order to precipitate the now fluid-fulled microbubbles. The
supernatant was removed and the tubes were again fulled with
poloxamer solution. The fluid-fulled microbubbles were resuspended
by vortexing and again centrifuged. After removing the supernatant
solution, 2 ml of a saturated, filtered solution of brilliant blue
G dye in 0.25% poloxamer 188 was added. The fluid-filled
microbubbles were allowed to soak for approximately 72 hours.
Microscopic examination revealed 90-95% of the fluid-filled
microbubbles to be filled with dye solution A 4 ml sample of a
lyophilization excipient was prepared, added to the microbubble
suspension, and mixed thoroughly. Two 10 ml serum vials were filled
with 3 ml each of the prepared mixture and then lyophilized. Both
vials and a portion of deionized water were purged with
perfluorobutane for 10 minutes. The vials were then reconstituted
with perfluorobutane purged deionized water and rinsed with two 40
ml portions of 0.25% poloxamer 188 on a vacuum filter. The
resulting microbubble suspension was split into two 3 ml portions.
One portion was sonicated in an ultrasonic bath to rupture the
bubbles. Both portions were diluted 1/10 with poloxamer solution
and placed into cuvettes. The cuvettes were centrifuged and a
visible spectra was taken.
TABLE-US-00003 Absorbance of Unsonicated sample (605 nm-800 nm):
0.136 Absorbance of Sonicated Sample: 0.193
The higher absorption after sonication indicates that encapsulated
dye was released upon insonation of the microbubbles.
Example 7
Comparison of Acoustic Response of Microbubbles Having Controlled
Fragility with Microbubbles Having an Arbitrary Wall Thickness
[0088] In a comparative study, microbubble agent having an
arbitrary wall thickness was tested along with two agents
exhibiting controlled fragility. The agent having an arbitrary wall
thickness was represented by a mixture of several microbubble
populations each prepared by means similar to Example 1 but
differing in their wall thickness to diameter ratio. This was
achieved by varying the concentration of wall forming material for
each prepared batch included in the mixture. All other parameters
remained the same, including wall material, mean diameter, and
spectral size distribution. The Doppler Flow Phantom and the HP
5500 Scanner were employed for this study according to the
procedures described in Example 2. Results are shown in FIG. 5.
[0089] Three plots are shown on the graph. Two of the plots are
results taken from microbubble agents having constant wall
thickness to diameter ratios, one representing a relatively durable
microbubble population (identified as "0.6.times.") and one of a
relatively fragile population (identified as "0.2.times."). The
third plot, identified as "blend", are the results derived from the
microbubble agent having a variable wall thickness to diameter
ratio and hence having an arbitrary wall thickness.
[0090] Upon evaluation of the results of the two agents having
constant h/d, it is evident that there is a good linear fut to the
data even up the point of intercept with the x-axis. This
relationship is typical for agents with constant hid. The critical
MI of an agent is determined by identifying the x-intercept. Using
linear regression techniques, the critical MI for the 0.2.times.
and 0.6.times. microbubble agents are calculated to be 0.36 and
0.64, respectively. Note that the 0.6.times. agent has a higher
critical MI than does the 0.2.times. microbubble agent. This is an
expected result since the 0.6.times. agent has a greater h/d value
and thus is the more durable of the two.
[0091] By contrast, the data points taken from the agent having the
variable h/d displays have neither a linear relationship nor a
clearly defined critical MI. The results demonstrate that a
microbubble agent having a variable h/d, or synonymously an
arbitrary wall thickness, begins to fail almost immediately as
those microbubbles in the blend having the thinnest walls would
begin to rupture almost immediately. As signal intensity is
increased a higher percentage of the agents become involved in the
rupture process. Thus, the blended data exhibits a curvilinear
relationship indicating the continuous failure of the new material
as power is increased. The result is a microbubble agent with no
critical MI and hence no controlled fragility.
* * * * *