U.S. patent application number 13/593747 was filed with the patent office on 2013-05-16 for targeting microbubbles.
This patent application is currently assigned to CALIFORNIA INSTITUTE OF TECHNOLOGY. The applicant listed for this patent is HOYONG CHUNG, ALISSA M. FITZGERALD, ROBERT H. GRUBBS, THOMAS W. KENNY, MARSHALL L. STOLLER, RENEE M. THOMAS. Invention is credited to HOYONG CHUNG, ALISSA M. FITZGERALD, ROBERT H. GRUBBS, THOMAS W. KENNY, MARSHALL L. STOLLER, RENEE M. THOMAS.
Application Number | 20130123781 13/593747 |
Document ID | / |
Family ID | 47746887 |
Filed Date | 2013-05-16 |
United States Patent
Application |
20130123781 |
Kind Code |
A1 |
GRUBBS; ROBERT H. ; et
al. |
May 16, 2013 |
TARGETING MICROBUBBLES
Abstract
This invention related to manufactured microbubbles, as well as
methods of using manufactured microbubbles, for example, in
medicinal applications. The invention pertains to the physical
structure and materials of the microbubbles, as well as to methods
for manufacturing microbubbles, methods for targeting microbubbles
for specific medicinal applications, and methods for delivering
microbubbles in medical treatment.
Inventors: |
GRUBBS; ROBERT H.; (SOUTH
PASADENA, CA) ; STOLLER; MARSHALL L.; (SAN FRANCISCO,
CA) ; CHUNG; HOYONG; (Pasadena, CA) ;
FITZGERALD; ALISSA M.; (SAN FRANCISCO, CA) ; KENNY;
THOMAS W.; (San Francisco, CA) ; THOMAS; RENEE
M.; (LOS ANGELES, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GRUBBS; ROBERT H.
STOLLER; MARSHALL L.
CHUNG; HOYONG
FITZGERALD; ALISSA M.
KENNY; THOMAS W.
THOMAS; RENEE M. |
SOUTH PASADENA
SAN FRANCISCO
Pasadena
SAN FRANCISCO
San Francisco
LOS ANGELES |
CA
CA
CA
CA
CA
CA |
US
US
US
US
US
US |
|
|
Assignee: |
CALIFORNIA INSTITUTE OF
TECHNOLOGY
PASADENA
CA
|
Family ID: |
47746887 |
Appl. No.: |
13/593747 |
Filed: |
August 24, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61527031 |
Aug 24, 2011 |
|
|
|
Current U.S.
Class: |
606/45 ; 424/400;
606/128 |
Current CPC
Class: |
A61K 41/0028 20130101;
A61B 17/22004 20130101; A61K 31/663 20130101; A61B 2017/22007
20130101; A61K 9/1075 20130101; A61K 9/0019 20130101; A61N 7/00
20130101; A61N 2007/0039 20130101; A61P 35/00 20180101; A61K
47/6911 20170801; A61B 17/22022 20130101; A61P 43/00 20180101; A61B
17/2202 20130101; A61K 9/0009 20130101; A61K 47/24 20130101; A61B
2017/22008 20130101; A61K 41/0033 20130101 |
Class at
Publication: |
606/45 ; 424/400;
606/128 |
International
Class: |
A61K 41/00 20060101
A61K041/00; A61B 17/22 20060101 A61B017/22 |
Claims
1. A targeting microbubble comprising: (a) a core containing a
fluid having a normal boiling point less than about 30.degree. C.;
(b) an anchoring moiety comprising a bio-lipid, protein,
surfactant, or synthetic polymer; and (c) a targeting moiety
comprising: (i) a chemical group having an affinity for a
metal-containing material; or (ii) a cell specific ligand.
2. The targeting microbubble of claim 1, wherein the anchoring
moiety and the targeting moiety are linked via a covalent, ionic,
or hydrogen-bonding linkage.
3. The targeting microbubbles of claim 2, wherein the anchoring
moiety covalently attached to the targeting moiety.
4. The targeting microbubble of claim 1, further comprising a
polymeric linker that covalently attaches the anchoring moiety to
the targeting moiety.
5. The targeting microbubble of claim 1, wherein the fluid is air,
CO.sub.2, a fluorinated or perfluorinated C.sub.1-6 hydrocarbon, or
a combination thereof.
6. The targeting microbubble of claim 5, wherein the fluorinated
hydrocarbon is perfluoropropane or perfluoropentane.
7. The targeting microbubble of claim 5, wherein the targeting
moiety comprises a chemical group having an affinity for a
metal-containing material.
8. The targeting microbubble of claim 1, wherein the chemical group
comprises a chelant having at least two amino, carboxy, hydroxyl,
phosphoryl, or thiol groups, or a combination thereof, said chelant
having an affinity for the metal-containing material.
9. The targeting microbubble of claim 1, wherein the targeting
moiety comprises a chemical group having an affinity for a
calcium-containing material
10. The targeting microbubble of claim 9, wherein the chemical
group comprises a bisphosphonate moiety.
11. The targeting microbubble of claim 9, wherein the
calcium-containing material is atheromatous plaque, biliary stone,
a calcified tissue or plaque, a cancerous tumor, or a urinary
stone.
12. The targeting microbubbles of claim 1, wherein the targeting
moiety is a cell specific ligand.
13. The targeting microbubbles of claim 12, wherein the cell
specific ligand is a cancer tumor cell specific ligand.
14. The targeting microbubbles of claim 13, wherein the cancer
tumor cell specific ligand is a folate.
15. A solution comprising a plurality of the targeting microbubbles
of claim 1 dispersed in a solvent.
16. The solution of claim 15, wherein the solvent is water.
17. The solution of claim 15, wherein the solvent is a
physiological fluid.
18. The solution claim 15, wherein the plurality of targeting
microbubbles have an average diameter in the solution in the range
of about 1 micron to about 10 microns.
19. A method for preparing a solution of microbubbles, the method
comprising delivering energy to a solution comprising a
bubble-forming material and a solvent, wherein: (a) the
bubble-forming material comprises an anchoring moiety and a
targeting moiety, said targeting moiety comprising: (i) a chemical
group having an affinity for a metal-containing material; or (ii) a
cell specific ligand; and (b) the energy is sufficient to cause the
bubble-forming material to form microbubbles in the solvent.
20. The method of claim 19, wherein the chemical group comprises a
chelant having at least two amino, carboxy, hydroxyl, phosphoryl,
or thiol groups, or a combination thereof, said chelant having an
affinity for the metal-containing material.
21. The method of claim 19, wherein the targeting moiety comprises
a chemical group having an affinity for a calcium-containing
material
22. The method of claim 21, wherein the targeting moiety comprises
a bisphosphonate moiety.
23. The method of claim 19, wherein the energy is in the form of
ultrasound, mechanical, or microwave energy.
24. The method of claim 19, wherein the anchoring moiety comprises
a bio-lipid, surfactant, synthetic polymer, protein, or combination
thereof.
25. The method of claim 19, wherein the anchoring moiety and the
targeting moiety are chemically linked.
26. The method of claim 25, wherein the chemical linkage is a
covalent polymeric linking moiety.
27. The method of claim 19, wherein the bubble-forming material
further comprises a bio-lipid, surfactant, synthetic polymer, or
protein, wherein the compound is not chemically linked to the
targeting moiety.
28. A method of treating a patient comprising applying energy to
microbubbles disposed within the patient, wherein the microbubbles
comprise a targeting moiety with a specific affinity to a target
within the patient, and wherein the energy is effective to cause
cavitation of the microbubbles.
29. The method of claim 28, wherein the energy is in the form of
ultrasound or electromagnetic energy.
30. The method of claim 28, further comprising administering the
microbubbles to the patient prior to applying the energy.
31. The method of claim 30, wherein the administering is via
injection, inhalation, or implantation.
32. The method of claim 28, wherein the target is a
calcium-containing mass, a cancerous cell, a tumor, or a
tissue.
33. The method of claim 28, wherein the target is a
calcium-containing mass, and where the cavitation causes damage to
the target.
34. The method of claim 33, wherein the target is a renal or
urinary stone, biliary stone, blood clot, fibroid, cancerous tumor,
or atheromatous plaque.
35. The method of claim 28, wherein the target is a cancerous cell,
and where the cavitation causes lysis of the target.
36. The method of claim 28, wherein the targeting moiety is
chemically attached to an anchoring moiety.
37. The method of claim 36, wherein the anchoring moiety comprises
a bio-lipid, synthetic polymer, protein, or surfactant, or
combination thereof.
38. The method of claim 36, wherein the chemical attachment is via
a linking polymeric moiety.
39. The method of claim 28, wherein the microbubbles further
comprise a bio-lipid, synthetic polymer, protein, or surfactant,
wherein the compound is lacking a targeting moiety.
40. The method of claim 28, wherein the microbubbles are attached
to the target.
41. The method of claim 28, wherein the microbubbles are in
proximity to the target, but are not attached to the target.
42. A method of treating a patient, the method comprising: (a)
delivering a solution comprising microbubbles to a site within the
patient; and (b) applying energy to the microbubbles, wherein the
energy is in the form of electromagnetic or ultrasound energy and
is sufficient to cause cavitation of the microbubbles, and wherein
the cavitation releases sufficient energy to cause destruction of
cell, tissue, or calcium-containing mass at the site within the
patient.
43. The method of claim 42, wherein the solution is delivered
directly to the site via implantation or via a catheter.
44. The method of claim 42, wherein the solution is delivered to
the patient via injection or inhalation, and wherein the
microbubbles comprise a targeting moiety having an affinity for a
cell, tissue, or calcium-containing mass at the site within the
patient.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Patent Application
Ser. No. 61/527,031, filed Aug. 24, 2011, which is incorporated by
reference in its entirety herein.
TECHNICAL FIELD
[0002] These inventions are directed toward compositions comprising
a bubble forming material, wherein the bubble-forming material
comprises an anchoring moiety and a targeting moiety having an
affinity for metal-containing, especially calcium-containing,
bodies and/or biological targets. In certain embodiments, these
compositions are useful for providing targeted placement of
microbubbles capable of cavitation on application of high frequency
energy.
BACKGROUND
[0003] Cavitation is a component of some currently used medical
interventions, such as a treatment for kidney stones. For example,
in extracorporeal shock wave lithotripsy, shock waves are focused
onto a stone in the kidney or ureter. The interaction between the
waves and the stone induces the formation of cavitation bubbles.
The collapse of cavitation bubbles releases energy at the stone,
and the energy fragments the stone into pieces small enough to be
passed via the ureter.
[0004] A large number of medical conditions are characterized at
least in part by the presence of an abnormal mass. Examples include
urinary stones, biliary stones, blood clots, fibroids, cancerous
tumors, and atheromatous plaques. Destruction or reduction of the
mass without injury to healthy tissue is a goal for many
therapeutic treatments. Minimally invasive treatments are preferred
as they reduce the pain, discomfort, and risks associated with
surgical or other invasive therapies.
SUMMARY
[0005] In one aspect, the disclosure provides a target microbubble
comprising: (a) a core containing a fluid having a normal boiling
point less than about 30.degree. C.; (b) an anchoring moiety
comprising a bio-lipid, protein, surfactant, synthetic polymer, or
combination thereof; and (c) a targeting moiety comprising a
chemical group having an affinity for a metal-containing material,
especially calcium-containing, materials, or a small molecule cell
specific ligand, including a small molecule tumor cell specific
ligand.
[0006] In other aspects, certain embodiments provide solutions
comprising a plurality of the targeting microbubbles dispersed in a
solvent, where the solvent may be water or a physiological
fluid.
[0007] In another aspect, the disclosure provides methods for
preparing a solution, each method comprising combining a
bubble-forming material and a solvent, wherein the bubble-forming
material comprises an anchoring moiety and a targeting moiety
comprising a chemical group having an affinity for a
metal-containing material, especially a calcium-containing,
material, or a small molecule cell specific ligand, including a
small molecule tumor cell specific ligand.
[0008] In yet another aspect, the disclosure provides methods for
preparing a solution of microbubbles, each method comprising
delivering energy to a solution comprising a bubble-forming
material and a solvent, wherein: (a) the bubble-forming material
comprises an anchoring moiety and a targeting moiety, said
targeting moiety comprising a chemical group having an affinity for
metal-containing materials, especially calcium-containing,
materials or a small molecule cell specific ligand, especially a
small molecule tumor cell specific ligand; and (b) the energy is
sufficient to cause the bubble-forming material to form
microbubbles in the solvent
[0009] In another aspect, the disclosure provides methods for
treating a patient, each method comprising applying energy to
microbubbles disposed within the patient, wherein the microbubbles
comprise a targeting moiety with a specific affinity to a target
within the patient, and wherein the energy is effective to cause
cavitation of the microbubbles. In another aspect, the disclosure
provides a method for treating a patient, the method comprising:
(a) delivering a solution comprising microbubbles to a site within
the patient; and (b) applying energy to the microbubbles, wherein
the energy is in the form of electromagnetic, ultrasound,
microwave, or other energies and is sufficient to cause cavitation
of the microbubbles, and wherein the cavitation releases sufficient
energy to cause destruction of a cell, tissue, or calculous mass at
the site within the patient.
[0010] These and other aspects will be apparent from the disclosure
provided herein, including the claims, figures, and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The present application is further understood when read in
conjunction with the appended drawings. For the purpose of
illustrating the subject matter, there are shown in the drawings
exemplary embodiments of the subject matter; however, the presently
disclosed subject matter is not limited to the specific methods,
devices, and systems disclosed. In addition, the drawings are not
necessarily drawn to scale. In the drawings:
[0012] FIG. 1 provides a photographic image of a kidney stone after
an in vivo treatment using a method according to the
disclosure.
[0013] FIG. 2 illustrates one embodiment of the present invention
in which a dipolar compound (bisphosphonic acid linked to a
quaternary ammonium compound) serves to conjoin a target material
with a negatively charged anchoring moiety (e.g., phospholipid)
attachable to a microbubble.
[0014] FIG. 3 provides a pictorial representation of treatment of a
kidney stone with HCl (FIG. 3A) and linked quaternary salt (FIG.
3B) as described in Example 5.
[0015] FIG. 4 shows attachment and cavitation of microbubble to
kidney stone, and damage caused thereby. See Example 5. FIG. 4A
illustrates the successful attachment of the microbubble to the
kidney stone (calculous). Without the pretreatment described in
Example 5, the microbubbles did not attach. FIG. 4B shows an in
situ picture of the microbubble bursting. FIG. 4C shows the
multiple pitting damage caused by the cavitation. The surface of
this stone was smooth before microbubble treatment.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0016] The present invention may be understood more readily by
reference to the following description taken in connection with the
accompanying Figures and Examples, all of which form a part of this
disclosure. It is to be understood that this invention is not
limited to the specific products, methods, conditions or parameters
described and/or shown herein, and that the terminology used herein
is for the purpose of describing particular embodiments by way of
example only and is not intended to be limiting of any claimed
invention. Similarly, unless specifically otherwise stated, any
description as to a possible mechanism or mode of action or reason
for improvement is meant to be illustrative only, and the invention
herein is not to be constrained by the correctness or incorrectness
of any such suggested mechanism or mode of action or reason for
improvement. Additionally, throughout this text, it is recognized
that the descriptions refer both to the compositions comprising and
methods of making and using targeting microbubbles. These certain
compositions or methods may be described in terms of certain
embodiments or features. Where the disclosure describes and/or
claims a particular feature in a composition or method, it is
appreciated that such a feature is intended to relate to all
compositions or methods described herein.
[0017] In the present disclosure the singular forms "a," "an," and
"the" include the plural reference, and reference to a particular
numerical value includes at least that particular value, unless the
context clearly indicates otherwise. Thus, for example, a reference
to "a material" is a reference to at least one of such materials
and equivalents thereof known to those skilled in the art, and so
forth.
[0018] When a value is expressed as an approximation by use of the
descriptor "about," it will be understood that the particular value
forms another embodiment. In general, use of the term "about"
indicates approximations that can vary depending on the desired
properties sought to be obtained by the disclosed subject matter
and is to be interpreted in the specific context in which it is
used, based on its function. The person skilled in the art will be
able to interpret this as a matter of routine. In some cases, the
number of significant figures used for a particular value may be
one non-limiting method of determining the extent of the word
"about." In other cases, the gradations used in a series of values
may be used to determine the intended range available to the term
"about" for each value. Where present, all ranges are inclusive and
combinable. That is, references to values stated in ranges include
every value within that range.
[0019] It is to be appreciated that certain features of the
invention which are, for clarity, described herein in the context
of separate embodiments, may also be provided in combination in a
single embodiment. That is, unless obviously incompatible or
specifically excluded, each individual embodiment is deemed to be
combinable with any other embodiment(s) and such a combination is
considered to be another embodiment. Conversely, various features
of the invention that are, for brevity, described in the context of
a single embodiment, may also be provided separately or in any
sub-combination. It is further noted that the claims may be drafted
to exclude any optional element. As such, this statement is
intended to serve as antecedent basis for use of such exclusive
terminology as "solely," "only" and the like in connection with the
recitation of claim elements, or use of a "negative" limitation.
Finally, while an embodiment may be described as part of a series
of steps or part of a more general structure, each said step may
also be considered an independent embodiment in itself.
[0020] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
described herein.
[0021] As used herein the term "microbubble" refers to any
container, coil, or other space conforming geometry. Unless
otherwise specified, the terms "microbubbles" and "bubbles" are
used interchangeably.
[0022] Definitions of other terms and concepts appear throughout
the detailed description below.
[0023] In some aspects of the disclosure, there is herein provided
methods and materials for synthesizing microbubbles for medical
applications. Chemical tags are attached to biocompatible
microbubbles and the microbubbles are then delivered to a patient.
The chemical tags have an affinity for a targeted mass, tissue, or
structure, such that microbubbles concentrate near the target.
Ultrasound or another suitable form of energy is then applied
causing the microbubbles to induce cavitation. Cavitation of
microbubbles causes the delivery of energy at or near the target.
For example, where the target is an unwanted mass, cavitation
causes the mass to break apart into smaller pieces that may be
removed from the patient or may pass from the patient via normal
biological processes. In another example, where the target is a
biological entity such as a tissue or cell, cavitation causes
destruction of the entity and/or disruption of biological processes
involving the entity.
[0024] Various embodiments of the present invention provide methods
for preparing a solution, each method comprising combining a
bubble-forming material and a solvent, wherein the bubble-forming
material comprises an anchoring moiety and a targeting moiety a
targeting moiety comprising: (i) a chemical group having an
affinity for a metal-containing material; or (ii) a cell specific
ligand.
[0025] Still other embodiments provide targeting microbubbles, each
microbubble comprising: (a) a core containing a fluid having a
normal boiling point less than about 30.degree. C.; (b) an
anchoring moiety comprising a bio-lipid, protein, surfactant, or
synthetic polymer; and (c) a targeting moiety comprising: (i) a
chemical group having an affinity for a metal-containing material;
or (ii) a cell specific ligand. In certain of these embodiments,
the targeting moiety may either have an affinity for a
metal-containing material; e.g., a calcium-containing material,
such as a atheromatous plaque, biliary stone, a calcified tissue or
plaque, a cancerous tumor, or a urinary stone; or, by virtue of a
small molecule cell specific ligand, have an affinity for blood
clots, fibroids, cancerous tumors, and/or atheromatous or other
plaques.
[0026] Other embodiments provide solutions, each comprising a
plurality of targeting microbubbles dispersed in a solvent, wherein
the solvent may include water or some other physiological fluid. As
used herein, the term "physiological fluid" refers to a fluid of
the body, for example, including blood, lymph fluid, saliva, bile,
urine, and interstitial fluid.
[0027] In these, and other embodiments throughout this disclosure,
such calcium-containing materials may be found within or outside
the body of a patient, for example, including atheromatous or other
calcium-containing plaque (e.g., dental plaque), biliary stone, a
calcified tissue or plaque, a cancerous tumor, or a urinary stone.
Also, as used herein, the term "targeting moiety having an affinity
to metal- or calcium-containing materials" refers to a chemical
moiety, which by virtue of its chemical affinity for metal- or
calcium salts (e.g., calcium carbonate, calcium oxalate, calcium
phosphate, or hydroxyapatite) has a tendency to complex with such
salts. Bisphosphonate is one such moiety particularly useful for
calcium-containing materials, and is a preferred embodiment of the
present invention.
[0028] As used herein, the term "small molecule cell specific
ligand" is intended to connote a ligand comprising a cell specific
ligand having a molecular weight less than about 1000 Daltons and
having an affinity for a particular type of cell or cells, and be
distinguished from antibody or protein-based ligand. In certain
embodiments, the cell specific ligand is a cancer tumor cell
specific ligand, such as a folate (see, e.g., Example 6, below),
which is known to be a very selective receptor to cancerous tumors
and it is not harmful to healthy cells.
[0029] In each of the embodiments contemplated herein, the
anchoring moiety and the targeting moiety of the various
embodiments may be linked by one or more covalent, ionic, or
hydrogen-bonding linkages. In such embodiments, the bubble-forming
material may further comprise a polymeric linking moiety that
covalently links the anchoring moiety with the targeting moiety, as
discussed below. In other embodiments, the anchoring moiety is
directly chemically attached to the targeting moiety. Further, the
anchoring moiety may comprise a bio-lipid, synthetic polymer,
surfactant, and/or a protein.
[0030] Where the targeting microbubbles comprise a core containing
a fluid having a normal boiling point less than about 30.degree. C.
or 35.degree. C., such may comprise air, CO.sub.2, a fluorinated or
perfluorinated C.sub.1-6 hydrocarbon (e.g., perfluoropropane), or a
combination thereof. In some embodiments, the core may comprise a
fluid comprising a condensed gas; i.e., the composition is at a
temperature below the boiling point of the fluid. For example,
pentafluoropentane, with a boiling point of 29.5.degree. C., may
exist as a liquid at ambient temperature, but as a gas at
physiological temperatures (e.g., 37.degree. C.). Such a fluid is
considered within the scope of the present invention.
[0031] This invention also teaches methods for preparing a solution
of microbubbles, each method comprising delivering energy to a
solution comprising a bubble-forming material and a solvent,
wherein the bubble-forming material comprises an anchoring moiety
and a targeting moiety, such that the energy is sufficient to cause
the bubble-forming material to form microbubbles in the solvent.
Again, in these embodiments, the targeting moiety also either have
an affinity for a calcium-containing material (or other metal
target) or, by virtue of a small molecule cell specific ligand,
have an affinity for blood clots, fibroids, cancerous tumors,
and/or atheromatous or other plaques. In various embodiments, the
bubble-forming material further comprises a bio-lipid, surfactant,
synthetic polymer, or protein, wherein the compound is not
chemically linked to the targeting moiety.
[0032] Additional embodiments provide methods of treating a
patient, each method comprising applying energy to microbubbles
disposed within the patient, wherein the microbubbles comprise a
targeting moiety with a specific affinity to a target within the
patient, and wherein the energy is effective to cause cavitation of
the microbubbles. In certain embodiments, the methods further
comprise administering the microbubbles to the patient prior to
applying the energy, for example via injection, inhalation, or
implantation. In some of these embodiments, the target is a
calcium-containing mass, a cancerous cell, a tumor, or a tissue. In
some embodiments where the target is a calcium-containing mass, the
cavitation causes damage to the target. In other embodiments where
the target is a cancerous cell, the cavitation causes lysis of the
target. In still other embodiments where the target is a renal or
urinary stone, biliary stone, blood clot, fibroid, cancerous tumor,
or atheromatous or other plaque, the cavitation causes damage to
the target. In certain of these embodiments, the microbubbles
further comprise a bio-lipid, synthetic polymer, protein, or
surfactant, but the compound is lacking a targeting moiety. The
microbubbles may be alternatively attached to the target, or not
attached, in the latter case being proximate to the target.
[0033] Still other embodiments include methods of treating a
patient, each method comprising: (a) delivering a solution
comprising microbubbles to a site within the patient; and (b)
applying energy to the microbubbles, wherein the energy is in the
form of electromagnetic or ultrasound energy and is sufficient to
cause cavitation of the microbubbles, and wherein the cavitation
releases sufficient energy to cause destruction (e.g., lysis or
fracture) of the target cell, tissue, tumor, or calcium-containing
or other mass at the site within the patient--e.g., to the target
renal or urinary stones, biliary stones, blood clots, fibroids,
cancerous tumors, and atheromatous plaques. In certain related
embodiments, the microbubbles do not contain a targeting moiety. In
these embodiments, the solution may be delivered by any method
described herein for such purpose, but especially via implantation,
inhalation, injection, or by catheter. Where by inhalation or
injection, it is envisioned that the microbubbles have an affinity
for a cell, tissue, or calcium-containing mass at that site within
the patient.
[0034] Various microbubble products are available commercially,
including microbubbles marketed under the trade names ALBUNEX.RTM.,
DEFINITY.RTM., and OPTISON.RTM.. In some embodiments, the
microbubbles used in the procedures described herein are selected
from such commercially available materials and are further modified
to include targeting moieties as described herein.
[0035] An example for illustrative purposes is provided as follows.
In one embodiment, microbubbles are prepared having chemical tags
that are suitable for binding to kidney stones. Such chemical tags
may be, for example, bisphosphonate pendants. The microbubbles are
administered to a patient suffering from kidney stones. Ultrasound
is applied to cause the microbubbles to cavitate and break apart
the kidney stones into smaller particles. The smaller particles
pass through the kidney/ureter naturally and with limited or no
discomfort to the patient.
[0036] Prior medical applications of cavitation have used
extracorporeal energy sources to create and collapse air bubbles in
the tissue. The methods disclosed herein differ from such
procedures by utilizing application-specific, gas-containing
bubbles that are manufactured ex-vivo. The manufactured bubbles are
specifically delivered to the surface or vicinity of the targeted
tissue or mass. Alternatively, the bubbles contain targeting tags
that allow them to concentrate on or near the targeted tissue or
mass. Energy from external sources (e.g. ultrasound, RF energy, or
the like) is then applied in order to induce cavitation. The
engineered bubbles act as a cavitation nucleus upon interaction
with ultrasound or by absorption of radio frequency energy causing
local heating and cavitation. Expansion of bubbles and their rapid
collapse causes a shock wave that can fragment or lyse the targeted
mass. For certain masses, the release of energy will cause
fragmentation, as in the case of kidney stones. For other
conditions, the energy release will cause the lysis of cells, as in
tumors.
Microbubble Characteristics
[0037] The microbubbles of interest include a shell surrounding a
hollow core. In some embodiments, the shell is composed of
bio-lipids, proteins (e.g., albumin), surfactants, biocompatible
polymers, or any combination thereof. Specific examples of such
materials are provided herein below. In some embodiments, the
hollow core is filled with a gas or low boiling fluid, and examples
of such gases and fluids are also provided herein below. The
microbubbles are designed with a shape and size to nucleate
cavitation, which refers to the formation and collapse of gaseous
bubbles. The violent collapse of cavitation bubbles releases energy
that can cause the fragmentation of an adjacent mass.
[0038] In some embodiments, the microbubbles described herein are
modified to carry chemical tags (referred to herein as "targeting
moieties" or "functional moieties") on or near their surface. Such
tags are selected to target specific locations, masses, or
structures in vivo. Because of the targeting, microbubbles
concentrate at the targeted location, mass, or structure and can be
used in therapeutic treatments as described herein.
[0039] Alternatively or in addition, the microbubbles can be used
to transport a load of material within the core to a specific mass,
location, or structure in vivo.
[0040] For example, gas-filled microbubbles are synthesized with
one or more tags for targeting a specific tissue, tumor, mass,
stone or bone. The bubbles are delivered to the target as part of a
pharmaceutically acceptable formulation. Upon attachment to or
association with the target, cavitation is induced with consequent
disruption or fragmentation of the target.
[0041] The contents of the bubble can vary with application. In
some embodiments, the bubble contains air, CO.sub.2, a fluorinated
or perfluorinated gas (e.g. a perfluorinated alkane such as
perfluoropropane), another gas, or mixtures thereof. In other
embodiments, the bubble may contain a low boiling (e.g., normal
boiling point less than about 30.degree. or 35.degree. C.). This
allows that a deflated bubble may be injected into the patient,
said bubble inflating as it heats to physiological temperatures
(ca. 37.degree. C.). In other embodiments, the bubbles can be
filled partially or completely with a payload other than a gas,
such as a pharmaceutically active agent, a cytotoxic agent, an
imaging agent, or the like.
[0042] The bubbles are intended for delivery to the site of a
targeted mass or tissue that is to be reduced in size or
eliminated. The bubbles are tagged with a targeting moiety so that
they selectively bind or associate with the target.
[0043] Various sizes and shapes of bubbles are suitable based on
the specific intended applications. In some embodiments, the
microbubbles are selected from spherical, ellipsoidal, disk-shaped,
and asymmetric shapes. In some embodiments, the shape of the
bubbles is not static. For example, in some embodiments, the
unperturbed bubbles may be spherical, but the bubbles may adopt a
different shape such as ellipsoidal or disk-shaped when an external
force (e.g., a flowing fluid such as blood) is present.
[0044] In some embodiments, the microbubbles have an average
diameter (wherein "average diameter" refers to the largest
dimension for non-spheroidal shapes) between 0.1 .mu.m and 10
.mu.m, or between 0.5 .mu.m and 10 .mu.m, or between 1 .mu.m and 10
.mu.m. In some embodiments, the average diameter is between 0.5
.mu.m and 3 .mu.m, or between 1 .mu.m and 2 .mu.m. In some
embodiments, the microbubbles have an average diameter less than 10
.mu.m, or less than 5 .mu.m, or less than 1 .mu.m, or less than 0.5
.mu.m, or less than 0.1 .mu.m. In some embodiments, the
microbubbles have an average diameter greater than 0.1 .mu.m, or
greater than 0.5 .mu.m, or greater than 1 .mu.m, or greater than 5
.mu.m, or greater than 10 .mu.m. The synthetic processes described
herein allow the production of bubbles of various sizes and
materials. It will be appreciated that use of the term
"microbubbles" is not intended to limit the size of the bubbles to
any particular range (e.g., micron diameters).
[0045] In some embodiments, the microbubbles are targeted to the
mass of interest by the attachment of a targeting agent or tag, for
example to the surface of the bubble. For example, microbubbles can
be chemically functionalized using a variety of techniques, the
details of such techniques being dependent on the exact chemical
moiety to be attached.
[0046] Examples of methods of attachment of the targeting moieties
include covalent and ionic bonds. The targeting moiety is chosen
based on properties of the target tissue or mass as well as the
structure and chemical properties of the microbubbles. A variety of
targeting moieties may be used, some of which are described in more
detail below.
[0047] Targeting moieties and other functional groups can be
attached asymmetrically or in patterns as needed for a particular
application. In some embodiments there is directional modification
of the surface of the bubbles. For some applications, only one part
of the surface of the microbubble is functionalized with a tagging
moiety in order to direct energy toward or away from the intended
target.
Delivery and Administration
[0048] Delivery into or near the targeted mass, tissue, tumor,
stone, bone or other site of interest can be achieved by a variety
of means, as appropriate for the application. Bubbles may be
introduced, as examples, by injection or spray. Depending on
specific formulations, preparations may be prepared using
surfactants or other additives for dispersal. In some embodiments,
bubbles are introduced to the blood, bile, urine, or cerebral
spinal fluid. In some embodiments, bubbles are introduced to organs
by percutaneous injection. In some embodiments, bubbles are
introduced via an orifice of the body. Orifices include any opening
such as the mouth, nose, eyes, vagina, urethra, and ears. In some
embodiments, bubbles are introduced under the skin.
[0049] In some embodiments, bubbles are introduced directly at the
target site, such as by direct implantation into a target tissue or
mass. In some such cases, it is not necessary for the bubbles to be
manufactured with targeting agents.
[0050] In other embodiments, bubbles are introduced at a remote
location (e.g., into the bloodstream via percutaneous injection)
and are allowed to concentrate at the targeted site.
[0051] In each of these methods it will be appreciated that the
bubbles are introduced as part of a pharmaceutical formulation
which may include, for example, solvents or other carriers,
additives (e.g., stabilizers and preservatives, colorants,
surfactants, pH-modifiers, etc.), and/or one or more
pharmaceutically active agents.
Treatment
[0052] After introduction of the bubbles and attachment or
association of the bubbles with the target, cavitation may be
initiated by a variety of means. In some embodiments such means
involve application of energy, where such energy is generated ex
vivo. Examples include application of directed ultrasound and radio
waves. In some embodiments, electromagnetic (EM) energy of
frequencies between 400 kHz and 10 MHz is suitable because it
propagates through tissue without strong interactions (due to low
electrical conductivity). In one example, standard ultrasound units
are applied within or adjacent to the body with sufficient power to
initiate cavitation of the pre-positioned bubbles.
Materials and Methods
[0053] In some embodiments, preparations of the microbubbles used
herein are carried out according to literature procedures, with
appropriate modifications as necessary. The functionalized (i.e.,
tagged) microbubbles may be prepared by functionalizing a
bubble-forming material. Alternatively, microbubbles can be
prepared from un-functionalized materials and then subsequently
functionalized after bubble formation.
[0054] In some embodiments, microbubbles suitable for medicinal
applications are prepared by adapting a process for creating hollow
spheres for use in paints and surface treatments (C. J. McDonald
and M. J. Devon, Advanced in Colloid and Interface Science, 2002,
99, 181-213).
[0055] In some embodiments, microbubbles (including multi-layered
microbubbles) are prepared using methods known in the art; for
example, according to the process reported in Liu et al., J.
Controlled Release, 114 (2006) 89-99, and references cited therein.
In some embodiments, microbubbles are prepared according to the
process reported in Hu et al., J. Controlled Release, 147 (2010)
154-162, and references cited therein. In some embodiments,
microbubbles are prepared according to the process reported in
Hernot et al., Adv. Drug Delivery Rev. 60 (2008) 1153-1166, and
references cited therein. In some embodiments, microbubbles are
prepared according to the process reported in Geers et al., J.
Controlled Release 148 (2010) e57-e73 (abstracts), and references
cited therein. In some embodiments, microbubbles are prepared
according to the process reported in Tinkov et al., J. Controlled
Release 143 (2010) 143-150, and reference cited therein. Additional
synthetic details for preparing (untagged) microbubbles can be
found in Mayer et al., Adv. Drug Delivery Rev. 60 (2008) 1177-1192.
The procedures from any of the above-cited references can be
modified according to the examples provided herein below so as to
prepare the targeting microbubbles of interest.
[0056] Various materials can be used in the manufacture of bubbles.
In some embodiments, the bubble-forming material includes an
anchoring moiety and a targeting moiety. In some embodiments, the
anchoring moiety is hydrophobic and the targeting moiety is
hydrophilic. Alternatively, the anchoring moiety is hydrophilic and
the targeting moiety is hydrophobic. It will be appreciated that in
either of these cases, the bubble-forming material is amphiphilic.
The bubble-forming material may further contain one or more
additional moieties as described below
[0057] In some embodiments, the various components of the
bubble-forming material are chemically bonded to each other via
covalent bonds, ionic bonds, hydrogen bonds, or a combination
thereof. In some embodiments, two or more of the various components
are separate molecules (not chemically bonded) but are associated
with each other as part of the same microbubble. For example, the
"anchoring moiety" may be a separate compound from the "targeting
moiety," and both compounds together form microbubbles.
[0058] In some embodiments, the targeting moiety comprises a
chemical group having an affinity for a metal-containing material.
As used herein, a metal-containing material comprises any of the
elements of Group 2 to Group 12, and the metals of Groups 13-15,
though materials comprising calcium are especially attractive
targets for the present invention. The variety of structured,
chemical, and other characteristics capable of providing an
affinity to a metal-containing material are too numerous to mention
here, but are known to those skilled in the art. For example, such
groups will generally include functional groups capable of
interacting with such surfaces; e.g., heteroatoms such as nitrogen,
oxygen, sulfur and phosphorus. One such a chemical group may be a
bi- or poly-dentate chelant having at least two amino, carboxy,
hydroxyl, phosphoryl, or thiol groups, or a combination thereof.
Examples include amino acids or polyamino acids, triols,
polyamines, polycarboxylates, or combinations thereof.
[0059] In some embodiments, the targeting moiety is a phosphonate
such as a bisphosphonate. Bisphosphonates are useful agents for
targeting renal or urinary stones, and are part of a family of
bone-targeting agents (any of which may be used herein as desired).
For example, neridronate and alendronate have the appropriate
functionality to attach to kidney stones and other calculous
masses. Other targeting moieties include antibodies and specific
antigens (e.g. biotin/streptavidin).
[0060] In some embodiments, the targeting moiety is a cytokine or
chemokine suitable for targeting the microbubbles to cells
expressing a corresponding receptor. Examples of suitable ligands
are provided in Hu et al., J. Controlled Release, 147 (2010)
154-162. Such ligands may be incorporated into the microbubbles via
attachment to a bubble-forming material, or may be used as a
bubble-forming material and thereby incorporated directly into the
microbubbles. Examples of suitable receptors that can be targeted
in this manner are also reported in Hu et al.
[0061] In some embodiments, the targeting moiety does not provide
targeting per se, but provides one or more functional properties.
For example, functionalizing markers include metal complexes, spin
labels, and fluorescent tags or radioactive labels to enhance
identification with routine radiographic, ultrasound or magnetic
resonance imaging. Such functional moieties are particularly
suitable where the microbubbles are intended for direct
implantation at or near the target. In some embodiments, a
combination of functional moieties and targeting moieties are
used.
[0062] In some embodiments, the anchoring moiety is selected from
bio-lipids (e.g. phospholipids), surfactants, proteins (e.g.,
denatured human serum albumin), or biocompatible synthetic
polymers, or combinations thereof.
[0063] For example, the anchoring moiety may be a synthetic
polymer. Some examples of suitable polymers include PEG,
polylactide, polyglycolide polyacrylates, polymethacrylates, and
vinyl polymers such as polystyrene, as well as co-polymers thereof
(e.g., poly(lactide-co-glycolide)). The structure and molecular
weight of the polymer can be adjusted based on the desired
application. In some embodiments, the molecular weight of the
polymer is less than about 10,000 Da, or less than about 5000 Da,
or less than about 1000 Da. In some embodiments, the molecular
weight of the polymer is greater than about 1000 Da, or greater
than about 5000 Da, or greater than about 10,000 Da. The polymer
may be linear or non-linear, such as branched or comb-like.
[0064] In some embodiments, the anchoring moiety is a surfactant or
phospholipid that further comprises an attached polymeric moiety.
In some such embodiments, the polymeric moiety functions as a
linker that links the targeting moiety to the anchoring moiety. For
example, PEG moieties of various lengths (e.g., 1-30 repeat units
as described above) can serve to provide a flexible linker
moiety.
[0065] Some examples of additional moieties that may be included in
the bubble-forming material include lipids and steroids. For
example, a cholesterol moiety may be included as described
below.
[0066] In some embodiments, the targeting moiety is chemically
attached to the anchoring moiety. Such chemical attachment includes
attachment via a covalent, ionic, or hydrogen bond. In some
embodiments, as described previously, a linking moiety is present,
and the targeting moiety and anchoring moiety are indirectly
chemically attached via the linking moiety.
[0067] Chemical attachment (also referred to as conjugation) of the
targeting moiety to the anchoring moiety (either directly or via a
linking moiety) may be carried out using any of the methods
described herein, as well as standard synthetic methods such as via
the use of thioether, amide, or disulfide bonding. The conjugation
reaction may be carried out prior to or after bubble formation.
[0068] In some embodiments, each molecule of bubble-forming
material contains a single targeting moiety, whereas in other
embodiments each molecule contains a plurality of targeting
moieties. For example, a bubble-forming material prepared from a
branched polymer may contain numerous targeting moieties (e.g., one
targeting moiety at the end of each branch in the polymer).
[0069] In some embodiments, the microbubbles are prepared from a
single bubble-forming material such as those described above. In
such embodiments, each microbubble contains at least as many
targeting moieties as individual molecules, because each
bubble-forming molecule contains at least one targeting moiety.
[0070] In some embodiments, the microbubbles are prepared from a
mixture of materials. In some such embodiments, one or more of the
bubble-forming materials may be functionalized with a targeting
moiety, while one or more of the bubble-forming materials does not
contain a targeting moiety. By mixing functionalized with
un-functionalized bubble-forming materials in this manner, the
density of targeting moieties on each microbubble can be adjusted
as desired.
[0071] In some embodiments, the targeting moieties are disposed
exclusively on the exterior surface of the microbubbles. In other
embodiments, some or all of the targeting moieties are disposed
beneath the exterior surface of the microbubbles. It will be
appreciated that the location of the targeting moieties may be
dependent upon environmental conditions such as solvent polarity,
pH, ionic strength, etc., and may change with changing
conditions.
[0072] An alternative method for manufacturing medical bubbles
incorporates techniques used in fabrication of titanium
micro-electromechanical systems (MEMS). MEMS technology is used to
form shaped spheres. The fabrication process uses well established
micro-processing techniques.
[0073] Suitable methods for storage of the bubbles are determined
according to properties and applications of specific bubbles and
may require water, surfactant, oil or other medium.
[0074] In one particular example, the bubble-forming material is a
bisphosphonate having the structure shown below:
##STR00001##
[0075] In this example, the PEG chain lengths may be varied from 1
to 30 or greater. This material may be synthesized analogously to
the procedure outline in Bhushan et al., Angewandte Chemie
International Edition 2007, 46, 7969-7971. A similar example is a
cholesterol derivative containing a phosphonate moiety with the
following structure:
##STR00002##
[0076] In another specific example, the microbubbles are formed
from a lipid shell. Between about 1% and about 25% of the lipid
molecules are covalently attached to polymer molecules, with the
percentage being selected based on a variety of factors such as
polymer molecular weight and the identity of the microbubble
components. The polymer molecules form a stabilizing layer around
the shell. Some or all of the stabilizing polymer molecules contain
an attached targeting moiety that is suitable for the desired
application. For example, the material described in Deelman et al.,
Adv. Drug Delivery Rev. 62 (2010) 1369-1377 can be modified
according to the procedures disclosed herein in order to contain
appropriate targeting moieties.
[0077] As illustrated in the Examples included below, solutions of
microbubbles may be prepared by combining the bubble-forming
material with a solvent and then applying energy to induce bubble
formation. In some embodiments, such energy is applied in the form
of mechanical (vibrational) energy by shaking or otherwise mixing
the solution. In some embodiments, such energy is applied in the
form of ultrasound energy sufficient to induce bubble formation
(but not sufficient to induce cavitation). As used herein, the term
"pre-bubble solution" refers to a solution comprising a
bubble-forming material and a solvent prior to the application of
energy sufficient to induce bubble formation. It will be
appreciated, however, that a solution of microbubbles may, over
time, revert back to the state of the pre-bubble solution (i.e.,
where bubble-forming material is present but no bubbles are
present). It will further be appreciated that the microbubbles can
be re-formed by applying additional bubble-forming energy.
Formulation
[0078] The manufactured bubbles can be prepared for introduction to
a human patient, for example by injection, spray, implantation, or
the like. As required for medical applications, bubbles are
prepared as effective amounts in a pharmaceutical preparation in a
pharmaceutically acceptable carrier.
[0079] In some embodiments, the microbubble product is prepared for
introduction to a patient or subject. The product may be dispersed
in fluid for injection or formulated as an aerosol spray for
introduction near the target.
[0080] In some embodiments, the microbubbles are prepared as a
slurry or emulsion suitable for injection, administration via an
aerosol spray, or introduction via a catheter.
[0081] In addition to the microbubbles and a pharmaceutically
acceptable carrier, various other agents may be added to the
formulations as desired. In some embodiments, one or more
surfactants are included in the formulation. In other embodiments,
no surfactants are added to the microbubble formulation. Other
additives that may be present include pH-modifying agents,
preservatives, labeling compounds and/or image enhancing compounds,
salts, and the like.
Applications
[0082] The methods and materials described herein are appropriate
for many applications. For example, medicinal bubbles as disclosed
herein are suitable to be used in both human and animal medicine as
well as in experimental models.
[0083] In some embodiments, the methods and materials of interest
provide minimally invasive treatment of medical conditions,
including treatments that do not require expensive and bulky
equipment for administration to a patient.
[0084] A large number of medical conditions are characterized at
least in part by the presence of an abnormal mass. Examples include
urinary stones, biliary stones, blood clots, fibroids, cancerous
tumors, and atheromatous plaques. The methods and materials
described herein provide destruction or reduction of the mass with
minimal injury to healthy tissue and thus provide therapeutic
benefit. The therapeutic methods are minimally invasive and are
characterized by reduced pain, discomfort, and risks that are
associated with open surgical or other invasive therapies.
[0085] In a specific example, the methods and materials disclosed
herein are suitable for the treatment of kidney stones. In one
example of such treatment, targeting microbubbles are injected into
the ureter, and upon binding to the kidney stone, ultrasound is
applied either locally or via an extracorporeal source to cause
cavitation of the microbubbles. This cavitation breaks apart the
kidney stone into small particles that can be released by the body,
for example following the administration of a diuretic.
[0086] The following are additional examples of uses of the
materials and methods disclosed herein. Such examples are not
intended as a limitation on the invention.
[0087] A microbubble solution may be prepared for injection into
excess adipose tissue to remodel or destroy intended targets.
[0088] A microbubble solution may be prepared for injection into
the lens capsule for subsequent removal in cataract surgery.
[0089] A microbubble solution may be prepared for injection into
joints to destroy offending cartilage or to facilitate remodeling
of bone.
[0090] A microbubble solution may be prepared for injection into
blood stream to target and lyse occlusive blood clots.
[0091] A microbubble solution may be prepared for injection into
blood streams to target and fracture atheromatous plaques.
[0092] A microbubble solution may be prepared for injection into
targeted tissue to facilitate fenestration.
[0093] A microbubble solution may be prepared for injection into
posterior pharynx to induce scarring to alleviate sleep apnea.
[0094] A microbubble solution may be prepared for injection into
mammary tissue to facilitate breast reductions.
[0095] A microbubble solution may be prepared for injection into
reproductive tract to facilitate sterilization.
[0096] A microbubble solution may be prepared for ex-vivo
applications to target selected sperm (male vs. female).
EXAMPLES
Example 1
Synthesis of Targeting Microbubbles
[0097] A solution of microbubbles is synthesized according to the
following procedure.
[0098] First, a modified bisphosphonate lipid is synthesized. The
amine group of compound 1, which is commercially available, is
protected as shown to give compound 2. The phosphonate hydroxyl of
2 will subsequently be methylated to yield 3, which is reacted with
trifluoroacetic acid in methylene chloride to generate product
4.
##STR00003## ##STR00004##
[0099] Concurrently, compound 5, which is commercially available,
is reacted to form 6, which is directly carried on to form compound
7. This procedure is done following a method discussed in Bhushan
et al., Angewandte Chemie Chemie Int. Ed. 2007, 46, 7969-7971.
##STR00005##
[0100] Products 4 and 7 are then coupled together in the presence
of base and heat to yield compound 8. Deprotection of the methoxy
functionalities to hydroxyl functionalities affords target product
9.
##STR00006## ##STR00007##
[0101] The microbubble solution is then prepared as follows. The
concentrations and solutions are prepared to mimic "Definity"
microbubbles (see Example 2 for general procedure). For a 50 mL
solution of the proper concentrations, a buffer solution is made as
follows. Propylene glycol (5.1750 g), glycerin (6.3100 g),
NaPhosphate monobasic.times.1H2O (0.1170 g), NaPhosphate dibasic
10-hydrate (0.1080 g), and NaCl (0.2435 g) are combined with 25 mL
of distilled water, measured with a volumetric flask. The lipid
solution is prepared in chloroform. Ten mL stock solutions of each
of the lipids are made in chloroform, and a 25 mL solution is made
from these stock solutions. For DPPA, 4.5 mg is combined with 10 mL
of chloroform in a 10 mL volumetric flask. For lipid 9, 26.67 mg is
added with 10 mL of chloroform in a 10 mL volumetric flask, and
likewise for MPEG5000 DPPE, 30.4 mg is combined with 10 mL of
chloroform in a 10 mL volumetric flask. Subsequently 5 mL of each
of the stock solutions is combined, and this combined solution is
diluted to a 25 mL volume of chloroform in a 25 mL volumetric
flask. This 25 mL lipid solution will then be added with the 25 mL
buffer solution to make the final microbubble solution in a total
volume of 50 mL. The solution is distributed to vials, and the
headspace is filled with octafluoropropane gas through a septum
cap. The vials is sealed and stored at a cool temperature. The
microbubbles are generated when desired by shaking using a Vialmix
shaker or an equivalent shaker.
[0102] In an alternative synthesis, targeting microbubble materials
are prepared according to the following scheme:
##STR00008## ##STR00009## ##STR00010##
Example 2
Synthesis of Targeting Microbubbles
[0103] A solution of microbubbles (untagged) was synthesized
according to the following procedure.
[0104] Material Source Information: For MPEG 5000 DPPE, the sodium
salt was used rather than the ammonium salt. Source: Genzyme
Pharmaceuticals. DPPA: 1,2-dipalmitoyl-sn-glycero-3-phosphate,
monosodium salt. Source: Avanti Polar Lipids. DPPC:
1,2-dipalmitoyl-rac-glycero-3-phosphocholine hydrate, approx 99%.
Source: Sigma.
[0105] Combined Phospholipids 2.times.Stock Solution (in water):
Prepared according to the following table.
TABLE-US-00001 Mg in 50 mL Lipids Conc. In Definity of 2X stock
soln. DPPA 0.45 mg/mL 4.5 mg DPPC 0.401 mg/mL 40.1 mg MPEG5000 DPPE
0.304 mg/mL 30.4 mg
[0106] The lipids were dissolved in water by heating to between
60-80 .degree. C. The lipid solution was stored at 3.degree. C.
after preparation.
[0107] Buffer Solution: Prepared according to the following
table.
TABLE-US-00002 Material Conc. in Definity Conc. in 2X stock
Propylene glycol 103.5 mg/mL 207.0 mg/mL Glycerin 26.2 mg/mL 252.4
mg/mL NaPhosphate monobasic x 1H2O 2.34 mg/mL 4.68 mg/mL
NaPhosphate dibasic, 10-hydrate 2.16 mg/mL 4.32 mg/mL NaCl 4.87
mg/mL 9.74 mg/mL Water to 50 mL
[0108] The buffer solution was prepared at room temperature. 400
.mu.L of lipid mix stock solution and 400 .mu.L of buffer solution
were transferred to amber vials, which were then sealed with
silicone injection septum and screw cap. The vial headspace was
flushed with octafluoropropane. As needed, the vial is shaken 30
seconds in VialMix shaker at reduced temperature.
[0109] Additional details for preparing Definity microbubbles
(untagged) are found in the procedure reported in Unger et al.,
Adv. Drug Delivery Rev., 56 (2004) 1291-1314, and references cited
therein.
Example 3
Synthesis of Targeting Microbubbles via Cross-Metathesis
[0110] Olefin metathesis is extensively used to rearrange
carbon-carbon double bonds in various organic syntheses.
Especially, the second generation Grubbs catalysts, which are
ruthenium atom centered and contain saturated mesityl-substituted
N-heterocyclic carbene ligand, is suitable to prepare proposed
microbubble chemical structures due to high catalytic efficiency
together with high tolerance of diverse functional groups, organic
solvents in the air. The chemical tag, bisphosphonate, can be
introduced to the phospholipid, bubble-forming material, by two
synthetic routes.
[0111] In the first method, the carbon double bond containing
phospholipid, 5, and bisphosphonate containing structure, 2, are
synthesized separately to produce product 2 and 5. Cross-metathesis
can be performed between product 5 and product 2 in the presence of
2nd generation Grubbs catalyst. The cross-metathesis can prevent
possible homodimerization of product 2 due to poor electron density
of carbon double bond of 2. Therefore, this selective
cross-metathesis will help to avoid additional purification step to
separate desired product 6 and undesired byproduct, homodimerized
products.
##STR00011## ##STR00012##
[0112] An alternative synthetic method to produce bisphosphonate
containing phospholipid is shown in the following scheme. Unlike
previous synthetic route, cross-metathesis reaction was carried out
prior to introduction of bisphosphonate to phospholipids. Highly
efficient reaction between NHS and primary amine can produce
compound 8 without other undesired products.
##STR00013## ##STR00014##
Example 4
In Vivo Administration of Microbubbles and Urinary Stone
Fragmentation
[0113] Rats (n=2) were anesthetized and a small intramuscular
pocket was developed to instill 1.5-2.0 ml of microbubbles followed
by placement of a single urinary stone composed of calcium
phosphate and calcium oxalate (1.5 grams). This was repeated in a
remote location with the same animal with a new stone fragment of
the same composition. The intramuscular pockets were closed with
3.0 dexon sutures and the skin was closed with sub-cuticular
sutures. A 7.5 mHz ultrasonic transducer was then applied to the
skin with coupling gel. The stone and microbubbles were easily
identified. Ultrasonic energy was applied for 15-20 minutes with
direct visualization of both the stone and associated microbubbles.
The stone was retrieved and post-procedure stone weight decreased
by approximately 0.2 gram (dry then wet weight). Gross
visualization revealed significant pitting on the stone surface
demonstrating proof of concept of urinary stone fragmentation with
microbubbles activated by ultrasonic energy. An image of the stone
is provided in FIG. 1.
Example 5
Attachment of Pendant Linker Groups to Attach Microbubbles to
Calcium-Containing Materials
[0114] A series of experiments were conducted using chemistry in
which the anchoring moiety and the targeting moiety are linked via
an ionic charge bond, demonstrating that microbubbles comprising,
e.g., phosphonate coating structures can be appended to, e.g.,
calcium-containing materials (in this case, kidney stones) which
have been modified to present a cationic surface (in this case,
acid or quarternary ammonium salts) using the methods described
herein. This principle is illustrated in FIG. 2.
[0115] In one set of experiments, a kidney stone (calculous) was
treated with 0.1 M HCl, as shown pictorially in FIG. 3A, thus
generating a positive charge on the surface of the stone
(calculous). When placed in the presence of microbubbles containing
a negatively charged phospholipid coating (DEFINITY.TM.
microbubbles), the microbubbles were shown to adhere to the
positively charged stones (see, e.g., FIG. 4A). Upon application of
ultrasonic energy, the microbubbles exhibited cavitation (FIG. 4B),
after which the stones where shown to exhibit fracture damage (FIG.
4C).
[0116] A similar strategy may use targeting groups linked to
cationic residues, thereby providing positively charged pendants
attached to the metal-containing, especially calcium-containing
materials which are attachable to microbubbles via ionic bonding to
negatively charged microbubbles (e.g., pictorially shown in FIG.
3B).
[0117] One such synthetic scheme available, using the methods
described herein, involves the preparation of a quaternary ammonium
compound based on, for example, alendronic acid:
##STR00015##
The bisphosphonate moiety (in this case, bisphophonic acid) has
been shown to exhibit a sufficiently strong attraction to kidney
stones to withstand cavitation and fracture of the stone.
[0118] Additional materials are available from related starting
materials, using chemistries recognized by the skilled artisan:
##STR00016##
[0119] Calcium-containing materials may be treated by such
quaternary ammonium compounds either before microbubble injection
or simultaneously with microbubble injection.
Example 6
Synthesis of Microbubbles Using Small Molecule Cancerous Tumor Cell
Specific Ligands
[0120] Microbubbles can also be prepared so as to target tumors
using small molecule targeting moieties, including small molecule
cancerous tumor cell specific ligands. Such microbubbles can
optionally comprise therapeutic pharmaceutical agents, or can be
used simply to direct ultrasonic energy to damage or break-up the
calculous or tumor body. There are many examples of small molecule
cell specific agents, though none of these appear to have been used
as described in the present context. In this non-limiting example,
folate--which is known to be a very selective receptor to cancerous
tumor and it is not harmful to healthy cells.--is shown to be
incorporated into a phospholipid moiety as the target of the
microbubble structure. An exemplary synthetic scheme is shown
below.
##STR00017## ##STR00018##
[0121] It should be appreciated that other small molecule targeting
moieties may be similarly attached, by methods known by the skilled
artisan, using the teachings described herein, and these are
considered within the scope of the present invention.
[0122] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, that the foregoing description and the examples that
follow are intended to illustrate and not limit the scope of the
invention. It will be understood by those skilled in the art that
various changes may be made and equivalents may be substituted
without departing from the scope of the invention, and further that
other aspects, advantages and modifications will be apparent to
those skilled in the art to which the invention pertains. In
addition to the embodiments described herein, the present invention
contemplates and claims those inventions resulting from the
combination of features of the invention cited herein and those of
the cited prior art references which complement the features of the
present invention. Similarly, it will be appreciated that any
described material, feature, or article may be used in combination
with any other material, feature, or article, and such combinations
are considered within the scope of this invention.
[0123] The disclosures of each patent, patent application, and
publication cited or described in this document are hereby
incorporated herein by reference, each in its entirety, for all
purposes.
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