U.S. patent number 5,215,680 [Application Number 07/550,620] was granted by the patent office on 1993-06-01 for method for the production of medical-grade lipid-coated microbubbles, paramagnetic labeling of such microbubbles and therapeutic uses of microbubbles.
This patent grant is currently assigned to Cavitation-Control Technology, Inc.. Invention is credited to Joseph S. D'Arrigo.
United States Patent |
5,215,680 |
D'Arrigo |
June 1, 1993 |
**Please see images for:
( Certificate of Correction ) ** |
Method for the production of medical-grade lipid-coated
microbubbles, paramagnetic labeling of such microbubbles and
therapeutic uses of microbubbles
Abstract
This invention relates to a large scale method for the
production of medical grade lipid-coated microbubbles, to the
paramagnetic labeling of such microbubbles and to therapeutic
applications for the microbubbles. More particularly, the invention
relates to a method of the production of medical grade,
concentrated suspensions of stable, paramagnetically derivatized or
underivatized microbubbles useful for ultrasonic and magnetic
resonance imaging and also relates to therapeutic interventions
such as selective tumor destruction.
Inventors: |
D'Arrigo; Joseph S.
(Farmington, CT) |
Assignee: |
Cavitation-Control Technology,
Inc. (Farmington, CT)
|
Family
ID: |
24197921 |
Appl.
No.: |
07/550,620 |
Filed: |
July 10, 1990 |
Current U.S.
Class: |
516/11; 424/405;
424/9.321; 424/9.34; 436/173; 514/938; 516/18; 516/19; 600/410;
600/458 |
Current CPC
Class: |
A61K
9/5015 (20130101); A61K 41/0047 (20130101); A61K
49/18 (20130101); A61K 49/1815 (20130101); A61K
49/223 (20130101); Y10S 514/938 (20130101); Y10T
436/24 (20150115) |
Current International
Class: |
A61K
49/06 (20060101); A61K 49/18 (20060101); A61K
41/00 (20060101); A61K 9/50 (20060101); A61K
49/22 (20060101); B01J 013/00 (); A61K 049/00 ();
A61K 009/107 (); B01F 003/04 () |
Field of
Search: |
;424/9 ;436/173
;128/662.02 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Carr et al, "Intravenous Chelated Gadolinium As A Contrast Agent In
NMR Imaging Of Cerebral Tumors", Lancet, pp. 484-486 (Mar. 3,
1984). .
Lauffer et al, "Preparation and Water Relaxation Properties of
Proteins Labeled with Paramagnetic Metal Chelates", Mag. Res.
Imaging, 3:11-16 (1985). .
Lauffer et al, "1/T.sub.1 NMRD Profiles of Solutions of Mn.sup.2+
and Gd.sup.3+ Protein-Chelate Conjugates", Mag. Res. Med.,
3:541-548 (1986). .
Quan et al, "Applicators for generating ultrasound-induced
hyperthermia in neoplastic tumours and for use in ultrasound
physiotherapy", Phys. Med. Biol., 34:1719-1731 (1989). .
Runge et al, "Initial Clinical Evaluation of Gadolinium DTPA For
Contrast-Enhanced Magnetic Resonance Imaging", Mag. Res. Imaging,
3:27-35 (1985). .
ter Haar et al, "High intensity focused ultrasound--a surgical
technique for the treatment of discrete liver tumours", Phys. Med.
Biol., 34:1743-50 (1989)..
|
Primary Examiner: Lovering; Richard D.
Assistant Examiner: Covert; John M.
Attorney, Agent or Firm: Kramer, Brufsky & Cifelli
Government Interests
This invention was made with Government support under SBIR Phase II
Grant No. 2 R44 NS25851-02 awarded by the National Institute of
Neurological Disorders and Stroke, National Institutes of Health.
The Government has certain rights in the invention.
Claims
What is claimed is:
1. A method for preparing an imaging about suitable for enhancement
of ultrasonic imaging and magnetic resonance imaging comprising the
steps of:
A. obtaining a moderately hydrophobic neutral amino acid
homopolymer or copolymer which is capable of readily incorporating
into a lipid monolayer, and is labeled with a paramagnetic complex
comprising a metal ion and organic chelating ligand;
B. dissolving the labeled, hydrophobic polymer in a saline solution
in an amount of about 10 mM;
C. adding a surfactant mixture comprising:
(a) a member selected from the group consisting of glycerol
monoesters of saturated carboxylic acids containing from about 10
to about 18 carbon atoms and aliphatic alcohols containing from
about 10 to about 18 carbon atoms;
(b) a sterol-aromatic acid ester;
(c) a member selected from the group consisting of sterols,
terpenes, bile acids and alkali metal salts of bile acids;
(d) a member selected from the group consisting of sterol esters of
aliphatic acids containing from one to about 18 carbon atoms;
sterol esters of sugar acids; esters of sugar acids and aliphatic
alcohols containing from about 10 to about 18 carbon atoms, esters
of sugars and aliphatic acids containing from about 10 to about 18
carbon atoms; sugar acids, saponins; and sapogenins; and
(e) a member selected from the group consisting of glycerol,
glycerol di or triesters of aliphatic acids containing from about
10 to about 18 carbon atoms and aliphatic alcohols containing from
about 10 to about 18 carbon atoms;
D. shaking said solution mechanically for from about 2 to about 10
seconds in gaseous atmosphere at room temperature, thereby forming
a concentrated gas-in-liquid emulsion; and
E. passing the solution obtained after step (D) through a sterile
polysulfone membrane filter having an average pore diameter of
about 0.40-6.0 .mu.m.
2. A method according to claim 1, further comprising the steps of
allowing undissolved lipid materials to settle out for a period of
about 15-60 minutes before passing the solution through the
polysulfone membrane filter.
3. A method according to claim 2, wherein the sterile polysulfone
membrane filter has an average pore diameter of about 0.40-0.60
.mu.m.
4. A method according to claim 3, wherein the sterile polysulfone
membrane filter has an average pore diameter of about 45 .mu.m.
5. A method according to claim 3, wherein the moderately
hydrophobic polymer is selected from the group consisting of
homopolymers of valine, glycine and alanine and copolymers
thereof.
6. A method according to claim 5, wherein the paramagnetic complex
is selected from the group consisting of gadolinium DTPA, manganese
DTPA, gadolinium EDTA and manganese EDTA.
7. A method according to claim 6 wherein the paramagnetic complex
is gadolinium DTPA.
8. A method according to claim 7, wherein the surfactant mixture
components are present in the surfactant mixture in a weight ratio
a:b:c:d:e of about 2-4:0.5-1.5:0.5-1.5:0.5-1.5:0-1.5.
9. A method according to claim 8, wherein the surfactant mixture
consists essentially of glycerol monolaurate, cholesterol benzoate,
cholesterol, cholesterol acetate and glycerol tripalmitate in a
weight ratio of 2-4:1:1:1:1.
10. A method according to claim 1, further comprising the steps of
mechanically shaking the solution at least two more times before
passing the solution through the polysulfone membrane filter.
Description
FIELD OF THE INVENTION
This invention relates to methods for the production of medical
grade lipid-coated microbubbles, to the paramagnetic labeling of
such microbubbles and to therapeutic applications for the
microbubbles. More particularly, the invention relates to methods
for the production of medical grade, concentrated suspensions of
stable, paramagnetically derivatized or underivatized microbubbles
useful for ultrasonic and magnetic resonance imaging and also for
therapeutic interventions such as selective tumor destruction.
BACKGROUND OF THE INVENTION
Various technologies exist in which parts of an animal or human
body may be imaged so as to aid in diagnosis and therapy of medical
disorders. One of techniques that is now widely used in diagnostic
imaging various parts of the body is ultrasound. This technique
involves the use of an ultrasound transducer to generate and
receive sound waves. The transducer is placed on the body surface
over an area to be imaged and sound waves generated by the
transducer are directed to that area. The transducer then detects
sound waves reflected from the underlying area and translates the
data into images.
The basis for ultrasound imaging is that, when ultrasonic energy is
transmitted through a substance, the acoustic properties of the
substance will depend upon the velocity of the transmissions and
the density of the substance. Changes in the substance's acoustic
properties will be most prominent at the interface of different
substances (i.e., solids, liquids and gases). As a consequence,
when ultrasound energy is directed through various media, the
changes in acoustic properties at such interfaces will change the
reflection characteristics, resulting in a more intense sound
reflection signal received by the ultrasound transducer.
Early ultrasound techniques suffered from a lack of clarity. As a
result, extensive efforts were undertaken to improve the ultrasonic
equipment. In addition, contrast agents were introduced into the
bloodstream in an effort to obtain enhanced images. Many of these
contrast agents were liquids containing microbubbles of gas. These
contrast agents themselves are intense sound wave reflectors
because of acoustic differences between the liquids and the gas
microbubbles enclosed therein. Hence, when these contrast agents
are injected into the bloodstream and perfuse the microvasculature
of the tissue, clearer images of the tissue may be produced.
A number of different contrast agents are known in the art. For
example, Feinstein discloses microbubbles formed from protein
solutions, such as those formed from albumin, in U.S. Pat. No.
4,774,958. Microbubbles formed from gelatin are described as
suitable contrast agents in U.S. Pat. No. 4,276,885. U.S. Pat. No.
4,684,479 discloses lipid-coated microbubbles which, because of
their excellent in vitro stability, were suspected and recently
confirmed to be very long-lived in vivo and, hence, are
particularly well suited for diagnostic and therapeutic ultrasound
applications. The method for preparing the microbubbles disclosed
in the '479 patent is not, however, sufficient to allow for the
large scale production of medical grade microbubbles.
One of the limitations of diagnostic ultrasound is that it is of
very limited use preoperatively in neurosurgical applications
because of the presence of bone, and in particular, the skull.
Accordingly, magnetic resonance imaging ("MRI"), which is also
quite sensitive to tissue pathology, has been rapidly accepted as a
technique for neurological diagnosis. MRI of the brain is conducted
preoperatively, to provide an image which the surgeon can then
consult during an operation.
Initially, MRI was conducted without the aid of a contrast agent.
However, the poor specificity of MRI in neurological diseases soon
became evident. Contrast agents are now also available to enhance
MRI imaging. The best known MRI contrast agents are paramagnetic
metal ion chelates with low toxicity. These include manganese, iron
and gadolinium metal ions which have been chelated with
diethylenetriaminepentaacetic acid or ethylenediaminetetraacetic
acid. See, for example, Carr et al, The Lancet, 1:484-486 (1984);
Runge et al, Magnetic Resonance Imaging, 3:27-35 (1985); Lauffer et
al, Magnetic Resonance Imaging, 3:11-16 (1985); and Lauffer et al,
Magnetic Resonance Imaging, 3:541-548 (1986).
The foregoing techniques provide medical personnel with the ability
to obtain accurate images under a broad range of conditions. There
is, however, still a need for a contrast agent which could be-used
for both ultrasonic imaging and MRI. For example, while MRI is the
method of choice in neurological preoperative diagnosis, real time
imaging with MRI during a surgical procedure is not possible. This
is because of the massive size of the equipment required for MRI.
Yet real time imaging during surgery is often desirable,
particularly when the surgeon has reason to believe there has been
a shift in position of tissue due to invasion by the surgical
procedure and/or change in intracranial pressure. Although
ultrasound imaging can be performed during surgery, the current
unavailability of a contrast agent which can be used in both
ultrasonic imaging and MRI renders anatomical correlation between
the preoperative and operative images less reliable.
In addition to diagnostic applications, low frequency ultrasound
has also been used therapeutically by physiotherapists to treat a
variety conditions. Ultrasound is now also being investigated in
the treatment of malignant tumors, through the effects of heat and
cavitation. Quan et al, Phys. Med. Biol., 34:1719-1731 (1989)
describe a five element ultrasound transducer array potentially
useful in the treatment of malignant tumors through the effects of
heat. When heat is used in tumor destruction, the tumors are heated
to a temperature between 42.degree. and 45.degree. C., producing
cellular damage. ter Haar et al, Phys. Med. Biol., 34:1743-1750
(1989), discuss the potential use of high intensity, focused
ultrasound in the selective destruction of tumors, without damage
to intervening tissues. Heretofore, no one has suggested a method
of enhancing the effects of ultrasound through the use of
gas-in-liquid microbubbles.
Accordingly, it is an object of the present invention to provide a
process from the production of concentrated suspensions of medical
grade, lipid-coated microbubbles.
Another object of the present invention is to provide
paramagnetically-labeled lipid coated microbubbles suitable for use
in ultrasonic imaging and MRI.
A still further object of the present invention is to provide a
method for enhancing the selective destruction of tumors by
ultrasound through the use of lipid-coated microbubbles.
SUMMARY OF THE INVENTION
These as well as other objects and advantages are achieved in
accordance with the present invention, as described hereinafter. In
one embodiment, the invention provides methods for the production
of underivatized lipid-coated microbubbles suitable for use in
medical applications. In another embodiment, the invention provides
paramagnetically labeled lipid-coated microbubbles useful in
ultrasonic and MRI imaging. In yet another embodiment, the present
invention is directed to a novel therapeutic use of the
lipid-coated microbubbles in the selective destruction of tumors
through the effects of heat and cavitation.
The composition of the lipid-coated microbubbles has previously
been described in U.S. Pat. No. 4,684,479. These microbubbles are
particularly suited for use in diagnostic and therapeutic medical
applications because of their small size and excellent long term
stability. When produced in accordance with the improved method
described herein, they are small enough to pass through capillaries
so as to perfuse tissue. Also, the resulting emulsion is
sufficiently free from solid lipid materials and other particulate
impurities to be particularly suited for medical applications.
The methods of producing the microbubbles differ from the methods
described in the '479 patent, in that the gas-in-liquid emulsion
which forms the microbubbles is saline based. In addition, the
methods employed herein require the filter sterilization of the
emulsion for use in medical applications, to remove particulate and
supracolloidal material which otherwise might become lodged in the
capillaries during tissue perfusion and cause serious harm.
Although filter sterilization is a method step conventionally used
for pharmaceutical compositions, in accordance with the present
invention, it has surprisingly been found that a very specific
filter, having an average pore size of about 0.40 to about 6.0
.mu.m and having a polysulfone membrane is required to obtain an
emulsion suitable as a contrast agent for imaging. Use of other
filters, outside the scope of the present invention, results in a
large, somewhat variable, statistically significant decrease in
microbubble concentration, and hence a decrease in image
enhancement, which is unacceptable both on functional grounds and
in view of federal regulatory quality-assurance expectations.
Employing the methods of the present invention, microbubbles having
a mean diameter of about 2.0 .mu.m are produced. 99% of the
microbubbles are smaller than 4.5 .mu.m, with 100% being less than
6 .mu.m.
In another embodiment, the invention provides paramagnetically
labelled, lipid-coated microbubbles suitable for use in ultrasonic
imaging and MRI. The paramagnetic label is incorporated into the
microbubbles by first forming a paramagnetic complex covalently
attached to the amino groups of a hydrophobic proteinaceous
polymer, such as polyalanine, and then incorporating the complex
into the lipid monolayer surrounding the microbubble. The
thus-prepared microbubbles can be injected intravenously into an
animal or human body, and then detected using conventional
ultrasdnic or MRI scanning equipment. The advantage of the
derivatized microbubbles is that they permit preoperative
microbubble enhanced MRI images of neurological disorders to be
clearly correlated anatomically with microbubble enhanced
ultrasound images taken during and after neurosurgery. In addition,
simultaneous microbubble enhanced MR and ultrasound images can be
obtained outside of the brain cavity in order to improve the
overall accuracy and information content of the imaging data, e.g.,
as a cross check for the complete removal of primary tumors and
metastases.
In another embodiment, the present invention provides methods for
actual therapeutic intervention with lipid-coated microbubbles.
Such therapeutic applications include the destruction of tumors by
ultrasound heating and/or cavitation of microbubbles at a tumor
site. As shown in the examples herein, it has now been demonstrated
that underivatized, lipid-coated microbubbles, when injected
intravenously into an animal body, will cross disruptions in the
blood/brain barrier and thereby pass into tumors in the animal's
brain. Accordingly, a selective enhancement of the tumor image is
seen during intraoperative ultrasound images of the brain. The
demonstrated pooling of the lipid-coated microbubbles at the tumor
site in the brain indicates that the tumors can be selectively
destroyed through the use of ultrasound, with enhanced destruction
of tumor tissue due to the presence of the gas-in-liquid
microbubbles present at the site.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a histogram graphically illustrating the particle size
distribution of stable lipid-coated microbubbles obtained in
accordance with the method of the present invention, as determined
by electroimpedance-sensed volumetric sizing.
FIG. 2 is a histogram graphically illustrating the particle size
distribution of a lipid-coated microbubble suspension prepared
using a 0.20 .mu.m filter, as determined in FIG. 1.
FIG. 3 is a histogram graphically illustrating the particle size
distribution of a suspension of lipid-coated microbubbles prepared
using a 0.45 .mu.m cellulose-ester membrane filter as also
determined in FIG. 1.
FIGS. 4A and 4B are representative ultrasound images showing that
the lipid-coated microbubbles will cross into and intensify the
ultrasonic image of gliomas in a rat brain. FIG. 4A is an image of
rat cerebral glioma several minutes before intravenous injection of
the lipid-coated microbubbles; FIG. 4B is an image several minutes
after the intravenous injection.
DETAILED DESCRIPTION OF THE INVENTION
The surfactant mixtures employed in accordance with the method of
the present invention to obtain the underivatized and
paramagnetically labeled, lipid-coated microbubbles are best
obtained in powdered form. The surfactant mixture is described in
detail in U.S. Pat. No. 4,684,479, which is hereby incorporated by
reference. Briefly, the surfactant mixture comprises (a) a member
selected from the group consisting of glycerol monoesters of
saturated carboxylic acids containing from about 10 to about 18
carbon atoms and aliphatic alcohols containing from about 10 to
about 18 carbon atoms; (b) a sterol-aromatic acid ester; (c) a
member selected from the group consisting of sterols, terpenes,
bile acids and alkali metal salts of bile acids; (d) a member
selected from the group consisting of sterol esters of aliphatic
acids containing from one to about 18 carbon atoms; sterol esters
of sugar acids; esters of sugar acids and aliphatic alcohols
containing from about 10 to about 18 carbon atoms, esters of sugars
and aliphatic acids containing from about 10 to about 18 carbon
atoms; sugar acids; saponins; and sapogenins; and (e) a member
selected from the group consisting of glycerol, glycerol di or
triesters of aliphatic acids containing from about 10 to about 18
carbon atoms and aliphatic alcohols containing from about 10 to
about 18 carbon atoms, said components being present in the mixture
in a weight ratio of a:b:c:d:e of
2-4:0.5-1.5:0.5-1.5:0.5-1.5:0-1.5:0-1.5. A more complete
description of these components can be found in U.S. Pat. No.
4,684,479. Preferably, the composition is formed from glycerol
monolaurate, cholesterol benzoate, cholesterol, cholesterol
acetate, and glycerol tripalmitate.
The emulsions are obtained by forming at least a substantially
saturated solution of the surfactant mixture in saline based media,
which is generally obtained by mixing from about 0.02 to about 0.4
grams of the surfactant mixture with about 100 cc of saline, The
resulting mixture is then shaken vigorously for about 2 to about 20
seconds, preferably about 10 seconds in air or other gaseous
material at room temperature. After about five minutes, the shaking
is repeated two or more times.
Following the shaking, the solution is preferably allowed to stand
for about 15 to 60 minutes, most preferably about 30 minutes, to
allow the undissolved lipid material to settle out of the solution.
Settling out is not essential, but does tend to prevent clogging of
the filter during the subsequent filtration step.
The resulting solution is then filtered through a sterile
polysulfone membrane filter, having an average pore diameter of
from about 40 .mu.m to about 6.0 .mu.m. Preferably, the polysulfone
membrane filter will have an average pore diameter of from about
0.40 to about 60 .mu.m and most preferably, about 45 .mu.m. The
average pore diameter of the filter used in the filter
sterilization step is critical to obtaining a medical grade
suspension in accordance with the present invention. Below about
0.40 .mu.m, the filter does not allow sufficient reformation of the
microbubbles to obtain suspension useful as an imaging contrast
agent. In particular, as the microbubbles (about 1-5 .mu.m in
diameter) pass through the filter, they are broken down, and, quite
surprisingly, reform after passage through the filter. Reformation
of the microbubbles following passage through the filter is
believed to be due to the cavitation or turbulence which occurs
during passage through the filter. In accordance with the present
invention, it has surprisingly been found that this reformation
only occurs satisfactorily when a polysulfone membrane filter
having an average pore diameter of at least about 0.40 .mu.m is
employed.
Use of a filter above about 6.0 .mu.m will allow the passage of
particulates approaching the size of red blood cells into the
microbubble suspension. This is undesirable because of the
increased possibility of complications due to blockage in the
circulation system. Preferably, the polysulfone membrane filter
will have an average pore diameter of about 0.40 to about 0.60
.mu.m. These preferred filters will allow sufficient reformation of
the microbubbles to provide a useful contrast agent, and will also
reduce the size of particulates in the suspension, which is most
desirable for medical and pharmaceutical applications.
Reformation is particularly enhanced when the filter is a 0.45
.mu.m, sterile polysulfone membrane filter, which has low
absorptivity for the microbubbles. Other low absorption materials
could similarly be employed. Preferred polysulfone membrane filters
are available commercially and can be obtained from Gelman
Sciences, Ann Arbor, MI.
The microbubble suspension obtained in accordance with the
inventive method remains stable for at least nine months.
Typically, the microbubble concentration in the suspension is about
540,000 bubbles .+-.15% per milliliter. Maximum bubble diameter
remains under 6 .mu.m. Particle size analysis is best determined by
electroimpedance-sensed volumetric sizing using, for example, a
Coulter Multisizer in conjunction with Coulter's AccuComp data
handling software. Using this method of analysis, it can be
consistently determined that the mean microbubble diameter of the
microbubbles produced in accordance with the present invention is
about 2 .mu.m. Over 99% of the microbubble population is under 4.5
.mu.m. 100% of the microbubbles are under 6 .mu.m in diameter,
which is an ideal limit for contrast agents of this nature.
The paramagnetic complexes that can be employed in accordance with
the present invention can be any one of several previously
described in the scientific literature for use as contrast agents
in MRI. For example, gadolinium (Gd.sup.3+) or manganese
(Mn.sup.2+) complexed with multidentate ligands, either
ethylenediaminetetraacetic acid (EDTA) or
diethylenetriaminepentaacetic acid (DTPA) have all previously been
used as MRI contrast agents. The Gd-DTPA complex is particularly
preferred, since it has been approved by the federal Food and Drug
Administration and has been approved for use in humans for the MRI
detection of tumors. In addition, the Gd-DTPA complex can readily
be attached to the free amine groups of different proteins, which
increases the ability of the complex in enhancing MRI 2 to 1 fold.
This effect stems from the decrease in molecular motion that occurs
upon attachment to a larger molecule. (Lauffer et al, Magnetic
Resonance Imaging, 3:541-548 (1986).)
In accordance with the present invention, the enhanced MRI contrast
can be mimicked and may in fact be further enhanced by the
association of the paramagnetic complex with the even larger
lipid-coated microbubbles. This association is achieved by first
covalently bonding the hydrophilic paramagnetic complex, e.g.,
Gd-DTPA, to a hydrophobic proteinaceous homopolymer or copolymer,
after which the surface-active, paramagnetic Gd-DTPA derivative is
associated with the lipid monolayer surrounding the stable
microbubbles.
The hydrophobic proteinaceous polymer should be water soluble, to
facilitate the synthesis of the paramagnetic surfactant. In
addition, it is important that the polymer be moderately
hydrophobic, so that it will readily incorporate into the lipid
monolayer surrounding the microbubbles. Particularly suitable
proteinaceous polymers are moderately hydrophobic, neutral amino
acid homopolymers, such as those formed from glycine, valine and
alanine and copolymers thereof. Polyalanine, having a molecular
weight of about 1000 to 5000 daltons, is particularly
preferred.
The method of synthesis of the surface active paramagnetic complex
is described in further detail in the examples herein. Preferably,
the surface active paramagnetic complex is obtained as a
lyophilized powder, which is then incorporated into the lipid
monolayer of the microbubbles by adding the derivative to the
powdered lipid-surfactant mixture in an amount of about 5-10% w/w.
Upon shaking in isotonic saline, the paramagnetic-labeled surface
active derivative readily incorporates into the monolayer
surrounding the microbubbles, with the paramagnetic label remaining
exposed to the aqueous exterior.
The paramagnetically labeled microbubbles of the present invention
are useful in the enhancement of both ultrasonic imaging and MRI.
The labeled microbubbles will be injected into the human patient or
animal intravenously in standard diagnostic doses and then imaged
using conventional imaging equipment. These
paramagnetically-labeled microbubbles should allow the neurosurgeon
to look at, in the operating room using real time ultrasonic
imaging, precisely what he or she looked at using MRI
preoperatively.
Image mapping procedures can be employed to correlate the
ultrasonic and MRI images. Standard image mapping procedures are
known and produce maps which allow for the reconstruction of an
ultrasound image from the lesion's actual size and shape. See, for
example, Le Roux et al, Neurosurg., 71:691-698 (1989). Image
mapping procedures may also involve a mapping from the tumor
histology to the MRI image. Using composition or direct mapping,
the relationship between the MRI and ultrasonic images can be
established.
In another embodiment of the present invention, there is provided a
method for enhancing the selective destruction of tumors using the
heating and cavitational effects of ultrasound. This method takes
advantage of the tendency of the medical-grade lipid microbubbles
to pool or concentrate in tumors and the established fact that gas
pockets, such as those provided by the microbubbles, are known to
act as heat sinks as well as cavitation nuclei. For example, this
inventor has recently found that the lipid-coated microbubbles of
the present invention will, in fact, cross into and intensify
ultrasonic images of tumors in the rat brain. Passage of the
ultrasmall lipid microbubbles is possible primarily because of the
alterations in capillary permeability and vascular structure in the
area of the tumor. These pathological alterations in intrinsic
tumor capillaries increase the likelihood for small particulate
matter to pass out of circulation into the cerebral tumor.
Virtually all classes of tumors are known to have similar
disruptions in the vascular endothelium, regardless of the site of
the tumor. Similarly, the lipid-coated microbubbles cross the
vascular endothelium into tumors elsewhere in the body as they do
in the brain.
It has previously been shown that ultrasound, in the absence of any
contrast agent, can be utilized therapeutically in the selective
destruction of tumor tissue. (Quan et al, Phys. Med. Biol.,
34:1719-1731 (1989); ter Haar et al, Phys. Med. Biol., 34:1743-1750
(1989).) In accordance with a third embodiment of the invention,
the known cavitational and heating effects of ultrasound may be
enhanced through the use of lipid-coated microbubbles which have
pooled at the tumor site.
The novel method involves intravenously injecting lipid-coated
microbubbles into a human or animal in order for the microbubbles
to accumulate or pool at a predetermined area which has been
ultrasonically scanned to obtain a diagnostic, confirmatory image
of the tumor area and then intensifying the ultrasound signal,
thereby providing a therapeutic heating and/or cavitational
effect.
The excellent uniformity and extremely small size, coupled with the
exceptional in vivo longevity of the lipid-coated microbubbles
enables them to: 1) safely traverse the pulmonary microcirculation,
and 2) endure passage across structural disruptions of the vascular
lining (or wall), as commonly occurs in the abnormal capillary beds
of many types of tumors. The resulting accumulation and persistence
of these lipid-coated microbubbles at the tumor site directly
causes a selective enhancement of the ultrasonographic image of the
tumor, as observed in Example 6 herein with brain tumors at this
microbubble dosage level. The selective image enhancement is best
displayed by taking ultrasound scans of the tumor site before and
again a few minutes after microbubble contrast injection
intravenously.
This selective accumulation of lipid-coated microbubbles at the
tumor site can be very useful for accentuating the following tw
therapeutic effects of ultrasound on tumors. First, the ability to
focus ultrasound precisely on a predetermined volume (e.g.,
specified from prior diagnostic imaging) provides the ability to
selectively destroy tissue at a tumor site without damage to
intervening tissues. The highly echogenic nature of the ultrasonic
lesions produced during in vivo studies and in excised liver
samples in vitro suggest the effect is produced by cavitation
damage. The extent of the ultrasonic lesion is dependent upon both
length and intensity of the focused ultrasound exposure, where the
intensity of the focused ultrasound is routinely higher than that
required simply for imaging (ter Haar et al, Phys. Med. Biol.,
34:1743-1750 (1989)). However, lower intensities of ultrasound can
still be effective for tumor therapy through its thermal effect on
tissue. Ultrasound in the low megahertz frequency range is used to
treat malignant tumors by heating them to temperatures between
42.degree. and 45.degree. C., producing cellular damage, the extent
of which is determined by the duration and number of treatments
(Quan et al, Phys. Med. Biol., 34:1719-1731 (1989)). Both the
cavitational and thermal mechanisms of ultrasonic tumor therapy can
be accentuated by the presence of the accumulated microbubbles in
the tumor, since microbubbles are known to serve very effectively
as cavitation nuclei (D'Arrigo, J. Chem. Phys., 71:1809-1813
(1979); ibid., 72:5133-5138 (1980); ibid., 75:962-968 (1981)) and
their contained gas readily provides a local source of heat
generation in an ultrasonic field.
Considering first the cavitation facilitation, the threshold for
macroscopic bubble formation in an ultrasonic field has been
reported to be similar in both agar gels (ter Haar et al, Phys.
Med. Biol., 34:1533-1542) and guinea pig hind legs (Daniels &
Ter Haar, Proc. I.O.A., 8:147-157). At the same time, it is also
known from extensive physicochemical experimentation with highly
purified gels that the ordinary cavitation threshold for
macroscopic bubble formation in aqueous-based gels can be lowered
drastically by the presence of moderate or even low concentrations
of stable microbubbles (D'Arrigo, Aviat. Space Environ. Med.,
49:358-361; D'Arrigo, J. Chem. Phvs., 71:1809-1813 (1979); ibid.,
72:5133-5138 (1980); ibid., 75:962-968 (1981)). As concerns the
thermal enhancement effect mediated by any accumulated microbubbles
in the tumor, this physical relationship stems from the fact that
free gas bubbles in a liquid are capable of strong oscillatory
motion (i.e., small amplitude, radial pulsations of the bubbles) in
an ultrasound field (Ophir & Parker, Ultrasound Med. Biol.,
15:319-333 (1989). This strong oscillatory motion of the
microbubbles provides a mechanism for local heat generation in the
region of the microbubble. The degree of non-linearity in the
ultrasound beam will also determine the temperature rise achieved.
Theory predicts more non-linear distortion for ultrasonic pulses
with higher peak positive pressure amplitudes. Associated with
this, there may be an enhancement of the predicted temperature rise
due to absorption of the higher harmonic components (Swindell,
Ultrasound Med. Biol., 11:121-130).
Following microbubble-assisted ultrasonic destruction of a tumor
site, the walls of the tissue cavity created by liquefaction and
drainage of the ultrasonic lesion site can be better defined with
use of more lipid-coated microbubble contrast and the progress of
resolution of this cavity can be tracked over time.
The ultrasound frequency and exposure time are parameters which
will have to be controlled in order to achieve the desired tumor
destruction. These parameters will, in turn, be dependent upon a
number of factors, including the size and location of the tumor and
the ultrasonic equipment used. In general, at constant intensity,
the longer the exposure time, the greater the extent of tumor
tissue damage. Similarly, if the intensity is increased at constant
exposure time, the extent of damage will again be increased.
Another therapeutic application for the medical grade microbubbles
produced in accordance with the method of the present invention
involves the targeted delivery of therapeutic compositions, such as
drugs and chemotherapeutic agents, to the tumor site. This
intervention also takes advantage of the natural tendency of the
lipid-coated microbubbles to pool in the tumor tissue. In this
application, cytotoxic agents are entrapped within the membrane
shell of the lipid-coated microbubbles, and are released upon
injection of the microbubbles into the body and the application of
high intensity ultrasound waves at the tumor site. Based upon the
known effects of ultrasound, the energy will cause the microbubbles
to burst at the site, thereby releasing the cytotoxic agent
directly at the tumor location.
The incorporation of the particulate, cytotoxic agent within the
membrane of the lipid-coated microbubbles can be approached in any
one of a number of ways. One approach involves the preparation of a
hydrophobic coating that is applied to the surface of the
particulate form of a cytotoxic agent. The thus-coated cytotoxic
agent is then introduced as an aerosol over or otherwise deposited
on the surface of a saturated solution of an appropriate lipid
surfactant mixture in a closed container. Vigorous shaking by hand
then traps macroscopic gas pockets, containing either the
particulate aerosol or particle adhering to the gas/liquid
interface, within the body of the aqueous lipid surfactant
solution. Thereafter, the macroscopic gas pockets shrink by gas
dissolution, and a tightly packed, lipid-surfactant monolayer forms
around the microbubbles, which have entrapped therein the
hydrophobically coated, cytotoxic drug particles.
The following examples further illustrate the present invention.
Unless otherwise stated, all percentages are by weight.
EXAMPLE 1
This example illustrates the preparation of a surfactant mixture
for use in accordance with the present invention. A surfactant
mixture was prepared in accordance with the present invention by
admixing glycerol monolaurate, cholesterol benzoate, cholesterol,
cholesterol acetate and glycerol tripalmitate in a weight ratio of
3:1:1:1:1, respectively, to obtain a dry powdery surfactant
mixture.
EXAMPLE 2
This example illustrates the method for production of medical grade
lipid-coated microbubbles. Saline solution was prepared by
dissolving ACS reagent grade NaCl (0.9% w/v) in distilled water.
The thus prepared solution was then filtered through a sterile 0.2
.mu.m membrane filter.
An excess (1.5 gm/liter) of the powdered lipid-surfactant mixture
prepared as in Example 1 was added to the above solution. The
resulting solution was shaken vigorously (mechanically; sonication
is unnecessary) for 10 seconds in air at room temperature. After
five minutes, this shaking step was repeated two more times. The
undissolved lipid material was then allowed to settle out for about
30 minutes and the resulting colloidal suspension (i.e.,
supernatant) was filtered through a sterile polysulfone membrane
0.45 .mu.m pore-diameter, low-absorption membrane filter (Gelman
Sciences, Ann Arbor, MI).
The resulting suspension of lipid-coated microbubbles was then
analyzed using a Coulter Multisizer in conjunction with Coulter's
AccuCorp Software (Coulter Electronics, Inc., Hialeah, FL).
Analysis was conducted in isotonic saline at approximately
21.degree. C. The results of this analysis are set forth in FIG. 1.
The analysis revealed microbubbles present in a concentration of
about 540,000 bubbles/ml. Mean microbubble diameter was 2.00 .mu.m
and greater than 99% of the microbubbles were below 4.35 .mu.m.
COMPARATIVE EXAMPLE 3
This example illustrates the criticality of using a filter having
an average pore size of at least about 0.40 .mu.m and polysulfone
membrane, by comparing the microbubble suspension formed in
accordance with the present invention with a suspension prepared
using a 20 .mu.m pore size filter and a cellulose-ester membrane
filter, respectively.
Filter size
A saturated solution of a surfactant mixture was prepared in
accordance with Example 2, except that a 0.20 .mu.m polysulfone
membrane filter was used in the filtration step instead of a 0.45
.mu.m polysulfone membrane filter. Particle size analysis was
conducted as in Example 2. The results of the analysis are set
forth in FIG. 2.
As can be seen from the analysis, while the size distribution
remained between 1-5 .mu.m in diameter, the microbubble
concentration dropped precipitously to about 210,160 microbubbles
per ml., or about 40% of the concentration obtained in accordance
with the present invention. This suggests that the 0.20 .mu.m
filter does not allow sufficient reformation of the microbubbles
after passage through the filter.
The suspension obtained using the 0.20 .mu.m polysulfone membrane
filter is very unsatisfactory as a contrast agent, in view of the
low concentration of microbubbles present therein and the
variability of microbubble concentration measured. The product
specifications developed for this medical-grade contrast agent, in
accord with federal regulatory quality-assurance expectations,
require a concentration of 540,000.+-.15% microbubbles/ml --which
obviously cannot be met in this case.
Composition
The composition of the filter employed in the second filtration
step is also an important aspect of the present invention, although
the composition is not as critical as filter pore size. A saturated
solution of a surfactant mixture was next prepared in accordance
with Example 2, except that a 0.45 .mu.m cellulose
acetate/cellulose nitrate membrane filter was utilized rather than
the preferred 0.45 .mu.m polysulfone (low-absorption) membrane
filter. Particle size analysis was conducted as in Example 2. The
results of the analysis are set forth in FIG. 3.
As can be seen from this analysis, the size distribution of the
microbubbles again remains at about 1-5 .mu.m in diameter. However,
the microbubble concentration dropped to about 441,740 microbubbles
per ml of suspension. This is approximately 19% lower than the
concentration of microbubbles obtained in accordance with the
present invention, which employs a polysulfone membrane filter.
Such a reduction is sufficient to seriously limit the suspension's
usefulness as a sonographic contrast agent. Other membrane filter
materials, such as PTFE, lead to even poorer suspension
quality.
EXAMPLE 4
This example illustrates the method of preparing paramagnetically
labeled, lipid-coated microbubbles obtained in accordance with the
present invention.
Fifteen grams of polyalanine (1000-5000 daltons mol. wt.) were
dissolved in 2500 ml of 1.0 M phosphate buffer and filtered through
a 0.5 .mu.m pore-diameter filter. A 20-fold molar excess of solid
DTPA (Sigma Chemical Co., St. Louis, MO) was added to the protein
solution, and the pH adjusted to pH 8 by addition of solid sodium
phosphate buffer. After 30 minutes of additional stirring, the pH
was adjusted to pH 5.6 with glacial acetic acid (or concentrated
HCl) and a 30-fold molar excess of GdCl.sub.3 (Aldrich Chemical
Co., Milwaukee, WI) to protein was added. The solution was then
dialyzed against 0.15 M saline at 5.degree. C. for 96 hours using
1000-dalton cut-off dialysis tubing. The resulting 1.8-2.0 liters
of solution was then lyophilized over several days to give the
white, solid derivative (approximately 20 gm).
Incorporation of the lyophilized Gd-DTPA derivative into
lipid-coated microbubbles was accomplished by adding the derivative
(5-10% w/w) to the powdered lipid-surfactant mixture of Example 1.
Upon shaking in the isotonic saline, the paramagnetic-labeled
surface-active derivative is incorporated into the microbubble's
surrounding lipid monolayer, with the Gd-DTPA label remaining
exposed to the aqueous exterior. Additional Gd-DTPA derivative may
remain dissolved in the isotonic saline, and co-exist with the
lipid-coated microbubbles.
EXAMPLE 5
This example illustrates that the paramagnetic-labeled and/or
unlabeled lipid-coated microbubbles, produced by the
above-described process, are generally safe for medical diagnostic
and therapeutic applications.
Six Sprague-Dawley male rats were given an intravenous infusion of
lipid coated microbubbles produced in accordance with Example 1 at
an average dosage of 0.14 ml/kg 3 times weekly for 6 weeks. Blood
samples were obtained at 2 and 6 weeks for a serum chemistry
profile of 19 parameters including total protein, LDH, cholesterol
and creatinine. At the conclusion the animals were sacrificed,
subjected to a gross autopsy, organ weights obtained for 7 critical
organs and the tissues fixed for histological examination.
A comparison of the 6 week versus 2 week chemistry values show some
differences and elevations in a number of the parameters examined,
with the most prominent difference occurring in the LDH values. The
gross and microscopic pathology examinations did not reveal any
lesions related to the administration of the test material.
Furthermore, there were no atheromata, mural thrombi or emboli seen
in any of the tissues examined. These results support the safety of
the lipid-coated microbubbles produced in accordance with the
method of the present invention for single or double administration
for clinical trial.
Thereafter, three "SPF" (New Zealand albino) male rabbits received
an intravenous infusion of the lipid-coated microbubbles three
times per week for 2 1/2 weeks at a dosage of 0.48 ml/kg. After the
initial 2 1/2 week period, lipid-coated microbubbles (0.48 ml/kg)
with added Gd-DTPA derivative (3-20 mg/kg) were continued at the
same dosage for 2.5 months. An extensive serum chemistry profile, a
hematology profile and four coagulation tests were performed on
blood samples taken: prior to injection, after 2.5 weeks, 7 weeks,
and 11 weeks after the initial injection, and prior to necropsy. At
the end of this period the animals were sacrificed, subjected to a
gross autopsy and tissues fixed for histological examination.
The clinical pathology parameters for serum chemistry, hematology
and coagulation revealed no findings that could be related to the
administration of the test material and were essentially within
normal limits at each examination period. The microscopic pathology
examination revealed no atheromati, mural thrombi or emboli or any
other findings that could be related to the administration of the
paramagnetically-labeled lipid-coated microbubbles. Therefore, the
derivatized suspension is safe for single or 2 times administration
in short term clinical trials.
EXAMPLE 6
This example demonstrates that the lipid-coated microbubbles
prepared in accordance with the present invention will cross into
and intensify the ultrasonic images of glioma tumors in the rat
brain.
Lipid-coated microbubbles were prepared in accordance with Example
2 and supplied in a concentration of 520,000-550,000 microbubbles
per ml in isotonic saline.
Bilateral craniectomies were performed on Sprague-Dawley 250-333 g
male rats. The mice were anesthetized with ketamine (90mg/kg),
supplemented with xylazine (10mg/kg). Using the coordinates from
the stereotaxic atlas of Pelligrino et al (A Stereotaxic Atlas of
the Rat Brain, N.Y., Plenum (1979)) for a deep brain parenchymal
target, a needle was introduced for direct injection of 0.1 ml of
isotonic saline containing 3.times.10.sup.6 cultured C-6 glioma
cells. This procedure provided a dense, subcortical, circumscribed
tumor which remained intraparenchymal, with no spill into the
ventricles or the subarachnoid space. The lesion was easily
detected by histological evaluation. Histological evaluation showed
a consistent progression in tumor size over time.
20 rats were given the intra-parenchymal injection of cultured
glioma cells, as described above and 11 of these rats received a
daily intravenous injection (via tail vein) of 0.05 ml of the
lipid-coated microbubbles. Ultrasound imaging was performed using a
7.5 MHz probe and images were recorded on a broadcast-grade
videotape recorder and subsequently transformed to image processing
software. The day of sonographic detection of the developing
cerebral glioma in each animal was recorded; the mean and S.E.M.
were calculated for the microbubble (n=11) and control (n=9)
groups.
All animals were sacrificed after the last image was taken and
brains were removed for histological sectioning to verify tumor
location and to determine actual tumor size.
FIG. 4 is a representative ultrasound image of a rat cerebral
glioma before (4A) and within a few minutes after intravenous
injection of the lipid-coated microbubbles (4B). The results of the
experiments showed that the lipid-coated microbubbles will cross
into the gliomas in the rat brain and intensify the ultrasonic
image obtained. The results of the experiment, set forth in Table 1
below, also show that the developing tumors can be detected earlier
when the microbubble contrast agent is employed, compared to the
control group.
TABLE 1 ______________________________________ First Sonographic
Detection of Brain Tumors (Days) Control (n = 9) Microbubble (n =
11) ______________________________________ 6 4 8 5 6 4 9 4 6 4 6 4
7 4 6 4 6 4 4 4 Mean 6.67 4.09 .+-. S.E.M. 0.35 0.08
______________________________________ p < .001 (F.sub.18.sup.1
= 54.19)
This Table lists, for each animal, the time necessary for first
sonographic detection of the developing cerebral glioma. The means
of 6.67.+-.0.35 and 4.09.+-.0.08 represent the mean time
(.+-.S.E.M.) for the separate control and microbubble groups,
respectively. Sonographic detection of the cerebral gliomas
appeared about 40% earlier in the rats injected with the
lipid-coated microbubbles than in the control group, which further
indicates the clinical potential of the lipid-coated
microbubbles.
* * * * *