U.S. patent application number 11/036834 was filed with the patent office on 2005-07-28 for methods of preparing gaseous precursor-filled microspheres.
This patent application is currently assigned to Bristol-Myers Squibb Medical Imaging, Inc.. Invention is credited to Fritz, Thomas A., Matsunaga, Terry, Ramaswami, Varadarajan, Unger, Evan C., Wu, Guanli, Yellowhair, David.
Application Number | 20050163716 11/036834 |
Document ID | / |
Family ID | 46280433 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050163716 |
Kind Code |
A1 |
Unger, Evan C. ; et
al. |
July 28, 2005 |
Methods of preparing gaseous precursor-filled microspheres
Abstract
Methods of and apparatus for preparing gas-filled microspheres
are described. Gas-filled microspheres prepared by these methods
are particularly useful, for example, in ultrasonic imaging
applications and in therapeutic drug delivery systems.
Inventors: |
Unger, Evan C.; (Tucson,
AZ) ; Fritz, Thomas A.; (Tucson, AZ) ;
Matsunaga, Terry; (Tucson, AZ) ; Ramaswami,
Varadarajan; (Tucson, AZ) ; Yellowhair, David;
(Tucson, AZ) ; Wu, Guanli; (Tucson, AZ) |
Correspondence
Address: |
BRISTOL - MYERS SQUIBB COMPANY
PO BOX 4000
PRINCETON
NJ
08543-4000
US
|
Assignee: |
Bristol-Myers Squibb Medical
Imaging, Inc.
|
Family ID: |
46280433 |
Appl. No.: |
11/036834 |
Filed: |
January 14, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11036834 |
Jan 14, 2005 |
|
|
|
10213600 |
Aug 6, 2002 |
|
|
|
10213600 |
Aug 6, 2002 |
|
|
|
09118329 |
Jul 17, 1998 |
|
|
|
6479034 |
|
|
|
|
09118329 |
Jul 17, 1998 |
|
|
|
08487230 |
Jun 6, 1995 |
|
|
|
5853752 |
|
|
|
|
08487230 |
Jun 6, 1995 |
|
|
|
08159687 |
Nov 30, 1993 |
|
|
|
5585112 |
|
|
|
|
08159687 |
Nov 30, 1993 |
|
|
|
08160232 |
Nov 30, 1993 |
|
|
|
5542935 |
|
|
|
|
08159687 |
Nov 30, 1993 |
|
|
|
08159674 |
Nov 30, 1993 |
|
|
|
Current U.S.
Class: |
424/9.52 |
Current CPC
Class: |
A61K 9/127 20130101;
A61K 49/227 20130101; A61K 41/0028 20130101; A61K 9/1277 20130101;
A61K 9/1278 20130101; A61P 43/00 20180101; A61M 5/3145 20130101;
A61K 41/0052 20130101; A61K 47/6925 20170801; A61K 49/223
20130101 |
Class at
Publication: |
424/009.52 |
International
Class: |
A61K 049/00 |
Claims
1-15. (canceled)
16. Gas-filled lipid microspheres that comprise a lipid monolayer
encapsulating a perfluorocarbon gas, said lipid comprising
dipalmatoylphosphatidic acid, at least one phosphatidylcholine
selected from the group consisting of dioleoylphosphatidylcholine,
dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and
distearoylphosphatidylcholine, and at least one
phosphatidylethanolamine selected from the group consisting of
dipalmitoylphosphatidylethanolamine- ,
dipalmitoylphosphatidylethanolamine-PEG 5,000,
dioleoyl-phosphatidyletha- nolamine, and
N-succinyl-dioleoyl-phosphatidylethanolamine, wherein said
gas-filled microspheres are prepared by a process that comprises
shaking an aqueous suspension comprising said lipids in the
presence of said perfluorocarbon gas at a temperature below the gel
state to liquid crystalline transition temperature of said lipids,
said shaking performed with sufficient intensity and duration to
produce said gas-filled lipid microspheres.
17. The gas-filled lipid microspheres of claim 16 wherein said
perfluorocarbon gas is selected from the group consisting of
perfluoroethane, perfluoropropane, perfluorobutane,
perfluoropentane, perfluorohexane, and perfluorocyclobutane.
18. The gas-filled lipid microspheres of claim 17 wherein said
perfluorocarbon gas is selected from the group consisting of
perfluoropropane, perfluorobutane, perfluoropentane, and
perfluorocyclobutane.
19. The gas-filled lipid microspheres of claim 18 wherein said
perfluorocarbon gas is perfluoropropane.
20. The gas-filled lipid microspheres of claim 16 wherein said
phosphatidylcholine is dipalmitoylphosphatidylcholine.
21. The gas-filled lipid microspheres of claim 16 wherein said
phosphatidylethanolamine is dipalmitoylphosphatidylethanolamine-PEG
5,000.
22. The gas-filled lipid microspheres of claim 16 wherein said
phosphatidylcholine is dipalmitoylphosphatidylcholine and said
phosphatidylethanolamine is dipalmitoylphosphatidylethanolamine-PEG
5,000.
23. The gas-filled lipid microspheres of claim 22 wherein said
perfluorocarbon gas is selected from the group consisting of
perfluoropropane, perfluorobutane, perfluoropentane, and
perfluorocyclobutane.
24. The gas-filled lipid microspheres of claim 23 wherein said
perfluorocarbon gas is perfluoropropane.
25. The gas-filled lipid microspheres of claim 24 wherein said
microspheres comprise dipalmitoylphosphatidylcholine,
dipalmatoylphosphatidic acid and
dipalmitoylphosphatidylethanolamine-PEG 5,000 in a mole percent
ratio of 82%:10%:8%.
26. A method of ultrasound imaging comprising: (i) shaking an
aqueous solution comprising dipalmatoylphosphatidic acid, at least
one phosphatidylcholine selected from the group consisting of
dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,
dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine,
and at least one phosphatidylethanolamine selected from the group
consisting of dipalmitoylphosphatidylethanolamine,
dipalmitoylphosphatidylethanolamine-- PEG 5,000,
dioleoyl-phosphatidylethanolamine, and N-succinyl-dioleoyl-phos-
phatidylethanolamine, in the presence of a perfluorocarbon gas at a
temperature below the gel state to liquid crystalline transition
temperature of said lipids, and with sufficient intensity and
duration to produce gas-filled lipid microspheres that comprise a
lipid monolayer encapsulating a perfluorocarbon gas; (ii)
administering said gas-filled microspheres to a patient; and (iii)
scanning the patient using ultrasonic imaging.
27. The method of claim 26 wherein the aqueous solution is provided
in a vessel, said shaking produces a gas-filled lipid
microsphere-containing foam above said aqueous solution, and said
method further comprises the step of extracting said foam from said
vessel for administration to said patient.
28. The method of claim 27 wherein said extraction is by
syringe.
29. The gas-filled lipid microspheres of claim 26 wherein said
perfluorocarbon gas is selected from the group consisting of
perfluoroethane, perfluoropropane, perfluorobutane,
perfluoropentane, perfluorohexane, and perfluorocyclobutane.
30. The gas-filled lipid microspheres of claim 29 wherein said
perfluorocarbon gas is selected from the group consisting of
perfluoropropane, perfluorobutane, perfluoropentane, and
perfluorocyclobutane.
31. The gas-filled lipid microspheres of claim 30 wherein said
perfluorocarbon gas is perfluoropropane.
32. The gas-filled lipid microspheres of claim 26 wherein said
phosphatidylcholine is dipalmitoylphosphatidylcholine.
33. The gas-filled lipid microspheres of claim 26 wherein said
phosphatidylethanolamine is dipalmitoylphosphatidylethanolamine-PEG
5,000.
34. The gas-filled lipid microspheres of claim 26 wherein said
phosphatidylcholine is dipalmitoylphosphatidylcholine and said
phosphatidylethanolamine is dipalmitoylphosphatidylethanolamine-PEG
5,000.
35. The gas-filled lipid microspheres of claim 34 wherein said
perfluorocarbon gas is selected from the group consisting of
perfluoropropane, perfluorobutane, perfluoropentane, and
perfluorocyclobutane.
36. The gas-filled lipid microspheres of claim 35 wherein said
perfluorocarbon gas is perfluoropropane.
37. The gas-filled lipid microspheres of claim 36 wherein said
microspheres comprise dipalmitoylphosphatidylcholine,
dipalmatoylphosphatidic acid and
dipalmitoylphosphatidylethanolamine-PEG 5,000 in a mole percent
ratio of 82%:10%:8%.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of copending U.S. Ser. No.
09/118,329, filed Jul. 17, 1998, now allowed, which in turn is a
divisional of U.S. Ser. No. 08/487,230, filed Jun. 6, 1995, now
U.S. Pat. No. 5,853,752, which is a divisional of U.S. Ser. No.
08/159,687, filed Nov. 30, 1993, now U.S. Pat. No. 5,585,112, which
is a continuation-in-part of U.S. Ser. No. 08/160,232 filed Nov.
30, 1993, now U.S. Pat. No. 5,542,935, and a continuation-in-part
of U.S. Ser. No. 08/159,674, filed Nov. 30, 1993, now abandoned.
Said 08/160,232 and 08/159,674 are continuations-in-part of U.S.
Ser. No. 08/076,239 filed Jun. 11, 1993, now U.S. Pat. No.
5,469,854, which is a continuation-in-part of U.S. Ser. No.
07/717,084, now U.S. Pat. No. 5,228,446, and U.S. Ser. No.
07/716,899, now abandoned, both of which were filed Jun. 18, 1991.
Said 07/717,084 and 07/716,899 are continuations-in-part of U.S.
Ser. No. 07/569,828, filed Aug. 20, 1990, now U.S. Pat. No.
5,088,499, which in turn is a continuation-in-part of application
U.S. Ser. No. 07/455,707, filed Dec. 22, 1989, now abandoned. The
disclosures of each of these patent applications are incorporated
herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to novel methods and apparatus for
preparing gaseous precursor-filled liposomes. Liposomes prepared by
these methods are particularly useful, for example, in ultrasonic
imaging applications and in therapeutic delivery systems.
[0004] 2. Background of the Invention
[0005] A variety of imaging techniques have been used to detect and
diagnose disease in animals and humans. X-rays represent one of the
first techniques used for diagnostic imaging. The images obtained
through this technique reflect the electron density of the object
being imaged. Contrast agents such as barium or iodine have been
used over the years to attenuate or block X-rays such that the
contrast between various structures is increased. X-rays, however,
are known to be somewhat dangerous, since the radiation employed in
X-rays is ionizing, and the various deleterious effects of ionizing
radiation are cumulative.
[0006] Another important imaging technique is magnetic resonance
imaging (MRI). This technique, however, has various drawbacks such
as expense and the fact that it cannot be conducted as a portable
examination. In addition, MRI is not available at many medical
centers.
[0007] Radionuclides, employed in nuclear medicine, provide a
further imaging technique. In employing this technique,
radionuclides such as technetium labelled compounds are injected
into the patient, and images are obtained from gamma cameras.
Nuclear medicine techniques, however, suffer from poor spatial
resolution and expose the animal or patient to the deleterious
effects of radiation. Furthermore, the handling and disposal of
radionuclides is problematic.
[0008] Ultrasound is another diagnostic imaging technique which is
unlike nuclear medicine and X-rays since it does not expose the
patient to the harmful effects of ionizing radiation. Moreover,
unlike magnetic resonance imaging, ultrasound is relatively
inexpensive and can be conducted as a portable examination. In
using the ultrasound technique, sound is transmitted into a patient
or animal via a transducer. When the sound waves propagate through
the body, they encounter interfaces from tissues and fluids.
Depending on the acoustic properties of the tissues and fluids in
the body, the ultrasound sound waves are partially or wholly
reflected or absorbed. When sound waves are reflected by an
interface they are detected by the receiver in the transducer and
processed to form an image. The acoustic properties of the tissues
and fluids within the body determine the contrast which appears in
the resultant image.
[0009] Advances have been made in recent years in ultrasound
technology. However, despite these various technological
improvements, ultrasound is still an imperfect tool in a number of
respects, particularly with regard to the imaging and detection of
disease in the liver and spleen, kidneys, heart and vasculature,
including measuring blood flow. The ability to detect and measure
these regions depends on the difference in acoustic properties
between tissues or fluids and the surrounding tissues or fluids. As
a result, contrast agents have been sought which will increase the
acoustic difference between tissues or fluids and the surrounding
tissues or fluids in order to improve ultrasonic imaging and
disease detection.
[0010] The principles underlying image formation in ultrasound have
directed researchers to the pursuit of gaseous contrast agents.
Changes in acoustic properties or acoustic impedance are most
pronounced at interfaces of different substances with greatly
differing density or acoustic impedance, particularly at the
interface between solids, liquids and gases. When ultrasound sound
waves encounter such interfaces, the changes in acoustic impedance
result in a more intense reflection of sound waves and a more
intense signal in the ultrasound image. An additional factor
affecting the efficiency or reflection of sound is the elasticity
of the reflecting interface. The greater the elasticity of this
interface, the more efficient the reflection of sound. Substances
such as gas bubbles present highly elastic interfaces. Thus, as a
result of the foregoing principles, researchers have focused on the
development of ultrasound contrast agents based on gas bubbles or
gas containing bodies and on the development of efficient methods
for their preparation.
[0011] Ryan et al., in U.S. Pat. No. 4,544,545, disclose
phospholipid liposomes having a chemically modified cholesterol
coating. The cholesterol coating may be a monolayer or bilayer. An
aqueous medium, containing a tracer, therapeutic, or cytotoxic
agent, is confined within the liposome. Liposomes, having a
diameter of 0.001 microns to 10 microns, are prepared by agitation
and ultrasonic vibration.
[0012] D'Arrigo, in U.S. Pat. Nos. 4,684,479 and 5,215,680, teaches
a gas-in-liquid emulsion and method for the production thereof from
surfactant mixtures. U.S. Pat. No. 4,684,479 discloses the
production of liposomes by shaking a solution of the surfactant in
a liquid medium in air. U.S. Pat. No. 5,215,680 is directed to a
large scale method of producing lipid coated microbubbles including
shaking a solution of the surfactant in liquid medium in air or
other gaseous mixture and filter sterilizing the resultant
solution.
[0013] WO 80/02365 discloses the production of microbubbles having
an inert gas, such as nitrogen; or carbon dioxide, encapsulated in
a gellable membrane. The liposomes may be stored at low
temperatures and warmed prior and during use in humans. WO 82/01642
describes microbubble precursors and methods for their production.
The microbubbles are formed in a liquid by dissolving a solid
material. Gas-filled voids result, wherein the gas is 1.) produced
from gas present in voids between the microparticles of solid
precursor aggregates, 2.) absorbed on the surfaces of particles of
the precursor, 3.) an integral part of the internal structure of
particles of the precursor, 4.) formed when the precursor reacts
chemically with the liquid, and 5.) dissolved in the liquid and
released when the precursor is dissolved therein.
[0014] In addition, Feinstein, in U.S. Pat. Nos. 4,718,433 and
4,774,958, teaches the use of albumin coated microbubbles for the
purposes of ultrasound.
[0015] Widder, in U.S. Pat. Nos. 4,572,203 and 4,844,882, discloses
a method of ultrasonic imaging and a microbubble-type ultrasonic
imaging agent.
[0016] Quay, in WO 93/05819, describes the use of agents to form
microbubbles comprising especially selected gases based upon a
criteria of known physical constants, including 1) size of the
bubble, 2) density of the gas, 3) solubility of the gas in the
surrounding medium, and 4) diffusivity of the gas into the
medium.
[0017] Kaufman et al., in U.S. Pat. No. 5,171,755, disclose an
emulsion comprising an highly fluorinated organic compound, an oil
having no substantial surface activity or water solubility and a
surfactant. Kaufman et al. also teach a method of using the
emulsion in medical applications.
[0018] Another area of significant research effort is in the area
of targeted drug delivery. Targeted delivery means are particularly
important where toxicity is an issue. Specific therapeutic delivery
methods potentially serve to minimize toxic side effects, lower the
required dosage amounts, and decrease costs for the patient.
[0019] The methods and materials in the prior art for introduction
of genetic materials, for example, to living cells is limited and
ineffective. To date several different mechanisms have been
developed to deliver genetic material to living cells. These
mechanisms include techniques such as calcium phosphate
precipitation and electroporation, and carriers such as cationic
polymers and aqueous-filled liposomes. These methods have all been
relatively ineffective in vivo and only of limited use for cell
culture transfection. None of these methods potentiate local
release, delivery and integration of genetic material to the target
cell.
[0020] Better means of delivery for therapeutics such as genetic
materials are needed to treat a wide variety of human and animal
diseases. Great strides have been made in characterizing genetic
diseases and in understanding protein transcription but relatively
little progress has been made in delivering genetic material to
cells for treatment of human and animal disease.
[0021] A principal difficulty has been to deliver the genetic
material from the extracellular space to the intracellular space or
even to effectively localize genetic material at the surface of
selected cell membranes. A variety of techniques have been tried in
vivo but without great success. For example, viruses such as
adenoviruses and retroviruses have been used as vectors to transfer
genetic material to cells. Whole virus has been used but the amount
of genetic material that can be placed inside of the viral capsule
is limited and there is concern about possible dangerous
interactions that might be caused by live virus. The essential
components of the viral capsule may be isolated and used to carry
genetic material to selected cells. In vivo, however, not only must
the delivery vehicle recognize certain cells but it also must be
delivered to these cells. Despite extensive work on viral vectors,
it has been difficult to develop a successfully targeted viral
mediated vector for delivery of genetic material in vivo.
[0022] Conventional, liquid-containing liposomes have been used to
deliver genetic material to cells in cell culture but have mainly
been ineffective in vivo for cellular delivery of genetic material.
For example, cationic liposome transfection techniques have not
worked effectively in vivo. More effective means are needed to
improve the cellular delivery of therapeutics such as genetic
material.
[0023] The present invention is directed to addressing the
foregoing, as well as other important needs in the area of contrast
agents for ultrasonic imaging and vehicles for the effective
targeted delivery of therapeutics.
SUMMARY OF THE INVENTION
[0024] The present invention provides methods and apparatus for
preparing temperature activated gaseous precursor-filled liposomes
suitable for use as contrast agents for ultrasonic imaging or as
drug delivery agents. The methods of the present invention provide
the advantages, for example, of simplicity and potential cost
savings during manufacturing of temperature activated gaseous
precursor-filled liposomes.
[0025] Preferred methods for preparing the temperature activated
gaseous precursor-filled liposomes comprise shaking an aqueous
solution comprising a lipid in the presence of a temperature
activated gaseous precursor, at a temperature below the gel state
to liquid crystalline state phase transition temperature of the
lipid.
[0026] Unexpectedly, the temperature activated gaseous
precursor-filled liposomes prepared in accordance with the methods
of the present invention possess a number of surprising yet highly
beneficial characteristics. For example, gaseous precursor-filled
liposomes are advantageous due to their biocompatibility and the
ease with which lipophilic compounds can be made to cross cell
membranes after the liposomes are ruptured. The liposomes of the
invention also exhibit intense echogenicity on ultrasound, are
highly stable to pressure, and/or generally possess a long storage
life, either when stored dry or suspended in a liquid medium. The
echogenicity of the liposomes is of importance to the diagnostic
and therapeutic applications of the liposomes made according to the
invention. The gaseous precursor-filled liposomes also have the
advantages, for example, of stable particle size, low toxicity and
compliant membranes. It is believed that the flexible membranes of
the gaseous precursor-filled liposomes may be useful in aiding the
accumulation or targeting of these liposomes to tissues such as
tumors.
[0027] The temperature activated gaseous precursor-filled liposomes
made according to the present invention thus have superior
characteristics for ultrasound contrast imaging. When inside an
aqueous or tissue media, the gaseous precursor-filled liposome
creates an interface for the enhanced absorption of sound. The
gaseous precursor-filled liposomes are therefore useful in imaging
a patient generally, and/or in diagnosing the presence of diseased
tissue in a patient as well as in tissue heating and the
facilitation of drug release or activation.
[0028] In addition to ultrasound, the temperature activated gaseous
precursor-filled liposomes made according to the present invention
may be used, for example, for magnetic imaging and as MRI contrast
agents. For example, the gaseous precursor-filled liposomes may
contain paramagnetic gases, such as atmospheric air, which contains
traces of oxygen 17; paramagnetic ions such as Mn.sup.+2,
Gd.sup.+2, Fe.sup.+3; iron oxides; or magnetite (Fe.sub.3O.sub.4)
and may thus be used as susceptibility contrast agents for magnetic
resonance imaging. Additionally, for example, the gaseous
precursor-filled liposomes made according to the present invention
may contain radioopaque metal ions, such as iodine, barium,
bromine, or tungsten, for use as x-ray contrast agents.
[0029] The temperature activated gaseous precursor-filled liposomes
are also particularly useful as drug carriers. Unlike liposomes of
the prior art that have a liquid interior suitable only for
encapsulating drugs that are water soluble, the gaseous
precursor-filled liposomes made according to the present invention
are particularly useful for encapsulating lipophilic drugs.
Furthermore, lipophilic derivatives of drugs may be incorporated
into the lipid layer readily, such as alkylated derivatives of
metallocene dihalides. Kuo et al., J. Am. Chem. Soc. 1991, 113,
9027-9045.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1 is a view, partially schematic, of a preferred
apparatus according to the present invention for preparing the
gaseous precursor-filled liposome microspheres of the present
invention.
[0031] FIG. 2 shows a preferred apparatus for filtering and/or
dispensing therapeutic containing gaseous precursor-filled liposome
microspheres of the present invention.
[0032] FIG. 3 shows a preferred apparatus for filtering and/or
dispensing therapeutic containing gaseous precursor-filled liposome
microspheres of the present invention.
[0033] FIG. 4 is an exploded view of a portion of the apparatus of
FIG. 3.
[0034] FIG. 5 is a micrograph which shows the sizes of gaseous
precursor-filled liposomes of the invention before (A) and after
(B) filtration.
[0035] FIG. 6 graphically depicts the size distribution of gaseous
precursor-filled liposomes of the invention before (A) and after
(B) filtration.
[0036] FIG. 7 is a micrograph of a lipid suspension before (A) and
after (B) extrusion through a filter.
[0037] FIG. 8 is a micrograph of gaseous precursor-filled liposomes
formed subsequent to filtering and autoclaving a lipid suspension,
the micrographs having been taken before (A) and after (B) sizing
by filtration of the gaseous precursor-filled liposomes.
[0038] FIG. 9 is a diagrammatic illustration of a temperature
activated gaseous precursor-filled liposome prior to temperature
activation. The liposome has a multilamellar membrane.
[0039] FIG. 10 is a diagrammatic illustration of a temperature
activated liquid gaseous precursor-filled liposome after
temperature activation of the liquid to gaseous state resulting in
a unilamellar membrane and expansion of the liposome diameter.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention is directed to methods and apparatus
for preparing temperature activated gaseous precursor-filled
liposomes. Unlike the methods of the prior art which are directed
to the formation of liposomes with an aqueous solution filling the
interior, the methods of the present invention are directed to the
preparation of liposomes which comprise interior gaseous precursor
and/or ultimately gas.
[0041] As used herein, the phrase "temperature activated gaseous
precursor" denotes a compound which, at a selected activation or
transition temperature, changes phases from a liquid to a gas.
Activation or transition temperature, and like terms, refer to the
boiling point of the gaseous precursor, the temperature at which
the liquid to gaseous phase transition of the gaseous precursor
takes place. Useful gaseous precursors are those gases which have
boiling ponts in the range of about -100.degree. C. to 70.degree.
C. The activation temperature is particular to each gaseous
precursor. This concept is illustrated in FIGS. 9 and 10. An
activation temperature of about 37.degree. C., or human body
temperature, is preferred for gaseous precursors of the present
invention. Thus, a liquid gaseous precursor is activated to become
a gas at 37.degree. C. However, the gaseous precursor may be in
liquid or gaseous phase for use in the methods of the present
invention. The methods of the present invention may be carried out
below the boiling point of the gaseous precursor such that a liquid
is incorporated into a microsphere. In addition, the methods may be
performed at the boiling point of the gaseous precursor such that a
gas is incorporated into a microsphere. For gaseous precursors
having low temperature boiling points, liquid precursors may be
emulsified using a microfluidizer device chilled to a low
temperature. The boiling points may also be depressed using
solvents in liquid media to utilize a precursor in liquid form.
Alternatively, an upper limit of about 70.degree. C. may be
attained with focused high energy ultrasound. Further, the methods
may be performed where the temperature is increased throughout the
process, whereby the process starts with a gaseous precursor as a
liquid and ends with a gas.
[0042] The gaseous precursor may be selected so as to form the gas
in situ in the targeted tissue or fluid, in vivo upon entering the
patient or animal, prior to use, during storage, or during
manufacture. The methods of producing the temperature-activated
gaseous precursor-filled microspheres may be carried out at a
temperature below the boiling point of the gaseous precursor. In
this embodiment, the gaseous precursor is entrapped within a
microsphere such that the phase transition does not occur during
manufacture. Instead, the gaseous precursor-filled microspheres are
manufactured in the liquid phase of the gaseous precursor.
Activation of the phase transition may take place at any time as
the temperature is allowed to exceed the boiling point of the
precursor. Also, knowing the amount of liquid in a droplet of
liquid gaseous precursor, the size of the liposomes upon attaining
the gaseous state may be determined.
[0043] Alternatively, the gaseous precursors may be utilized to
create stable gas-filled microspheres which are pre-formed prior to
use. In this embodiment, the gaseous precursor is added to a
container housing a suspending and/or stabilizing medium at a
temperature below the liquid-gaseous phase transition temperature
of the respective gaseous precursor. As the temperature is then
exceeded, and an emulsion is formed between the gaseous precursor
and liquid solution, the gaseous precursor undergoes transition
from the liquid to the gaseous state. As a result of this heating
and gas formation, the gas displaces the air in the head space
above the liquid suspension so as to form gas-filled lipid spheres
which entrap the gas of the gaseous precursor, ambient gas (e.g.
air) or coentrap gas state gaseous precursor and ambient air. This
phase transition can be used for optimal mixing and stabilization
of the contrast medium. For example, the gaseous precursor,
perfluorobutane, can be entrapped in liposomes and as the
temperature is raised, beyond 3.degree. C. (boiling point of
perfluorobutane) liposomally entrapped fluorobutane gas results. As
an additional example, the gaseous precursor fluorobutane, can be
suspended in an aqueous suspension containing emulsifying and
stabilizing agents such as glycerol or propylene glycol and
vortexed on a commercial vortexer. Vortexing is commenced at a
temperature low enough that the gaseous precursor is liquid and is
continued as the temperature of the sample is raised past the phase
transition temperature from the liquid to gaseous state. In so
doing, the precursor converts to the gaseous state during the
microemulsification process. In the presence of the appropriate
stabilizing agents, surprisingly stable gas-filled liposomes
result.
[0044] Accordingly, the gaseous precursors of the present invention
may be selected to form a gas-filled liposome in vivo or designed
to produce the gas-filled liposome in situ, during the
manufacturing process, on storage, or at some time prior to
use.
[0045] As a further embodiment of this invention, by pre-forming
the liquid state of the gaseous precursor into an aqueous emulsion
and maintaining a known size, the maximum size of the microbubble
may be estimated by using the idea gas law, once the transition to
the gaseous state is effectuated. For the purpose of making gaseous
microspheres from gaseous precursors, the gas phase is assumed to
form instantaneously and no gas in the newly formed microbubble has
been depleted due to diffusion into the liquid (generally aqueous
in nature). Hence, from a known liquid volume in the emulsion, one
actually would predict an upper limit to the size of the gaseous
liposome.
[0046] Pursuant to the present invention, a emulsion of lipid
gaseous precursor-containing liquid droplets of defined size may be
formulated, such that upon reaching a specific temperature, the
boiling point of the gaseous precursor, the drpolets will expand
into gas liposomes of defined size. the defined size represents an
upper limit to the actual size because factors such as gas
diffusing into solution, loss of gas to the atmosphere, and the
effects of increased pressure are factors for which the ideal gas
law cannot account.
[0047] The ideal gas law and the equation for calculating the
increase in volume of the gas bubbles on transition from the liquid
to gaseous states follows:
[0048] The ideal gas law predicts the following:
PV=nRT
[0049] where
[0050] P=pressure in atmospheres
[0051] V=volume in liters
[0052] n=moles of gas
[0053] T=temperature in .degree. K
[0054] R=ideal gas constant=22.4 L atmospheres deg.sup.-1
mole.sup.-1
[0055] With knowledge of volume, density, and temperature of the
liquid in the emulsion of liquids, the amount (e.g. number of
moles) of liquid precursor as well as the volume of liquid
precursor, a priori, may be calculated, which when converted to a
gas, will expand into a liposome of known volume. The calculated
volume will reflect an upper limit to the size of the gaseous
liposome assuming instantaneous expansion into a gas liposome and
negligible diffusion of the gas over the time of the expansion.
[0056] Thus, stabilization of the precursor in the liquid state in
an emulsion whereby the precursor droplet is spherical, the volume
of the precursor droplet may be determined by the equation:
Volume(sphere)=4/3 .pi.r.sup.3
[0057] where
[0058] r=radius of the sphere
[0059] Thus, once the volume is predicted, and knowing the density
of the liquid at the desired temperature, the amount of liquid
(gaseous precursor) in the droplet may be determined. In more
descriptive terms, the following can be applied:
V.sub.gas=4/3 .pi.(r.sub.gas).sup.3
[0060] by the ideal gas law,
PV=nRT
[0061] substituting reveals,
V.sub.gas=nRT/P.sub.gas
[0062] or,
[0063] (A) n=4/3 [.pi.r.sub.gas.sup.3]P/RT
[0064] amount n=4/3 [.pi.r.sub.gas.sup.3 P/RT]*MW.sub.n
[0065] Converting back to a liquid volume
[0066] (B) V.sub.liq=[4/3[.pi.r.sub.gas.sup.3]P/RT]*MW.sub.n/D]
[0067] where D=the density of the precursor
[0068] Solving for the diameter of the liquid droplet,
[0069] (C)
diameter/2=[3/4.pi.[4/3*[.pi.r.sub.gas.sup.3]P/RT]MW.sub.n/D].s-
up.1/3
[0070] which reduces to
[0071] Diameter=2 [[r.sub.gas.sup.3]P/RT [MW.sub.n/D]].sup.1/3
[0072] As a further embodiment of the present invention, with the
knowledge of the volume and especially the radius, the
appropriately sized filter sizes the gaseous precursor droplets to
the appropriate diameter sphere.
[0073] A representative gaseous precursor may be used to form a
microsphere of defined size, for example, 10 microns diameter. In
this example, the microshpere is formed in the bloodstream of a
human being, thus the typical temperature would be 37.degree. C. or
310.degree. K. At a pressure of 1 atmosphere and using the equation
in (A), 7.54.times.10.sup.-17 moles of gaseous precursor would be
required to fill the volume of a 10 micron diameter
microsphere.
[0074] Using the above calculated amount of gaseous precursor, and
1-fluorobutane, which possesses a molecular weight of 76.11, a
boiling point of 32.5.degree. C. and a density of 6.7789
grams/mL.sup.-1 at 20.degree. C., further calculations predict that
5.74.times.10.sup.-15 grams of this precursor would be required for
a 10 micron microsphere. Extrapolating further, and armed with the
knowledge of the density, equation (B) further predicts that
8.47.times.10.sup.-16 mLs of liquid precursor are necessary to form
a microsphere with an upper limit of 10 microns.
[0075] Finally, using equation (C), an emulsion of lipid droplets
with a radius of 0.0272 microns or a corresponding diameter of
0.0544 microns need be formed to make a gaseous precursor filled
microsphere with an upper limit of a 10 micron microsphere.
[0076] An emulsion of this particular size could be easily achieved
by the use of an appropriately sized filter. In addition, as seen
by the size of the filter necessary to form gaseous precursor
droplets of defined size, the size of the filter would also suffice
to remove any possible bacterial contaminants and, hence, can be
used as a sterile filtration as well.
[0077] This embodiment of the present invention may be applied to
all gaseous precursors activated by temperature. In fact,
depression of the freezing point of the solvent system allows the
use gaseous precursors which would undergo liquid-to-gas phase
transitions at temperatures below 0.degree. C. The solvent system
can be selected to provide a medium for suspension of the gaseous
precursor. For example, 20% propylene glycol miscible in buffered
saline exhibits a freezing point depression well below the freezing
point of water alone. By increasing the amount of propylene glycol
or adding materials such as sodium chloride, the freezing point can
be depressed even further.
[0078] The selection of appropriate solvent systems may be
explained by physical methods as well. When substances, solid or
liquid, herein referred to as solutes, are dissolved in a solvent,
such as water based buffers for example, the freezing point is
lowered by an amount that is dependent upon the composition of the
solution. Thus, as defined by Wall, one can express the freezing
point depression of the solvent by the following:
Inx.sub.a=In(1-x.sub.b)=.DELTA.H.sub.fus/R(1/T.sub.o-1/T)
[0079] where:
[0080] x.sub.a=mole fraction of the solvent
[0081] x.sub.b=mole fraction of the solute
[0082] .DELTA.H.sub.fus=heat of fusion of the solvent
[0083] T.sub.o=Normal freezing point of the solvent
[0084] The normal freezing point of the solvent results. If X.sub.b
is small relative to x.sub.a, then the above equation may be
rewritten:
x.sup.b=.DELTA.H.sub.fus/R[T-T.sub.o/T.sub.oT].apprxeq..DELTA.H.sub.fus.DE-
LTA.T/RT.sub.o.sup.2
[0085] The above equation assumes the change in temperature
.DELTA.T is small compared to T.sub.2. The above equation can be
simplified further assuming the concentration of the solute (in
moles per thousand grams of solvent) can be expressed in terms of
the molality, m. Thus,
X.sub.b=m/[m+1000/m.sub.a].apprxeq.mMa/1000
[0086] where:
[0087] Ma=Molecular weight of the solvent, and
[0088] m=molality of the solute in moles per 1000 grams.
[0089] Thus, substituting for the fraction x.sub.b:
.DELTA.T=[M.sub.aRT.sub.o.sup.2/1000.DELTA.H.sub.fus].sub.m
[0090] or .DELTA.T=K.sub.fm, where
K.sub.f=M.sub.aRT.sub.o.sup.2/1000.DELTA.H.sub.fus
[0091] K.sub.f is referred to as the molal freezing point and is
equal to 1.86 degrees per unit of molal concentration for water at
one atmosphere pressure. The above equation may be used to
accurately determine the molal freezing point of gaseous-precursor
filled microsphere solutions of the present invention.
[0092] Hence, the above equation can be applied to estimate
freezing point depressions and to determine the appropriate
concentrations of liquid or solid solute necessary to depress the
solvent freezing temperature to an appropriate value.
[0093] Methods of preparing the temperature activated gaseous
precursor-filled liposomes include:
[0094] vortexing an aqueous suspension of gaseous precursor-filled
liposomes of the present invention; variations on this method
include optionally autoclaving before shaking, optionally heating
an aqueous suspension of gaseous precursor and lipid, optionally
venting the vessel containing the suspension, optionally shaking or
permitting the gaseous precursor liposomes to form spontaneously
and cooling down the gaseous precursor filled liposome suspension,
and optionally extruding an aqueous suspension of gaseous precursor
and lipid through a filter of about 0.22 .mu.m, alternatively,
filtering may be performed during in vivo administration of the
resulting liposomes such that a filter of about 0.22 .mu.m is
employed;
[0095] a microemulsification method whereby an aqueous suspension
of gaseous precursor-filled liposomes of the present invention are
emulsified by agitation and heated to form microspheres prior to
administration to a patient; and
[0096] forming a gaseous precursor in lipid suspension by heating,
and/or agitation, whereby the less dense gaseous precursor-filled
microspheres float to the top of the solution by expanding and
displacing other microspheres in the vessel and venting the vessel
to release air.
[0097] Freeze drying is useful to remove water and organic
materials from the lipids prior to the shaking gas instillation
method. Drying-gas instillation method may be used to remove water
from liposomes. By pre-entrapping the gaseous precursor in the
dried liposomes (i.e. prior to drying) after warming, the gaseous
precursor may expand to fill the liposome. Gaseous precursors can
also be used to fill dried liposomes after they have been subjected
to vacuum. As the dried liposomes are kept at a temperature below
their gel state to liquid crystalline temperature the drying
chamber can be slowly filled with the gaseous precursor in its
gaseous state, e.g. perfluorobutane can be used to fill dried
liposomes composed of dipalmitoylphosphatidylcholine (DPPC) at
temperatures between 3.degree. C. (the boiling point of
perfluorobutane) and below 40.degree. C., the phase transition
temperature of the lipid. In this case, it would be most preferred
to fill the liposomes at a temperature about 4.degree. C. to about
5.degree. C.
[0098] Preferred methods for preparing the temperature activated
gaseous precursor-filled liposomes comprise shaking an aqueous
solution having a lipid in the presence of a gaseous precursor at a
temperature below the gel state to liquid crystalline state phase
transition temperature of the lipid. The present invention also
provides a method for preparing gaseous precursor-filled liposomes
comprising shaking an aqueous solution comprising a lipid in the
presence of a gaseous precursor, and separating the resulting
gaseous precursor-filled liposomes for diagnostic or therapeutic
use. Liposomes prepared by the foregoing methods are referred to
herein as gaseous precursor-filled liposomes prepared by a gel
state shaking gaseous precursor installation method.
[0099] Conventional, aqueous-filled liposomes are routinely formed
at a temperature above the phase transition temperature of the
lipid, since they are more flexible and thus useful in biological
systems in the liquid crystalline state. See, for example, Szoka
and Papahadjopoulos, Proc. Natl. Acad. Sci. 1978, 75, 4194-4198. In
contrast, the liposomes made according to preferred embodiments of
the methods of the present invention are gaseous precursor-filled,
which imparts greater flexibility since gaseous precursors after
gas formation are more compressible and compliant than an aqueous
solution. Thus, the gaseous precursor-filled liposomes may be
utilized in biological systems when formed at a temperature below
the phase transition temperature of the lipid, even though the gel
phase is more rigid.
[0100] The methods of the present invention provide for shaking an
aqueous solution comprising a lipid in the presence of a
temperature activated gaseous precursor. Shaking, as used herein,
is defined as a motion that agitates an aqueous solution such that
gaseous precursor is introduced from the local ambient environment
into the aqueous solution. Any type of motion that agitates the
aqueous solution and results in the introduction of gaseous
precursor may be used for the shaking. The shaking must be of
sufficient force to allow the formation of foam after a period of
time. Preferably, the shaking is of sufficient force such that foam
is formed within a short period of time, such as 30 minutes, and
preferably within 20 minutes, and more preferably, within 10
minutes. The shaking may be by microemulsifying, by
microfluidizing, for example, swirling (such as by vortexing),
side-to-side, or up and down motion. In the case of the addition of
gaseous precursor in the liquid state, sonication may be used in
addition to the shaking methods set forth above. Further, different
types of motion may be combined. Also, the shaking may occur by
shaking the container holding the aqueous lipid solution, or by
shaking the aqueous solution within the container without shaking
the container itself. Further, the shaking may occur manually or by
machine. Mechanical shakers that may be used include, for example,
a shaker table, such as a VWR Scientific (Cerritos, Calif.) shaker
table, a microfluidizer, Wig-L-Bug.TM. (Crescent Dental
Manufacturing, Inc., Lyons, Ill.) and a mechanical paint mixer, as
well as other known machines. Another means for producing shaking
includes the action of gaseous precursor emitted under high
velocity or pressure. It will also be understood that preferably,
with a larger volume of aqueous solution, the total amount of force
will be correspondingly increased. Vigorous shaking is defined as
at least about 60 shaking motions per minute, and is preferred.
Vortexing at at least 1000 revolutions per minute, an example of
vigorous shaking, is more preferred. Vortexing at 1800 revolutions
per minute is most preferred.
[0101] The formation of gaseous precursor-filled liposomes upon
shaking can be detected by the presence of a foam on the top of the
aqueous solution. This is coupled with a decrease in the volume of
the aqueous solution upon the formation of foam. Preferably, the
final volume of the foam is at least about two times the initial
volume of the aqueous lipid solution; more preferably, the final
volume of the foam is at least about three times the initial volume
of the aqueous solution; even more preferably, the final volume of
the foam is at least about four times the initial volume of the
aqueous solution; and most preferably, all of the aqueous lipid
solution is converted to foam.
[0102] The required duration of shaking time may be determined by
detection of the formation of foam. For example, 10 ml of lipid
solution in a 50 ml centrifuge tube may be vortexed for
approximately 15-20 minutes or until the viscosity of the gaseous
precursor-filled liposomes becomes sufficiently thick so that it no
longer clings to the side walls as it is swirled. At this time, the
foam may cause the solution containing the gaseous precursor-filled
liposomes to raise to a level of 30 to 35 ml.
[0103] The concentration of lipid required to form a preferred foam
level will vary depending upoli the type of lipid used, and may be
readily determined by one skilled in the art, once armed with the
present disclosure. For example, in preferred embodiments, the
concentration of 1,2-dipalimitoylphosphatidylcholine (DPPC) used to
form gaseous precursor-filled liposomes according to the methods of
the present invention is about 20 mg/ml to about 30 mg/ml saline
solution. The concentration of distearoylphosphatidylcholine (DSPC)
used in preferred embodiments is about 5 mg/ml to about 10 mg/ml
saline solution.
[0104] Specifically, DPPC in a concentration of 20 mg/ml to 30
mg/ml, upon shaking, yields a total suspension and entrapped
gaseous precursor volume four times greater than the suspension
volume alone. DSPC in a concentration of 10 mg/ml, upon shaking,
yields a total volume completely devoid of any liquid suspension
volume and contains entirely foam.
[0105] It will be understood by one skilled in the art, once armed
with the present disclosure, that the lipids or liposomes may be
manipulated prior and subsequent to being subjected to the methods
of the present invention. For example, the lipid may be hydrated
and then lyophilized, processed through freeze and thaw cycles, or
simply hydrated. In preferred embodiments, the lipid is hydrated
and then lyophilized, or hydrated, then processed through freeze
and thaw cycles and then lyophilized, prior to the formation of
gaseous precursor-filled liposomes.
[0106] According to the methods of the present invention, the
presence of gas, such as and not limited to air, may also be
provided by the local ambient atmosphere. The local ambient
atmosphere may be the atmosphere within a sealed container, or in
an unsealed container, may be the external environment.
Alternatively, for example, a gas may be injected into or otherwise
added to the container having the aqueous lipid solution or into
the aqueous lipid solution itself in order to provide a gas other
than air. Gases that are not heavier than air may be added to a
sealed container while gases heavier than air may be added to a
sealed or an unsealed container. Accordingly, the present invention
includes co-entrapment of air and/or other gases along with gaseous
precursors.
[0107] The preferred methods of the invention are carried out at a
temperature below the gel state to liquid crystalline state phase
transition temperature of the lipid employed. By "gel state to
liquid crystalline state phase transition temperature", it is meant
the temperature at which a lipid bilayer will convert from a gel
state to a liquid crystalline state. See, for example, Chapman et
al., J. Biol. Chem. 1974, 249, 2512-2521. The gel state to liquid
crystalline state phase transition temperatures of various lipids
will be readily apparent to those skilled in the art and are
described, for example, in Gregoriadis, ed., Liposome Technology,
Vol. I, 1-18 (CRC Press, 1984) and Derek Marsh, CRC Handbook of
Lipid Bilayers (CRC Press, Boca Raton, Fla. 1990), at p. 139. See
also Table I, below. Where the gel state to liquid crystalline
state phase transition temperature of the lipid employed is higher
than room temperature, the temperature of the container may be
regulated, for example, by providing a cooling mechanism to cool
the container holding the lipid solution.
[0108] Since gaseous precursors (e.g. perfluorobutane) are less
soluble and diffusable than other gases, such as air, they tend to
be more stable when entrapped in liposomes even when the liposomes
are composed of lipids in the liquid-crystalline state. Small
liposomes composed of liquid-crystalline state lipid such as egg
phosphatidyl choline may be used to entrap a nanodroplet of
perfluorobutane. For example, lipid vesicles with diameters of
about 30 nm to about 50 nm may be used to entrap nanodroplets of
perfluorobutane with with mean diameter of about 25 nm. After
temperature activated conversion, the precursor filled liposomes
will create microspheres of about 10 microns in diameter. The lipid
in this cae, serves the purpose of defining the size of the
microsphere via the small liposome. The lipids also serve to
stabilize the resultant microsphere size. In this case, techniques
such as microemulsification are preferred for forming the small
liposomes which entrap the precursor. A microfluidizer
(Microfluidics, Newton, Mass.) is particularly useful for making an
emulsion of small liposomes which entrap the gaseous precursor.
1TABLE I Saturated Diacyl-sn-Glycero-3-Phosphocholi- nes Main Chain
Gel State to Liquid Crystalline State Phase Transition Temperatures
Liquid Crystalline # Carbons in Acyl Phase Transition Chains
Temperature (.degree. C.) 1,2-(12:0) -1.0 1,2-(13:0) 13.7
1,2-(14:0) 23.5 1,2-(15:0) 34.5 1,2-(16:0) 41.4 1,2-(17:0) 48.2
1,2-(18:0) 55.1 1,2-(19:0) 61.8 1,2-(20:0) 64.5 1,2-(21:0) 71.1
1,2-(22:0) 74.0 1,2-(23:0) 79.5 1,2-(24:0) 80.1
[0109] Conventional, aqueous-filled liposomes are routinely formed
at a temperature above the gel to liquid crystalline phase
transition temperature of the lipid, since they are more flexible
and thus useful in biological systems in the liquid crystalline
state. See, for example, Szoka and Papahadjopoulos, Proc. Natl.
Acad. Sci. 1978, 75, 4194-4198. In contrast, the liposomes made
according to preferred embodiments of the methods of the present
invention are gaseous precursor-filled, which imparts greater
flexibility since gaseous precursor is more compressible and
compliant than an aqueous solution. Thus, the gaseous
precursor-filled liposomes may be utilized in biological systems
when formed at a temperature below the phase transition temperature
of the lipid, even though the gel phase is more rigid.
[0110] A preferred apparatus for producing the temperature
activated gaseous precursor-filled liposomes using a gel state
shaking gaseous precursor instillation process is shown in FIG. 1.
A mixture of lipid and aqueous media is vigorously agitated in the
process of gaseous precursor installation to produce gaseous
precursor-filled liposomes, either by batch or by continuous feed.
Referring to FIG. 1, dried lipids 51 from a lipid supply vessel 50
are added via conduit 59 to a mixing vessel 66 in either a
continuous flow or as intermittent boluses. If a batch process is
utilized, the mixing vessel 66 may comprise a relatively small
container such as a syringe, test tube, bottle or round bottom
flask, or a large container. If a continuous feed process is
utilized, the mixing vessel is preferably a large container, such
as a vat.
[0111] Where the gaseous precursor-filled liposomes contain a
therapeutic compound, the therapeutic compound may be added, for
example, in a manner similar to the addition of the lipid described
above before the gaseous precursor installation process.
Alternatively, the therapeutic compound may be added after the
gaseous precursor installation process when the liposomes are
coated on the outside with the therapeutic compound.
[0112] In addition to the lipids 51, an aqueous media 53, such as a
saline solution, from an aqueous media supply vessel 52, is also
added to the vessel 66 via conduit 61. The lipids 51 and the
aqueous media 53 combine to form an aqueous lipid solution 74.
Alternatively, the dried lipids 51 could be hydrated prior to being
introduced into the mixing vessel 66 so that lipids are introduced
in an aqueous solution. In the preferred embodiment of the method
for making liposomes, the initial charge of solution 74 is such
that the solution occupies only a portion of the capacity of the
mixing vessel 66. Moreover, in a continuous process, the rates at
which the aqueous lipid solution 74 is added and gaseous
precursor-filled liposomes produced are removed is controlled to
ensure that the volume of lipid solution 74 does not exceed a
predetermined percentage of the mixing vessel 66 capacity.
[0113] The shaking may be accomplished by introducing a high
velocity jet of a pressurized gaseous precursor directly into the
aqueous lipid solution 74. Alternatively, the shaking may be
accomplished by mechanically shaking the aqueous solution, either
manually or by machine. Such mechanical shaking may be effected by
shaking the mixing vessel 66 or by shaking the aqueous solution 74
directly without shaking the mixing vessel itself. As shown in FIG.
1, in the preferred embodiment, a mechanical shaker 75, is
connected to the mixing vessel 66. The shaking should be of
sufficient intensity so that, after a period of time, a foam 73
comprised of gaseous precursor-filled liposomes is formed on the
top of the aqueous solution 74, as shown in FIG. 1. The detection
of the formation of the foam 73 may be used as a means for
controlling the duration of the shaking; that is, rather than
shaking for a predetermined period of time, the shaking may be
continued until a predetermined volume of foam has been
produced.
[0114] The apparatus of FIG. 1 may also contain a means for
controlling temperature such that the apparatus may be maintained
at one temperature for the method of making the liposomes. For
example, in the preferred embodiment, the methods of making
liposomes are performed at a temperature below the boiling point of
the gaseous precursor. In the preferred embodiment, a liquid
gaseous precursor fills the internal space of the liposomes.
Alternatively, the apparatus may be maintained at about the
temperature of the liquid to gas transition temperature of the
gaseous precursor such that a gas is contained in the liposomes.
Further, the temperature of the apparatus may be adjusted
throughout the method of making the liposomes such that the gaseous
precursor begins as a liquid, however, a gas is incorporated into
the resulting liposomes. In this embodiment, the temperature of the
apparatus is adjusted during the method of making the liposomes
such that the method begins at a temperature below the phase
transition temperature and is adjusted to a temperature at about
the phase transition temperature of the gaseous precursor.
Accordingly, the vessel may be closed and periodically vented, or
open to the ambient atmosphere.
[0115] In a preferred embodiment of the apparatus for making
gaseous precursor-filled liposomes in which the lipid employed has
a gel to liquid crystalline phase transition temperature below room
temperature, a means for cooling the aqueous lipid solution 74 is
provided. In the embodiment shown in FIG. 1, cooling is
accomplished by means of a jacket 64 disposed around the mixing
vessel 66 so as to form an annular passage surrounding the vessel.
As shown in, FIG. 1, a cooling fluid 63 is forced to flow through
this annular passage by means of jacket inlet and outlet ports 62
and 63, respectively. By regulating the temperature and flow rate
of the cooling fluid 62, the temperature of the aqueous lipid
solution 74 can be maintained at the desired temperature.
[0116] As shown in FIG. 1, a gaseous precursor 55, which may be
1-fluorobutane, for example, is introduced into the mixing vessel
66 along with the aqueous solution 74. Air may be introduced by
utilizing an unsealed mixing vessel so that the aqueous solution is
continuously exposed to environmental air. In a batch process, a
fixed charge of local ambient air may be introduced by sealing the
mixing vessel 66. If a gaseous precursor heavier than air is used,
the container need not be sealed. However, introduction of gaseous
precursors that are not heavier than air will require that the
mixing vessel be sealed, for example by use of a lid 65, as shown
in FIG. 1. The gaseous precursor 55 may be pressurized in the
mixing vessel 66, for example, by connecting the mixing vessel to a
pressurized gas supply tank 54 via a conduit 57, as shown in FIG.
1.
[0117] After the shaking is completed, the gaseous precursor-filled
liposome containing foam 73 may be extracted from the mixing vessel
66. Extraction may be accomplished by inserting the needle 102 of a
syringe 100, shown in FIG. 2, into the foam 73 and drawing a
predetermined amount of foam into the barrel 104 by withdrawing the
plunger 106. As discussed further below, the location at which the
end of the needle 102 is placed in the foam 73 may be used to
control the size of the gaseous precursor-filled liposomes
extracted.
[0118] Alternatively, extraction may be accomplished by inserting
an extraction tube 67 into the mixing vessel 66, as shown in FIG.
1. If the mixing vessel 66 is pressurized, as previously discussed,
the pressure of the gaseous precursor 55 may be used to force the
gaseous precursor-filled liposomes 77 from the mixing vessel 66 to
an extraction vessel 76 via conduit 70. In the event that the
mixing vessel 66 is not pressurized, the extraction vessel 76 may
be connected to a vacuum source 58, such as a vacuum pump, via
conduit 78, that creates sufficient negative pressure to suck the
foam 73 into the extraction vessel 76, as shown in FIG. 1. From the
extraction vessel 76, the gaseous precursor-filled liposomes 77 are
introduced into vials 82 in which they may be shipped to the
ultimate user. A source of pressurized gaseous precursor 56 may be
connected to the extraction vessel 76 as aid to ejecting the
gaseous precursor-filled liposomes. Since negative pressure may
result in increasing the size of the gaseous precursor-filled
liposomes, positive pressure is preferred for removing the gaseous
precursor-filled liposomes.
[0119] Filtration may be carried out in order to obtain gaseous
precursor-filled liposomes of a substantially uniform size. In
certain preferred embodiments, the filtration assembly contains
more than one filter, and preferably, the filters are not
immediately adjacent to each other, as illustrated in FIG. 4.
Before filtration, the gaseous precursor-filled liposomes range in
size from about 1 micron to greater than 60 microns (FIGS. 5A and
6A). After filtration through a single filter, the gaseous
precursor-filled liposomes are generally less than 10 microns but
particles as large as 25 microns in size remain. After filtration
through two filters (10 micron followed by 8 micron filter), almost
all of the liposomes are less than 10 microns, and most are 5 to 7
microns (FIGS. 5B and 6B).
[0120] As shown in FIG. 1, filtering may be accomplished by
incorporating a filter element 72 directly onto the end of the
extraction tube 67 so that only gaseous precursor-filled liposomes
below a pre-determined size are extracted from the mixing vessel
66. Alternatively, or in addition to the extraction tube filter 72,
gaseous precursor-filled liposome sizing may be accomplished by
means of a filter 80 incorporated into the conduit 79 that directs
the gaseous precursor-filled liposomes 77 from the extraction
vessel 76 to the vials 82, as shown in FIG. 1. The filter 80 may
contain a cascade filter assembly 124, such as that shown in FIG.
4. The cascade filter assembly 124 shown in FIG. 4 comprises two
successive filters 116 and 120, with filter 120 being disposed
upstream of filter 116. In a preferred embodiment, the upstream
filter 120 is a "NUCLEPORE" 10 .mu.m filter and the downstream
filter 116 is a "NUCLEPORE" 8 .mu.m filter. Two 0.15 mm metallic
mesh discs 115 are preferably installed on either side of the
filter 116. In a preferred embodiment, the filters 116 and 120 are
spaced apart a minimum of 150 .mu.m by means of a Teflon.TM.
O-ring, 118.
[0121] In addition to filtering, sizing may also be accomplished by
taking advantage of the dependence of gaseous precursor-filled
liposome buoyancy on size. The gaseous precursor-filled liposomes
have appreciably lower density than water and hence may float to
the top of the mixing vessel 66. Since the largest liposomes have
the lowest density, they will float most quickly to the top. The
smallest liposomes will generally be last to rise to the top and
the non gaseous precursor-filled lipid portion will sink to the
bottom. This phenomenon may be advantageously used to size the
gaseous precursor-filled liposomes by removing them from the mixing
vessel 66 via a differential flotation process. Thus, the setting
of the vertical location of the extraction tube 67 within the
mixing vessel 66 may control the size of the gaseous
precursor-filled liposomes extracted; the higher the tube, the
larger the gaseous precursor-filled liposomes extracted. Moreover,
by periodically or continuously adjusting the vertical location of
the extraction tube 67 within the mixing vessel 66, the size of the
gaseous precursor-filled liposomes extracted may be controlled on
an on-going basis. Such extraction may be facilitated by
incorporating a device 68, which may be a threaded collar 71 mating
with a threaded sleeve 72 attached to the extraction tube 67, that
allows the vertical location of the extraction tube 66 within the
extraction vessel 66 to be accurately adjusted.
[0122] The gel state shaking gaseous precursor installation process
itself may also be used to improve sizing of the gaseous
precursor-filled lipid based microspheres. In general, the greater
the intensity of the shaking energy, the smaller the size of the
resulting gaseous precursor-filled liposomes.
[0123] The current invention also includes novel methods for
preparing drug-containing gaseous precursor-filled liposomes to be
dispensed to the ultimate user. Once gaseous precursor-filled
liposomes are formed, they generally cannot be sterilized by
heating at a temperature that would cause rupture. Therefore, it is
desirable to form the gaseous precursor-filled liposomes from
sterile ingredients and to perform as little subsequent
manipulation as possible to avoid the danger of contamination.
According to the current invention, this may be accomplished, for
example, by sterilizing the mixing vessel containing the lipid and
aqueous solution before shaking and dispensing the gaseous
precursor-filled liposomes 77 from the mixing vessel 66, via the
extraction vessel 76, directly into the barrel 104 of a sterile
syringe 100, shown in FIG. 2, without further processing or
handling; that is, without subsequent sterilization. The syringe
100, charged with gaseous precursor-filled liposomes 77 and
suitably packaged, may then be dispensed to the ultimate user.
Thereafter, no further manipulation of the product is required in
order to administer the gaseous precursor-filled liposomes to the
patient, other than removing the syringe from its packaging and
removing a protector (not shown) from the syringe needle 102 and
inserting the needle into the body of the patient, or into a
catheter. Moreover, the pressure generated when the syringe plunger
106 is pressed into the barrel 104 will cause the largest gaseous
precursor-filled liposomes to collapse, thereby achieving a degree
of sizing without filtration.
[0124] Where it is desired to filter the gaseous precursor-filled
liposomes at the point of use, for example because they are removed
from the extraction vessel 76 without filtration or because further
filtration is desired, the syringe 100 may be fitted with its own
filter 108, as shown in FIG. 2. This results in the gaseous
precursor-filled liposomes being sized by causing them to be
extruded through the filter 108 by the action of the plunger 106
when the gaseous precursor-filled liposomes are injected. Thus, the
gaseous precursor-filled liposomes may be sized and injected into a
patient in one step.
[0125] In order to accommodate the use of a single or dual filter
in the hub housing of the syringe, a non-standard syringe with hub
housing is necessary. As shown in FIG. 3, the hub that houses the
filter(s) are of a dimension of approximately 1 cm to approximately
2 cm in diameter by about 1.0 cm to about 3.0 cm in length with an
inside diameter of about 0.8 cm for which to house the filters. The
abnormally large dimensions for the filter housing in the hub are
to accommodate passage of the microspheres through a hub with
sufficient surface area so as to decrease the pressure that need be
applied to the plunger of the syringe. In this manner, the
microspheres will not be subjected to an inordinately large
pressure head upon injection, which may cause rupture of the
microspheres.
[0126] As shown in FIG. 3, a cascade filter housing 110 may be
fitted directly onto a syringe 112, thereby allowing cascade
filtration at the point of use. As shown in FIG. 4, the filter
housing 110 is comprised of a cascade filter assembly 124,
previously discussed, incorporated between a lower collar 122,
having male threads, and a female collar 114, having female
threads. The lower collar 122 is fitted with a Luer lock that
allows it to be readily secured to the syringe 112 and the upper
collar 114 is fitted with a needle 102.
[0127] In preferred embodiments, the lipid solution is extruded
through a filter and the lipid solution is heat sterilized prior to
shaking. Once gaseous precursor-filled liposomes are formed, they
may be filtered for sizing as described above. These steps prior to
the formation of gaseous precursor-filled liposomes provide the
advantages, for example, of reducing the amount of unhydrated lipid
and thus providing a significantly higher yield of gaseous
precursor-filled liposomes, as well as and providing sterile
gaseous precursor-filled liposomes ready for administration to a
patient. For example, a mixing vessel such as a vial or syringe may
be filled with a filtered lipid suspension, and the solution may
then be sterilized within the mixing vessel, for example, by
autoclaving. A gaseous precursor may be instilled into the lipid
suspension to form gaseous precursor-filled liposomes by shaking
the sterile vessel. Preferably, the sterile vessel is equipped with
a filter positioned such that the gaseous precursor-filled
liposomes pass through the filter before contacting a patient.
[0128] The first step of this preferred method, extruding the lipid
solution through a filter, decreases the amount of unhydrated lipid
by breaking up the dried lipid and exposing a greater surface area
for hydration. Preferably, the filter has a pore size of about 0.1
to about 5 .mu.m, more preferably, about 0.1 to about 4 .mu.m, even
more preferably, about 0.1 to about 2 .mu.m, and even more
preferably, about 1 .mu.m, most preferably 0.22 .mu.m. As shown in
FIG. 7, when a lipid suspension is filtered (FIG. 7B), the amount
of unhydrated lipid is reduced when compared to a lipid suspension
that was not pre-filtered (FIG. 7A). Unhydrated lipid appears as
amorphous clumps of non-uniform size and is undesirable.
[0129] The second step, sterilization, provides a composition that
may be readily administered to a patient. Preferably, sterilization
is accomplished by heat sterilization, preferably, by autoclaving
the solution at a temperature of at least about 100.degree. C., and
more preferably, by autoclaving at about 100.degree. C. to about
130.degree. C., even more preferably, about 110.degree. C. to about
130.degree. C., even more preferably, about 120.degree. C. to about
130.degree. C., and most preferably, about 130.degree. C.
Preferably, heating occurs for at least about 1 minute, more
preferably, about 1 to about 30 minutes, even more preferably,
about 10 to about 20 minutes, and most preferably, about 15
minutes.
[0130] Where sterilization occurs by a process other than heat
sterilization at a temperature which would cause rupture of the
gaseous precursor-filled liposomes, sterilization may occur
subsequent to the formation of the gaseous precursor-filled
liposomes, and is preferred. For example, gamma radiation may be
used before and/or after gaseous precursor-filled liposomes are
formed.
[0131] Sterilization of the gaseous precursor may be achieved via
passage through a 0.22 .mu.m filter or a smaller filter, prior to
emulsification in the aqueous media. This can be easily achieved
via sterile filtration of the contents directly into a vial which
contains a predetermined amount of likewise sterilized and
sterile-filled aqueous carrier.
[0132] FIG. 8 illustrates the ability of gaseous precursor-filled
liposomes to successfully form after autoclaving, which was carried
out at 130.degree. C. for 15 minutes, followed by vortexing for 10
minutes. Further, after the extrusion and sterilization procedure,
the shaking step yields gaseous precursor-filled liposomes with
little to no residual anhydrous lipid phase. FIG. 8A shows gaseous
precursor-filled liposomes generated after autoclaving but prior to
filtration, thus resulting in a number of gaseous precursor-filled
liposomes having a size greater than 10 .mu.m. FIG. 8B shows
gaseous precursor-filled liposomes after a filtration through a 10
.mu.m "NUCLEPORE" filter, resulting in a uniform size around 10
.mu.m.
[0133] The materials which may be utilized in preparing the gaseous
precursor-filled lipid microspheres include any of the materials or
combinations thereof known to those skilled in the art as suitable
for liposome preparation. Gas precursors which undergo phase
transition from a liquid to a gas at their boiling point may be
used in the present invention. The lipids used may be of either
natural or synthetic origin. The particular lipids are chosen to
optimize the desired properties, e.g., short plasma half-life
versus long plasma half-life for maximal serum stability. It will
also be understood that certain lipids may be more efficacious for
particular applications, such as the containment of a therapeutic
compound to be released upon rupture of the gaseous
precursor-filled lipid microsphere.
[0134] The lipid in the gaseous precursor-filled liposomes may be
in the form of a single bilayer or a multilamellar bilayer, and are
preferably multilamellar.
[0135] Gaseous precursors which may be activated by temperature may
be useful in the present invention. Table II lists examples of
gaseous precursors which undergo phase transitions from liquid to
gaseous states at close to normal body temperature (37.degree. C.)
and the size of the emulsified droplets that would be required to
form a microsphere having a size of 10 microns. The list is
composed of potential gaseous precursors that may be used to form
temperature activated gaseous precursor-containing liposomes of a
defined size. The list should not be construed as being limiting by
any means, as to the possibilities of gaseous precursors for the
methods of the present invention.
2TABLE II Physical Characteristics of Gaseous Precursors and
Diameter of Emulsified Droplet to Form a 10 .mu.m Microsphere
Diameter (.mu.m) of Emulsified droplet Molecular Boiling Point to
make 10 micron Compound Weight (.degree. C.) Density microsphere 1-
76.11 32.5 6.7789 1.2 fluorobutane 2-methyl 72.15 27.8 0.6201 2.6
butane (isopentane) 2-methyl 1- 70.13 31.2 0.6504 2.5 butene
2-methyl-2- 70.13 38.6 0.6623 2.5 butene 1-butene-3- 66.10 34.0
0.6801 2.4 yne-2-methyl 3-methyl-1- 68.12 29.5 0.6660 2.5 butyne
perfluoro 88.00 -129 3.034 3.3 methane perfluoro 138.01 -79 1.590
1.0 ethane perfluoro 238.03 3.96 1.6484 2.8 butane perfluoro 288.04
57.73 1.7326 2.9 pentane octafluoro 200.04 -5.8 1.48 2.8
cyclobutane decafluoro 238.04 -2 1.517 3.0 butane hexafluoro 138.01
-78.1 1.607 2.7 ethane docecafluoro 288.05 29.5 1.664 2.9 pentane
octafluoro-2- 200.04 1.2 1.5297 2.8 butene perfluoro 200.04 -5.8
1.48 2.8 cyclobutane octafluoro 212.05 27 1.58 2.7 cyclopentene
perfluoro 162 5 1.602 2.5 cyclobutene *Source: Chemical Rubber
Company Handbook of Chemistry and Physics Robert C. Weast and David
R. Lide, eds. CRC Press, Inc. Boca Raton, Florida. (1989-1990).
[0136] Examples of gaseous precursors are by no means limited to
Table II. In fact, for a variety of different applications,
virtually any liquid can be used to make gaseous precursors so long
as it is capable of undergoing a phase transition to the gas phase
upon passing through the appropriate activation temperature.
Examples of gaseous precursors that may be used include, and are by
no means limited to, the following: hexafluoro acetone; isopropyl
acetylene; allene; tetrafluoroallene; boron trifluoride;
1,2-butadiene; 1,3-butadiene; 1,3-butadiene; 1,2,3-trichloro,
2-fluoro-1,3-butadiene; 2-methyl,1,3 butadiene;
hexafluoro-1,3-butadiene; butadiyne; 1-fluoro-butane;
2-methyl-butane; decafluoro butane; 1-butene; 2-butene;
2-methy-1-butene; 3-methyl-1-butene; perfluoro-1-butene;
perfluoro-1-butene; perfluoro-2-butene; 1,4-phenyl-3-butene-2-one;
2-methyl-1-butene-3-yne; butyl nitrate; 1-butyne; 2-butyne;
2-chloro-1,1,1,4,4,4-hexafluoro-butyne- ; 3-methyl-1-butyne;
perfluoro-2-butyne; 2-bromo-butyraldehyde; carbonyl sulfide;
crotononitrile; cyclobutane; methyl-cyclobutane;
octafluoro-cyclobutane; perfluoro-cyclobutene;
3-chloro-cyclopentene; perfluoro ethane; perfluoro propane;
perfluoro butane; perfluoro pentane; perfluoro hexane;
cyclopropane; 1,2-dimethyl-cyclopropane; 1,1-dimethyl cyclopropane;
1,2-dimethyl cyclopropane; ethyl cyclopropane; methyl cyclopropane;
diacetylene; 3-ethyl-3-methyl diaziridine;
1,1,1-trifluorodiazoethane; dimethyl amine; hexafluoro-dimethyl
amine; dimethylethylamine; -bis-(Dimethyl phosphine)amine;
2,3-dimethyl-2-norbornane; perfluorodimethylamine; dimethyloxonium
chloride; 1,3-dioxolane-2-one; perfluorocarbons such as and not
limited to 4-methyl,1,1,1,2-tetrafluoro ethane;
1,1,1-trifluoroethane; 1,1,2,2-tetrafluoroethane;
1,1,2-trichloro-1,2,2-trifluoroethane; 1,1 dichloroethane;
1,1-dichloro-1,2,2,2-tetrafluoro ethane; 1,2-difluoro ethane;
1-chloro-1,1,2,2,2-pentafluoro ethane; 2-chloro,
1,1-difluoroethane; 1-chloro-1,1,2,2-tetrafluoro ethane; 2-chloro,
1,1-difluoroethane; chloroethane; chloropentafluoro ethane;
dichlorotrifluoroethane; fluoro-ethane; hexafluoro-ethane;
nitro-pentafluoro ethane; nitroso-pentafluoro ethane; perfluoro
ethane; perfluoro ethylamine; ethyl vinyl ether; 1,1-dichloro
ethylene; 1,1-dichloro-1,2-difluoro ethylene; 1,2-difluoro
ethylene; Methane; Methane-sulfonyl chloride-trifluoro;
Methanesulfonyl fluoride-trifluoro;
Methane-(pentafluorothio)trifluoro; Methane-bromo difluoro nitroso;
Methane-bromo fluoro; Methane-bromo chloro-fluoro;
Methanebromo-trifluoro; Methane-chloro difluoro nitro;
Methane-chloro dinitro; Methanechloro fluoro; Methane-chloro
trifluoro; Methane-chloro-difluoro; Methane dibromo difluoro;
Methane-dichloro difluoro; Methane-dichloro-fluoro;
Methanedifluoro; Methane-difluoro-iodo; Methane-disilano;
Methane-fluoro; Methaneiodo; Methane-iodo-trifluoro;
Methane-nitro-trifluoro; Methane-nitroso-trifluor- o;
Methane-tetrafluoro; Methane-trichlorofluoro; Methane-trifluoro;
Methanesulfenylchloride-trifluoro; 2-Methyl butane; Methyl ether;
Methyl isopropyl ether; Methyl lactate; Methyl nitrite; Methyl
sulfide; Methyl vinyl ether; Neon; Neopentane; Nitrogen (N.sub.2);
Nitrous oxide; 1,2,3-Nonadecane tricarboxylic
acid-2-hydroxytrimethylester; 1-Nonene-3-yne; Oxygen (O.sub.2);
1,4-Pentadiene; n-Pentane; Pentane-perfluoro;
2-Pentanone-4-amino-4-methyl; 1-Pentene; 2-Pentene [cis]; 2-Pentene
(trans); 1-Pentene-3-bromo; 1-Pentene-perfluoro; Phthalic
acid-tetrachloro; Piperidine-2,3,6-trimethyl; Propane, Propane-1,
1, 1, 2, 2,3-hexafluoro; Propane-1,2-epoxy; Propane-2,2 difluoro;
Propane 2-amino, Propane-2-chloro; Propane-heptafluoro-1-nitro;
Propane-heptafluoro-1-nitroso; Propane-perfluoro; Propene;
Propyl-1,1,1,2,3,3-hexafluoro-2,3 dichloro; Propylene-1-chloro;
Propylenechloro-(trans); Propylene-2-chloro; Propylene-3-fluoro;
Propylene-perfluoro; Propyne; Propyne-3,3,3-trifluoro;
Styrene-3-fluoro; Sulfur hexafluoride; Sulfur
(di)-decafluoro(S2F10); Toluene-2,4-diamino; Trifluoroacetonitrile;
Trifluoromethyl peroxide; Trifluoromethyl sulfide; Tungsten
hexafluoride; Vinyl acetylene; Vinyl ether; Xenon; Nitrogen; air;
and other ambient gases.
[0137] Perfluorocarbons are the preferred gases of the present
invention, fluorine gas, perfluoromethane, perfluoroethane,
perfluorobutane, perfluoropentane, perfluorohexane; even more
preferrably perfluoroethane, perfluoropropane and perfluorobutane;
most preferrably perfluoropropane and perfluorobutane as the more
inert perfluorinated gases are less toxic.
[0138] Microspheres of the present invention include and are not
limited to liposomes, lipid coatings, emulsions and polymers.
[0139] Lipids which may be used to create lipid microspheres
include but are not limited to: lipids such as fatty acids,
lysolipids, phosphatidylcholine with both saturated and unsaturated
lipids including dioleoylphosphatidylcholine;
dimyristoylphosphatidylcholine;
dipentadecanoylphosphatidyl-choline, dilauroylphosphatidylcholine,
dioleoylphosphatidyl-choline, dipalmitoylphosphatidylcholine;
distearoyl-phosphatidylcholine; phosphatidylethanolamines such as
dioleoylphosphatidylethanolamine; phosphatidylserine;
phosphatidylglycerol; phosphatidylinositol, sphingolipids such as
sphingomyelin; glycolipids such as ganglioside GM1 and GM2;
glucolipids; sulfatides; glycosphingolipids; phosphatidic acid;
palmitic acid; stearic acid; arachidonic acid; oleic acid; lipids
bearing polymers such as polyethyleneglycol, chitin, hyaluronic
acid or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-,
oligo- or polysaccharides; cholesterol, cholesterol sulfate and
cholesterol hemisuccinate; tocopherol hemisuccinate, lipids with
ether and ester-linked fatty acids, polymerized lipids, diacetyl
phosphate, stearylamine, cardiolipin, phospholipids with short
chain fatty acids of 6-8 carbons in length, synthetic phospholipids
with asymmetric acyl chains (e.g., with one acyl chain of 6 carbons
and another acyl chain of 12 carbons),
6-(5-cholesten-3.beta.-yloxy)-1-thio-.beta.-D-galactopyranoside,
digalactosyldiglyceride,
6-(5-cholesten-3.beta.-yloxy)hexyl-6-amino-6-deo-
xy-1-thio-.beta.-D-galactopyranoside,
6-(5-cholesten-3.beta.-yloxy)hexyl-6-
-amino-6-deoxyl-1-thio-.alpha.-D-mannopyranoside,
12-(((7'-diethylaminocou-
marin-3-yl)carbonyl)methylamino)-octadecanoic acid;
N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)
octadecanoyl]-2-aminopalmitic acid;
cholesteryl)4'-trimethyl-ammonio)buta- noate;
N-succinyldioleoylphosphatidylethanol-amine;
1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol;
1,3-dipalmitoyl-2-succinylglycerol;
1-hexadecyl-2-palmitoylglycerophospho- ethanolamine; and
palmitoylhomocysteine; and/or combinations thereof. The liposomes
may be formed as monolayers or bilayers and may or may not have a
coating.
[0140] Lipids bearing hydrophilic polymers such as
polyethyleneglycol (PEG), including and not limited to PEG 2,000
MW, 5,000 MW, and PEG 8,000 MW, are particularly useful for
improving the stability and size distribution of the gaseous
precursor-containing liposomes. Various different mole ratios of
PEGylated lipid, dipalmitoylphosphatidylethanola- mine (DPPC)
bearing PEG 5,000 MW, for example, are also useful; 8 mole percent
DPPC is preferred. A preferred product which is highly useful for
entrapping gaseous precursors contains 83 mole percent DPPC, 8 mole
percent DPPE-PEG 5,000 MW and 5 mole percent
dipalmitoylphosphatidic acid.
[0141] In addition, examples of compounds used to make mixed
systems include, but by no means are limited to
lauryltrimethylammonium bromide (dodecyl-), cetyltrimethylammonium
bromide (hexadecyl-), myristyltri-methylammonium bromide
(tetradecyl-), alkyldimethylbenzylammo- nium chloride
(alkyl=C12,C14,C16), benzyldimethyldodecylammonium
bromide/chloride, benzyldimethylhexadecylammonium bromide/chloride,
benzyldimethyltetradecylammonium bromide/chloride,
cetyldimethylethylammonium bromide/chloride, or cetylpyridinium
bromide/chloride. Likewise perfluorocarbons such as pentafluoro
octadecyl iodide, perfluorooctylbromide (PFOB), perfluorodecalin,
perfluorododecalin, perfluorooctyliodide, perfluorotripropylamine,
and perfluorotributylamine. The perfluorocarbons may be entrapped
in liposomes or stabilized in emulsions as is well know in the art
such as U.S. Pat. No. 4,865,836. The above examples of lipid
suspensions may also be sterilized via autoclave without
appreciable change in the size of the suspensions.
[0142] If desired, either anionic or cationic lipids may be used to
bind anionic or cationic pharmaceuticals. Cationic lipids may be
used to bind DNA and RNA analogues with in or on the surface of the
gaseous precursor-filled microsphere. A variety of lipids such as
DOTMA, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
chloride; DOTAP, 1,2-dioleoyloxy-3-(trimethylammonio)propane; and
DOTB, 1,2-dioleoyl-3-(4'-trimethyl-ammonio)butanoyl-sn-glycerol may
be used. In general the molar ratio of cationic lipid to
non-cationic lipid in the liposome may be, for example, 1:1000,
1:100, preferably, between 2:1 to 1:10, more preferably in the
range between 1:1 to 1:2.5 and most preferably 1:1 (ratio of mole
amount cationic lipid to mole amount non-cationic lipid, e.g.,
DPPC). A wide variety of lipids may comprise the non-cationic lipid
when cationic lipid is used to construct the microsphere.
Preferably, this non-cationic lipid is
dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine
or dioleoylphosphatidyl-ethanolamine. In lieu of cationic lipids as
described above, lipids bearing cationic polymers such as
polylysine or polyarginine may also be used to construct the
microspheres and afford binding of a negatively charged
therapeutic, such as genetic material, to the outside of the
microspheres. Additionally, negatively charged lipids may be used,
for example, to bind positively charged therapeutic compounds.
Phosphatidic acid, a negatively charged lipid, can also be used to
complex DNA. This is highly surprising, as the positively charged
lipids were heretofore thought to be generally necessary to bind
genetic materials to liposomes. 5 to 10 mole percent phosphatidic
acid in the liposomes improves the stability and size distribution
of the gaseous precursor-filled liposomes.
[0143] Other useful lipids or combinations thereof apparent to
those skilled in the art which are in keeping with the spirit of
the present invention are also encompassed by the present
invention. For example, carbohydrate-bearing lipids may be employed
for in vivo targeting, as described in U.S. Pat. No. 4,310,505, the
disclosures of which are hereby incorporated herein by reference in
their entirety.
[0144] The most preferred lipids are phospholipids, preferably DPPC
and DSPC, and most preferably DPPC.
[0145] Saturated and unsaturated fatty acids that may be used to
generate gaseous precursor-filled microspheres preferably include,
but are not limited to molecules that have between 12 carbon atoms
and 22 carbon atoms in either linear or branched form. Examples of
saturated fatty acids that may be used include, but are not limited
to, lauric, myristic, palmitic, and stearic acids. Examples of
unsaturated fatty acids that may be used include, but are not
limited to, lauroleic, physeteric, myristoleic, palmitoleic,
petroselinic, and oleic acids. Examples of branched fatty acids
that may be used include, but are not limited to, isolauric,
isomyristic, isopalmitic, and isostearic acids and isoprenoids.
[0146] Cationic polymers may be bound to the lipid layer through
one or more alkyl groups or sterol groups which serve to anchor the
cationic polymer into the lipid layer surrounding the gaseous
precursor. Cationic polymers that may be used in this manner
include, but are not limited to, polylysine and polyarginine, and
their analogs such as polyhomoarginine or polyhomolysine. The
positively charged groups of cationic lipids and cationic polymers,
or perfluoroalkylated groups bearing cationic groups, for example,
may be used to complex negatively charged molecules such as sugar
phosphates on genetic material, thus binding the material to the
surface of the gaseous precursor-filled lipid sphere. For example,
cationic analogs of amphiphilic perfluoroalkylated bipyridines, as
described in Garelli and Vierling, Biochim. Biophys Acta, 1992,
1127, 41-48, the disclosures of which are hereby incorporated
herein by reference in their entirety, may be used. Alternatively,
for example, negatively charged molecules may be bound directly to
the head groups of the lipids via ester, amide, ether, disulfide or
thioester linkages.
[0147] Bioactive materials, such as peptides or proteins, may be
incorporated into the lipid layer provided the peptides have
sufficient lipophilicity or may be derivatized with alkyl or sterol
groups for attachment to the lipid layer. Negatively charged
peptides may be attached, for example, using cationic lipids or
polymers as described above.
[0148] One or more emulsifying or stabilizing agents may be
included with the gaseous precursors to formulate the temperature
activated gaseous precursor-filled microspheres. The purpose of
these emulsifying/stabilizing agents is two-fold. Firstly, these
agents help to maintain the size of the gaseous precursor-filled
microsphere. As noted above, the size of these microspheres will
generally affect the size of the resultant gas-filled microspheres.
Secondly the emulsifying and stabilizing agents may be used to coat
or stabilize the microsphere which results from the precursor.
Stabilization of contrast agent-containing microspheres is
desirable to maximize the in vivo contrast effect. Although
stabilization of the microsphere is preferred this is not an
absolute requirement. Because the gas-filled microspheres resulting
from these gaseous precursors are more stable than air, they may
still be designed to provide useful contrast enhancement; for
example, they pass through the pulmonary circulation following
peripheral venous injection, even when not specifically stabilized
by one or more coating or emulsifying agents. One or more coating
or stabilizing agents is preferred however, as are flexible
stabilizing materials. Gas microspheres stabilized by
polysaccharides, gangliosides, and polymers are more effective than
those stabilized by albumin and other proteins. Liposomes prepared
using aliphatic compounds are preferred as microspheres stabilized
with these compounds are much more flexible and stable to pressure
changes.
[0149] Solutions of lipids or gaseous precursor-filled liposomes
may be stabilized, for example, by the addition of a wide variety
of viscosity modifiers, including, but not limited to carbohydrates
and their phosphorylated and sulfonated derivatives; polyethers,
preferably with molecular weight ranges between 400 and 8000; di-
and trihydroxy alkanes and their polymers, preferably with
molecular weight ranges between 800 and 8000. Glycerol propylene
glycol, polyethylene glycol, polyvinyl pyrrolidone, and polyvinyl
alcohol may also be useful as stabilizers in the present invention.
Particles which are porous or semi-solid such as hydroxyapatite,
metal oxides and coprecipitates of gels, e.g. hyaluronic acid with
calcium may be used to formulate a center or nidus to stabilize the
gaseous precursors.
[0150] Emulsifying and/or solubilizing agents may also be used in
conjunction with lipids or liposomes. Such agents include, but are
not limited to, acacia, cholesterol, diethanolamine, glyceryl
monostearate, lanolin alcohols, lecithin, mono- and di-glycerides,
mono-ethanolamine, oleic acid, oleyl alcohol, poloxamer,
polyoxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 10
oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate,
polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80,
propylene glycol diacetate, propylene glycol monostearate, sodium
lauryl sulfate, sodium stearate, sorbitan mono-laurate, sorbitan
mono-oleate, sorbitan mono-palmitate, sorbitan monostearate,
stearic acid, trolamine, and emulsifying wax. All lipids with
perfluoro fatty acids as a component of the lipid in lieu of the
saturated or unsaturated hydrocarbon fatty acids found in lipids of
plant or animal origin may be used. Suspending and/or
viscosity-increasing agents that may be used with lipid or liposome
solutions include but are not limited to, acacia, agar, alginic
acid, aluminum mono-stearate, bentonite, magma, carbomer 934P,
carboxymethylcellulose, calcium and sodium and sodium 12, glycerol,
carrageenan, cellulose, dextrin, gelatin, guar gum, hydroxyethyl
cellulose, hydroxypropyl methylcellulose, magnesium aluminum
silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl
alcohol, povidone, propylene glycol, alginate, silicon dioxide,
sodium alginate, tragacanth, and xanthum gum. A preferred product
of the present invention incorporates lipid as a mixed solvent
system in a ratio of 8:1:1 or 9:1:1 normal saline:
glycerol:propylene glycol.
[0151] The gaseous precursor-filled liposomes of the present
invention are preferably comprised of an impermeable material.
Impermeable material is defined a material that does not permit the
passage of a substantial amount of the contents of the liposome in
typical storage conditions. Substantial is defined as greater than
about 50% of the contents, the contents being both the gas as well
as any other component encapsulated within the interior of the
liposome, such as a therapeutic. Preferably, no more than about 25%
of the gas is released, more preferably, no more than about 10% of
the gas is released, and most preferably, no more than about 1% of
the gas is released during storage and prior to administration to a
patient.
[0152] At least in part, the gas impermeability of gaseous
precursor-filled liposomes has been found to be related to the gel
state to liquid crystalline state phase transition temperature. It
is believed that, generally, the higher gel state to liquid
crystalline state phase transition temperature, the more gas
impermeable the liposomes are at a given temperature. See Table I
above and Derek Marsh, CRC Handbook of Lipid Bilayers (CRC Press,
Boca Raton, Fla. 1990), at p. 139 for main chain melting
transitions of saturated diacyl-sn-glycero-3-phosphocholine- s.
However, it should be noted that a lesser degree of energy can
generally be used to release a therapeutic compound from gaseous
precursor-filled liposomes composed of lipids with a lower gel
state to liquid crystalline state phase transition temperature.
[0153] In certain preferred embodiments, the phase transition
temperature of the lipid is greater than the internal body
temperature of the patient to which they are administered. For
example, lipids having a phase transition temperature greater than
about 37.degree. C. are preferred for administration to humans. In
general, microspheres having a gel to liquid phase transition
temperature greater than about 20.degree. C. are adequate and those
with a phase transition temperature greater than about 37.degree.
C. are preferred.
[0154] In preferred embodiments, the liposomes made by the methods
of the present invention are stable, stability being defined as
resistance to rupture from the time of formation until the
application of ultrasound. The lipids used to construct the
microspheres may be chosen for stability. For example, gaseous
precursor-filled liposomes composed of DSPC
(distearoylphosphatidylcholine) are more stable than gaseous
precursor-filled liposomes composed of DPPC
(dipalmitoylphosphatidylcholi- ne) and that these in turn are more
stable than gaseous precursor-filled liposomes composed of egg
phosphatidylcholine (EPC). Preferably, no more than about 50% of
the liposomes rupture from the time of formation until the
application of ultrasound, more preferably, no more than about 25%
of the liposomes rupture, even more preferably, no more than about
10% of the liposomes, and most preferably, no more than about 1% of
the liposomes.
[0155] The subject liposomes tend to have greater gas
impermeability and stability during storage than other gas-filled
liposomes produced via known procedures such as pressurization or
other techniques. At 72 hours after formation, for example,
conventionally prepared liposomes often are essentially devoid of
gas, the gas having diffused out of the liposomes and/or the
liposomes having ruptured and/or fused, resulting in a concomitant
loss in reflectivity. In comparison, gaseous precursor-filled
liposomes of the present invention maintained in aqueous solution
generally have a shelf life stability of greater than about three
weeks, preferably a shelf life stability of greater than about four
weeks, more preferably a shelf life stability of greater than about
five weeks, even more preferably a shelf life stability of greater
than about three months, and often a shelf life stability that is
even much longer, such as over six months, twelve months, or even
two years.
[0156] In addition, it has been found that the gaseous
precursor-filled liposomes of the present invention can be
stabilized with lipids covalently linked to polymers of
polyethylene glycol, commonly referred to as PEGylated lipids. It
has also been found that the incorporation of at least a small
amount of negatively charged lipid into any liposome membrane,
although not required, is beneficial to providing liposomes that do
not have a propensity to rupture by aggregation. By at least a
small amount, it is meant about 1 to about 10 mole percent of the
total lipid. Suitable negatively charged lipids, or lipids bearing
a net negative charge, will be readily apparent to those skilled in
the art, and include, for example, phosphatidylserine,
phosphatidylglycerol, phosphatidic acid, and fatty acids. Liposomes
prepared from dipalmitoylphosphatidylcholine are most preferred as
they are selected for their ability to rupture on application of
resonant frequency ultrasound, radiofrequency energy, (e.g.
microwave), and/or echogenicity in addition to their stability
during delivery.
[0157] Further, the liposomes of the invention are preferably
sufficiently stable in the vasculature such that they withstand
recirculation. The gaseous precursor-filled liposomes may be coated
such that uptake by the reticuloendothelial system is minimized.
Useful coatings include, for example, gangliosides, glucuronide,
galacturonate, guluronate, polyethyleneglycol, polypropylene
glycol, polyvinylpyrrolidone, polyvinylalcohol, dextran, starch,
phosphorylated and sulfonated mono, di, tri, oligo and
polysaccharides and albumin. The liposomes may also be coated for
purposes such as evading recognition by the immune system.
[0158] The lipid used is also preferably flexible. Flexibility, as
defined in the context of gaseous precursor-filled liposomes, is
the ability of a structure to alter its shape, for example, in
order to pass through an opening having a size smaller than the
liposome.
[0159] Provided that the circulation half-life of the liposomes is
sufficiently long, the liposomes will generally pass through the
target tissue while passing through the body. Thus, by focusing the
sound waves on the selected tissue to be treated, the therapeutic
will be released locally in the target tissue. As a further aid to
targeting, antibodies, carbohydrates, peptides, glycopeptides,
glycolipids, lectins, and synthetic and natural polymers may also
be incorporated into the surface of the liposomes. Other aids for
targeting include polymers such as polyethyleneglycol,
polyvinylpyrrolidone, and polyinylalcohol, which may be
incorporated onto the surface via alkylation, acylation, sterol
groups or derivatized head groups of phospholipids such as
dioleoylphosphatidylethanolamine (DOPE),
dipalmitoylphosphatidylethanolam- ine (DPPE), or
distearoylphosphatidylethanolamine (DSPE). Peptides, antibodies,
lectins, glycopeptides, oligonucleotides, and glycoconjugates may
also be incorporated onto the surfaces of the gaseous
precursor-filled lipid spheres.
[0160] In certain preferred embodiments, as an aid to the gaseous
precursor instillation process as well as to maintain the stability
of the gaseous precursor-filled liposomes, for example, emulsifiers
may be added to the lipid. Examples of emulsifiers include, but are
not limited to, glycerol, cetyl alcohol, sorbitol, polyvinyl
alcohol, polypropylene glycol, propylene glycol, ethyl alcohol,
sodium lauryl sulfate, Laureth 23, polysorbates (all units), all
saturated and unsaturated fatty acids, triethanolamine, Tween 20,
tween 40, Tween 60, tween 80, Polysorbate 20, Polysorbate 40,
Polysorbate 60, and Polysorbate 80.
[0161] For storage prior to use, the liposomes of the present
invention may be suspended in an aqueous solution, such as a saline
solution (for example, a phosphate buffered saline solution), or
simply water, and stored preferably at a temperature of between
about 2.degree. C. and about 10.degree. C., preferably at about
4.degree. C. Preferably, the water is sterile.
[0162] Typical storage conditions are, for example, a non-degassed
aqueous solution of 0.9% NaCl maintained at 4.degree. C. for 48
hours. The temperature of storage is preferably below the gel state
to liquid crystalline state phase transition temperature of the
material forming the liposomes.
[0163] Most preferably, the liposomes are stored in an isotonic
saline solution, although, if desired, the saline solution may be
hypotonic (e.g., about 0.3 to about 0.5% NaCl). The solution also
may be buffered, if desired, to provide a pH range of about pH 5 to
about pH 7.4. Suitable buffers include, but are not limited to,
acetate, citrate, phosphate, bicarbonate, and phosphate-buffered
saline, 5% dextrose, and physiological saline (normal saline).
[0164] Bacteriostatic agents may also be included with the
liposomes to prevent bacterial degradation on storage. Suitable
bacteriostatic agents include but are not limited to benzalkonium
chloride, benzethonium chloride, benzoic acid, benzyl alcohol,
butylparaben, cetylpyridinium chloride, chlorobutanol,
chlorocresol, methylparaben, phenol, potassium benzoate, potassium
sorbate, sodium benzoate and sorbic acid.
[0165] By "gas-filled", as used herein, it is meant liposomes
having an interior volume that is at least about 10% gas,
preferably at least about 25% gas, more preferably at least about
50% gas, even more preferably at least about 75% gas, and most
preferably at least about 90% gas. It will be understood by one
skilled in the art, once armed with the present disclosure, that a
gaseous precursor may also be used, followed by activation to form
a gas.
[0166] Various biocompatible gases may be employed in the
gas-filled liposomes of the present invention. Such gases include
air, nitrogen, carbon dioxide, oxygen, argon, fluorine, xenon,
neon, helium, or any and all combinations thereof. Other suitable
gases will be apparent to those skilled in the art once armed with
the present disclosure. In addition to the gaseous precursors
disclosed herein, the precursors may be co-entrapped with other
gases. For example, during the transition from the gaseous
precursor to a gas in an enclosed environment containing ambient
gas (as air), the two gases may mix and upon agitation and
formation of microspheres, the gaseous content of the microspheres
results in a mixture of two or more gases, dependent upon the
densities of the gases mixed.
[0167] The size of the liposomes of the present invention will
depend upon the intended use. With the smaller liposomes, resonant
frequency ultrasound will generally be higher than for the larger
liposomes. Sizing also serves to modulate resultant liposomal
biodistribution and clearance. In addition to filtration, the size
of the liposomes can be adjusted, if desired, by procedures known
to one skilled in the art, such as extrusion, sonication,
homogenization, the use of a laminar stream of a core of liquid
introduced into an immiscible sheath of liquid. See, for example,
U.S. Pat. No. 4,728,578; U.K. Patent Application GB 2193095 A; U.S.
Pat. No. 4,728,575; U.S. Pat. No. 4,737,323; International
Application PCT/US85/01161; Mayer et al., Biochimica et Biophysica
Acta 1986, 858, 161-168; Hope et al., Biochimica et Biophysica Acta
1985, 812, 55-65; U.S. Pat. No. 4,533,254; Mayhew et al., Methods
in Enzymology 1987, 149, 64-77; Mayhew et al., Biochimica et
Biophysica Acta 1984, 755, 169-74; Cheng et al, Investigative
Radiology 1987, 22, 47-55; PCT/US89/05040; U.S. Pat. No. 4,162,282;
U.S. Pat. No. 4,310,505; U.S. Pat. No. 4,921,706; and Liposomes
Technology, Gregoriadis, G., ed., Vol. I, pp. 29-37, 51-67 and
79-108 (CRC Press Inc, Boca Raton, Fla., 1984). The disclosures of
each of the foregoing patents, publications and patent applications
are incorporated by reference herein, in their entirety. Extrusion
under pressure through pores of defined size is a preferred method
of adjusting the size of the liposomes.
[0168] Since liposome size influences biodistribution, different
size liposomes may be selected for various purposes. For example,
for intravascular application, the preferred size range is a mean
outside diameter between about 30 nanometers and about 10 microns,
with the preferable mean outside diameter being about 5
microns.
[0169] More specifically, for intravascular application, the size
of the liposomes is preferably about 10 .mu.m or less in mean
outside diameter, and preferably less than about 7 .mu.m, and more
preferably no smaller than about 5 nanometers in mean outside
diameter. Preferably, the liposomes are no smaller than about 30
nanometers in mean outside diameter.
[0170] To provide therapeutic delivery to organs such as the liver
and to allow differentiation of tumor from normal tissue, smaller
liposomes, between about 30 nanometers and about 100 nanometers in
mean outside diameter, are preferred.
[0171] For embolization of a tissue such as the kidney or the lung,
the liposomes are preferably less than about 200 microns in mean
outside diameter.
[0172] For intranasal, intrarectal or topical administration, the
microspheres are preferably less than about 100 microns in mean
outside diameter.
[0173] Large liposomes, e.g., between 1 and 10 microns in size,
will generally be confined to the intravascular space until they
are cleared by phagocytic elements lining the vessels, such as the
macrophages and Kuppfer cells lining capillary sinusoids. For
passage to the cells beyond the sinusoids, smaller liposomes, for
example, less than about a micron in mean outside diameter, e.g.,
less than about 300 nanometers in size, may be utilized.
[0174] The route of administration of the liposomes will vary
depending on the intended use. As one skilled in the art would
recognize, administration of therapeutic delivery systems of the
present invention may be carried out in various fashions, such as
intravascularly, intralymphatically, parenterally, subcutaneously,
intramuscularly, intranasally, intrarectally, intraperitoneally,
interstitially, into the airways via nebulizer, hyperbarically,
orally, topically, or intratumorly, using a variety of dosage
forms. One preferred route of administration is intravascularly.
For intravascular use, the therapeutic delivery system is generally
injected intravenously, but may be injected intraarterially as
well. The liposomes of the invention may also be injected
interstitially or into any body cavity.
[0175] The delivery of therapeutics from the liposomes of the
present invention using ultrasound is best accomplished for tissues
which have a good acoustic window for the transmission of
ultrasonic energy. This is the case for most tissues in the body
such as muscle, the heart, the liver and most other vital
structures. In the brain, in order to direct the ultrasonic energy
past the skull a surgical window may be necessary. For body parts
without an acoustic window, e.g. through bone, radiofrequency or
microwave energy is preferred.
[0176] Additionally, the invention is especially useful in
delivering therapeutics to a patient's lungs. Gaseous
precursor-filled liposomes of the present invention are lighter
than, for example, conventional liquid-filled liposomes which
generally deposit in the central proximal airway rather than
reaching the periphery of the lungs. It is therefore believed that
the gaseous precursor-filled liposomes of the present invention may
improve delivery of a therapeutic compound to the periphery of the
lungs, including the terminal airways and the alveoli. For
application to the lungs, the gaseous precursor-filled liposomes
may be applied through nebulization, for example.
[0177] 2 cc of liposomes (lipid=83% DPPC/8% DPPE-PEG 5,000/5% DPPA)
entrapping air was placed in a nebulizer and nebulized. The
resulting liposomes post nebulization, were around 1 to 2 microns
in size and were shown to float in the air. These size particles
appear ideal for delivering drugs, peptides, genetic materials and
other therapeutic compounds into the far reaches of the lung (i.e.
terminal airways and alveoli). Because the gas filled liposomes are
almost as light as air, much lighter than conventional water filled
liposomes, they float longer in the air, and as such are better for
delivering compounds into the distal lung. When DNA is added to
these liposomes, it is readily adsorbed or bound to the liposomes.
Thus, liposomes and microspheres filled by gas and gaseous
precursors hold vast potential for pulmonary drug delivery.
[0178] In applications such as the targeting of the lungs, which
are lined with lipids, the therapeutic may be released upon
aggregation of the gaseous precursor-filled liposome with the
lipids lining the targeted tissue. Additionally, the gaseous
precursor-filled liposomes may burst after administration without
the use of ultrasound. Thus, ultrasound need not be applied to
release the drug in the above type of administration.
[0179] Further, the gaseous precursor-filled liposomes of the
invention are especially useful for therapeutics that may be
degraded in aqueous media or upon exposure to oxygen and/or
atmospheric air. For example, the liposomes may be filled with an
inert gas such as nitrogen or argon, for use with labile
therapeutic compounds. Additionally, the gaseous precursor-filled
liposomes may be filled with an inert gas and used to encapsulate a
labile therapeutic for use in a region of a patient that would
normally cause the therapeutic to be exposed to atmospheric air,
such as cutaneous and ophthalmic applications.
[0180] The gaseous precursor-filled liposomes are also especially
useful for transcutaneous delivery, such as a patch delivery
system. The use of rupturing ultrasound may increase transdermal
delivery of therapeutic compounds. Further, a mechanism may be used
to monitor and modulate drug delivery. For example, diagnostic
ultrasound may be used to visually monitor the bursting of the
gaseous precursor-filled liposomes and modulate drug delivery
and/or a hydrophone may be used to detect the sound of the bursting
of the gaseous precursor-filled liposomes and modulate drug
delivery.
[0181] In preferred embodiments, the gas-filled liposomes are
administered in a vehicle as individual particles, as opposed to
being embedded in a polymeric matrix for the purposes of controlled
release.
[0182] For in vitro use, such as cell culture applications, the
gaseous precursor-filled liposomes may be added to the cells in
cultures and then incubated. Subsequently sonic energy, microwave,
or thermal energy (e.g. simple heating) can be applied to the
culture media containing the cells and liposomes.
[0183] Generally, the therapeutic delivery systems of the invention
are administered in the form of an aqueous suspension such as in
water or a saline solution (e.g., phosphate buffered saline).
Preferably, the water is sterile. Also, preferably the saline
solution is an isotonic saline solution, although, if desired, the
saline solution may be hypotonic (e.g., about 0.3 to about 0.5%
NaCl). The solution may also be buffered, if desired, to provide a
pH range of about pH 5 to about pH 7.4. In addition, dextrose may
be preferably included in the media. Further solutions that may be
used for administration of gaseous precursor-filled liposomes
include, but are not limited to almond oil, corn oil, cottonseed
oil, ethyl oleate, isopropyl myristate, isopropyl palmitate,
mineral oil, myristyl alcohol, octyl-dodecanol, olive oil, peanut
oil, persic oil, sesame oil, soybean oil, squalene, myristyl
oleate, cetyl oleate, myristyl palmitate, as well as other
saturated and unsaturated alkyl chain alcohols (C=2-22) esterified
to alkyl chain fatty acids (C=2-22).
[0184] The useful dosage of gaseous precursor-filled microspheres
to be administered and the mode of administration will vary
depending upon the age, weight, and mammal to be treated, and the
particular application (therapeutic/diagnostic) intended.
Typically, dosage is initiated at lower levels and increased until
the desired therapeutic effect is achieved.
[0185] For use in ultrasonic imaging, preferably, the liposomes of
the invention possess a reflectivity of greater than 2 dB, more
preferably between about 4 dB and about 20 dB. Within these ranges,
the highest reflectivity for the liposomes of the invention is
exhibited by the larger liposomes, by higher concentrations of
liposomes, and/or when higher ultrasound frequencies are
employed.
[0186] For therapeutic drug delivery, the rupturing of the
therapeutic containing liposomes of the invention is surprisingly
easily carried out by applying ultrasound of a certain frequency to
the region of the patient where therapy is desired, after the
liposomes have been administered to or have otherwise reached that
region. Specifically, it has been unexpectedly found that when
ultrasound is applied at a frequency corresponding to the peak
resonant frequency of the therapeutic containing gaseous
precursor-filled liposomes, the liposomes will rupture and release
their contents.
[0187] The peak resonant frequency can be determined either in vivo
or in vitro, but preferably in vivo, by exposing the liposomes to
ultrasound, receiving the reflected resonant frequency signals and
analyzing the spectrum of signals received to determine the peak,
using conventional means. The peak, as so determined, corresponds
to the peak resonant frequency (or second harmonic, as it is
sometimes termed).
[0188] Preferably, the liposomes of the invention have a peak
resonant frequency of between about 0.5 mHz and about 10 mHz. Of
course, the peak resonant frequency of the gaseous precursor-filled
liposomes of the invention will vary depending on the outside
diameter and, to some extent, the elasticity or flexibility of the
liposomes, with the larger and more elastic or flexible liposomes
having a lower resonant frequency than the smaller and less elastic
or flexible liposomes.
[0189] The therapeutic-containing gaseous precursor-filled
liposomes will also rupture when exposed to non-peak resonant
frequency ultrasound in combination with a higher intensity
(wattage) and duration (time). This higher energy, however, results
in greatly increased heating, which may not be desirable. By
adjusting the frequency of the energy to match the peak resonant
frequency, the efficiency of rupture and therapeutic release is
improved, appreciable tissue heating does not generally occur
(frequently no increase in temperature above about 2.degree. C.),
and less overall energy is required. Thus, application of
ultrasound at the peak resonant frequency, while not required, is
most preferred.
[0190] For diagnostic or therapeutic ultrasound, any of the various
types of diagnostic ultrasound imaging devices may be employed in
the practice of the invention, the particular type or model of the
device not being critical to the method of the invention. Also
suitable are devices designed for administering ultrasonic
hyperthermia, such devices being described in U.S. Pat. Nos.
4,620,546, 4,658,828, and 4,586,512, the disclosures of each of
which are hereby incorporated herein by reference in their
entirety. Preferably, the device employs a resonant frequency (RF)
spectral analyzer. The transducer probes may be applied externally
or may be implanted. Ultrasound is generally initiated at lower
intensity and duration, and then intensity, time, and/or resonant
frequency increased until the liposome is visualized on ultrasound
(for diagnostic ultrasound applications) or ruptures (for
therapeutic ultrasound applications).
[0191] Although application of the various principles will be
readily apparent to one skilled in the art, once armed with the
present disclosure, by way of general guidance, for gaseous
precursor-filled liposomes of about 1.5 to about 10 microns in mean
outside diameter, the resonant frequency will generally be in the
range of about 1 to about 10 megahertz. By adjusting the focal zone
to the center of the target tissue (e.g., the tumor) the gaseous
precursor-filled liposomes can be visualized under real time
ultrasound as they accumulate within the target tissue. Using the
7.5 megahertz curved array transducer as an example, adjusting the
power delivered to the transducer to maximum and adjusting the
focal zone within the target tissue, the spatial peak temporal
average (SPTA) power will then be a maximum of approximately 5.31
mW/cm.sup.2 in water. This power will cause some release of
therapeutic from the gaseous precursor-filled liposomes, but much
greater release can be accomplished by using higher power.
[0192] By switching the transducer to the doppler mode, higher
power outputs are available, up to 2.5 watts per cm.sup.2 from the
same transducer. With the machine operating in doppler mode, the
power can be delivered to a selected focal zone within the target
tissue and the gaseous precursor-filled liposomes can be made to
release their therapeutics. Selecting the transducer to match the
resonant frequency of the gaseous precursor-filled liposomes will
make this process of therapeutic release even more efficient.
[0193] For larger diameter gaseous precursor-filled liposomes,
e.g., greater than 3 microns in mean outside diameter, a lower
frequency transducer may be more effective in accomplishing
therapeutic release. For example, a lower frequency transducer of
3.5 megahertz (20 mm curved array model) may be selected to
correspond to the resonant frequency of the gaseous
precursor-filled liposomes. Using this transducer, 101.6 milliwatts
per cm.sup.2 may be delivered to the focal spot, and switching to
doppler mode will increase the power output (SPTA) to 1.02 watts
per cm.sup.2.
[0194] To use the phenomenon of cavitation to release and/or
activate the drugs/prodrugs within the gaseous precursor-filled
liposomes, lower frequency energies may be used, as cavitation
occurs more effectively at lower frequencies. Using a 0.757
megahertz transducer driven with higher voltages (as high as 300
volts) cavitation of solutions of gaseous precursor-filled
liposomes will occur at thresholds of about 5.2 atmospheres.
[0195] Table II shows the ranges of energies transmitted to tissues
from diagnostic ultrasound on commonly used instruments such as the
Piconics Inc. (Tyngsboro, Mass.) Portascan general purpose scanner
with receiver pulser 1966 Model 661; the Picker (Cleveland, Ohio)
Echoview 8L Scanner including 80C System or the Medisonics
(Mountain View, Calif.) Model D-9 Versatone Bidirectional Doppler.
In general, these ranges of energies employed in pulse repetition
are useful for diagnosis and monitoring the gas-filled liposomes
but are insufficient to rupture the gas-filled liposomes of the
present invention.
3TABLE III Power and Intensities Produced by Diagnostic Equipment*
Average Intensity Pulse repetition Total ultrasonic at transducer
face rate (Hz) power output P (mW) I.sub.TD (W/m.sup.2) 520 4.2 32
676 9.4 71 806 6.8 24 1000 14.4 51 1538 2.4 8.5 *Values obtained
from Carson et al., Ultrasound in Med. & Biol. 1978, 3,
341-350, the disclosures of which are hereby incorporated herein by
reference in their entirety.
[0196] Higher energy ultrasound such as commonly employed in
therapeutic ultrasound equipment is preferred for activation of the
therapeutic containing gaseous precursor-filled liposomes. In
general, therapeutic ultrasound machines employ as much as 50% to
100% duty cycles dependent upon the area of tissue to be heated by
ultrasound. Areas with larger amounts of muscle mass (i.e., backs,
thighs) and highly vascularized tissues such as heart may require
the larger duty cycle, e.g., 100%.
[0197] In diagnostic ultrasound, one or several pulses of sound are
used and the machine pauses between pulses to receive the reflected
sonic signals. The limited number of pulses used in diagnostic
ultrasound limits the effective energy which is delivered to the
tissue which is being imaged.
[0198] In therapeutic ultrasound, continuous wave ultrasound is
used to deliver higher energy levels. In using the liposomes of the
present invention, the sound energy may be pulsed, but continuous
wave ultrasound is preferred. If pulsing is employed, the sound
will preferably be pulsed in echo train lengths of at least about 8
and preferably at least about 20 pulses at a time.
[0199] Either fixed frequency or modulated frequency ultrasound may
be used. Fixed frequency is defined wherein the frequency of the
sound wave is constant over time. A modulated frequency is one in
which the wave frequency changes over time, for example, from high
to low (PRICH) or from low to high (CHIRP). For example, a PRICH
pulse with an initial frequency of 10 MHz of sonic energy is swept
to 1 MHz with increasing power from 1 to 5 watts. Focused,
frequency modulated, high energy ultrasound may increase the rate
of local gaseous expansion within the liposomes and rupturing to
provide local delivery of therapeutics.
[0200] The frequency of the sound used may vary from about 0.025 to
about 100 megahertz. Frequency ranges between about 0.75 and about
3 megahertz are preferred and frequencies between about 1 and about
2 megahertz are most preferred. Commonly used therapeutic
frequencies of about 0.75 to about 1.5 megahertz may be used.
Commonly used diagnostic frequencies of about 3 to about 7.5
megahertz may also be used. For very small liposomes, e.g., below
0.5 micron in mean outside diameter, higher frequencies of sound
may be preferred as these smaller liposomes will absorb sonic
energy more effectively at higher frequencies of sound. When very
high frequencies are used, e.g., over 10 megahertz, the sonic
energy will generally have limited depth penetration into fluids
and tissues. External application may be preferred for the skin and
other superficial tissues, but for deep structures, the application
of sonic energy via interstitial probes or intravascular ultrasound
catheters may be preferred.
[0201] Where the gaseous precursor-filled liposomes are used for
therapeutic delivery, the therapeutic compound to be delivered may
be embedded within the wall of the liposome, encapsulated in the
liposome and/or attached to the liposome, as desired. The phrase
"attached to" or variations thereof, as used herein in connection
with the location of the therapeutic compound, means that the
therapeutic compound is linked in some manner to the inside and/or
the outside wall of the microsphere, such as through a covalent or
ionic bond or other means of chemical or electrochemical linkage or
interaction. The phrase "encapsulated in variations thereof" as
used in connection with the location of the therapeutic compound
denotes that the therapeutic compound is located in the internal
microsphere void. The phrase "embedded within" or variations
thereof as used in connection with the location of the therapeutic
compound, signifies the positioning of the therapeutic compound
within the microsphere wall. The phrase "comprising a therapeutic"
denotes all of the varying types of therapeutic positioning in
connection with the microsphere. Thus, the therapeutic can be
positioned variably, such as, for example, entrapped within the
internal void of the gaseous precursor-filled microsphere, situated
between the gaseous precursor and the internal wall of the gaseous
precursor-filled microsphere, incorporated onto the external
surface of the gaseous precursor-filled microsphere and/or enmeshed
within the microsphere structure itself.
[0202] Any of a variety of therapeutics may be encapsulated in the
liposomes. By therapeutic, as used herein, it is meant an agent
having beneficial effect on the patient. As used herein, the term
therapeutic is synonymous with the terms contrast agent and/or
drug.
[0203] Examples of drugs that may be delivered with gaseous
precursor-filled liposomes may contain for drug delivery purposes,
but by no means is limited to; hormone products such as,
vasopressin and oxytocin and their derivatives, glucagon, and
thyroid agents as iodine products and anti-thyroid agents;
cardiovascular products as chelating agents and mercurial diuretics
and cardiac glycosides; respiratory products as xanthine
derivatives (theophylline & aminophylline); anti-infectives as
aminoglycosides, antifungals (amphotericin), penicillin and
cephalosporin antibiotics, antiviral agents as Zidovudine,
Ribavirin, Amantadine, Vidarabine, and Acyclovir, anti-helmintics,
antimalarials, and antituberculous drugs; biologicals as immune
serums, antitoxins and antivenins, rabies prophylaxis products,
bacterial vaccines, viral vaccines, toxoids; antineoplastics as
nitrosureas, nitrogen mustards, antimetabolites (fluorouracil,
hormones as progestins and estrogens and antiestrogens; antibiotics
as Dactinomycin; mitotic inhibitors as Etoposide and the Vinca
alkaloids, Radiopharmaceuticals as radioactive iodine and
phosphorus products; as well as Interferon, hydroxyurea,
procarbazine, Dacarbazine, Mitotane, Asparaginase and
cyclosporins.
[0204] Genetic and bioactive materials may be incorporated into the
internal gaseous precursor-filled space of these liposomes during
the gaseous precursor installation process or into or onto the
lipid membranes of these particles. Incorporation onto the surface
of these particles is preferred. Genetic materials and bioactive
products with a high octanol/water partition coefficient may be
incorporated directly into the lipid layer surrounding the gaseous
precursor but incorporation onto the surface of the gaseous
precursor-filled lipid spheres is more preferred. To accomplish
this, groups capable of binding genetic materials or bioactive
materials are generally incorporated into the lipid layers which
will then bind these materials. In the case of genetic materials
(DNA, RNA, both single stranded and double stranded and antisense
and sense oligonucleotides) this is readily accomplished through
the use of cationic lipids or cationic polymers which may be
incorporated into the dried lipid starting materials.
[0205] It is the surprising discovery of the invention that
liposomes, gas-filled and gas precursor-filled, when produced with
phosphatidic acid, e.g. dipalmitoylphosphatidic acid in molar
amounts in excess of 5 mole % and preferably about 10 mole %,
function as highly effective binders of genetic material. Such
liposomes bind DNA avidly. This is surprising since positively
charged liposomes were heretofore recognized as most useful for
binding DNA. Liposomes with 5 mole % to 10 mole % DPPA function as
highly effective gas and gaseous precursor retaining structures.
Compositions incorporating phosphatidic acid are more robust for
diagnostic ultrasound and useful for carrying DNA as well as other
pharmaceuticals.
[0206] It is believed that nanoparticles, microparticles, and
emulsions of certain precursors are particularly effective at
accumulating in ischemic and diseased tissue. Such precursors can
be used for detecting ischemic and diseased tissue via ultrasound
and also for delivering drugs to these tissues. By co-entrapping
drugs with the emulsions or nanoparticles comprising the gaseous
precursors said drugs can then be delivered to the diseased
tissues. For example, emulsions of, sulfur hexafluoride,
hesafluoropropylene, bromochlorofluoromethane, octafluoropropane,
1,1dichloro, fluoro ethane, hexafluoroethane, hesafluoro-2-butyne,
perfluoropentane, perfluorobutane, octafluoro-2-butene or
hexafluorobuta-1,3-diene or octafluorocyclopentene (27.degree. C.)
can be used to deliver drugs such as cardiac glycosides, angiogenic
factors and vasoactive compounds to ischemic regions of the
myocardium. Similarly, emulsions of the above precursors may also
be used to deliver antisense DNA or chemotherapeutics to tumors. It
is postulated that subtle changes in temperature, pH and oxygen
tension are responsible for the accumulation of certain precursors
preferentially by diseased and ischemic tissues. These precursors
can be used as a delivery vehicle or in ultrasound for drug
delivery.
[0207] Suitable therapeutics include, but are not limited to
paramagnetic gases, such as atmospheric air, which contains traces
of oxygen 17; paramagnetic ions such as Mn.sup.+2, Gd.sup.+2
Fe.sup.+3; iron oxides or magnetite (Fe.sub.3O.sub.4) and may thus
be used as susceptibility contrast agents for magnetic resonance
imaging (MRI), radioopaque metal ions, such as iodine, barium,
bromine, or tungsten, for use as x-ray contrast agents, gases from
quadrupolar nuclei, may have potential for use as Magnetic
Resonance contrast agents, antineoplastic agents, such as platinum
compounds (e.g., spiroplatin, cisplatin, and carboplatin),
methotrexate, fluorouracil, adriamycin, taxol, mitomycin,
ansamitocin, bleomycin, cytosine arabinoside, arabinosyl adenine,
mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan
(e.g., PAM, L-PAM or phenylalanine mustard), mercaptopurine,
mitotane, procarbazine hydrochloride dactinomycin (actinomycin D),
daunorubicin hydrochloride, doxorubicin hydrochloride, mitomycin,
plicamycin (mithramycin), aminoglutethimide, estramustine phosphate
sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen
citrate, testolactone, trilostane, amsacrine (m-AMSA), asparaginase
(L-asparaginase) Erwina asparaginase, etoposide (VP-16), interferon
.alpha.-2a, interferon .alpha.-2b, teniposide (VM-26), vinblastine
sulfate (VLB), vincristine sulfate, bleomycin, bleomycin sulfate,
methotrexate, adriamycin, and arabinosyl, hydroxyurea,
procarbazine, and dacarbazine; mitotic inhibitors such as etoposide
and the vinca alkaloids, radiopharmaceuticals such as radioactive
iodine and phosphorus products; hormones such as progestins,
estrogens and antiestrogens; anti-helmintics, antimalarials, and
antituberculosis drugs; biologicals such as immune serums,
antitoxins and antivenins; rabies prophylaxis products; bacterial
vaccines; viral vaccines; aminoglycosides; respiratory products
such as xanthine derivatives theophylline and aminophylline;
thyroid agents such as iodine products and anti-thyroid agents;
cardiovascular products including chelating agents and mercurial
diuretics and cardiac glycosides; glucagon; blood products such as
parenteral iron, hemin, hematoporphyrins and their derivatives;
biological response modifiers such as muramyldipeptide,
muramyltripeptide, microbial cell wall components, lymphokines
(e.g., bacterial endotoxin such as lipopolysaccharide, macrophage
activation factor), sub-units of bacteria (such as Mycobacteria,
Corynebacteria), the synthetic dipeptide
N-acetyl-muramyl-L-alanyl-D-isoglutamine; anti-fungal agents such
as ketoconazole, nystatin, griseofulvin, flucytosine (5-FC),
miconazole, amphotericin B, ricin, cyclosporins, and .beta.-lactam
antibiotics (e.g., sulfazecin); hormones such as growth hormone,
melanocyte stimulating hormone, estradiol, beclomethasone
dipropionate, betamethasone, betamethasone acetate and
betamethasone sodium phosphate, vetamethasone disodium phosphate,
vetamethasone sodium phosphate, cortisone acetate, dexamethasone,
dexamethasone acetate, dexamethasone sodium phosphate, flunsolide,
hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate,
hydrocortisone sodium phosphate, hydrocortisone sodium succinate,
methylprednisolone, methylprednisolone acetate, methylprednisolone
sodium succinate, paramethasone acetate, prednisolone, prednisolone
acetate, prednisolone sodium phosphate, prednisolone terbutate,
prednisone, triamcinolone, triamcinolone acetonide, triamcinolone
diacetate, triamcinolone hexacetonide and fludrocortisone acetate,
oxytocin, vassopressin, and their derivatives; vitamins such as
cyanocobalamin neinoic acid, retinoids and derivatives such as
retinol palmitate, and .alpha.-tocopherol; peptides, such as
manganese super oxide dimutase; enzymes such as alkaline
phosphatase; anti-allergic agents such as amelexanox;
anti-coagulation agents such as phenprocoumon and heparin;
circulatory drugs such as propranolol; metabolic potentiators such
as glutathione; antituberculars such as para-aminosalicylic acid,
isoniazid, capreomycin sulfate cycloserine, ethambutol
hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin
sulfate; antivirals such as acyclovir, amantadine azidothymidine
(AZT or Zidovudine), ribavirin and vidarabine monohydrate (adenine
arabinoside, ara-A); antianginals such as diltiazem, nifedipine,
verapamil, erythrityl tetranitrate, isosorbide dinitrate,
nitroglycerin (glyceryl trinitrate) and pentaerythritol
tetranitrate; anticoagulants such as phenprocoumon, heparin;
antibiotics such as dapsone, chloramphenicol, neomycin, cefaclor,
cefadroxil, cephalexin, cephradine erythromycin, clindamycin,
lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin,
dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,
nafcillin, oxacillin, penicillin, including penicillin G,
penicillin V, ticarcillin rifampin and tetracycline;
antiinflammatories such as difunisal, ibuprofen, indomethacin,
meclofenamate, mefenamic acid, naproxen, oxyphenbutazone,
phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and
salicylates; antiprotozoans such as chloroquine,
hydroxychloroquine, metronidazole, quinine and meglumine
antimonate; antirheumatics such as penicillamine; narcotics such as
paregoric; opiates such as codeine, heroin, methadone, morphine and
opium; cardiac glycosides such as deslanoside, digitoxin, digoxin,
digitalin and digitalis; neuromuscular blockers such as atracurium
besylate, gallamine triethiodide, hexafluorenium bromide,
metocurine iodide, pancuronium bromide, succinylcholine chloride
(suxamethonium chloride), tubocurarine chloride and vecuronium
bromide; sedatives (hypnotics) such as amobarbital, amobarbital
sodium, aprobarbital, butabarbital sodium, chloral hydrate,
ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide,
methotrimeprazine hydrochloride, methyprylon, midazolam
hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium,
phenobarbital sodium, secobarbital sodium, talbutal, temazepam and
triazolam; local anesthetics such as bupivacaine hydrochloride,
chloroprocaine hydrochloride, etidocaine hydrochloride, lidocaine
hydrochloride, mepivacaine hydrochloride, procaine hydrochloride
and tetracaine hydrochloride; general anesthetics such as
droperidol, etomidate, fentanyl citrate with droperidol, ketamine
hydrochloride, methohexital sodium and thiopental sodium; and
radioactive particles or ions such as strontium, iodide rhenium and
yttrium.
[0208] In certain preferred embodiments, the therapeutic is a
monoclonal antibody, such as a monoclonal antibody capable of
binding to melanoma antigen.
[0209] Other preferred therapeutics include genetic material such
as nucleic acids, RNA, and DNA, of either natural or synthetic
origin, including recombinant RNA and DNA and antisense RNA and
DNA. Types of genetic material that may be used include, for
example, genes carried on expression vectors such as plasmids,
phagemids, cosmids, yeast artificial chromosomes (YACs), and
defective or "helper" viruses, antigene nucleic acids, both single
and double stranded RNA and DNA and analogs thereof, such as
phosphorothioate, phosphoroamidate, and phosphorodithioate
oligodeoxynucleotides. Additionally, the genetic material may be
combined, for example, with proteins or other polymers.
[0210] Examples of genetic therapeutics that may be applied using
the liposomes of the present invention include DNA encoding at
least a portion of an HLA gene, DNA encoding at least a portion of
dystrophin, DNA encoding at least a portion of CFTR, DNA encoding
at least a portion of IL-2, DNA encoding at least a portion of TNF,
an antisense oligonucleotide capable of binding the DNA encoding at
least a portion of Ras.
[0211] DNA encoding certain proteins may be used in the treatment
of many different types of diseases. For example, adenosine
deaminase may be provided to treat ADA deficiency; tumor necrosis
factor and/or interleukin-2 may be provided to treat advanced
cancers; HDL receptor may be provided to treat liver disease;
thymidine kinase may be provided to treat ovarian cancer, brain
tumors, or HIV infection; HLA-B7 may be provided to treat malignant
melanoma; interleukin-2 may be provided to treat neuroblastoma,
malignant melanoma, or kidney cancer; interleukin-4 may be provided
to treat cancer; HIV env may be provided to treat HIV infection;
antisense ras/p53 may be provided to treat lung cancer; and Factor
VIII may be provided to treat Hemophilia B. See, for example,
Thompson, L., Science, 1992, 258, 744-746.
[0212] If desired, more than one therapeutic may be applied using
the liposomes. For example, a single liposome may contain more than
one therapeutic or liposomes containing different therapeutics may
be co-administered. By way of example, a monoclonal antibody
capable of binding to melanoma antigen and an oligonucleotide
encoding at least a portion of IL-2 may be administered at the same
time. The phrase "at least a portion of," as used herein, means
that the entire gene need not be represented by the
oligonucleotide, so long as the portion of the gene represented
provides an effective block to gene expression.
[0213] Similarly, prodrugs may be encapsulated in the liposomes,
and are included within the ambit of the term therapeutic, as used
herein. Prodrugs are well known in the art and include inactive
drug precursors which, when exposed to high temperature,
metabolizing enzymes, cavitation and/or pressure, in the presence
of oxygen or otherwise, or when released from the liposomes, will
form active drugs. Such prodrugs can be activated from, or released
from, gas-filled lipid spheres in the method of the invention, upon
the application of ultrasound or radiofrequency microwave energy to
the prodrug-containing liposomes with the resultant cavitation,
heating, pressure, and/or release from the liposomes. Suitable
prodrugs will be apparent to those skilled in the art, and are
described, for example, in Sinkula et al., J. Pharm. Sci. 1975, 64,
181-210, the disclosure of which are hereby incorporated herein by
reference in its entirety.
[0214] Prodrugs, for example, may comprise inactive forms of the
active drugs wherein a chemical group is present on the prodrug
which renders it inactive and/or confers solubility or some other
property to the drug. In this form, the prodrugs are generally
inactive, but once the chemical group has been cleaved from the
prodrug, by heat, cavitation, pressure, and/or by enzymes in the
surrounding environment or otherwise, the active drug is generated.
Such prodrugs are well described in the art, and comprise a wide
variety of drugs bound to chemical groups through bonds such as
esters to short, medium or long chain aliphatic carbonates,
hemiesters of organic phosphate, pyrophosphate, sulfate, amides,
amino acids, azo bonds, carbamate, phosphamide, glucosiduronate,
N-acetylglucosamine and .beta.-glucoside.
[0215] Examples of drugs with the parent molecule and the
reversible modification or linkage are as follows: convallatoxin
with ketals, hydantoin with alkyl esters, chlorphenesin with
glycine or alanine esters, acetaminophen with caffeine complex,
acetylsalicylic acid with THAM salt, acetylsalicylic acid with
acetamidophenyl ester, naloxone with sulfate ester,
15-methylprostaglandin F.sub.2.alpha. with methyl ester, procaine
with polyethylene glycol, erythromycin with alkyl esters,
clindamycin with alkyl esters or phosphate esters, tetracycline
with betaine salts, 7-acylaminocephalosporins with ring-substituted
acyloxybenzyl esters, nandrolone with phenylproprionate decanoate
esters, estradiol with enol ether acetal, methylprednisolone with
acetate esters, testosterone with n-acetylglucosaminide
glucosiduronate (trimethylsilyl) ether, cortisol or prednisolone or
dexamethasone with 21-phosphate esters.
[0216] Prodrugs may also be designed as reversible drug derivatives
and utilized as modifiers to enhance drug transport to
site-specific tissues. Examples of parent molecules with reversible
modifications or linkages to influence transport to a site specific
tissue and for enhanced therapeutic effect include isocyanate with
haloalkyl nitrosurea, testosterone with propionate ester,
methotrexate (3-5'-dichloromethotrexa- te) with dialkyl esters,
cytosine arabinoside with 5'-acylate, nitrogen mustard
(2,2'-dichloro-N-methyldiethylamine), nitrogen mustard with
aminomethyl tetracycline, nitrogen mustard with cholesterol or
estradiol or dehydroepiandrosterone esters and nitrogen mustard
with azobenzene.
[0217] As one skilled in the art would recognize, a particular
chemical group to modify a given drug may be selected to influence
the partitioning of the drug into either the membrane or the
internal space of the liposomes. The bond selected to link the
chemical group to the drug may be selected to have the desired rate
of metabolism, e.g., hydrolysis in the case of ester bonds in the
presence of serum esterases after release from the gaseous
precursor-filled liposomes. Additionally, the particular chemical
group may be selected to influence the biodistribution of the drug
employed in the gaseous precursor-filled drug carrying liposome
invention, e.g., N,N-bis(2-chloroethyl)-phosphorodiamid- ic acid
with cyclic phosphoramide for ovarian adenocarcinoma.
[0218] Additionally, the prodrugs employed within the gaseous
precursor-filled liposomes may be designed to contain reversible
derivatives which are utilized as modifiers of duration of activity
to provide, prolong or depot action effects. For example, nicotinic
acid may be modified with dextran and carboxymethlydextran esters,
streptomycin with alginic acid salt, dihydrostreptomycin with
pamoate salt, cytarabine (ara-C) with 5'-adamantoate ester,
ara-adenosine (ara-A) with 5-palmitate and 5'-benzoate esters,
amphotericin B with methyl esters, testosterone with
17-.beta.-alkyl esters, estradiol with formate ester, prostaglandin
with 2-(4-imidazolyl)ethylamine salt, dopamine with amino acid
amides, chloramphenicol with mono- and bis(trimethylsilyl) ethers,
and cycloguanil with pamoate salt. In this form, a depot or
reservoir of long-acting drug may be released in vivo from the
gaseous precursor-filled prodrug bearing liposomes.
[0219] In addition, compounds which are generally thermally labile
may be utilized to create toxic free radical compounds. Compounds
with azolinkages, peroxides and disulfide linkages which decompose
with high temperature are preferred. With this form of prodrug,
azo, peroxide or disulfide bond containing compounds are activated
by cavitation and/or increased heating caused by the interaction of
high energy sound with the gaseous precursor-filled liposomes to
create cascades of free radicals from these prodrugs entrapped
therein. A wide variety of drugs or chemicals may constitute these
prodrugs, such as azo compounds, the general structure of such
compounds being R--N.dbd.N--R, wherein R is a hydrocarbon chain,
where the double bond between the two nitrogen atoms may react to
create free radical products in vivo.
[0220] Exemplary drugs or compounds which may be used to create
free radical products include azo containing compounds such as
azobenzene, 2,2'-azobisisobutyronitrile, azodicarbonamide,
azolitmin, azomycin, azosemide, azosulfamide, azoxybenzene,
aztreonam, sudan III, sulfachrysoidine, sulfamidochrysoidine and
sulfasalazine, compounds containing disulfide bonds such as
sulbentine, thiamine disulfide, thiolutin, thiram, compounds
containing peroxides such as hydrogen peroxide and benzoylperoxide,
2,2'-azobisisobutyronitrile, 2,2'-azobis(2-amidopropane)
dihydrochloride, and 2,2'-azobis(2,4-dimethyl- valeronitrile).
[0221] A gaseous precursor-filled liposome filled with oxygen gas
should create extensive free radicals with cavitation. Also, metal
ions from the transition series, especially manganese, iron and
copper can increase the rate of formation of reactive oxygen
intermediates from oxygen. By encapsulating metal ions within the
liposomes, the formation of free radicals in vivo can be increased.
These metal ions may be incorporated into the liposomes as free
salts, as complexes, e.g., with EDTA, DTPA, DOTA or
desferrioxamine, or as oxides of the metal ions. Additionally,
derivatized complexes of the metal ions may be bound to lipid head
groups, or lipophilic complexes of the ions may be incorporated
into a lipid bilayer, for example. When exposed to thermal
stimulation, e.g., cavitation, these metal ions then will increase
the rate of formation of reactive oxygen intermediates. Further,
radiosensitizers such as metronidazole and misonidazole may be
incorporated into the gaseous precursor-filled liposomes to create
free radicals on thermal stimulation.
[0222] By way of an example of the use of prodrugs, an acylated
chemical group may be bound to a drug via an ester linkage which
would readily cleave in vivo by enzymatic action in serum. The
acylated prodrug is incorporated into the gaseous precursor-filled
liposome of the invention. The derivatives, in addition to
hydrocarbon and substituted hydrocarbon alkyl groups, may also be
composed of halo substituted and perhalo substituted groups as
perfluoroalkyl groups. Perfluoroalkyl groups should possess the
ability to stabilize the emulsion. When the gaseous
precursor-filled liposome is popped by the sonic pulse from the
ultrasound, the prodrug encapsulated by the liposome will then be
exposed to the serum. The ester linkage is then cleaved by
esterases in the serum, thereby generating the drug.
[0223] Similarly, ultrasound may be utilized not only to rupture
the gaseous precursor-filled liposome, but also to cause thermal
effects which may increase the rate of the chemical cleavage and
the release of the active drug from the prodrug.
[0224] The liposomes may also be designed so that there is a
symmetric or an asymmetric distribution of the drug both inside and
outside of the liposome.
[0225] The particular chemical structure of the therapeutics may be
selected or modified to achieve desired solubility such that the
therapeutic may either be encapsulated within the internal gaseous
precursor-filled space of the liposome, attached to the liposome or
enmeshed in the liposome. The surface-bound therapeutic may bear
one or more acyl chains such that, when the bubble is popped or
heated or ruptured via cavitation, the acylated therapeutic may
then leave the surface and/or the therapeutic may be cleaved from
the acyl chains chemical group. Similarly, other therapeutics may
be formulated with a hydrophobic group which is aromatic or sterol
in structure to incorporate into the surface of the liposome.
[0226] The present invention is further described in the following
examples, which illustrate the preparation and testing of the
gaseous precursor-filled liposomes. Examples 1-5, and 22-24 are
actual; Examples 6-21 are prophetic. The following examples should
not be construed as limiting the scope of the appended claims.
EXAMPLE 1
Preparation of Gas-Filled Lipid Spheres from Perfluorobutane
[0227] Gaseous precursor-containing liposomes were prepared using
perfluorobutane (Pfaltz and Bauer, Waterbury, Conn.) as follows: A
5 mL solution of lipid, 5 mg per ml, lipid=87 mole percent DPPC, 8
mole percent DPPE-PEG 5,000, 5 mole percent dipalmitoylphosphatidic
acid (all lipids from Avanti Polar Lipids, Alabaster, Ala.), in
8:1:1 normal saline:glycerol:propylene glycol, was placed in a
glass bottle with a rubber stopper (volume of bottle=15.8 ml). Air
was evacuated from the bottle using a vacuum pump, Model Welch
2-Stage DirecTorr Pump (VWR Scientific, Cerritos, Calif.) by
connecting the hose to the bottle through a 18 gauge needle which
perforated the rubber stopper. After removing the gas via vacuum,
perfluorobutane was placed in the stoppered bottle via another 18
gauge needle connected to tubing attached to the canister of
perfluorobutane. This process was repeated 5 time such that any
traces of air were removed from the stoppered bottle and the space
above the lipid solution was completely filled with
perfluorobutane. The pressure inside the glass bottle was
equilibrated to ambient pressure by allowing the 18 gauge needle to
vent for a moment or two before removing the 18 gauge needle from
the stopper. After filling the bottle with perfluorobutane the
bottle was secured to the arms of a Wig-L-Bug (Crescent Dental Mfg.
Co., Lyons, Ill.) using rubber bands to fasten the bottle. The
bottle was then shaken by the Wig-L-Bug.TM. for 60 seconds. A
frothy suspension of foam resulted and it was noted that it took
several minutes for any appreciable separation of the foam layer
from the clear solution at the bottom. After shaking, the volume of
the material increased from 5 cc to about 12 cc, suggesting that
the liposomes entrapped about 7 cc of the perfluorocarbon gaseous
precursor. The material was sized using an Accusizer (Model 770,
Particle Sizing System, Santa Barbara, Calif.) and also examined by
a light polarizing microscope (Nikon TMS, Nikon) at 150.times.
magnification power. The liposomes appeared as rather large
spherical structure with mean diameter of about 20 to 50 microns. A
portion of these liposomes was then injected via a syringe through
a Costar filter (Syrfil 800938, Costar, Pleasanton, Calif.) with
pore sizes of 8 microns. The liposomes were again examined via
light microscope and the Accusizer System. The mean size of the
liposomes was about 3 microns and the volume weighted mean was
about 7 microns. Greater that 99.9 percent of the liposomes were
under 11 microns in size. The above experiment exhibits the use of
a gaseous precursor gas, perfluorobutane, can be used to make very
desirable sized liposomes by a process of shaking and
filtration.
[0228] The above was substantially repeated except that after
filling the bottle with perfluorobutane at room temperature the
bottle was then transferred to a freezer and the material subjected
to a temperature of -20.degree. C. At this temperature the
perfluorobutane became liquid. Because of the glycerol and
propylene glycol, the lipid solution did not freeze. The bottle was
quickly transferred to the Wig-L-Bug and subjected to shaking as
described above for three cycles, one minute each, at room
temperature. During this time the contents of the bottle
equilibrated to room temperature and was noted to be slightly warm
to the touch secondary to the energy imparted through shaking by
the Wig-L-Bug. At the end of vortexing a large volume of foam was
again noted similar to that described above. The resulting
liposomes were again studied by light microscopy and Accusizer. A
portion was then subjected to filtration sizing through an 8 micron
filter as described above and again studied by microscope and
Accusizer. The results from sizing were substantially the same as
with the gaseous precursor as described above.
[0229] Imaging was performed in a New Zealand White rabbit weighing
about 3.5 kg. The animal was sedated with rabbit mix (Xylene 10
mg/ml; Ketamine 100 mg/ml and Acepromazine 20 mg/ml) and scanned
with an Acoustic Imaging, Model No. 7200, clinical ultrasound
machine, scanning the kidney by color doppler with a 7.5 MHz
transducer. Simultaneously while the kidney was scanned the rabbits
heart was also scanned using a second Acoustic Imaging ultrasound
machine, model n. 5200, with a 7.5 MHz transducer for grey scale
imaging of the heart. Injection of the perfluorobutane-filled
liposomes was administered via ear vein through a syringe fitted
with a 8 micron filter (see above). After injection of 0.5 cc (0.15
cc per kg) of liposomes containing the gaseous precursor
perfluorobutane, dramatic and sustained enhancement of the kidney
was observed for over 30 minutes. This was shown as brilliant color
within the renal parenchyma reflecting increased signal within the
renal arcuate arteries and microcirculation. The simultaneous
imaging of the heart demonstrated shadowing for the first several
minutes which precluded visualization of the heart, i.e. the
reflections were so strong the ultrasound beam was completely
reflected and absorbed. After several minutes, however, brilliant
and sustained ventricular and blood pool enhancement was observed
which also persisted for more than 50 minutes. Images were also
obtained of the liver using the grey scale ultrasound machine.
These showed parenchymal and vascular enhancement at the same time
as the cardiac and blood pool enhancement.
[0230] In summary, this experiment demonstrates how liposomes can
be used to entrap a gaseous precursor and create very stable
liposomes of defined and ideal size. The invention has vast
potential as an ultrasound contrast agent and for drug delivery.
Because the liposomes are so stable they will pass through the
target tissue (a tumor for example) via the circulation. Energy can
then be focused on the target tissue using ultrasound, microwave
radiofrequency or magnetic fields to pop the liposomes and perform
local drug delivery.
EXAMPLE 2
Preparation of Gaseous Precursors Via Microfluidization
[0231] Gaseous precursor-filled lipid bilayers were prepared as in
Example 1 except, after addition of the gaseous precursor, the
contents were microfluidized through six passes on a Microfluidics
microfluidizer (Microfluidics Inc., Newton, Mass.). The stroke
pressure ranged between 10,000 and 20,000 psi. Continuing with the
preparation as per Example 1, produced gas-filled lipid bilayers
with gaseous precursor encapsulated.
EXAMPLE 3
Formulation of Gas-Filled Lipid Bilayers using Phosphatidic Acid
and Dipalmitoyphosphatidylcholine
[0232] Gas-filled lipid bilayers were prepared as set forth in
Example 6 except for the fact that DPPC was used in combination
with 5 mole % phosphatidic acid (Avanti Polar Lipids, Alabaster,
Ala.). Formulation of gas-filled lipid bilayers resulted in an
increase in solubility as exemplified by a decrease in the amount
of lipid particulate in the lower aqueous vehicle layer. Resultant
sizing appeared to decrease the overall mean size vs. DPPC alone to
less than 40 .mu.m.
EXAMPLE 4
Formulation of Gas-Filled Lipid Bilayers using Phosphatidic Acid,
Dipalmitoylphosphatidylethanolamine-PEG 5,000 and
Dipalmitoylphosphatidyl- choline
[0233] Perfluorobutane encapsulated lipid bilayers were formed as
discussed in Example 3 except that the lipid formulation contained
82% dipalmitoylphosphatidylcholine, 10 mole %
dipalmitoylphosphatidic acid, and 8 mole %
dipalmitoylphosphatidylethanolamine-PEG 5,000 (Avanti Polar Lipids,
Alabaster, Ala.) in a vehicle consisting of 8:1:1 (v:v:v) normal
saline:propylene glycol:glycerol, yielding a foam and a lower
vehicle layer that was predominantly devoid of any particulate.
Variations of this vehicle yielded varying degrees of clarity to
the lower vehicle layer. The formulation was prepared identically
as in Example 3 to yield gas-filled lipid bilayers containing
perfluorobutane. Prior to filtration, the gas-filled microspheres
were sized on a Particle Sizing SYstems Model 770 optical sizer
(Particle Sizing Systems, Santa Barbara, Calif.). Sizing resulted
in 99% of all particles residing below 34 .mu.m. The resultant
product ws then filtered through an 8 .mu.m filter to yield
microspheres of uniform size. Sizing of the subsequent microspheres
resulted in 99.5% of all particles residing below 10 .mu.m. This
product was used in the in vivo experiments in Example 1.
[0234] It is noted that the vehicle was altered with other
viscosity modifiers and solubilizers in varying proportions which
resulted in greater or lesser degrees of clarity and particulate.
Amongst a variety of lipids and lipid analogs used in combination,
it was subsequently found that the introduction of DPPE-PEG lipid
significantly improved the size distribution and apparent stability
of the gas-filled lipid bilayers.
EXAMPLE 4
Binding of DNA by Gas-Filled Lipid Bilayers
[0235] Binding of DNA by liposomes containing phosphatidic acid and
gaseous precursor and gas containing liposomes. A 7 mM solution of
distearoyl-sn-glycerophospate (DSPA) (Avanti Polar Lipids,
Alabaster, Ala.) was suspended in normal saline and vortexed at
50.degree. C. The material was allowed to cool to room temperature.
40 micrograms of pBR322 plasmid DNA (International Biotechnologies,
Inc., New Haven, Conn.) was added to the lipid solution and shaken
gently. The solution was centrifuged for 10 minutes in a Beckman
TJ-6 Centrifuge (Beckman, Fullerton, Calif.). The supernatant and
the precipitate were assayed for DNA content using a Hoefer TKO-100
DNA Fluorometer (Hoefer, San Francisco, Calif.). This method only
detects double stranded DNA as it uses an intercalating dye,
Hoechst 33258 which is DNA specific. It was found that the
negatively charged liposomes, or lipids with a net negative charge,
prepared with phosphatidic acid surprisingly bound the DNA. This
experiment was repeated using neutral liposomes composed of DPPC as
a control. No appreciable amount of DNA was detected with the DPPC
liposomes. The experiment was repeated using gas-filled liposomes
prepared from an 87:8:5 mole percent of DPPC to DPPE-PEG 500 to
DPPA mixture of lipids in a microsphere. Again, the DNA bound to
the gas-filled liposomes containing dipalmitoylphosphatidic
acid.
EXAMPLE 5
Microemulsification of Precursor
[0236] A Microfluidizer (Microfluidics, Newton, Mass.) was placed
in a cold room at -20.degree. C. A stoppered glass flask containing
a head space of 35 cc of perfluorobutane and 25 cc of lipid
solution was taken into the cold room. The lipid solution contained
an 83:8:5 molar ratio of DPPC:DPPE+PEG 5,000:DPPA in 8:1:1
phosphate buffered saline (pH 7.4):glycerol:propylene glycol. The
solution did not freeze in the cold room but the perfluorobutane
became liquid.
[0237] The suspension of lipids and liquid gaseous precursor was
then placed into the chamber of the Microfluidizer and subjected to
20 passes at 16,000 psi. Limited size vesicles, having a size of
about 30 nm to about 50 nm, resulted. Upon warming to room
temperature, stabilized microspheres of about 10 microns
resulted.
EXAMPLE 6
Preparation of Gaseous Precursor-filled Liposomes
[0238] Fifty mg of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine (MW:
734.05, powder, Lot No. 160 pc-183) (Avanti-Polar Lipids,
Alabaster, Ala.) is weighed and hydrated with 5.0 ml of saline
solution (0.9% NaCl) or phosphate buffered saline (0.8% sodium
chloride, 0.02% potassium chloride, 0.115% dibasic sodium phosphate
and 0.02% monobasic potassium phosphate, pH adjusted to 7.4) in a
centrifuge tube. To this suspension is added 165 .mu.L mL.sub.-1 of
2-methyl-2-butene. The hydrated suspension is then shaken on a
vortex machine (Scientific Industries, Bohemia, N.Y.) for 10
minutes at an instrument setting of 6.5. A total volume of 12 ml is
then noted. The saline solution is expected to decrease from 5.0 ml
to about 4 ml.
[0239] The gaseous precursor-filled liposomes made via this new
method are then sized by optical microscopy. It will be determined
that the largest size of the liposomes ranged from about 50 to
about 60 .mu.m and the smallest size detected is about 8 .mu.m. The
average size ranges from about 15 to about 20 .mu.m.
[0240] The gaseous precursor-filled liposomes are then filtered
through a 10 or 12 .mu.m "NUCLEPORE" membrane using a Swin-Lok
Filter Holder, (Nuclepore Filtration Products, Costar Corp.,
Cambridge, Mass.) and a 20 cc syringe (Becton Dickinsion & Co.,
Rutherford, N.J.). The membrane is a 10 or 12 .mu.m "NUCLEPORE"
membrane (Nuclepore Filtration Products, Costar Corp., Cambridge,
Mass.). The 10.0 .mu.m filter is placed in the Swin-Lok Filter
Holder and the cap tightened down securely. The liposome solution
is shaken up and transferred to the 20 cc syringe via an 18 gauge
needle. Approximately 12 ml of liposome solution is placed into the
syringe, and the syringe is screwed onto the Swin-Lok Filter
Holder. The syringe and the filter holder assembly are inverted so
that the larger of the gaseous precursor-filled liposomes vesicles
could rise to the top. Then the syringe is gently pushed up and the
gaseous precursor-filled liposomes are filtered in this manner.
[0241] The survival rate (the amount of the gaseous
precursor-filled liposomes that are retained after the extrusion
process) of the gaseous precursor-filled liposomes after the
extrusion through the 10.0 .mu.m filter is about 83-92%. Before
hand extrusion, the volume of foam is about 12 ml and the volume of
aqueous solution is about 4 ml. After hand extrusion, the volume of
foam is about 10-11 ml and the volume of aqueous solution is about
4 ml.
[0242] The optical microscope is used again to determine the size
distribution of the extruded gaseous precursor-filled liposomes. It
will be determined that the largest size of the liposomes range
from about 25 to about 30 .mu.m and the smallest size detected is
about 5 .mu.m. The average size range is from about 8 to about 15
.mu.m.
[0243] It is found that after filtering, greater than 90% of the
gaseous precursor-filled liposomes are smaller than 15 .mu.m.
EXAMPLE 7
Preparation of Gaseous Precursor-Filled Liposomes Incorporating
Lyophilization
[0244] Fifty mg of 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine,
(MW: 734.05, powder) (Avanti-Polar Lipids, Alabaster, Ala.) is
weighed and placed into a centrifuge tube. The lipid is then
hydrated with 5.0 ml of saline solution (0.9% NaCl). To this
suspension is added 165 .mu.L mL.sup.-1 of 2-methyl-2-butene. The
lipid is then vortexed for 10 minutes at an instrument setting of
6.5. After vortexing, the entire solution is frozen in liquid
nitrogen. Then the sample is put on the lyophilizer for freeze
drying. The sample is kept on the lyophilizer for 18 hours. The
dried lipid is taken off the lyophilizer and rehydrated in 5 ml of
saline solution and vortexed for ten minutes at a setting of 6.5. A
small sample of this solution is pipetted onto a slide and the
solution is viewed under a microscope. The size of the gaseous
precursor-filled liposomes will then be determined. It will be
determined that the largest size of the liposomes is about 60 .mu.m
and the smallest size detected is about 20 .mu.m. The average size
range is from about 30 to about 40 .mu.m.
EXAMPLE 8
Example of Gaseous Precursor-Filled Liposome Preparation Above the
Phase Transition Temperature of the Lipid
[0245] Fifty mg of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine (MW:
734.05, powder) (Avanti-Polar Lipids, Alabaster, Ala.) is weighed
and placed into a centrifuge tube. To this suspension is added 165
.mu.L mL.sup.-1 of 2-methyl-2-butene. Approximately two feet of
latex tubing (0.25 in. inner diameter) is wrapped around a conical
centrifuge tube in a coil-like fashion. The latex tubing is then
fastened down to the centrifuge tube with electrical tape. The
latex tubing is then connected to a constant temperature
circulation bath (VWR Scientific Model 1131). The temperature of
the bath is set to 60.degree. C. and the circulation of water is
set to high speed to circulate through the tubing. A thermometer is
placed in the lipid solution and found to be between 42.degree. C.
and 50.degree. C.
[0246] The lipid solution is vortexed for a period of 10 minutes at
a vortex instrument setting of 6.5. It will be noted that very
little foaming of the lipid (phase transition temp.=41.degree. C.)
and that the suspension did not appreciably form gaseous
precursor-filled liposomes. Optical microscopy revealed large
lipidic particles in the solution. The number of gaseous
precursor-filled liposomes that form at this temperature is less
than 3% of the number that form at a temperature below the phase
transition temperature. The solution is allowed to sit for 15
minutes until the solution temperature equilibrated to room
temperature (25.degree. C.). The solution is then vortexed for a
duration of 10 minutes. After 10 minutes, it will be noted that
gaseous precursor-filled liposomes formed.
EXAMPLE 9
Preparation of Gaseous Precursor-Filled Liposomes Incorporating a
Freeze-Thaw Procedure
[0247] 50 mg of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine (MW:
734.05, powder) (Avanti-Polar Lipids, Alabaster, Ala.) is weighed
and placed into a centrifuge tube. The lipid is then hydrated with
5.0 ml of 0.9% NaCl added. To this suspension is added 165 .mu..pi.
mL.sup.-1 of 2-methyl-2-butene. The aqueous lipid solution is
vortexed for 10 minutes at an instrument setting of 6.5. After
vortexing, the entire solution is frozen in liquid nitrogen. The
entire solution is then thawed in a water bath at room temperature
(25.degree. C.). The freeze thaw procedure is then repeated eight
times. The hydrated suspension is then vortexed for 10 minutes at
an instrument setting of 6.5. Gaseous precursor-filled liposomes
are then detected as described in Example 6.
EXAMPLE 10
Preparation of Gaseous Precursor-Filled Liposomes with an
Emulsifying Agent (Sodium Lauryl Sulfate)
[0248] Two centrifuge tubes are prepared, each having 50 mg of
DPPC. 1 mol % (.sup..about.0.2 mg of Duponol C lot No. 2832) of
sodium lauryl sulfate is added to one of the centrifguge tubes, and
the other tube receives 10 mol % (2.0 mg of Duponol C lot No.
2832). Five ml of 0.9% NaCl is added to both centrifuge tubes. 165
.mu.L mL.sup.-1 of 2-methyl-2-butene is added to both tubes. Both
of the tubes are frozen in liquid nitrogen and lyophilized for
approximately 16 hours. Both samples are removed from the
lyophilizer and 5 ml of saline is added to both of the tubes. Both
of the tubes are vortexed at position 6.5 for 10 minutes.
[0249] It will be determined that the largest size of the gaseous
precursor-filled liposomes with 1 mol % of sodium lauryl sulfate is
about 75 .mu.m and the smallest size detected is about 6 .mu.m. The
average size range is from about 15 to about 40 .mu.m. It will be
determined that the largest size of the gaseous precursor-filled
liposomes with 10 mol % of sodium lauryl sulfate is about 90 .mu.m
and the smallest size detected is about 6 .mu.m. The average size
range is from about 15 to about 35 .mu.m.
[0250] The volume of foam in the solution containing gaseous
precursor-filled liposomes with 1 mol % sodium lauryl sulfate is
about 15 ml and the volume of aqueous solution is about 3-4 ml. The
volume of foam in the solution containing gaseous precursor-filled
liposomes with 10 mol % sodium lauryl sulfate is also about 15 ml
and the volume of aqueous solution is about 3-4 ml.
EXAMPLE 11
Determination of Whether Gaseous Precursor-Filled Liposomes can be
Generated by Sonication
[0251] 50 mg of lipid, 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine
(Avanti-Polar Lipids, Alabaster, Ala.), is weighed out and hydrated
with 5 ml of 0.9% NaCl. To this suspension is added 165
.mu.L/mL.sup.-1 of 2-methyl-2-butene. Instead of vortexing, the
aqueous solution is sonicated using a Heat Systems Sonicator
Ultrasonic Processor XL (Heat Systems, Inc., Farmingdale, N.Y.)
Model XL 2020. The sonicator, with a frequency of 20 KHz, is set to
continuous wave, at position 4 on the knob of the sonicator. A
micro tip is used to sonicate for 10 minutes at a temperature of
4.degree. C. Following sonication, the temperature is increased to
40.degree. C. and the solution is viewed under an optical
microscope. There will be evidence of gaseous precursor-filled
liposomes having been produced.
[0252] Next, the above is repeated with sonication at a temperature
of 50.degree. C. and 165 .mu.L mL.sup.-1 of 2-methyl 2-butene is
added. The micro tip of the sonicator is removed and replaced with
the end cap that is supplied with the sonicator. Another solution
(50 mg of lipid per 5 ml of saline) is prepared and sonicated with
this tip. After 10 minutes, the solution is viewed under the
microscope. The production of gas-filled liposomes with sonication
above the temperature of the transition of the gas resulted in a
lower yield of gas-filled lipid spheres.
EXAMPLE 12
Determination of Concentration Effects on Gaseous Precursor-Filled
Liposome Production
[0253] This example determined whether a lower concentration limit
of the lipid halts the production of gaseous precursor-filled
liposomes. Ten mg of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine
(Avanti-Polar Lipids, Alabaster, Ala.) is added to 10 ml of saline.
To this suspension is added 165 .mu.L/mL.sup.-1 of
2-methyl-2-butene. The lipid/saline/gas precursor solution is
vortexed at position 6.5 for 10 minutes. The solution is viewed
under an optical microscope for sizing. It will be determined that
the largest size of the liposomes ranges from about 30 to about 45
.mu.m and the smallest size detected is about 7 .mu.m. The average
size range is from about 30 to about 45 .mu.m.
[0254] It appears that the gaseous precursor-filled liposomes are
more fragile as they appear to burst more rapidly than previously
shown. Thus, it appears that concentration of the lipid is a factor
in the generation and stability of gaseous precursor-filled
liposomes.
EXAMPLE 13
Cascade Filtration
[0255] Unfiltered gaseous precursor-filled liposomes may be drawn
into a 50 ml syringe and passed through a cascade of a "NUCLEPORE"
10 .mu.m filter and 8 .mu.m filter that are a minimum of 150 .mu.m
apart (FIGS. 3 and 4). Alternatively, for example, the sample may
be filtered through a stack of 10 .mu.m and 8 .mu.m filters that
are immediately adjacent to each other. Gaseous precursor-filled
liposomes are passed through the filters at a pressure whereby the
flow rate is 2.0 ml min.sup.-1. The subsequently filtered gaseous
precursor-filled liposomes are then measured for yield of gaseous
precursor-filled lipid liposomes which results in a volume of
80-90% of the unfiltered volume.
[0256] The resulting gaseous precursor-filled liposomes are sized
by four different methods to determine their size and distribution.
Sizing is performed on a Particle Sizing Systems Model 770 Optical
Sizing unit, a Zeiss Axioplan optical microscope interfaced to
image processing software manufactured by Universal Imaging, and a
Coulter Counter (Coulter Electronics Limited, Luton, Beds.,
England). As seen in FIGS. 5 and 6, the size of the gaseous
precursor-filled liposomes are more uniformly distributed around
8-10 .mu.m as compared to the unfiltered gaseous precursor-filled
liposomes. Thus, it can be seen that the filtered gaseous
precursor-filled liposomes are of much more uniform size.
EXAMPLE 14
Preparation of Filtered DPPC Suspension
[0257] 250 mg DPPC (dipalmitoylphosphatidylcholine) and 10 ml of
0.9% NaCl are added to a 50 ml Falcon centrifuge tube
(Becton-Dickinson, Lincoln Park, N.J.) and maintained at an ambient
temperature (approx. 20.degree. C.). To this suspension is added
165 .mu.L/mL.sup.-1 of 2-methyl-2-butene. The suspension is then
extruded through a 1 .mu.m Nuclepore (Costar, Pleasanton, Calif.)
polycarbonate membrane under nitrogen pressure. The resultant
suspension is sized on a Particle Sizing Systems (Santa Barbara,
Calif.) Model 370 laser light scattering sizer. All lipid particles
are 1 .mu.m or smaller in mean outside diameter.
[0258] In addition, the same amount of DPPC/gas precursor
suspension is passed five times through a Microfluidics.TM.
(Microfluidics Corporation, Newton, Mass.) microfluidizer at 18,000
p.s.i. The suspension, which becomes less murky, is sized on a
Particle Sizing Systems (Santa Barbara, Calif.) Sub Micron Particle
Sizer Model 370 laser light scattering sizer where it is found that
the size is uniformly less than 1 .mu.m. The particle size of
microfluidized suspensions is known to remain stable up to six
months.
EXAMPLE 15
Preparation of Filtered DSPC Suspension
[0259] 100 mg DSPC (distearoylphosphatidylcholine) and 10 ml of
0.9% NaCl are added to a 50 ml Falcon centrifuge tube
(Becton-Dickinson, Lincoln Park, N.J.). To this suspension is added
165 .mu.L/mL.sup.-1 of 2-methyl-2-butene. The suspension is then
extruded through a 1 .mu.m "NUCLEPORE" (Costar, Pleasanton, Calif.)
polycarbonate membrane under nitrogen pressure at 300-800 p.s.i.
The resultant suspension is sized on a Particle Sizing Systems
(Santa Barbara, Calif.) Sub Micron Particle Sizer Model 370 laser
light scattering sizer. It will be found that all particles are 1
.mu.m or smaller in size.
[0260] In addition, the same amount of DPPC/gas precursor
suspension is passed five times through a Microfluidics.TM.
(Microfluidics Corporation, Newton, Mass.), microfluidizer at
18,000 p.s.i. The resultant suspension, which is less murky, is
sized on a Sub Micron Particle Sizer Systems Model 370 laser light
scattering sizer and it is found that the size is uniformly less
than 1 .mu.m.
EXAMPLE 16
Sterilization of Filtered Lipid Suspensions by Autoclaving
[0261] The previously sized suspensions of DPPC/gas precursor and
DSPC/gas precursor of Examples 9 and 10 are subjected to
autoclaving for twenty minutes on a Barnstead Model C57835
autoclave (Barnstead/Thermolyne, Dubuque, Iowa) and then subjected
to shaking. A filtration step may be performed immediately prior to
use through an in line filter. Also, the gaseous precursor may be
autoclaved before sizing and shaking.
[0262] After equilibration to room temperature (approx. 20.degree.
C.), the sterile suspension is used for gaseous precursor
instillation.
EXAMPLE 17
Gaseous Precursor Instillation of Filtered, Autoclaved Lipids Via
Vortexing
[0263] 10 ml of a solution of 1,2-dipalmitoyl-phosphatidylcholine
at 25 mg/ml in 0.9% NaCl, which had previously been extruded
through a 1 .mu.m filter and autoclaved for twenty minutes, is
added to a Falcon 50 ml centrifuge tube (Becton-Dickinson, Lincoln
Park, N.J.). To this suspension is added 165 .mu.L/mL.sup.-1 of
2-methyl-2-butene. After equilibration of the lipid suspension to
room temperature (approximately 20.degree. C.), the liquid is
vortexed on a VWR Genie-2 (120V, 0.5 amp, 60 Hz.) (Scientific
Industries, Inc., Bohemia, N.Y.) for 10 minutes or until a time
that the total volume of gaseous precursor-filled liposomes is at
least double or triple the volume of the original aqueous lipid
solution. The solution at the bottom of the tube is almost totally
devoid of anhydrous particulate lipid, and a large volume of foam
containing gaseous precursor-filled liposomes results. Thus, prior
autoclaving does not affect the ability of the lipid suspension to
form gaseous precursor-filled liposomes. Autoclaving does not
change the size of the liposomes, and it does not decrease the
ability of the lipid suspensions to form gaseous precursor-filled
liposomes.
EXAMPLE 18
Gaseous Precursor Instillation of Filtered, Autoclaved Lipids Via
Shaking on Shaker Table
[0264] 10 ml of a solution of 1,2-dipalmitoyl-phosphatidylcholine
at 25 mg/ml in 0.9% NaCl, which has previously been extruded
through a 1 .mu.m filter and autoclaved for twenty minutes, is
added to a Falcon 50 ml centrifuge tube (Becton-Dickinson, Lincoln
Park, N.J.). To this suspension is added 165 .mu.L/mL.sup.-1 of
perfluoropentane (PCR Research Chemicals, Gainesville, Fla.). After
equilibration of the lipid suspension to room temperature
(approximately 20.degree. C.), the tube is then placed upright on a
VWR Scientific Orbital shaker (VWR Scientific, Cerritos, Calif.)
and shaken at 300 r.p.m. for 30 minutes. The resultant agitation on
the shaker table results in the production of gaseous
precursor-filled liposomes.
EXAMPLE 18A
[0265] The above experiment may be performed replacing
perfluoropentane with sulfur hexafluoride, hexafluoropropylene,
bromochlorofluoromethane, octafluoropropane, 1,1 dichloro, fluoro
ethane, hexafluoroethane, hexafluoro-2-butyne, perfluoropentane,
perfluorobutane, octafluoro-2-butene or hexafluorobuta-1,3-diene or
octafluorocyclopentene, all with the production of gaseous
precursor filled liposomes.
EXAMPLE 19
Gaseous Precursor Instillation of Filtered, Autoclaved Lipids Via
Shaking on Shaker Table Via Shaking on Paint Mixer
[0266] 10 ml of a solution of 1,2-dipalmitoyl-phosphatidylcholine
at 25 mg/ml in 0.9% NaCl, which has previously been extruded
through a 1 .mu.m filter and autoclaved for twenty minutes, is
added to a Falcon 50 ml centrifuge tube (Becton-Dickinson, Lincoln
Park, N.J.). To this suspension is added 165 .mu.L/mL.sup.-1 of
2-methyl-2-butene. After equilibration of the lipid suspension to
room temperature (approximately 20.degree. C.), the tube is
immobilized inside a 1 gallon empty household paint container and
subsequently placed in a mechanical paint mixer employing a
gyrating motion for 15 minutes. After vigorous mixing, the
centrifuge tube is removed, and it is noted that gaseous
precursor-filled liposomes form.
EXAMPLE 20
Gaseous Precursor Instillation of Filtered, Autoclaved Lipids Via
Shaking by Hand
[0267] 10 ml of a solution of 1,2-dipalmitoyl-phosphatidylcholine
at 25 mg/ml in 0.9% NaCl, which had previously been extruded
through a 1 .mu.m nuclepore filter and autoclaved for twenty
minutes, is added to a Falcon 50 ml centrifuge tube
(Becton-Dickinson, Lincoln Park, N.J.). To this suspension is added
165 .mu.L/mL.sup.-1 of 2-methyl-2-butene. After equilibration of
the lipid suspension to room temperature (approximately 20.degree.
C.), the tube is shaken forcefully by hand for ten minutes. Upon
ceasing agitation, gaseous precursor-filled liposomes form.
EXAMPLE 21
Sizing Filtration of Autoclaved Gaseous Precursor-Filled Liposomes
Via Cascade or Stacked Filters
[0268] Gaseous precursor-filled liposomes are produced from DPPC as
described in Example 17. The resultant unfiltered liposomes are
drawn into a 50 ml syringe and passed through a cascade filter
system consisting of a "NUCLEPORE" (Costar, Pleasanton, Calif.) 10
.mu.m filter followed by an 8 .mu.m filter spaced a minimum of 150
.mu.m apart. In addition, on a separate sample, a stacked 10 .mu.m
and 8 .mu.m filtration assembly is used, with the two filters
adjacent to one another. Gaseous precursor-filled liposomes are
passed through the filters at a pressure such that they are
filtered a rate of 2.0 ml/min. The filtered gaseous
precursor-filled liposomes yields a volume of 80-90% of the
unfiltered volume.
[0269] The resultant gaseous precursor-filled liposomes are sized
by four different methods to determine their size distribution.
Sizing is performed on a Particle Sizing Systems (Santa Barbara,
Calif.) Model 770 Optical Sizing unit, and a Zeiss (Oberkochen,
Germany) Axioplan optical microscope interfaced to image processing
software (Universal Imaging, West Chester, Pa.) and a Coulter
Counter (Coulter Electronics Limited, Luton, Beds., England). As
illustrated in FIG. 8, the size of the gaseous precursor-filled
liposomes is more uniformly distributed around 8-10 .mu.m as
compared to the unfiltered gaseous precursor-filled liposomes.
EXAMPLE 22
Extra Efficient Production of Gas-Precursor Filled Lipid
Spheres
[0270] The same procedure as in Example 6 is performed except that
the shaker used is a Crescent "Wig-L-Bug (Crescent Manufacturing
Dental Co., Lyons, Ill.). The formulation is then agitated for 60
seconds instead of the usual 5 minutes to 10 minutes as described
previously. Gas-filled lipid spheres are produced.
EXAMPLE 23
[0271] 100 .mu.L of perfluoropentane (bp 29.5.degree. C., PCR
Research Chemicals, Gainesville, Fla.) was added to a 5 mg/mL lipid
suspension and vortexed on a Genie II mixer (Scientific Industries,
Inc., Bohemia, N.Y.) at room temperature at power setting of 6.5. A
Richmar (Richmar Industries, Inola, Okla.) 1 MHz therapeutic
ultrasound device was then used to perform hyperthermia, elevating
the temperature to above 42.degree. C. as measured by a
thermometer. Upon reaching the phase transition temperature, gas
microspheres were noted. A simultaneous scanning was performed with
a diagnostic ultrasound (Acoustic Imaging, Phoenix, Ariz.).
Acoustic signals from the gas microspheres could also be visualized
on the clinical diagnostic ultrasound.
[0272] The same exeriment was conducted with octafluorocyclopentene
(bp 27.degree. C., PCR Research Chemicals, Gainesville, Fla.).
EXAMPLE 24
[0273] An experiment identical to Example 23 was performed where
the suspension was vortexed and injected into a Harlan-Sprague
Dawley rat, 300 grams, previously given a C5A tumor cell line in
the left femoral region. A Richmar 1 MHz therapeutic ultrasound was
then placed over the tumor region and an adriamycin embedded lipid
suspension injected intravenously. The therapeutic ultrasound was
then placed on a continuous wave (100% duty cycle) setting and the
tumor heated. A second rat, having a C5A tumor cell line in the
left femoral region, was given an identical dose of the adriamycin
emulsion, however, no ultrasound was utilized in this animal.
Within three weeks it was noted that the tumor, compared to the
control without the use of ultrasound, was noticeably smaller.
[0274] Various modifications of the invention in addition to those
shown and described herein will be apparent to those skilled in the
art from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
* * * * *