U.S. patent application number 11/568936 was filed with the patent office on 2008-06-05 for compositions and methods for enhancing contrast in imaging.
This patent application is currently assigned to MARVEL THERAPEUTICS. Invention is credited to Ananth Annapragada, Ravi V. Bellamkonda, Ketan Ghaghada, Eric Hoffman, Chen-Yu Kao, Chandra Vijayalakshmi.
Application Number | 20080131369 11/568936 |
Document ID | / |
Family ID | 35136670 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131369 |
Kind Code |
A1 |
Annapragada; Ananth ; et
al. |
June 5, 2008 |
Compositions And Methods For Enhancing Contrast In Imaging
Abstract
Example compositions of liposomes with hydrophilic polymers on
their surface, and containing relatively high concentrations of
contrast-enhancing agents for computed tomography are provided.
Example pharmaceutical compositions of such liposomes, when
administered to a subject, provide for increased contrast of
extended duration, as measured by computed tomography, in the
bloodstream and other tissues of the subject. Also provided are
example methods for making the liposomes containing high
concentrations of contrast-enhancing agents, and example methods
for using the compositions.
Inventors: |
Annapragada; Ananth;
(Manvel, TX) ; Bellamkonda; Ravi V.; (Marietta,
GA) ; Hoffman; Eric; (Iowa City, IA) ;
Vijayalakshmi; Chandra; (Manvel, TX) ; Kao;
Chen-Yu; (Atlanta, GA) ; Ghaghada; Ketan;
(Houston, TX) |
Correspondence
Address: |
BENESCH, FRIEDLANDER, COPLAN & ARONOFF LLP;ATTN: IP DEPARTMENT DOCKET
CLERK
2300 BP TOWER, 200 PUBLIC SQUARE
CLEVELAND
OH
44114
US
|
Assignee: |
MARVEL THERAPEUTICS
Manvel
TX
|
Family ID: |
35136670 |
Appl. No.: |
11/568936 |
Filed: |
January 12, 2005 |
PCT Filed: |
January 12, 2005 |
PCT NO: |
PCT/US05/00876 |
371 Date: |
December 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10830190 |
Apr 21, 2004 |
|
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11568936 |
|
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Current U.S.
Class: |
424/9.1 |
Current CPC
Class: |
A61K 49/0438 20130101;
A61K 49/0466 20130101; A61K 49/0093 20130101; A61K 9/1271
20130101 |
Class at
Publication: |
424/9.1 |
International
Class: |
A61K 49/00 20060101
A61K049/00 |
Claims
1-17. (canceled)
18. A method for making a composition, comprising: selecting one or
more solutions containing one or more nonradioactive
contrast-enhancing agents; and forming sterically stabilized
liposomes in the presence of the one or more solutions containing
one or more nonradioactive contrast-enhancing agents to provide a
solution of sterically stabilized liposomes that is associated with
the one or more nonradioactive contrast-enhancing agents.
19. The method of claim 26, wherein the concentrating comprises one
or more of: (i) precipitating at least some of the nonradioactive
contrast-enhancing agents from the solution containing one or more
nonradioactive contrast-enhancing agents and suspending the
precipitated nonradioactive contrast-enhancing agents in a liquid
at a higher concentration, and (ii) evaporating at least some of a
liquid from the solution containing one or more nonradioactive
contrast-enhancing agents.
20. The method of claim 18, further comprising concentrating the
solution of sterically stabilized liposomes that is associated with
the one or more nonradioactive contrast-enhancing agents to produce
a more concentrated solution of sterically stabilized liposomes
that is associated with the one or more nonradioactive
contrast-enhancing agents, the more concentrated solution of
sterically stabilized liposomes that is associated with the one or
more nonradioactive contrast-enhancing agents containing from about
37 to about 200 milligrams of iodine per milliliter of the more
concentrated solution of sterically stabilized liposomes that is
associated with the one or more nonradioactive contrast-enhancing
agents.
21. The method of claim 20, wherein the step of concentrating the
solution of sterically stabilized liposomes that is associated with
the one or more nonradioactive contrast-enhancing agents to produce
a more concentrated solution of sterically stabilized liposomes
that is associated with the one or more nonradioactive
contrast-enhancing agents comprises one or more of, dewatering, ion
exchange, washing, and dialysis.
22. The method of claim 18, wherein forming the sterically
stabilized liposomes comprises one or more of: hydrating dried
lipids in the presence of the one or more solutions containing one
or more nonradioactive contrast-enhancing agents; mixing a volatile
organic solution of lipids with the one or more solutions
containing one or more nonradioactive contrast-enhancing agents;
and dialyzing an aqueous solution of one or more of lipids,
detergents, and surfactants in the presence of the one or more
solutions containing one or more nonradioactive contrast-enhancing
agents to remove the one or more lipids, detergents, and
surfactants.
23-25. (canceled)
26. The method of claim 18, further comprising concentrating at
least one of the solutions containing one or more nonradioactive
contrast-enhancing agents by at least about 10% to produce a
concentrated solution of nonradioactive contrast-enhancing
agents.
27-37. (canceled)
38. A method comprising: selecting one or more nonradioactive
contrast-enhancing agents; and forming sterically stabilized
liposomes in the presence of the nonradioactive contrast-enhancing
agents to provide liposomes containing or associated with one or
more contrast-enhancing agents.
39. The method of claim 38, where one milliliter of a suspension of
the sterically stabilized liposomes has at least 30 milligrams of
iodine.
40. The method of claim 38, where an average diameter of the
sterically stabilized liposomes is less than about 120
nanometers.
41. The method of claim 38, where liposomes are found in the
presence of one or more contrast-enhancing agents using a method
selected from the group consisting of hydration of dried lipids in
the presence of one or more contrast-enhancing agents, mixing a
volatile organic solution of lipids with an aqueous solution of one
or more contrast-enhancing agents causing evaporation of the
organic solution, and dialysis of an aqueous solution of lipids and
detergents and/or surfactants to remove the detergents and/or
surfactants and form liposomes in the presence of one or more
contrast-enhancing agents.
42. A method comprising: forming sterically stabilized liposomes;
and drawing one or more nonradioactive contrast-enhancing agents
into the liposomes to provide liposomes containing or associated
with one or more contrast-enhancing agents.
43. The method of claim 42, where one milliliter of a suspension of
the sterically stabilized liposomes has at least 30 milligrams of
iodine.
44. The method of claim 42, where an average diameter of the
sterically stabilized liposomes is less than about 120 nanometers.
Description
BACKGROUND
[0001] Some medical X-ray imaging techniques can detect variations
in contrast of regions of interest in a subject, including
different organs, tissues, cells and the like. To increase the
contrast of regions of interest, some of the imaging techniques
utilize the administration of one or more contrast-enhancing agents
to a subject. The contrast-enhancing agents can accentuate existing
differences in contrast between different areas of interest, or can
produce differences in contrast where such differences do not exist
without use of the agents.
[0002] There have been advancements in medical X-ray imaging,
specifically relating to the instruments or machines used to detect
the differences in contrast. These advancements include increases
in the speed of the instruments, increases in the resolution of the
instruments, and the like. These advancements have provided, in
part, for new medical imaging methods. One example method,
whole-body imaging, can yield information on the vasculature of the
entire body of a subject.
[0003] Compared to advances in the instruments used for X-ray
imaging, advances in contrast-enhancing agents have not been as
forthcoming. Current contrast-enhancing agents for medical imaging
using X-rays can have limitations for applications such as
whole-body imaging due to, among other things, rapid clearance from
the body of a subject, greater than desired extravasation, renal
toxicity and inability to target specific areas of the body of a
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] In the accompanying drawings, which are incorporated in and
constitute a part of the specification, embodiments of
contrast-enhancing agent formulations, pharmaceutical compositions
containing the formulations, methods for making the formulations
and methods for using the formulations in imaging are illustrated
which, together with the detailed description given below, serve to
describe the example embodiments of formulations, compositions,
methods, and so on. It will be appreciated that the embodiments
illustrated in the drawings are shown for the purpose of
illustration and not for limitation. It will be appreciated that
changes, modifications and deviations from the embodiments
illustrated in the drawings can be made without departing from the
spirit and scope of the invention, as disclosed below.
[0005] FIG. 1 illustrates an example method 100 of preparing
liposomes containing or associated with contrast-enhancing
agents;
[0006] FIG. 2 illustrates another example method 200 of preparing
liposomes containing or associated with contrast-enhancing
agents;
[0007] FIG. 3 illustrates another example method 300 of preparing
liposomes containing or associated with contrast-enhancing
agents;
[0008] FIG. 4 shows example results 400 from an in vitro stability
test of one embodiment of a liposomal iohexyl formulation when
dialyzed with PBS at 4.degree. C. The total iodine amount is 30 mg
iodine;
[0009] FIG. 5 shows example results 500 from an in vitro plasma
stability test of one embodiment of a liposomal iohexyl formulation
when dialyzed against PBS at 37.degree. C. The total iodine content
is 28 mg iodine;
[0010] FIG. 6 shows example time-attenuation curves 600 of various
regions of interest at different post-injection times after
intravenous administration of one embodiment of a liposomal iohexyl
formulation (injection to 2.2 kg rabbit vein at a dose of 475 mg
I/kg) given in two incremental injections;
[0011] FIG. 7 shows example pre- and post-enhancement computed
tomography (CT) images 700 of one embodiment of liposomal iohexyl:
2.2 kg rabbit with 34.8 mg/ml iodine IV injection. Left Panels 705,
715: pre-contrast; Right Panels 710, 720: 2 hours 18 minutes post
injection. Upper panels 705, 710 are images taken at the level of
the liver. Lower panels 715, 720 are images taken at mid heart
level;
[0012] FIG. 8 shows example volume-rendered CT images 800 of a
rabbit torso. Left panel 805: right lateral view before contrast
injection; Right panel 810: right lateral view 2 hours 18 minutes
after injection of 475 mg I/kg of one embodiment of a liposomal
iohexyl formulation. Note the enhanced vascular bed seen in the
right panel 815;
[0013] FIG. 9 shows example volume-rendered CT images 900 of an in
vivo rabbit heart imaged before 905 and at multiple time sequences
post injection 910, 915, 920, 925, 930 of one embodiment of
liposomal iohexyl. All volume-rendering parameters and display
parameters were held constant across time points;
[0014] FIG. 10 shows an example of a thick-slab rendering 1000 of
ultra-high resolution CT scan (24 line pair per cm) of post-mortem
rabbit (no cardiac motion). Rabbit was sacrificed 3.5 hours after
the second injection of one embodiment of liposomal iohexyl. Images
were reconstructed to fit a 1,024.times.1,024 matrix with a 0.5-cm
field of view;
[0015] FIG. 11 shows an example image 1100 of the left coronary
artery of the rabbit under high magnification;
[0016] FIG. 12 shows example time-lapse coronal images 1200 of a
mouse heart obtained by micro CT at 10 millisecond intervals 1205,
1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245; and
[0017] FIG. 13 shows an example image 1300, obtained by micro CT,
of the abdominal region of a nude mouse containing a tumor (human
squamous cell carcinoma) 1305 in the right flank and an inflamed
lymph node 1310 on the left side.
DETAILED DESCRIPTION
Definitions
[0018] Definitions of selected terms or phrases are contained
immediately following, and throughout the disclosure. The
definitions include examples of various embodiments and/or forms of
components that fall within the scope of a term and that may be
used for implementation. The examples are not intended to be
limiting and other embodiments may be implemented. Both singular
and plural forms of all terms fall within each meaning.
[0019] "X-ray imaging," as used herein, generally refers to any of
a number of procedures using a source producing X-rays. Examples of
X-ray imaging include computed tomography and the like.
[0020] "Computed tomography" or "CT" or "CAT," as used herein,
generally refers to procedures using a rotating X-ray instrument or
machine to produce X-ray radiation and direct it through areas of a
subject as the instrument rotates. The radiation that is not
absorbed by the subject generally is detected and recorded as data.
Generally, the data are sent to a computer which creates detailed
cross-sectional images, or slices, of organs and body parts based
on differential absorption of X-rays by different areas of the
subject. CT of high resolution may be called "micro CT."
[0021] "Whole body imaging," as used herein, generally refers to
methodologies for obtaining images, using CT for example, of the
entire body of a subject. In one type of whole body imaging, the
entire vasculature system may be examined. Generally, imaging where
the vasculature system is examined is called "blood pool
imaging."
DESCRIPTION
[0022] This application describes example compositions comprising
liposomes which contain or are associated with one or more
contrast-enhancing agents. In one example, the liposomes contain or
are associated with relatively high concentrations of
contrast-enhancing agents. In one example, the liposomes contain
one or more contrast-enhancing agents for X-ray imaging (e.g., CT
imaging). In one example, the contrast-enhancing agents are not
radioactive.
[0023] In one example, the liposomes have one or more hydrophilic
polymers attached to or associated with the liposomes. In one
example, the hydrophilic polymers are attached to or associated
with the surface of the liposomes. When administered to a subject,
the liposomes can provide increased contrast in the body of a
subject. In one example, the increased contrast lasts for an
extended period of time.
[0024] This application also describes example pharmaceutical
compositions that contain the liposomes and contrast-enhancing
agents, and example methods of making the compositions of liposomes
containing contrast-enhancing agents. The application also
describes example methods of using the compositions in X-ray
imaging.
Contrast-Enhancing Agents
[0025] "Contrast-enhancing agent," as used herein, generally refers
to a substance that affects the attenuation, or the loss of
intensity or power, of radiation as it passes through and interacts
with a medium. It will be appreciated that contrast-enhancing
agents may increase or decrease the attenuation. Generally, the
contrast-enhancing agents referred to herein may increase the
attenuation of radiation. In one example, the contrast-enhancing
agents described herein are contrast-enhancing agents for X-ray
imaging. In one example, the contrast-enhancing agents can be used
for CT. In one example, the contrast-enhancing agents used herein
are nonradioactive. In one embodiment, the contrast-enhancing
agents can contain iodine and may be called "iodinated.".
[0026] Contrast-enhancing agents may be classified in various ways.
In one classification, for example, iodinated contrast-enhancing
agents can be water soluble (e.g., monoiodinated pyridine
derivatives, di-iodinated pyridine derivatives, tri-iodinated
benzene ring compounds, and the like), water-insoluble (e.g.,
propyliodone and the like) or oily (e.g., iodine in poppy seed oil,
ethyl esters of iodinated fatty acids of poppy seed oil containing
iodine, and the like).
[0027] In one example, a grouping of iodinated contrast-enhancing
agents are water soluble. Present water soluble iodinated
contrast-enhancing agents can be derivatives of tri-iodinated
benzoic acid. These compounds can have one or more benzene rings.
These compounds can be ionic or nonionic. Suitable, nonionic
compounds include, but are not limited to, metrizamide, iohexyl,
iopamidol, iopentol, iopromide, ioversol, iotrolan, iodixanol and
others.
[0028] Suitable ionic compound contrast-enhancing agents may be
weakly acidic (pK.sub.a of from approximately 4.0 to 6.5) or weakly
basic (pKa of from approximately 6.5 to 8.5). Generally, acids are
capable of giving up or donating one or more protons. In their
protonated form, the acids are generally substantially electrically
neutral or uncharged. In their unprotonated form, the acids are
generally substantially negatively charged. Suitable weakly acidic
agents can have one or more carboxyl groups. The carboxyl groups
are capable of donating a proton. The carboxyl groups may be
attached to a benzene ring and/or may be part of a benzoic acid.
Examples of such benzoic acids include, but are not limited to,
acetrizoate, diatrizoate, iodamide, ioglicate, iothalamate,
ioxithalamate, metrizoate, sodium meglumine ioxaglate and
others.
[0029] Generally, bases are capable of accepting one or more
protons. In their protonated form, the bases are generally
substantially positively charged. In their unprotonated form, the
bases are generally substantially neutral or uncharged. Suitable
weakly basic agents may have one or more primary amine groups. The
amines are capable of accepting a proton. The weakly basic agents
may be amides.
Liposomes
[0030] "Liposomes," as used herein, generally refer to spherical or
roughly spherical particles containing an internal cavity. The
walls of liposomes can include a bilayer of lipids. These lipids
can be phospholipids. Numerous lipids and/or phospholipids may be
used to make liposomes. One example are amphipathic lipids having
hydrophobic and polar head group moieties which may form
spontaneously into bilayer vesicles in water, as exemplified by
phospholipids, or which may be stably incorporated into lipid
bilayers, with their hydrophobic moiety in contact with the
interior, hydrophobic region of the bilayer membrane, and their
polar head group moiety oriented toward the exterior, polar surface
of the membrane.
[0031] As used herein, "phospholipids" include, but are not limited
to, phosphatidic acid (PA), phosphatidylglycerol (PG),
phosphatidylcholine (PC), egg phosphatidylcholine (EPC),
lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE),
phosphatidylinositol (PI), phosphatidylserine (PS), and mixtures of
two or more thereof. The vesicle-forming lipids of this type may be
lipids having two hydrocarbon chains, typically acyl chains, and a
polar head group. Included in this class are phospholipids, such as
phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidic acid (PA), phosphatidylglycerol (PG),
phosphatidylinositol (PI), and sphingomyelin (SM), plus others.
These phospholipids can be fully saturated or partially saturated.
They can be naturally occurring or synthetic. In another example,
lipids that can be included in the liposomes can be
glycolipids.
[0032] The phospholipids used in the example liposomes described
herein can be those where the two hydrocarbon chains are between
about 14 and about 24 carbon atoms in length, and have varying
degrees of unsaturation. Some examples of these phospholipids are
given below. Although the phospholipids listed below may be used,
alone or in combination with other phospholipids, the list is not
intended to be complete. Other phospholipids not listed herein can
also be used.
[0033] Phospholipids [0034]
1-Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine, [0035]
1-Myristoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine, [0036]
1-Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine, [0037]
1-Myristoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine, [0038]
1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphate (POPA), [0039]
1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine, [0040]
1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE),
[0041] 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)]
(POPG), [0042] 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-L-Serine]
(POPS), [0043] 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphate,
[0044] 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine, [0045]
1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphoethanolamine, [0046]
1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)],
[0047] 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-[Phospho-L-Serine],
[0048] 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphate, [0049]
1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphocholine, [0050]
1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphoethanolamine, [0051]
1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)],
[0052] 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-[Phosph-L-Serine],
[0053] 1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphate, [0054]
1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphocholine, [0055]
1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphoethanolamine,
[0056]
1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)],
[0057]
1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-[Phospho-L-Serine],
[0058] 1-Stearoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine, [0059]
1-Stearoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine, [0060]
1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphate, [0061]
1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine, [0062]
1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine, [0063]
1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol], [0064]
1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-L-Serine], [0065]
1-Stearoyl-2-Linoleoyl-sn-Glycero-3-Phosphate, [0066]
1-Stearoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine, [0067]
1-Stearoyl-2-Linoleoyl-sn-Glycero-3-Phosphoethanolamine, [0068]
1-Stearoyl-2-Linoleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)],
[0069] 1-Stearoyl-2-Linoleoyl-sn-Glycero-3-[Phospho-L-Serine],
[0070] 1-Stearoyl-2-Arachidonoyl-sn-Glycero-3-Phosphate, [0071]
1-Stearoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine, [0072]
1-Stearoyl-2-Arachidonoyl-sn-Glycero-3-Phosphoethanolamine, [0073]
1-Stearoyl-2-Arachidonoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)],
[0074] 1-Stearoyl-2-Arachidonoyl-sn-Glycero-3-[Phospho-L-Serine],
[0075] 1-Stearoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphate, [0076]
1-Stearoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphocholine, [0077]
1-Stearoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphoethanolamine,
[0078]
1-Stearoyl-2-Docosahexaenoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)],
[0079]
1-Stearoyl-2-Docosahexaenoyl-sn-Glycero-3-[Phospho-L-Serine],
[0080] 1-Oleoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine, [0081]
1-Oleoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine, [0082]
1-Oleoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine, [0083]
1,2-Dimyristoyl-sn-Glycero-3-Phosphate (DMPA), [0084]
1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), [0085]
1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine (DMPE), [0086]
1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG),
[0087] 1,2-Dimyristoyl-sn-Glycero-3-[Phospho-L-Serine] (DMPS),
[0088] 1,2-Dipentadecanoyl-sn-Glycero-3-Phosphocholine, [0089]
1,2-Dipalmitoyl-sn-Glycero-3-Phosphate (DPPA), [0090]
1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC), [0091]
1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE), [0092]
1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DPPG),
[0093] 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-L-Serine) (DPPS),
[0094] 1,2-Diphytanoyl-sn-Glycero-3-Phosphate, [0095]
1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine, [0096]
1,2-Diphytanoyl-sn-Glycero-3-Phosphoethanolamine, [0097]
1,2-Diphytanoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)], [0098]
1,2-Diphytanoyl-sn-Glycero-3-[Phospho-L-Serine], [0099]
1,2-Diheptadecanoyl-sn-Glycero-3-Phosphocholine, [0100]
1,2-Distearoyl-sn-Glycero-3-Phosphate (DSPA), [0101]
1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC), [0102]
1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine (DSPE), [0103]
1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG),
[0104] 1,2-Distearoyl-sn-Glycero-3-[Phospho-L-Serine], [0105]
1,2-Dibromostearoyl-sn-Glycero-3-Phosphocholine, [0106]
1,2-Dinonadecanoyl-sn-Glycero-3-Phosphocholine, [0107]
1,2-Diarachidoyl-sn-Glycero-3-Phosphocholine, [0108]
1,2-Diheneicosanoyl-sn-Glycero-3-Phosphocholine, [0109]
1,2-Dibehenoyl-sn-Glycero-3-Phosphocholine, [0110]
1,2-Ditricosanoyl-sn-Glycero-3-Phosphocholine, [0111]
1,2-Dilignoceroyl-sn-Glycero-3-Phosphocholine, [0112]
1,2-Dimyristoleoyl-sn-Glycero-3-Phosphocholine, [0113]
1,2-Dimyristelaidoyl-sn-Glycero-3-Phosphocholine, [0114]
1,2-Dipalmitoleoyl-sn-Glycero-3-Phosphocholine, [0115]
1,2-Dipalmitelaidoyl-sn-Glycero-3-Phosphocholine, [0116]
1,2-Dipalmitoleoyl-sn-Glycero-3-Phosphoethanolamine, [0117]
1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), [0118]
1,2-Dioleoyl-sn-Glycero-3-Phosphate (DOPA), [0119]
1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), [0120]
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE), [0121]
1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DOPG), [0122]
1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] (DOPS), [0123]
1,2-Dielaidoyl-sn-Glycero-3-Phosphocholine, [0124]
1,2-Dielaidoyl-sn-Glycero-3-Phosphoethanolamine, [0125]
1,2-Dielaidoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)], [0126]
1,2-Dilinoleoyl-sn-Glycero-3-Phosphate, [0127]
1,2-Dilinoleoyl-sn-Glycero-3-Phosphocholine, [0128]
1,2-Dilinoleoyl-sn-Glycero-3-Phosphoethanolamine, [0129]
1,2-Dilinoleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)], [0130]
1,2-Dilinoleoyl-sn-Glycero-3-[Phospho-L-Serine], [0131]
1,2-Dilinolenoyl-sn-Glycero-3-Phosphocholine, [0132]
1,2-Dilinolenoyl-sn-Glycero-3-Phosphoethanolamine, [0133]
1,2-Dilinolenoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)], [0134]
1,2-Dieicosenoyl-sn-Glycero-3-Phosphocholine, [0135]
1,2-Diarachidonoyl-sn-Glycero-3-Phosphate, [0136]
1,2-Diarachidonoyl-sn-Glycero-3-Phosphocholine, [0137]
1,2-Diarachidonoyl-sn-Glycero-3-Phosphoethanolamine, [0138]
1,2-Diarachidonoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)], [0139]
1,2-Diarachidonoyl-sn-Glycero-3-[Phospho-L-Serine], [0140]
1,2-Dierucoyl-sn-Glycero-3-Phosphocholine, [0141]
1,2-Didocosahexaenoyl-sn-Glycero-3-Phosphate, [0142]
1,2-Didocosahexaenoyl-sn-Glycero-3-Phosphocholine, [0143]
1,2-Didocosahexaenoyl-sn-Glycero-3-Phosphoethanolamine, [0144]
1,2-Docosahexaenoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)], [0145]
1,2-Didocosahexaenoyl-sn-Glycero-3-[Phospho-L-Serine], and [0146]
1,2-Dinervonoyl-sn-Glycero-3-Phosphocholine.
[0147] The liposome composition can be formulated to include
amounts of fatty alcohols, fatty acids, and/or cholesterol esters
or other pharmaceutically acceptable excipients. For example, the
liposomes can include lipids that can stabilize a vesicle or
liposome composed predominantly of phospholipids. For example,
cholesterol between about 25 to 40 mole percent may be used.
[0148] In one embodiment, the type of liposomes used may be
"sterically stabilized liposomes." Sterically stabilized liposomes
can include a surface that contains or is coated with flexible
water soluble (hydrophilic) polymer chains. These polymer chains
may prevent interaction between the liposomes and blood plasma
components, the plasma components playing a role in uptake of
liposomes by cells of the blood and removal of the liposomes from
the blood. Sterically stabilized liposomes may avoid uptake by the
organs of the mononuclear phagocyte system, primarily the liver and
spleen (reticulendothelial system or RES). Such sterically
stabilized liposomes may also be called "long circulating
liposomes."
[0149] Sterically stabilized liposomes can contain lipids or
phospholipids that are derivatized with a polymer chain. The lipids
or phospholipids that may be used generally may be any of those
described above. One exemplary phospholipid is
phosphatidylethanolamine (PE) with a reactive amino group which may
be convenient for coupling to the activated polymers. An exemplary
PE may be distearyl PE (DSPE).
[0150] Examples of polymers that are suitable for use in sterically
stabilized liposomes include, but are not limited to, the
hydrophilic polymers polyvinylpyrrolidone, polymethyloxazoline,
polyethyloxazoline, polyhydroxypropyl methacrylamide,
polymethacrylamide, polydimethylacrylamide, polylactic acid,
polyglycolic acid, and derivatized celluloses, like
hydroxymethylcellulose or hydroxyethylcellulose. Polylysine may be
used. Lipid-polymer conjugates containing these polymers attached
to a suitable lipid, such as PE, may be used. Other example
polymers can be used.
[0151] In one embodiment, the polymer in the derivatized lipid or
phospholipid can be polyethylene glycol (PEG). The PEG can have any
of a variety of molecular weights. In one example, the PEG chain
may have a molecular weight between about 1,000-10,000 daltons.
Once a liposome is formed, the PEG chains may provide a surface
coating of hydrophilic chains sufficient to extend the blood
circulation time of the liposomes in the absence of such a coating.
Such liposomes may be called "PEGylated liposomes." PEGylated
liposomes can include so-called STEALTH.RTM. liposomes, provided by
ALZA Corporation.
[0152] PEGylated liposomes may also include liposomes with PEG on
their surface, where the PEG may be released from the liposome at
some time after administration of the liposomes to a subject. In
one example, there can be one or more bonds or linkages attaching
the PEG, or other hydrophilic polymer, to the liposome surface
and/or lipid molecules comprising the liposome surface. In one
example, the bonds or linkages can be cleaved, providing for
separation of the PEG from the liposome. For example, PEG may be
attached to a lipid by one or more disulfide bonds. The disulfide
bonds may be cleaved by free thiol, releasing the PEG from the
liposome. Other types of cleavable links or bonds can be used to
attach the polymers to the liposomes. Other types of agents or
compounds can be used to cleave the bonds or linkages.
[0153] In one example, the liposomes used can have a composition of
between about 60 and 75 mole % of one or more of the phospholipids
with carbon chains between about 14-24 in length, as described
above. A fraction of these phospholipids may be attached to one or
more hydrophilic polymers such that between about 1 and 20 mole %
of the liposome composition is phospholipid derivatized with
polymer chains. In addition, the liposomes used may have between
about 25 and 40 mole % cholesterol, or fatty alcohols, fatty acids,
and/or other cholesterol esters or other pharmaceutically
acceptable excipients, generally for the purpose of stabilizing the
liposomes.
[0154] In another example, the liposomes can have a molecule or
molecules, commonly called a "ligand," which may be accessible from
the surface of the liposome, that may specifically bind or attach
to, for example, one or more molecules or antigens. These ligands
may direct or target the liposomes to a specific cell or tissue and
may bind to a molecule or antigen on or associated with the cell or
tissue. The ligand may be an antibody or antibody fragment. The
antibody may be a monoclonal antibody or fragment. Such liposomes
may be of a type called "targeted liposomes."
[0155] In one example, targeted liposomes can have lipids or
phospholipids which have been modified for coupling antibody
molecules to the liposome outer surface. These modified lipids may
be of different types. The modified lipid may contain a spacer
chain attached to the lipid. The spacer chain may be a hydrophilic
polymer. The hydrophilic polymer may typically be
end-functionalized for coupling antibody to its functionalized end.
The functionalized end group may be a maleimide group, for
selective coupling to antibody sulfhydryl groups. Other
functionalized end groups may include bromoacetamide and disulfide
groups for reaction with antibody sulfhydryl groups, activated
ester and aldehyde groups for reaction with antibody amine groups.
Hydrazide groups are reactive toward aldehydes, which may be
generated on numerous biologically relevant compounds. Hydrazides
may also be acylated by active esters or carbodiimide-activated
carboxyl groups. Acyl azide groups reactive as acylating species
may be easily obtained from hydrazides and permit the attachment of
amino containing ligands.
[0156] In another example, the phospholipid can be modified by a
biotin molecule. To attach the antibody molecule to the
biotinylated liposome surface, once the liposome is formed, the
antibody molecule may also be modified with biotin and then
incubated in the presence of the avidin. Biotinylated lipids, such
as biotinylated PE, may be commercially available.
[0157] In another example, lipids can be modified by a substrate
for use in binding a targeting molecule to a liposome surface.
Typically, substrates, as exemplified with biotin, may be
relatively small, less than about 5,000 daltons for example, to
allow their incorporation into multilamellar liposomes with a
minimum of disruption of the lipid bilayer structures. The
substrate may be one capable of binding irreversibly to a targeting
molecule, to ensure that the targeting molecule remains bound to
the liposomes over its lifetime in the bloodstream.
Preparation of Liposomes Containing Contrast-Enhancing Agents
[0158] Liposomes can be prepared by a variety of methods. Example
methods include, but are not limited to, hydration of dried lipids,
introduction of a volatile organic solution of lipids into an
aqueous solution causing evaporation of the organic solution, and
dialysis of an aqueous solution of lipids and detergents or
surfactants to remove the detergents or surfactants, and other
methods.
[0159] Liposomes can contain or may be associated with one or more
contrast-enhancing agents. In one example, the liposomes may
contain the contrast-enhancing agents. In the process of making
liposomes, the contrast-enhancing agents may be added at any
desired time. For example, contrast-enhancing agents may be
associated with components of liposomes before liposomes are
formed. Contrast-enhancing agents may be combined with liposome
components at the time the liposomes are made. Contrast-enhancing
agents may also be added after the liposomes are formed. Other
methods of associating contrast-enhancing agents with liposomes may
exist. Generally, contrast-enhancing agents which are hydrophilic
in nature may be located or associated with the internal cavity of
the liposome particles. Contrast-enhancing agents which are
lipophilic in nature may be located or associated with the lipid
bilayer of liposome particles. Generally, the contrast-enhancing
agents herein are located or associated with the internal cavity of
the liposome. The example liposomes contain at least 30 mg
iodine/milliliter (I/ml) of liposome suspension when iodinated
contrast enhancing agents are used. One example of the liposomes
can contain between about 35 and about 250 mg I/ml of liposome
suspension. One example of the liposomes can contain between about
37 and about 200 mg I/ml of liposome suspension. One example of the
liposomes can contain between about 80 and about 160 mg I/ml of
liposome suspension. One example of the liposomes can contain
between about 100 and about 120 mg I/ml of liposome suspension. One
example of the liposomes can contain between about 85 and about 100
mg I/ml of liposome suspension. One example of the liposomes can
contain more than about 100 mg I/ml of liposome suspension.
[0160] There are a variety of methods for loading the
contrast-enhancing agents into the liposomes. Example methods may
be better appreciated with reference to the flow diagrams of FIGS.
1-3. While for purposes of simplicity of explanation, the
illustrated methodologies are shown and described as a series of
blocks, it is to be appreciated that the methodologies are not
limited by the order of the blocks, as some blocks may occur in
different orders and/or concurrently with other blocks from that
shown and described. Moreover, less than all the illustrated blocks
may be required to implement an example methodology. Blocks may be
combined or separated into multiple components. Furthermore,
additional and/or alternative methodologies can employ additional,
not illustrated blocks. While the figures illustrate various
actions occurring in serial, it is to be appreciated that various
actions could occur concurrently, substantially in parallel, and/or
at substantially different points in time. The diagrams of FIGS.
1-3 are not intended to limit the implementation of the described
examples.
[0161] Illustrated in FIG. 1 is an example method 100 for preparing
liposomes containing or associated with contrast-enhancing agents.
The method may include selecting one or more contrast-enhancing
agents to be used (block 105). The method may also include forming
liposomes in the presence of the one or more contrast-enhancing
agents (block 110). Generally, the step illustrated as block 110
may be performed using the methods described earlier for preparing
liposomes. These methods may include hydration of dried lipids,
introduction of a volatile organic solution of lipids into an
aqueous solution causing evaporation of the organic solution,
dialysis of an aqueous solution of lipids and detergents or
surfactants to remove the detergents or surfactants, and
others.
[0162] Illustrated in FIG. 2 is another example method 200 for
preparing liposomes containing or associated with
contrast-enhancing agents. The method may include selecting one or
more contrast-enhancing agents to be used (block 205). The method
may also include concentrating the one or more contrast-enhancing
agents (block 210). The method may also include forming liposomes
in the presence of the one or more contrast-enhancing agents (block
215). The method may also include concentrating the liposomes
(block 220).
[0163] Concentrating the one or more contrast-enhancing agents
(block 210) can be performed using a variety of methods. In one
example, a commercially available solution of one or more
contrast-enhancing agents may be concentrated using the methods. In
one example, the contrast-enhancing agents may be precipitated from
a solution and the precipitated contrast-enhancing agents suspended
in a liquid at a concentration higher than in the original
solution. In another example, the contrast-enhancing agents in a
solution may be concentrated by evaporation. One example of
evaporation may be rotary evaporation. Other methods may be used.
In one example, a solution of contrast-enhancing agents may be
concentrated by at least 10%. In one example, a solution of
contrast enhancing agents may be concentrated by 100% (i.e.,
2-fold) or more. In another example, solid forms of the contrast
enhancing agents may be dissolved in a liquid at a relatively high
concentration (e.g., at a higher concentration than in commercially
available solutions). In one example, heating may be used to
increase the solubility of the contrast-enhancing agents in the
solution. In another example, a solvent may be used in which the
contrast-enhancing agents may be more soluble than in another
solvent.
[0164] It will be appreciated that the viscosity of a liposome
suspension generally is determined by the concentration of
liposomes and generally is not determined by the viscosity of the
liposome contents. For example, contrast-enhancing agents that have
been encapsulated into liposomes may form a gel phase or even
crystallize inside the liposomes (e.g., if the temperature is
lowered). Generally, this may not affect the liposome suspension
and may facilitate the stability of the liposome suspension (e.g.,
by reducing the probability of leakage of the contrast-enhancing
agents from the liposomes).
[0165] After the liposomes are made and are in solution, the
solution of liposomes may be concentrated to obtain a more
concentrated solution of liposomes by decreasing the volume of the
solution without substantially changing the number of liposomes in
the solution. Concentrating the liposomes (block 220) can be
performed using a variety of methods. When the liposomes are in an
aqueous solution, concentration by removal of water may be called
dewatering. One example method of dewatering can be diafiltration.
In one example of diafiltration, a suspension of liposomes in a
liquid may be passed through a filter or membrane to decrease the
amount of liquid in which an amount of liposomes is suspended.
Other example methods can include ion exchange, washing of the
liposomes using ultracentrifugation, dialysis, and so on. These
methods can result in example liposome suspensions with
concentrations of between about 35-250 mg I/ml of liposome
suspension. One example of the liposomes can contain between 37 and
200 mg I/ml of liposome suspension. One example of the liposomes
can contain more than 100 mg I/ml of liposome suspension. These
methods may also remove impurities from a suspension of liposomes.
In one example, the impurities may include contrast-enhancing
agents that have not been encapsulated into or associated with
liposomes.
[0166] Illustrated in FIG. 3 is another example method 300 for
preparing liposomes containing or associated with
contrast-enhancing agents. The method 300 may include forming
liposomes in the presence of a loading agent (block 305). The
method may also include establishing an ion gradient between the
interface and exterior of the liposomes (block 310). The method may
also include loading one or more ionic iodinated benzenes into the
liposomes (block 315).
[0167] The method illustrated in FIG. 3 may be of a type or class
referred to as active or remote loading methods. In one example of
active or remote loading, the contrast-enhancing agent or agents to
be contained by or within the liposomes (e.g., contrast-enhancing
agents) may enter liposomes after the liposomes have been formed or
partially formed. Such formed liposomes generally are those whose
process of making is completed. Partially formed liposomes may not
have completed the making process.
[0168] In one example method, an ion gradient can be established
from or between the outside of the liposome and the inside of the
liposome (e.g., the concentration of one or more ions outside the
liposomes is different than the concentration inside the liposomes)
of the formed liposomes. The contrast-enhancing agent to be loaded
into the liposomes can move from the outside of the liposomes to
the inside of the liposomes. This movement may be due to movement
of the contrast-enhancing agent through the membranes of the
liposomes. Generally, contrast-enhancing agents capable of moving
through membranes may be substantially neutral in electrical charge
or uncharged. This movement may be based on a concentration
gradient (e.g., a greater concentration of the contrast-enhancing
agent outside the liposomes than inside the liposomes). This
movement may be based on an ion gradient. This movement may be
based on other factors or combinations of various factors. Once
inside the liposomes, the different ion concentration inside the
liposomes as compared to outside the liposomes may retard or
prevent the contrast-enhancing agent from moving out of the
liposomes. In one example, the different ion concentration inside
the liposomes as compared to outside the liposomes can chemically
alter the contrast-enhancing agent such that its movement out of
the liposomes is retarded or prevented.
[0169] One example ion gradient can be a pH gradient. Hydrated
liposomes may have a selected internal and external pH. This pH may
have been selected based on the pH of the environment in which the
liposomes were formed. The external solution in which the hydrated
liposomes are present may then be titrated until a selected pH
different from the internal pH is obtained. The external solution
may also be exchanged with another solution of a selected pH
different from the internal pH. For example, the original external
solution in which the liposomes are present may have a pH of 5.5
and then be titrated or exchanged for a solution that may have a pH
of 8.5. Once a contrast-enhancing agent enters into the liposomes,
a contrast-enhancing agent inside the liposome may be chemically
altered by accepting or donating one or more protons. A
contrast-enhancing agent that has accepted or donated one or more
protons may be charged. The charged contrast-enhancing agents may
be unable or inhibited in their ability to pass through the
liposome membrane. In these liposomes, the contrast-enhancing
agents may be unable to exit or have a reduced ability to exit the
liposomes.
[0170] In another example of active or remote loading, the formed
or partially formed liposomes may contain a loading agent. For
example, the liposomes may be formed in the presence of the loading
agent. The loading agent may assist or facilitate entry of
contrast-enhancing agents into the liposomes. The loading agent may
facilitate establishing a certain condition inside the liposomes,
such as a concentration of hydrogen ions for example. The loading
agent may facilitate chemical alteration of a contrast-enhancing
agent, such as facilitating the contrast-enhancing agent accepting
or donating one or more protons. The loading agent may prevent or
retard contrast-enhancing agents that enter the liposomes from
leaving the liposomes.
[0171] In one example approach, a weakly acidic contrast-enhancing
agent (pK.sub.a of from approximately 4.0 to 6.5) is loaded into
liposomes. Such an agent may be weakly amphiphatic. The weakly
acidic agent may be substantially uncharged in its protonated form.
The weakly acidic agent may be substantially negatively charged in
its unprotonated form. Generally, such weakly acidic agents may
have one or more free carboxyl groups. Such free carboxyl groups
may be ionizable in that they may donate a proton. Example weakly
acidic contrast-enhancing agents may include acetrizoate,
diatrizoate, iodamide, ioglicate, iothalamate, ioxithalamate,
metrizoate, ioxaglate, and others
[0172] In one example of this approach, liposomes can be formed in
the presence of calcium acetate (e.g., (CH.sub.3COO).sub.2Ca). The
calcium acetate may be a loading agent. Calcium acetate is present
inside the liposomes and in the external solution. The calcium
acetate may then be removed from the phase exterior to the
liposomes, by dilution for example. Calcium acetate inside the
liposomes may dissociate into calcium ion and acetate ions. The
acetate ions may combine with water inside the liposomes to yield
acetic acid and hydroxide ion. Dilution of the solution external to
the liposomes may cause acetic acid inside the liposomes to diffuse
out of the liposomes, into the external solution, leaving hydroxide
ions inside the liposomes. This may create a pH gradient in which
the interior of the liposomes are more basic than the exterior of
the liposomes. Addition of a weakly acidic contrast-enhancing agent
to an exterior phase at a pH where a significant amount of the
weakly acidic contrast-enhancing agent is protonated and uncharged
may result in the contrast-enhancing agent moving into the interior
of the liposomes. Such movement may be due to an outside-to-inside
concentration gradient of the agent. Such movement may be due to
forces favoring osmolar equilibrium as ammonia moves out of the
liposomes. Such movement may be due to other or additional forces
or combinations of such forces. When the contrast-enhancing agent
moves to the interior of the liposomes, the contrast-enhancing
agent may donate one or more protons, becoming negatively charged,
and may be retarded or prevented from moving out of the liposome.
Additions to, substitutions and variations of this approach may
exist.
[0173] In one example approach, a weakly basic contrast-enhancing
agent (pK.sub.a of from approximately 6.5 to 8.5) agent is loaded
into liposomes. Such an agent may be weakly amphiphatic. The weakly
basic agent generally is uncharged at or around neutral pH. The
weakly basic agent may be substantially uncharged in its
unprotonated form. The weakly basic agent may be substantially
positively charged in its protonated form. Generally, such weakly
basic agents may have one or more primary amine groups. Such
primary amine groups may be ionizable in that they may accept a
proton. Such weakly basic agents may be amides.
[0174] In one example of this approach, liposomes can be formed in
the presence of ammonium sulfate ((NH.sub.4)SO.sub.4). The ammonium
sulfate may be a loading agent. Ammonium sulfate is present inside
the liposomes and in the external solution. The ammonium sulfate
may then be removed from the phase exterior to the liposomes, by
dilution for example. Ammonium sulfate inside the liposomes may
dissociate into ammonium ions (NH.sub.4.sup.+) and sulfate ions
(SO.sub.4.sup.-). Ammonium ions inside the liposomes may dissociate
into ammonia and hydrogen ions. Dilution of the solution external
to the liposomes may cause ammonia inside the liposomes to diffuse
out of the liposomes, into the external solution, leaving hydrogen
ions inside the liposomes. This may create a pH gradient in which
the interior of the liposomes are more acidic than the exterior of
the liposomes. Addition of a weakly basic contrast-enhancing agent
to an exterior phase at a pH where a significant amount of the
weakly basic contrast-enhancing agent is unprotonated and uncharged
may result in the contrast-enhancing agent moving into the interior
of the liposomes. Such movement may be due to an outside-to-inside
concentration gradient of the contrast-enhancing agent. Such
movement may be due to forces favoring osmolar equilibrium as
ammonia moves out of the liposomes. Such movement may be due to
other or additional forces or combinations of such forces. When the
contrast-enhancing agent moves to the interior of the liposomes,
the contrast-enhancing agent may accept one or more protons,
becoming positively charged, and may be retarded or prevented from
moving out of the liposome. Additions to, substitutions and
variations of this approach may exist. A variety of other active or
remote loading methods may also exist.
[0175] After liposomes are made, techniques for manipulating the
liposomes can be used. For example, a preparation of liposomes made
by standard techniques may vary in size and lamellarity (i.e., wall
thickness) after it is made. Techniques like subjecting the
liposomes to a high shearing force, extrusion of the liposomes
through membranes, or sonication of the liposomes may be used
either to select liposomes of a desired size or modify the
liposomes so that they have a desired size. After manipulation of
liposomes by these methods, the size distribution of the liposomes
may be measured to ensure that liposomes of the desired size have
been obtained. Techniques like as Fraunhofer diffraction and
dynamic light scattering (DLS) may be used to measure the size
distribution of the liposomes. These techniques generally measure
an equivalent spherical diameter which, in the case of Fraunhofer
diffraction, may be the diameter of a sphere with the same light
scattering properties as the measured liposomes. In the case of
DLS, equivalent spherical diameter may be the diameter of a sphere
with the same diffusion coefficient as the measured liposomes.
Generally, the example liposomes have an average diameter of 150 nm
or less. Example preparations of liposomes may have an average
diameter of approximately 120 nm or less. Example preparations of
liposomes may have an average diameter of approximately 100 nm or
less. It will be appreciated that other sizes can be used.
[0176] In one embodiment, a nano-scale liposomal formulation
carrying over 30 mg of iohexyl per ml of liposome is formulated
using passive loading. In this formulation, the lipid composition
of the bilayer is adjusted as described below to allow this amount
of contrast-enhancing agent to be encapsulated. In one example,
using pure DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine)
of C16 chain length, with about 40 mole % cholesterol and 5 mole %
mPEG-DSPE (N-(carbonylmethoxypolyethyleneglycol
2000)-1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine) (the
polyethylene glycol-conjugated lipid that confers long circulating
properties), the encapsulation of active molecules inside the
liposomes is increased by 20% over what is possible using
hydrogenated Soy PC(HSPC), a mixture of C16 and C18 lipids, or pure
DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) of C18 chain
length. Using a formulation of 55 mole % DPPC, 40 mole %
cholesterol and 5 mole % mPEG-DSPE and an iohexyl solution of 350
mg I/ml, an overall concentration of over 30 mg I/ml is achieved,
with an average liposomal diameter of 100.6.+-.3 nm, as determined
by DLS.
[0177] In another embodiment, a liposomal formulation carrying over
80 mg of iohexyl per ml of liposome is formulated using passive
loading. In this formulation, an iohexyl solution of 350 mg I/ml is
concentrated to at least 400-450 mg I/ml and used to prepare
liposomes as described in the previous paragraph. After the
liposomes are obtained, the suspension of the liposomes is
concentrated. Using this formulation, liposome suspensions with a
concentration of over 85 mg I/ml are obtained.
Pharmaceutical Compositions and Administration to Subjects
[0178] The liposomes containing and/or associated with one or more
contrast-enhancing agents can be part of a pharmaceutical
composition suitable for administration to a subject. The
compositions generally are administered using a route that delivers
the composition to an area of interest. In one example, the
compositions of contrast-enhancing agents are administered
parenterally to the subject, such as through intravenous,
intraarterial, subcutaneous, or other route of injection.
[0179] The formulation of the particular pharmaceutical composition
generally will depend on the method by which the composition is
administered to a patient. It will be appreciated that the
pharmaceutical compositions can include salt, buffering agents,
preservatives, other vehicles and, optionally, other agents.
Compositions suitable for parenteral administration may comprise a
sterile, pyrogen-free, aqueous or oleaginous preparation which is
generally isotonic with the blood of the subject. This aqueous
preparation may be formulated according to known methods using
suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation also may be a sterile injectable
solution or suspension in a non-toxic parenterally-acceptable
diluent or solvent. Among acceptable vehicles and solvents that may
be employed are water, Ringer's solution, and isotonic sodium
chloride or other salt, dextrose, phosphate buffered saline and the
like, or combinations thereof.
[0180] The pharmaceutical compositions used may also contain
stabilizers, preservatives, buffers, antioxidants, or other
additives. In addition, sterile, fixed oils may be employed as a
solvent or suspending medium. In addition, fatty acids such as
oleic acid may be used in the preparation of injectables. Carrier
formulations suitable for the administrations may be found in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa. The pharmaceutical compositions may conveniently be presented
in unit dosage form.
[0181] Parenteral administration contemplates the use of a syringe,
catheter or similar device, which delivers the pharmaceutical
composition to a site. Delivery may result, at least initially, in
the pharmaceutical composition being systemically distributed
throughout the circulatory system of the subject.
[0182] Generally, the pharmaceutical compositions are administered
to the subject at a point in time before the imaging of the subject
is performed, although the compositions may also be administered
during the imaging. The amount of the pharmaceutical compositions
administered preferably results in increased contrast of one or
more tissues of the subject. Ultimately, the attending physician or
technician generally will decide the amount of pharmaceutical
composition to administer to the subject. Generally, the increase
in contrast can be any level above what is present without use of
the contrast-enhancing agents in the pharmaceutical compositions.
Example increases in contrast of at least about 50 HU, at least
about 100 HU or more, to one or more organ systems, including the
vasculature, may be obtained.
Applications
[0183] The compositions of liposomes containing contrast-enhancing
agents or pharmaceutical compositions thereof, when administered to
a subject, can maintain a level of contrast-enhancing agent in the
blood and/or organs of a subject that results in an increased
contrast and is detectable by X-ray imaging techniques. The
increase in contrast may be detectable for an extended period of
time. Depending on the particular application, the compositions
described herein may have half lives in the circulation of from
minutes to hours, to even days. In one example, half lives in the
circulation of from 8 to 24 hours may be obtained. In one example,
an administered composition provides an enhanced contrast that may
remain detectable at least 30 minutes after administration. In
another example, an administered composition provides an enhanced
contrast that may remain detectable at least 5 minutes after
administration. Many applications, including those in anatomic,
functional and molecular imaging may be possible. For example, use
of the compositions described herein may have applications in
cardiology, oncology, neurology and other areas.
[0184] In one embodiment, blood pool imaging can be used to detect
and, in some cases, quantify ischemia. For example, because
injection of the pharmaceutical compositions generally alters the
contrast of the entire vasculature, reduced blood flow as is
present in ischemia may be detected. A variety of types of ischemia
may be detected, including that causing ischemic bowel disease,
pulmonary embolism, and types of ischemia that produce
cardiomyopathy, and others. In other applications, aneurysms may
also be detected.
[0185] In one embodiment, the compositions described herein can be
used in cardiac imaging to detect, examine and/or assess stenosis,
and the therapy or remediation of stenosis, as occurs in
angioplasty, for example. The utility of such techniques may be
enhanced through the use of contrast-enhancing agent preparations,
such as those described herein.
[0186] In one embodiment, the compositions described herein can be
used to detect myocardial microcirculatory insufficiencies.
Myocardial microcirculation is known to display signs of
obstruction before the epicardial arteries show signs of
obstruction. Therefore, detection of obstruction in the myocardial
microcirculation may be an earlier detector of atherosclerosis in
presymptomatic, at-risk patients, than conventional methods. The
compositions described herein may facilitate detection of
obstructions in the myocardial microcirculation.
[0187] In another embodiment, the compositions described herein can
be used to detect and characterize a wide range of tumors and
cancers. These applications may be facilitated by the property of
sterically stabilized liposomes being present for extended periods
of time in the circulation and to extravasate at regions where the
vasculature is "leaky," such as in tumors, for example. The
leakiness of the vasculature in tumors may be attributed to the
high proportion of neovasculature, the result of continuing
angiogenesis as the tumor grows in size. Upon encountering such
leaky vasculature, liposomes may leave the circulation, driven with
the extravasate fluid, by hydrostatic pressure. Such liposomes
generally do not return to the circulation after extravasation
since the pressure gradient opposes such return. Such methods may
be used to detect both primary and metastatic tumors.
[0188] In other embodiments, the compositions can be used for
"staging" and/or classification of tumors. These applications may
depend on, among other things, differences in the "leakiness" of
the vasculature of a given tumor or cancer at different stages of
progression.
[0189] In one embodiment, the compositions can be used in the area
of monitoring and characterizing injury and healing of damaged
spinal cords. In a typical spinal cord injury, as occurs in an
automobile accident for example, there may also be damage to tissue
surrounding the spinal cord. It is thought that the process of
healing of the surrounding tissue may be deleterious to healing of
the spinal cord. It is thought that formation of neovasculature in
the surrounding tissue, as occurs in healing of the surrounding
tissue, may inhibit healing of the spinal cord. It is thought that
by inhibiting healing of the surrounding tissue, and the formation
of neovasculature in the surrounding tissue, the spinal cord may
heal. Subsequently, the surrounding tissue may heal. The
compositions of contrast-enhancing agents described here may be
useful for monitoring the healing and inhibition of healing of the
tissue surrounding the spinal cord.
[0190] There may be a variety of other applications for the
compositions described herein. For example, the compositions may be
used in detection and monitoring of inflammation, reperfusion
injuries, and the like.
[0191] Additionally, the liposomes which comprise the compositions
of contrast-enhancing agents can be targeted to desired cells and
tissues in the body of a subject by, for example, attaching
antibodies to the surface of the liposomes. This targeting may
result in enhanced contrast to the targeted areas of the body.
[0192] The compositions of contrast-enhancing agents may have a
relatively long residence time in the body, low extravasation,
except in those areas of the vasculature that are leaky as
described above, may be relatively nontoxic to the kidneys and may
be used to target specific areas of the body. Additionally, the
traditional osmolality related toxicity problems associated with
ionic contrast-enhancing media generally are not an issue with the
liposomal encapsulates since the high osmolality phase is interior
to the liposomes and generally is not exposed to the blood.
EXAMPLES
Example 1
Preparation of PEGylated Liposomes Containing Iohexyl
[0193] Example liposomal iohexyl formulations can be produced as
follows. Briefly, a lipid mixture (200 mM) of
1,2-Dipalmitoly-sn-glycero-3-phosphocholine (DPPC), cholesterol
(chol) and N-(carbonyl-methoxypolyethyleneglycol
2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine
(DSPE-MPEG2000), in a 55:40:5 molar ratio, was dissolved in ethanol
at 65.degree. C. The ethanol solution was then hydrated with
iohexyl (350 mg I/ml) for 1.5-2 hours. Liposomes were extruded on a
10 ml Lipex Thermoline extruder (Northern Lipids, Vancouver,
British Columbia, Canada) with 5 passes through a 0.2 .mu.m
Nucleopore membrane (Waterman Inc., Newton Mass.) and 7 passes
through a 0.1 .mu.m Nucleopore membrane (Waterman Inc., Newton
Mass.). Liposomes were then be dialyzed in a 300,000 molecular
weight cutoff (MWCO) dialysis bag against phosphate buffer saline
(PBS) overnight to remove the free iohexyl.
[0194] The size of the resulting example liposomal iohexyl
formulations can be determined by dynamic light scattering (DLS)
using a modified BI-90 goniometer, a JDS uniphase 532 nm laser,
Hamamastu photomultiplier and Brookhaven DLS Software Version 3.16.
The average diameter of the liposomal iohexyl capsules was 100.6 nm
(STD=3.0 nm), which is in nano-scale range, as determined by
DLS.
[0195] The iohexyl concentrations of example liposomal iohexyl
formulations can be determined by measuring the absorption at 245
nm using a UV-Vis spectrophotometer. Equivalent iodine
concentrations can then be calculated. In the example preparations,
different lipid hydration times (1.5 hours and 2 hours) resulted in
different iohexyl loading concentrations (30 and 34.8 mg I/ml
respectively). The 30 mg I/ml iohexyl liposomal formulation was
used in the in vitro stability tests described below, and the 34.8
mg I/ml iohexyl liposomal formulation were used in the in vivo CT
imaging experiment described below.
[0196] The osmolarity of liposomal iohexyl formulation can be
measured by, for example, Vapro.RTM. vapor pressure osmometer
(Wescor Inc.). The osmolarity of the example iohexyl formulations
ranged between 305 to 315 mmol/kg.
Example 2
In Vitro Stability of PEGylated Liposomes Containing Iohexyl
[0197] The in vitro stability of example liposomal iohexyl
formulations can be determined by measuring the leakage of iohexyl
from liposomal iohexyl formulations both in PBS at 4.degree. C. and
in plasma at 37.degree. C. In the procedure, 1 ml of an example
liposomal iohexyl formulation was placed in a 300,000 MWCO dialysis
bag and dialyzed against 250 ml PBS at 4.degree. C. At each time
point (0, 1, 2, 3, 8, 24 hours, and 3, 4, 5, 6, 8, 10, 18 days), 1
ml of the dialysate was removed for a UV absorption-based iohexyl
measurement. At least three data points were obtained at each time
point. After measurement, samples were returned to the PBS to
maintain constant volume.
[0198] To measure stability in plasma, the example liposomal
iohexyl formulations can be dialyzed against 250 ml PBS at
25.degree. C. for 1 hour to remove the free iohexyl. In these
experiments, 1 ml liposomal iohexyl formulations was placed in a
300,000 MWCO dialysis bag with 4 ml of human plasma, and dialyzed
against 250 ml PBS at 37.degree. C. (1:4 ratio was chosen). One ml
of the external phase was removed at 0, 1, 2, 3, 4, 5, 6 and 8
hours respectively, and analyzed by the UV-vis absorption. Since
plasma components also leak from the dialysis bag and have a finite
absorbance at 245 nm, a control experiment, where a PBS-plasma
mixture is dialyzed against PBS, was also performed. The absorbance
of the external phase was subtracted from that for the liposomal
iohexyl formulation experiments and the resulting absorbance traces
can be representative of the leakage of iohexyl from liposomal
iohexyl formulations. The results showed that the liposomal iohexyl
formulation was stable in PBS and in human plasma.
[0199] The example leakage curves 400 of iohexyl is shown in FIG.
4. The example liposomal iohexyl formulation (30 mg I/ml) was
dialyzed against 250 ml of PBS at 4.degree. C. At example time
points 405 of 0, 1, 2, 3, 8, 24 hours, and 3, 4, 5, 6, 8, 10 and 18
days, the dialysate was tested for the amount of iohexyl. The
example leakage curve 410 was obtained by drawing a line through
the data at each time point. The data show that the curve
stabilized after 1 hour of dialysis. Liposomal iohexyl exhibited a
leakage of 7.4% of the total encapsulated iohexyl over 8 hours, and
7.8% for 18 days by equilibrium dialysis at 4.degree. C. The shelf
life of liposomal iohexyl formulation therefore can be longer than
18 days.
[0200] The leakage curves 500 of an example iohexyl-plasma mixture
is shown in FIG. 5. Liposomal iohexyl that had previously been
dialyzed against PBS for 1 hour was used in this study to determine
the contribution of plasma to leakage of iohexyl from the
liposomes. At example time points 505 of 0, 1, 2, 3, 8, 24 hours,
and 3, 4, 5, 6, 8, 10 and 18 days, the dialysate was tested for the
amount of iohexyl. The example leakage curve 510 was obtained by
drawing a line through the data at each time point. The data show
that the curve stabilized after 3 hours, and the liposomal iohexyl
formulation exhibited a leakage of 2.3% of the total encapsulated
iohexyl for the 8 hour period, beyond the leakage observed during
storage in PBS. Together, these results indicate that the liposomal
iohexyl formulation can be about 90% encapsulated when stored for
18 days and then injected.
Example 3
In Vivo Studies Using Imaging of PEGylated Liposomes Containing
Iohexyl in a Rabbit
[0201] A female rabbit weighing 2.2 kg was anesthetized with 35
mg/kg ketamine and 5 mg/kg xylazine given intramuscularly, followed
by 2% isoflurane vapor given by face cone. After tracheal
intubation and placement of venous catheter in an ear vein, 20 mg
pentobarbital was given intravenously. The animal's lungs were
ventilated using a pressure control ventilator set to peak airway
pressure of 15 cm H.sub.2O, and 25 breaths min.sup.-1. After
transport to the CT scanner, the animal was given 0.25 mg of
pancuronium (muscle relaxant) to insure minimal motion during the
image acquisition. Supplemental pentobarbital was given every 30-60
minutes, 10-20 mg per dose. An initial volume image of the chest
and abdomen was obtained using a 4 slice Phillips Mx8000 MDCT
scanner in spiral scanning mode, (100 mAs, 120 keV) with a single
slice equivalent pitch of 1.25, and a slice collimation and
thickness of 1.3 mm. Images were reconstructed into a 512.times.512
matrix using a standard reconstruction kernel (the "B" kernel). A
0.5 second gantry rotation speed was used. During each imaging
protocol, the rabbit was held apneic with airway pressure fixed at
20 cm H.sub.2O (e.g. near total lung capacity) using an underwater
bubbler tube on the exhalation port. Next, 15 ml of 34 mg I/ml
liposomal iohexyl formulation was hand-injected followed by a
repeat volume image, then a second injection of 15 ml of liposomal
iohexyl formulation suspension was followed by a third volume
image. A total dose of 475 mg iodine per kg was given in the two
injections. Repeat volume images were then initiated at
approximately 12, 60, 90, 120, 150 and 180 minutes after the second
contrast injection. Following the last image acquisition (-3.5 hr
post injection of contrast agent), the animal was euthanized with
an overdose of pentobarbital and a final, high resolution image was
obtained with no motion artifact (with the same airway pressure and
image acquisition settings). Finally, an ultrahigh resolution scan
was obtained using an ultra sharp reconstruction kernal ("D" kernal
and a 1024.times.1024 image matrix) to evaluate anatomic detail
without the presence of cardiogenic motion.
Example 4
Image Reconstitution
[0202] Subsequent offline example reconstructions were performed
for each of the scans obtained as described in Example 3 with the
smallest field of view (5 cm.times.5 cm, 0.1 mm voxel size) for 3D
viewing of the heart. The enhanced heart chambers were visualized
by selecting appropriate settings of the volume rendering software
present on the Philips MXV workstation software (version 4.1). Once
the settings were established, the same rendering and display
settings were used for all time points. Additional structures were
segmented at various time points.
[0203] Quantitative analysis was performed by locating regions of
interest (ROI) in the aorta, heart, kidney (core and cortex),
liver, muscle and spleen. Mean Hounsfield units (HU) were
determined at each time point to enable tracking of any decay in
contrast concentration with time in each of these structures. Slice
and slice location of the ROI's were adjusted for minor variations
in anatomic configuration of the rabbit from time point to time
point.
Example 5
Time-Attenuation of PEGylated Liposomes Containing Iohexyl In
Vivo
[0204] The example image analysis described in Example 4 was
performed at regions of interest in the aorta, kidney (medulla and
cortex), liver parenchyma, back muscle, left main coronary artery,
pulmonary artery, and in the main stem bronchus (as a control
value) and plotted over time in a graph 600 (FIG. 6). Mean
attenuations (Hounsfield units) were determined at the time points
stated in Example 3 to quantify the decay in contrast with time in
each of these locations. The data show the enhancement and
maintenance of contrast over time in various regions of interest.
The average attenuation in the aorta 605, pulmonary artery 615 and
liver cortex 3.5 hours post contrast injection attenuation was 200
HU (enhancement 130 HU), and in the kidney cortex 625 the
attenuation was 75 HU (enhancement 25 HU). Attenuation in the blood
pool rose rapidly post-injection, and remained virtually constant
for the 3.5 hours of study. A slight increase in attenuation in the
liver parenchyma 620 was observed. A transient increase in the
kidney core 630 was observed, indicating early clearance with
little to no clearance later in the study. The small region of
interest placed over the left main coronary artery indicated
attenuation of 9 HU at base line and peaked at a value of 118 HU.
FIG. 7 shows 0 hour baseline 705 and peak enhanced 710 images
obtained 2 hours 18 minutes post liposomal injection at the level
of the liver. FIG. 7 also shows 0 hour baseline 715 and peak
enhanced 720 images obtained 2 hours 18 minutes post liposomal
injection at the level of the mid-heart.
[0205] These data indicate the residence time of example PEGylated
liposome formulations, which provided contrast enhancement, to be
more than 3 hours. Additionally, the data show that contrast
enhancement in muscle can be low, indicating the liposomal iohexyl
can be retained in the blood vessels and does not rapidly
extravasate. Additionally, the contrast enhancement in the liver
parenchyma indicated that clearance of the composition may
substantially be due to the liver, and not the kidneys.
Example 6
In Vivo Images of Heart After Administration of PEGylated Liposomes
Containing Iohexyl
[0206] Additionally, example images 800 (FIG. 8), 900 (FIG. 9),
1000 (FIG. 10) and 1100 (FIG. 11) of the rabbit heart were
analyzed. FIG. 8 shows volume rendered images 800 of the whole
rabbit, before 805 and 2 hours 18 minutes after injection of the
liposomal iohexyl formulation 810. Enhancement to the vasculature
815 due to the liposomes can be seen. The results show that, even
more than 2 hours after injection, the blood vessels can be visible
815 while, using the same display and rendering parameters, they
may not be visible before liposome administration. This enhancement
can persist up until the time that the animal is euthanized at more
than 3 hours after injection of the second dose of liposomes.
[0207] FIG. 9 shows volume images 900 of the rabbit heart acquired
pre-contrast 905 and at 20 minutes 910, 1 hour 15 minutes 915, 1
hour 51 minutes 920, 2 hour 38 minutes 925, and 3 hour 23 minutes
930 after administration of the liposomal iohexyl formulation. All
display and rendering parameters are identical for all images. The
anatomies of all four heart chambers can be distinctly visualized
along with the associated great vessels. Note that there may be
absence of blood pool in the upper left panel 905 and the
persistent enhanced opacity of the blood pool up to the final panel
representing 3 hours 23 minutes post injection 930. Visible
structures include: right ventricle 935 (RV); left ventricle 940
(LV); Aorta 945 (Ao); pulmonary artery 950 (PA); and the inferior
vena cava 855 (IVC). These images demonstrated sustained contrast
even 3 hours after administration of the liposomal iohexyl.
[0208] FIG. 10 shows a thick-slab rendering 1000 of the heart
obtained at ultrahigh resolution after the rabbit was euthanized
and thus cardiac motion was eliminated. Labeled structures include
the right ventricle 1005 (RV); left ventricle 1010 (LV); and aorta
1015 (Ao).
[0209] FIG. 11 shows images 1100 of the left coronary artery of a
rabbit under high magnification conditions at 3 hours after the
second injection of the liposomal iohexyl formulation. The left
panel 1105 shows a 1.3 mm thick CT slice of in vivo rabbit heart
imaged 3 hours 18 minutes after the second injection of one
embodiment of liposomal iohexyl. The right panel 1110 shows a
volume rendered view of the same data set. The left coronary artery
(shown as 1115 in 1110) was enhanced by 109 HU.
Example 7
Preparation of PEGylated Liposomes Containing Iohexyl or
Iodixanol
[0210] Example liposomal formulations were produced as follows.
Iohexyl or iodixanol solutions of approximately 350 mg I/ml were
concentrated by rotary evaporation to concentrations of
approximately 400-450 mg I/ml. The iohexyl or iodixanol solutions
were then used to prepare liposomes as described in Example 1. The
suspensions of liposomes that were obtained were then extruded
through a series of nucleopore track-etch membranes to obtain
uniformly sized 100 nm liposomes, as described in Example 1. The
liposome suspensions were then cleaned and the liposomes
concentrated approximately 2.5-fold by diafiltration using
Microkros.RTM. modules of 100,000 Dalton cutoff. Liposome
suspensions with iodine concentrations of between 85 and 100 mg
I/ml were obtained.
Example 8
In Vivo Images of Heart and Tumor in a Mouse After Administration
of PEGylated Liposomes Containing Iohexyl
[0211] Imaging of the mouse used a specially constructed micro CT
system. In this system, the animal is vertically positioned in a
rotatable cradle and a stationary X-ray source and detector are
used. In the system, there is a high flux rotating anode X-ray tube
(Philips SRO 09 50) with a dual 0.3/1.0 mm focal spot. The flux
from the system is sufficient to support exposures as short as 10
ms to limit the motion blur from the heart. A high-resolution
detector with 50.times.50 micron pixels covering an image matrix of
2048.times.2048 (Microphotonics X-ray Image Star camera, Photonics
Science, East Sussex, UK) was used over an active area input of
106.times.106 mm. A hardware feature was used that combines pixels
to a 2.times.2 array that reduced the effective detector pitch to
100 microns.
[0212] Imaging was performed using the following X-ray parameters:
typically 80 kVp, 170 mA, and 10 ms. Projections were acquired over
a circular orbit of 1900 (i.e. 1800+ fan angle) with a step angle
of 0.50 using a total of 260 projections. Each projection set took
approximately 8-10 minutes to acquire. Scanning was done with the
animal placed at a source-to-object distance (sod=400 mm), an
object-to-detector distance (odd=40 mm), and a source-to-detector
distance (sdd=440 mm), resulting in a geometric blur of the focal
spot that matched the Nyquist sample at the detector. This resulted
in measured exposure for each image set of 17.64 R.
[0213] These projection images were used to reconstruct tomograms
with a Feldkamp algorithm using Parker weighting. For this purpose,
Cobra EXXIM software package (EXXIM Computing Corp, Livermore,
Calif.) was used. Data were reconstructed as isotropic
1024.times.1024.times.1024 arrays with effective digital sampling
in the image plane of 90 microns, since the magnification factor
for the used geometry was 1.1.
[0214] All datasets were acquired with ventilatory synchronization
(on end expiration) and cardiac gating on different points of the
ECG cycle. Both temperature (36.5.+-.1.degree. C.) and heart rate
(RR=90-100 ms) were relatively stable during the imaging
studies.
[0215] To perform the studies, one-half milliliter of a liposome
suspension as described in Example 7 was injected into the tail
vein of a mouse. Imaging and image reconstruction were performed as
described above. The data indicated a stable opacification of 700
HU in the blood. The stable opacification facilitated, for example,
cardiac and respiratory gated imaging, allowing time-lapse images.
Example time-lapse coronal images 1200 of the mouse heart, taken at
10 millisecond intervals, are illustrated in FIG. 12. Enhancement
of the cardiac chambers is visible.
[0216] In another study, a liposome suspension as described in
Example 7 was injected into a nude mouse into which had been
implanted a human squamous cell carcinoma (FaDu) in the right
flank. FIG. 13 illustrates a micro CT coronal image 1300 of the
abdominal region of the mouse 4 hours after injection of the
liposome suspension. The tumor 1305 is visible in the illustrated
image as is vasculature in the tumor and graded opacification in
the tissue surrounding the vessels. Also visible is extravasation
(leakage of blood from the vessels into the tissue) of the blood in
the tumor. The positions of vessels in the tumor, and
nonvascularized portions of the tumor (in the center) were
confirmed by histological examination after necropsy. Also visible
is an inflamed lymph node (metastatic) 1310 on the left side of the
mouse.
[0217] The above descriptions have referred to the preferred
embodiments and selected alternate embodiments. Modifications and
alterations will become apparent to persons skilled in the art upon
reading and understanding the preceding detailed description. It is
intended that the embodiments described herein be construed as
including all such alterations and modifications insofar as they
come within the scope of the appended claims or the equivalence
thereof.
* * * * *