U.S. patent application number 09/245828 was filed with the patent office on 2002-05-23 for diagnostic imaging of lymph structures.
Invention is credited to MATTREY, ROBERT F..
Application Number | 20020061280 09/245828 |
Document ID | / |
Family ID | 22928244 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020061280 |
Kind Code |
A1 |
MATTREY, ROBERT F. |
May 23, 2002 |
DIAGNOSTIC IMAGING OF LYMPH STRUCTURES
Abstract
In accordance with the present invention, there are provided
methods for identifying the sentinel lymph node in a drainage field
for a tissue or organ in a subject. In select embodiments, the
invention allows for the identification of the first or sentinel
lymph node that drains the tissue or organ, particularly those
tissues associated with neoplastic or infectious diseases and
disorders, and within the pertinent lymph drainage basin. Once the
drainage basin from the tissue or organ, i.e., the sentinel lymph
node, is identified, a pre-operative or intraoperative mapping of
the affected lymphatic structure can be carried out with a contrast
agent. Identification of the first or sentinel lymph node, on the
most direct drainage pathway in the drainage field, can be
accomplished by a variety of imaging techniques, including
ultrasound, MRI, CT, nuclear and others. Moreover, once the
lymphatic structure is identified as being associated with
neoplastic or infectious diseases and disorders, the affected
lymphatic structure can be removed surgically or by a suitable
minimally invasive procedure to allow pathological analysis to be
performed to determine whether certain diseases or disorders exist,
without resort to more radical lymphadenectomy. Further, the agent
can be made to carry diagnostic or therapeutic probes to be
activated and/or delivered to the injection site or any part of the
lymphatic pathway downstream from the injection site.
Inventors: |
MATTREY, ROBERT F.; (SAN
DIEGO, CA) |
Correspondence
Address: |
GRAY CARY WARE & FREIDENRICH
4365 EXECUTIVE DRIVE
SUITE 1600
SAN DIEGO
CA
921212189
|
Family ID: |
22928244 |
Appl. No.: |
09/245828 |
Filed: |
February 5, 1999 |
Current U.S.
Class: |
424/9.52 ;
424/9.51; 600/441; 600/458 |
Current CPC
Class: |
Y10S 977/928 20130101;
A61K 49/225 20130101; Y10S 977/929 20130101; A61K 49/223
20130101 |
Class at
Publication: |
424/9.52 ;
424/9.51; 600/458; 600/441 |
International
Class: |
A61B 005/055; A61B
008/00; A61B 008/12; A61B 008/14 |
Claims
That which is claimed is:
1. A method for identifying, diagnosing, or treating one or more
lymph structures in a subject, said method comprising:
administering to said subject a diagnostically effective amount of
a particulate contrast agent, said contrast agent having a mean
particle size in the range of about 1 micron up to about 10 microns
in diameter wherein at least a portion of said contrast agent
associates with said lymph structure; and imaging said subject
whereby said lymph structure is detected.
2. A method according to claim 1, wherein said lymph structure
comprises a lymph vessel or lymph node.
3. A method according to claim 2, wherein said agent is
percutaneously administered to said subject to localize to a lymph
vessel.
4. A method according to claim 3, wherein said method further
comprises massaging the injection site or stimulating the injection
site by exercise.
5. A method according to claim 1, wherein said lymph structure is
the sentinel lymph node.
6. A method according to claim 1, wherein said particulate contrast
agent comprises a microbubble preparation having dispersed therein
a plurality of microbubbles.
7. A method according to claim 6, wherein said microbubbles
comprise a fluorocarbon gas or gas precursor.
8. A method according to claim 1, wherein said imaging comprises a
technique selected from the group consisting of X-ray imaging,
magnetic resonance (MR) imaging, computed tomography (CT) imaging,
ultrasound (US) imaging and optical imaging.
9. A method according to claim 1, wherein said imaging comprises
ultrasound imaging.
10. A method according to claim 9, wherein said ultrasound imaging
comprises harmonic ultrasound imaging.
11. A method according to claim 1, wherein said contrast agent
further comprises a dye.
12. A method according to claim 1, wherein said contrast agent
carries diagnostic probes that are released at any site from the
point of injection to any portion of the lymphatic chain.
13. A method according to claim 1, wherein said contrast agent
carries a therapeutic substance to treat or infect the target
tissue at the site of injection or anywhere along the lymphatic
pathway.
14. A method for identifying a sentinel lymph node in a subject
comprising the steps of: administering to said subject a
diagnostically effective amount of a contrast agent comprising a
plurality of microbubbles, wherein at least a portion of said
contrast agent associates with said sentinel lymph node; and
imaging said subject whereby said sentinel lymph node is
detected.
15. A method according to claim 14, wherein said microbubbles
comprise a fluorocarbon gas or gas precursor.
16. A method according to claim 14, wherein said imaging comprises
a technique selected from the group consisting of X-ray imaging,
magnetic resonance (MR) imaging, computed tomography (CT) imaging,
ultrasound (US) imaging and optical imaging.
17. A method according to claim 14, wherein said imaging comprises
ultrasound imaging.
18. A method according to claim 14, wherein said sentinel lymph
node is downstream from tissue that is neoplastic or suspected to
be neoplastic.
19. A method for identifying, diagnosing, or treating one or more
lymph structures in a subject, said method comprising:
administering to said subject a diagnostically effective amount of
microbubble contrast agent, wherein at least a portion of said
contrast agent associates with said lymph structure; and imaging
said subject whereby said lymph structure is detected.
20. A method according to claim 16, wherein said agent is modified
to promote macrophage uptake.
Description
FIELD OF THE INVENTION
[0001] In a broad aspect, the present invention is directed to
methods of imaging lymphocytic structures. More particularly, the
present invention advantageously uses contrast agents to identify
and image lymphatic ducts, sentinel nodes and/or other lymph nodes
by various means, including ultrasound and MRI.
BACKGROUND OF THE INVENTION
[0002] The lymphatic system is made of vessels or ducts that begin
in tissues and are designed to carry lymph fluid to local lymph
nodes where the fluid is filtered and processed and sent to the
next lymph node down the line until the fluid reaches the thoracic
duct where it enters the blood stream. Lymph vessels infiltrate all
tissues and organs of the body. Lymph fluid is generated from
capillaries which because of tissue motion and hydrostatic
pressure, enters the lymph vessels carrying with it local and
foreign substances and materials from the tissues. These local and
foreign molecular, micromolecular and macromolecular substances
include antigens, infectious agents, particles and cells. Lymph
nodes, the lymph "filters", consist of essentially two major
compartments: the fluid spaces, or sinuses, and the cellular
elements. There is one major sinus at the outer margin of the node
that feeds a maze of sinuses that serve to percolate the fluid
slowly towards the hilum of the node from where it is carried
downstream. The sinuses are lined by macrophages that phagocytose
materials carried by the fluid, particularly if the materials have
certain surface charges or specific shapes. The remainder of the
cellular elements in the lymph node performs the immunologic
function of the node. In this regard, the lymph nodes process fluid
by sieving and phagocytosis to remove particulate and cell
materials delivered by the lymphatic vessels, thereby cleaning it
before it is returned to the blood stream.
[0003] The local particulate materials carried to the node are
typically proteins that either escaped the capillary beds or were
produced by tissues or organs. Foreign particulates are
micromolecular (less than 1 micrometer) or macromolecular (greater
than one micrometer), and include viruses, bacteria, and injectable
suspensions such as contrast media or radiopharamaceuticals. The
particles enter the lymph vessel from the interstitium through gaps
between lymphatic endothelial cells or by transcellular
endo-exocytosis. The gaps change in caliber with physiologic or
pathologic conditions. Entry of the particle into the gap is
believed to be a hit-or-miss affair and should be weakly related to
particle size at dimensions less than the size of the gap.
[0004] On average, smaller particles (10-50 nm) are more likely to
enter than larger particles. As particles approach 1000 nm, their
uptake into lymphatics is so poor that they become ineffective.
Very large particles in the interstitial space must be carried away
by phagocytes or reduced in size by local processes. In fact over
95% of particles larger than 400 nm were found to remain at the
injection site (Oussoren et al., Pharm Res. (1997) 4(10):1479-1484
and Oussoren et al., Biochim Biophys Acta. (1997) 1328(2):261-272)
whereas 74% of particles 10 times smaller (40 nm) were absorbed.
Small particles (less than 5nm) and particles without appropriate
surface characteristics are not retained by the first lymph node
and are carried to downstream lymph nodes or directly into the
blood stream by the capillaries.
[0005] Several attempts have been made to improve retention of
diagnostic agents in the lymph node. One is to administer a viscous
substance such as ethiodol directly into the lymph vessel thereby
plugging the sinuses and hindering the forward progress of the
liquid. This technique, called direct lymphangiography, is
performed to detect tumor deposits in lymph nodes but is limited to
the few regions of the body where direct canulation of the lymph
vessel is possible. Another technique is to administer particulate
suspensions such as emulsions with specific size distributions that
are phagocytosed by macrophages (Wolf et al., U.S. Pat. No.
5,496,536 and Bergquist, et al., Sem. Nucl. Med (1983) 13: 9-19).
These agents, although phagocytosed, are not sufficiently retained
in the lymph node to halt their progress and highlight several
lymph nodes in the local chain. The third technique to promote
retention is the placement of surface active substances that
promote phagocytosis (Vera et al., J Nucl Med (1997)
38(4):530-5).
[0006] The cellular elements carried in lymph or that gain access
to lymph nodes are typically the circulating white cells or
phagocytes that are involved in the cellular defense mechanism.
Although these cells are very large (several tens of micrometers in
diameter) they gain access to the lymph space by their ability to
deform and migrate through tiny openings. These cells patrol the
extracellular space, phagocytose materials and carry such materials
into the lymph and the lymph nodes for further action by the immune
system. When cancer occurs in tissues or organs, its loose matrix
allows the dislodging of cells that gain access to the lymph space.
However, because they lack the functionality of white blood cells,
they can become trapped in the lymph node and grow. In the early
stages of cancer development in the node, the cancer remains
limited to the node. However, in time, the nodal deposit can grow
to totally replace the node or can spread downstream to the next
node. The lymph nodes that drain the tissue or organ of interest
(i.e., the cancerous tissue), called the regional nodes, and the
first node that traps the cancer is called the sentinel node.
[0007] Unfortunately, although certain patterns in the spread of
tumors are recognized, these patterns are complicated. Metastasis
of neoplastic cells does not simply result in the spread of the
neoplastic cells to the next physically nearest node. Nodes in
close physical proximity to the primary tumor are more likely to
contain the sentinel node, however, the sentinel node may be in a
more distant nodal group. This can occur due to normal anatomic
pathways that can bypass adjacent nodal clusters. Complex patterns
can also arise because tumors, current or prior infections, injury
or previous treatments can block the lymph vessels that directly
drain the tissue or organ, promoting the development of collateral
and aberrant pathways.
[0008] Lymphadenectomy is a common procedure that provides local
control and staging of breast cancer patients as well as establish
prognosis and method of treatment. The degree of involvement of
axillary lymph nodes remains the most important prognostic
indicator (Cancer 1993:71). These nodes are positive in as many as
40% of breast cancers including those cancers between 5-10mm in
size. Because of the associated morbidity with axillary dissection
in as many as 20% of patients (Coburn M C and Bland K I, Curr Opin
Oncol 1995; 7:506; Petrek JA and Blackwood M M. Curr Prob Surg
1995; 267), attempts at limiting dissection have led to the
development of sentinel node resection. The concept of the sentinel
node dissection was popularized by Morton D L (Arch Surg 1992;
127:392-99) for staging melanoma and Giuliano A E applied it to
breast cancer (Ann Surg 1994; 220:391-401). They showed that
limiting the dissection to the sentinel node can predict the status
of the remainder of the nodal system. When the sentinel node was
negative, the remainder of the nodes were negative in 126 out 127
cases. When the node was positive, it was the only positive node in
over 60% of cases (Morton D L et al., Arch Surg 1992; 127:392) and
contained five times more micrometastasis than nonsentinel nodes
(Giuliano A E et al., Ann Surg 1995; 394). So, in addition to
staging, sentinel node resection provides some therapeutic benefit,
as all micrometastases would be removed in a majority of cases.
[0009] Sentinel lymphadenectomy begins with the injection of 3 to 5
mL of isosulfan blue in the breast mass and surrounding tissue
(Giuliano A E et al., Ann Surg 1994; 220:391). Approximately 5
minutes later blunt dissection is made to locate a blue lymphatic
channel or a blue node. Although all blue nodes are removed, an
attempt is made to identify the feeding lymphatic channel, which is
then followed proximally toward the breast to ensure the
identification of the first and true sentinel node. The resected
node(s) is assessed histologically. If the sentinel node is free of
disease, dissection is terminated. If the sentinel node is not
detected or if it is positive, classic axillary dissection is
performed. This technique is much less invasive than
lymphadenectomy and is especially useful for patients with low risk
for axillary metastasis. Much in this technique bears refinement,
however, since Giuliano, the most experienced investigator,
reported that the sentinel node was detected in 58% in the first 87
cases and 78% in the next 87 cases.
[0010] The failure to identify sentinel nodes results from the fact
that they are indistinguishable from breast tissue unless colored
blue. Unfortunately, the dye has unpredictable and rapid clearance,
and, possibly, the drainage pattern varies. Moreover, as it is
water soluble, the blue dye provides a short time-window to
identify lymph nodes intraoperatively. Thus, only a few minutes are
available between operating too early (where no nodes are stained)
or too late (where too many nodes are stained). Yet, other than the
blue dye, only radiopharmaceutical agents are available that
provide images as they flow through the lymphatic chain (Uren R F
et al., J Nucl Med 1995; 36:1775; Krag D N et al., Surg Oncol 1993;
2:335). Although radiolabelled colloids have a more delayed
transit, they are invisible intraoperatively, limiting guidance;
they only provide a skin-marking option, and they contaminate the
operative field with radioactivity, decreasing the target to
background ratio and increasing the complexity of the procedure.
Further, only 3 to 4% of the colloid is entrapped, requiring larger
dosages than necessary, which flood the field and enhance all
nodes, decreasing sentinel node specificity (Ege G N and Warbick A,
Br J Radiol 1979; 52:124; Kapteijn B A E et al., J Nucl Med 1995;
35:222P). At present, most procedures utilize both blue dye and
radiolabeled colloids to gain sensitivity. The consequences of
failed or difficult sentinel node detection is extensive
exploration analogous to or potentially more extensive that the
standard lymphadenectomy.
[0011] The technique proposed by Wolf et al. (U.S. Pat. No.
5,496,536), indirect lymphography, delivers particle to the tissue
or organ to image the draining lymph nodes. However, this technique
preferably employs contrast agents that are less than 1 micrometer
in diameter. Moreover, it has been reported that such agents are
most effective when there is an interval of time between the
administration of the contrast agent and imaging for sufficient
accumulation of the contrast agent in the nodes (Saunders H B et
al., Radiology. 1993; 189(P):295). As such, they may complicate
normal procedures in the operating room.
[0012] Another approach that has been utilized to image the lymph
nodes is intravenous administration of suspensions of ultrasmall
particles that leak through normal capillaries and become trapped
in the lymph nodes by macrophages. Although these agents can
visualize nodes, they do not provide any specificity as they
visualize all nodes that drain or do not drain a tissue of
interest. Therefore, they do not allow the recognition of the
sentinel node in the regional nodal chain. Although it is possible
to enhance the blood within nodes with intravascular agents to
promote the visualization of their intravascular space (not
lymphatic space) and aid in the recognition of benign from
malignant nodes (Maurer et al., Invest. Radiol. 1997; 32:441), this
technique enhances the blood pool in all small lesions whether or
not they are in nodes. Moreover, this technique does not permit the
recognition of the sentinel node in regional nodes as it enhances
the vascular space of all nodes.
[0013] Accordingly, there is a need, particularly in oncology, for
a method that clearly delineates lymphatic structures employing
contrast agents suitable to identify the sentinel lymph node. It
would be desirable to further reduce the morbidity of sentinel
lymph node biopsy. This would be possible if suitable methodology
were available to allow the sentinel lymph node to be identified
and removed with as little effect as possible to the surrounding
tissue structure. It would be especially desirable to be able to
locally administer suitable contrast agents percutaneously and
recognize the sentinel node immediately, pre-operatively or
intraoperatively, to direct the surgeon to the sentinel lymph node
without significant exploration.
SUMMARY OF INVENTION
[0014] In a broad aspect, the present invention provides methods
and systems for identifying and imaging lymphatic structures in a
subject. To this end, the present invention preferably comprises
the administration of contrast agents that are preferentially taken
up by the lymphatic system and allow for enhanced visualization of
the afferent lymphatics and regional lymph nodes. Unlike prior art
imaging agents that have been used for such imaging purposes,
contrast agents compatible with the present invention are not
limited to relatively small particle sizes. Rather, preferred
contrast agents of the present invention comprise microbubble
preparations having mean bubble sizes on the order of from about
one micron to about ten microns. Surprisingly, it has been found
that such agents are readily taken up by the lymphatic structures
in relatively short order and may easily be detected and imaged
using common imaging modalities such as ultrasound or magnetic
resonance. Particularly preferred embodiments of the invention will
comprise the use of microbubble preparations incorporating an
insoluble gas, such as a fluorocarbon, that provides for relatively
long half-lives. Those skilled in the art will appreciate that such
compositions will preferably be administered interstitially in the
region of the lymph node to be imaged.
[0015] Significantly, the present invention allows for the
identification of the first or sentinel lymph node that drains the
tissue or organ of interest, particularly those tissues associated
with neoplastic or infectious diseases and disorders, within the
pertinent lymph structure. Once the drainage basin from the tissue
or organ, i.e., the sentinel lymph node, is identified, a
pre-operative or intraoperative mapping of the affected lymphatic
structure can be carried out with a contrast agent. Identification
of the first or sentinel lymph node, on the most direct drainage
pathway in the drainage field, can be accomplished by a variety of
imaging techniques, including ultrasound, MRI, CT, nuclear and
others. Moreover, once the lymphatic structure is identified as
being associated with neoplastic or infectious diseases and
disorders, the affected lymphatic structure can be removed
surgically or by a suitable minimally invasive procedure to allow
pathological analysis to be performed to determine whether certain
diseases or disorders exist, without resort to more radical
lymphadenectomy.
[0016] In yet other aspects the present invention allows for the
detection or diagnosis of irregularities associated with one or
more components of a lymphatic structure. Further, the contrast
agent may be associated with therapeutic or additional diagnostic
agents that may be activated and/or delivered to any part of the
lymphatic pathway downstream from the injection site. For example,
the microbubble preparation could be mixed or associated with an
MRI imaging agent such as gadolinium or with a visible dye. The
resulting composition could then be imaged using more than one
modality (i.e. MRI and ultrasound) or, in the case of dye, could be
visually detected during an operative procedure.
[0017] Other objects, features and advantages of the present
invention will be apparent to those skilled in the art from a
consideration of the following detailed description of preferred
exemplary embodiments thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0018] While the present invention may be embodied in many
different forms, disclosed herein are specific illustrative
embodiments thereof that exemplify the principles of the invention.
It should be emphasized that the present invention is not limited
to the specific embodiments illustrated.
[0019] As discussed above, the present invention provides improved
methods and systems for imaging lymphatic structures in a subject.
In this respect, it has suprisingly been found that selected
contrast agents, particularly microbubble contrast agents, are
especially effective in providing enhanced images of lymphatic
structures. Unlike prior art contrast agents and methods that were
inconvenient or required relatively small particle sizes, the
present invention allows for the use of relatively inexpensive
contrast agents having larger particle sizes. Preferably the
selected contrast agent will incorporate a plurality of
microbubbles comprising a fluorocarbon gas or gas precusor.
[0020] Accordingly, in selected embodiments the present invention
provides methods for identifying, diagnosing, or treating one or
more lymph structures in a subject, said methods comprising:
[0021] administering to said subject a diagnostically effective
amount of a particulate contrast agent, said contrast agent
typically having a mean particle size in the range of about 1
micron up to about 10 microns in diameter wherein at least a
portion of said contrast agent associates with said lymph
structure; and
[0022] imaging said subject whereby said lymph structure is
detected.
[0023] As indicated above, particularly preferred contrast agents
for use in the present invention comprise microbubble contrast
agents. It will be appreciated that microbubble contrast agents
typically comprise a liquid preparation incorporating a plurality
of microbubbles. Preferably, microbubble preparations compatible
with the present invention will be relatively stable and, in
particularly preferred embodiments, will incorporate microbubbles
comprising a fluorocarbon gas, vapor or gaseous precursor.
[0024] In this regard, the present invention comprises methods for
identifying, diagnosing, or treating one or more lymph structures
in a subject, said methods comprising:
[0025] administering to said subject a diagnostically effective
amount of a microbubble contrast agent, wherein at least a portion
of said contrast agent associates with said lymph structure;
and
[0026] imaging said subject whereby said lymph structure is
detected.
[0027] Those skilled in the art will appreciate that the lymph
structure preferably comprises a sentinel lymph node. In other
embodiments the lymph structure will preferably be downstream from
tissue that is neoplastic or suspected of being neoplastic.
[0028] Accordingly, another aspect of the present invention
provides methods for identifying the sentinel lymph node in a
drainage field for a tissue or organ in a subject. The invention is
useful for the identification and localization of the sentinel
lymph node, the diagnosis of whether the lymph node is normal or
affected by disease, which can subsequently lead to resection,
treatment and or prevention of lymphatic diseases and disorders
(e.g., lymphatic metastases of cancers, lymphomas, lymph node
hyperplasia etc.); for differentiation of the above diagnoses; for
studying the structure and function of the lymphatic system; for
immunomodulation or immunization; for ecological monitoring; and
the like.
[0029] Thus, a further embodiment of the invention comprises
methods for identifying a sentinel lymph node in a subject, said
method comprising:
[0030] administering to said subject a diagnostically effective
amount of a contrast agent comprising a plurality of microbubbles,
wherein at least a portion of said contrast agent associates with
said sentinel lymph node; and
[0031] imaging said subject whereby said sentinel lymph node is
detected.
[0032] It will be appreciated that the identification of the first
or sentinel lymph node, on the most direct drainage pathway in the
drainage field, can be accomplished by a variety of imaging
techniques, including ultrasound, MRI, CT, nuclear and others.
Moreover, once the lymphatic structure is identified as being
associated with neoplastic or infectious diseases and disorders,
the affected lymphatic structure can be removed surgically or by a
suitable minimally invasive procedure to allow pathological
analysis to be performed to determine whether certain diseases or
disorders exist, without resort to more radical
lymphadenectomy.
Contrast Agents
[0033] As described herein, the invention may be performed with a
wide variety of contrast agents using a number of detection
procedures. The present invention utilizes contrast agents that
have particular characteristics that facilitate their uptake into
lymphatics and retention in the regional lymph nodes or the
prevention of the their propagation downstream. As used herein, the
terms "contrast agent" and "imaging agent" are used interchangeably
and broadly encompass any compound, composition or formulation that
enhances, contrasts or improves the visualization or detection of
an object or system in any way. Exemplary contrast agents include,
for example, contrast agents, further described herein, for use in
connection with ultrasound, magnetic resonance imaging, X-ray,
x-ray computed tomography, nuclear imaging techniques, and the
like. Preferred contrast agents will comprise particulate
preparations incorporating solid, liquid, or gas particulates. For
example, the contrast agent may comprise a suspension or emulsion.
Particularly preferred imaging agents comprise microbubble
preparations typically used as ultrasound contrast agents. Those
skilled in the art will recognize that a large number of suitable
contrast or imaging agents have been described in the literature or
are commercially available. As long as they meet the criteria set
forth herein, any of these agents are contemplated for use in the
disclosed invention.
[0034] Contrast agents suitable for imaging by one or more imaging
techniques in accordance with the present invention, are preferably
in particulate form and are adapted to be preferentially taken up
by the lymphatic system upon percutaneous administration. These
contrast agents can be radiopaque materials, MRI imaging agents,
ultrasound imaging agents, radiopharmaceuticals and any other
contrast agent suitable for detection by a device that images an
animal body. They are preferably nontoxic, and generally should
have an average particle size between about 1 micron and about 20
microns. Of course, in any given particulate system, particle sizes
usually form a distribution. Typically, the mean particle sizes
fall within the range of about 1-10 microns, preferably within the
range of about 2-8 microns.
[0035] As used herein, the term "micron" refers to a unit of
measure of one one-thousandth of a millimeter or one .mu.m.
[0036] In preferred embodiments of the present invention, contrast
agents will be elastic or deformable or capable of being elastic
under certain conditions, e.g., temperature, pH, light, application
of energy (e.g., ultrasound, and the like), and the like. As
employed herein, the term "elastic" refers to the ability of
contrast agents employed in the present invention to be non-rigid
and/or to alter their shape, for example, to pass through an
opening that is smaller than the diameter of the contrast
agent.
[0037] In contrast to the elastic contrast agents described above,
it may be desirable, in certain circumstances, to formulate
contrast agents from substantially impermeable materials such as
polymer materials, including, for example, polymethyl methacrylate.
This would generally result in the formation of contrast agents
which may be substantially impermeable and relatively inelastic and
even brittle. In embodiments involving diagnostic imaging, for
example, ultrasound, contrast media which comprise such vesicles
with limited elasticity would generally not provide the desirable
reflectivity nor the ability to adequately gain access to the lumen
of the lymphatic vessel. However, by increasing the power output on
ultrasound or by applying another energy source, the vesicle can be
made to rupture, thereby causing acoustic emissions which can be
detected by an ultrasound transducer, can be deformed in such a way
as to gain access to the lumen of the lymph vessel, or
alternatively, release its contents that can then gain access to
the lumen of the lymph vessel. Further, the contents of the vesicle
can have diagnostic or therapeutic function that can be released in
an extravascular site of interest such as a tumor, a lymph node, or
any normal or abnormal region of a patient.
[0038] In accordance with preferred embodiments of the present
invention, the contrast agents utilized in the present invention
are useful for diagnostic imaging, including, for example, X-ray,
x-ray computed tomography (CT) imaging, including CT angiography
(CTA) imaging, magnetic resonance (MR) imaging, magnetic resonance
angiography (MRA), nuclear medicine, ultrasound (US) imaging,
optical imaging, elastography, infrared imaging, microwave imaging,
and the like.
[0039] For x-ray or computed tomography imaging, the contrast agent
should have a different electron density than surrounding tissues
(either more or less electron density) to render it visible with
these techniques. With respect to contrast agents for CT, it is
generally sought to employ agents that will increase electron
density in certain areas of a region of the body (positive contrast
agents). Suitable electron density is achieved, for example, in
compounds with bromine, flourine or iodine moieties, and in
materials comprising or including radiopaque metal atoms. With
respect to contrast agents for CT, is also sought to employ agents
that will decrease electron density in certain areas of a region of
the body (negative contrast agents). Suitable agents can be fat or
air or any substance with lower electron density than water.
[0040] For MRI, contrast agents which are suitable for use in
accordance with invention methods should have adequate nuclear or
relaxation properties or susceptibility effect for imaging that are
different from the corresponding properties of the tissue being
imaged. Either an imageable nucleus (such as .sup.19F),
radionuclides, diamagnetic, paramagnetic, ferromagnetic,
superparamagnetic substances, and the like, can be used with
appropriate MRI equipment.
[0041] Ultrasound and x-ray imaging, including the use of digital
subtraction techniques, may also be utilized according to another
embodiment of the present invention. Ultrasound contrast agents can
be selected on the basis of density or acoustical properties.
Preferably, the contrast agent is echogenic. As employed herein,
the term "echogenic" refers to a contrast agent that may be capable
of reflecting or emitting sound waves. Echogenic contrast agents
may be particularly useful to alter, for example, the acoustic
properties of a lymph tissue, organ or region of a patient,
preferably the sentinel lymph node, thereby resulting in improved
contrast in diagnostic imaging techniques, such as those described
herein. As previously alluded to, microbubble preparations are
particularly compatible with ultrasound imaging.
[0042] In this regard, any contrast agent that can be selectively
rendered more detectable within a lymphatic structure by exposure
to a particular treatment or energy may be employed in the present
invention. Moreover, the selected contrast agent may be imaged
using other than traditional detection procedures. For example,
contrast agents that are traditionally used in conjunction with
ultrasound detection methods (i.e., microbubble agents) may, in the
context of the present invention, be used with magnetic resonance
visualization methods. That is, the contrast agent may be imaged
using ultrasonic energy and the infusion of intact, detectable
contrast agents monitored through magnetic resonance. While any
imaging agent possessing the requisite properties may be employed,
preferred embodiments of the invention comprise the use of
ultrasound contrast agents and/or magnetic resonance imaging.
agents. For example, a preferred contrast agent may comprise a
microbubble preparation wherein the microbubbles are associated
with an MRI agent such as a paramagnetic material.
[0043] As indicated above, a number of contrast agents may be
employed in the practice of the present invention, including
droplets, bubbles, microbubbles, polymer particles, microspheres,
microballoons, microcapsules, suspensions, emulsions, and the like.
As discussed herein, particularly preferred embodiments of the
present invention comprise the use of microbubble-based imaging
agents. Accordingly, microbubble formulations suitable for use in
the methods of the present invention are described in some detail
below. However, it will be appreciated that the present methods are
not limited, in any way, to the use of these particular microbubble
formulations.
[0044] In one preferred embodiment, the contrast agents are adapted
to return a signal at a frequency different from the frequency of
the ultrasonic pulse emitted by the transducer, such as a harmonic
frequency of the ultrasonic pulse. That is, the contrast agents are
adapted for harmonic imaging such as is described in U.S. Pat. No.
5,540,909 which is incorporated herein by reference in its
entirety. Yet, it must be emphasized that, while the present
invention may often be described in the context of the preferred
embodiments, the invention is not limited to the use of such
formulations.
[0045] Contrast agents, and particularly microbubble contrast
agents, contemplated for use in the present invention may be
formulated, for example, from lipids, polymeric materials,
proteins, and the like. The lipids, polymers, and/or proteins may
be natural, synthetic or semi-synthetic. It should be noted that
for medical uses, the selected contrast agent should be
biocompatible or not be physiologically deleterious or injurious to
biological functions, and which will not result in any degree of
unacceptable toxicity, including allergenic responses and disease
states. For example, ultimately, components comprising the contrast
agent may decay wherein the components will preferably be released
into the blood either as dissolved particles or gas or as submicron
droplets of condensed liquid. It will be understood that gases will
primarily be removed from the body through lung respiration or
through a combination of respiration and other metabolic pathways
including the reticuloendothelial system.
[0046] As previously indicated, microbubbles contemplated for use
in the practice of the present invention are those that are free,
or are surrounded or comprise an elastic or rigid shell, wall, or
membrane. As employed herein, the term "shell" (used
interchangeably with the terms, "wall" or "membrane") is used to
refer to the material surrounding or defining a microbubble,
whether it be a surfactant, another film forming liquid, a solid or
semisolid outer layer, and the like. The walls may be concentric or
otherwise. In a presently preferred microbubble, the shell is
formulated from lipids (i.e., phospholipids), natural and synthetic
polymeric materials, proteinaceous materials, carbohydrates,
sacchirides, and the like. In addition, in presently preferred
microbubbles, the shells may be in the form of a monolayer or
bilayer, and the mono- or bilayer may be used to form one or more
mono- or bilayers. In the case of more than one mono- or bilayer,
the mono- or bilayers may be concentric, if desired. Preferably,
lipids may be used to form a unilamellar microbubble (comprised of
one monolayer or bilayer), an oligolamellar microbubble (comprised
of about two or about three monolayers or bilayers) or a
multilamellar microbubble (comprised of more than about three
monolayers or bilayers). Similarly, the microbubbles prepared from
polymers or proteins may comprise one or more walls or membranes,
concentric or otherwise. The walls or membranes of microbubbles
prepared from lipids, polymers, or proteins may be substantially
solid (uniform), or they may be porous or semi-porous.
[0047] When referring to the pressure of dissolved gas in a liquid,
the more familiar term "pressure" may be used interchangeably with
"tension.""Gas osmotic pressure" is more fully defined below, but
in a simple approximation can be thought of as the difference
between the partial pressure of a gas inside a contrast agent and
the pressure or tension of that gas (either in a gas phase or
dissolved in a liquid phase) outside of the contrast agent, when
the membrane is permeable to the gas. More precisely, it relates to
differences in gas diffusion rates across a membrane.
[0048] It will be appreciated that the contrast agents utilized in
the present methods should have a lifetime sufficient to enable
them to persist for the time period during which images and/or
measurements are taken. In accordance with preferred embodiments of
the present invention, several ultrasound contrast agents that are
commercially available or under development may be used to provide
the desired images.
[0049] In this respect, suitable contrast agents include Imagent
(AFO150), Alliance Pharmaceutical Corp., San Diego, Calif.; AI-700,
Acusphere, Inc., Cambridge, Mass., AIP201 by UofV; Albunex and
Optison (FS069), both by Molecular Biosystems, Inc., San Diego,
Calif.; Echogen and QW7437 both by Sonus Pharmaceuticals Bothell,
Wash.; Levovist (SH/TA-508), Echovist and Sonovist (SHU563), all by
Schering AG, Berlin, Germany; Aerosomes-DMP115 and MRX115, by ImaRx
Pharmaceuticals, Tucson, Ariz.; BR1 and BR14, both by Bracco
International B.V., Amsterdam, NL; Quantison and Quantison Depot,
both by Andaris, Ltd. Nottingham, GB; and NC100100, Nycomed Imaging
AS, Oslo, Norway, and the like. Contrast agents and methods of
forming contrast agents usable in the present invention are
disclosed in U.S. Pat. Nos. 4,957,656, 5,141,738, 4,657,756,
5,558,094, 5,393,524, 5,558,854, 5,573,751, 5,558,853, 5,595,723,
5,558,855, 5,409,688, 5,567,413, 5,558,856, 5,556,610, 5,578,292,
5,271,928, 5,531,980 5,562,893, 5,837.221, 4,572,203, 4,844,882,
5,552,133, 5,536,489 and 5,558,092, each of which is incorporated
herein by reference in its entirety. International applications WO
96/40282, WO 95/01187 and WO 96/40278 further describe compatible
contrast agent preparations and are also incorporated herein.
Additional suitable contrast agents, as well as their compatible
characteristics are described, for example, in Calliada et al., Eur
J Radio (1998) Suppl 2:S157-160, the disclosures of which are
hereby incorporated herein by reference in their entirety.
[0050] The microbubble contrast agents employed in the methods of
the present invention preferably contain a gas and/or vapor (or
precursor thereof. When referring to a "gas," it will be understood
that mixtures of gases together having the requisite properties
fall within the definition, except where the context otherwise
requires. A "vapor," on the other hand, is the gaseous phase of a
material that is a liquid at ambient temperature and pressure, but
that has an appreciable vapor pressure at the relevant temperature,
e.g., body temperature. The gas and/or vapor may provide the
contrast agent with enhanced imaging capabilities, such as
reflectivity of ultrasound, particularly in connection with
microbubble compositions in which the gas is entrapped within the
microbubble. Those skilled in the art will appreciate that the term
"gas" as used herein shall be held to mean gases, gas mixtures or
vapors unless otherwise specified.
[0051] As suggested above, fluorocarbon or fluorinated gases or
vapors are particularly preferred as osmotic or stabilizing agents
for microbubble preparations. The term "fluorocarbon" is used
herein in its broadest sense and includes fully fluorinated
compounds (perfluorocarbons) as well as partially fluorinated
hydrocarbon materials (fluorochemicals or fluorinated compounds),
including unsubstituted chains or those substituted with another
halogen such as Br, Cl, or I or another substituent, such as O, OH,
S, NO, and the like. For example, sulfur hexafluoride would be
considered a fluorocarbon gas for the purposes of the present
invention and may be used to provide stabilized microbubble
preparations in accordance with the teachings herein. In selected
embodiments, microbubbles useful with the present invention may
contain materials that can change state from a gas to a liquid or
solid at body temperature, (generally from about 35.5.degree. C. to
about 40.degree. C.), and at useful pressures (generally about 1-2
atm). Similarly, fluorocarbons or other compounds that are not
gases at room or body temperature can be used, provided that they
have sufficient vapor pressure, preferably at least about 10-20
Torr, and more preferably 30, 40, 50 or 100 Torr at body
temperature, or more preferably at least about 150 or 200 Torr.
[0052] In particular, substances possessing suitable solubility
and/or vapor pressure criteria for the formation and use of
microbubbles in accordance with the invention include
fluoroheptanes, fluorocycloheptanes, fluoromethylcycloheptanes,
fluorohexanes, fluorocyclohexanes, fluoropentanes,
fluorocyclopentanes, fluoromethylcyclopentanes,
fluorodimethylcyclopentanes, fluoromethylcyclobutanes,
fluorodimethylcyclobutanes, fluorotrimethylcyclobutanes,
fluorobutanes, fluorocyclobutanse, fluoropropanes, fluoroethers,
fluoropolyethers, fluorotriethylamines, and the like. Particularly
preferred embodiments of the present invention employ microbubbles
comprising perfluorohexanes, perfluoropentanes, perfluorobutanes,
perfluoropropanes, sulfur hexafluoride, and the like. One
particular preferable class of compatible compounds comprises
flouroethers. Other useful gases or vapors comprise Freon 113,
methylene chloride, Freon 12 (dichlorodifluoromethane), Freon 11
(trichloromonofluoromethane), butanes, pentanes, hexanes, propane,
methane, ethane, and the like.
[0053] Whichever gases or osmotic agents are ultimately selected,
it will be appreciated that microbubbles comprising mixtures of
gases and/or vapors may be used with the disclosed methods as can
microbubbles comprising pure gases. For example, both mixtures of
fluorocarbon gases (i.e. fluorohexane and fluorobutane) and
fluorocarbon gases mixed with nonfluorocarbon compounds (i.e.
fluoropentane and nitrogen) can form particularly stable
microbubbles. It will further be appreciated that several types of
gas or vapor are compatible with either microbubble configuration,
i.e. they are useful as a component of a mixture or in a pure
state.
[0054] In this regard, mixtures of gases and/or vapors may be used
to form particularly long lasting contrast agents. This is because
contrast agents of a primary modifier gas such as air or nitrogen
(including fluorocarbon gases) saturated with a selected
perfluorocarbon osmotic agent can grow rather than shrink when
exposed to air dissolved in a liquid due to the osmotic pressure
exerted by the perfluorocarbon gas or vapor. Preferably, the
osmotic agent is relatively impermeable to the contrast agent film
and thus remains inside the contrast agent. Air or other gases
inside the contrast agent are diluted by the perfluorocarbon, which
acts to slow the air diffusion flux out of the contrast agent. This
gas osmotic pressure is proportional to the concentration gradient
of the perfluorocarbon vapor across the contrast agent film, the
concentration of air surrounding the contrast agent, and the ratio
of the contrast agent film permeability to air and to
perfluorocarbon.
[0055] Further, as disclosed in U.S. Pat. No. 5,315,997, gases and
perfluorocarbon vapors have magnetic susceptibilities substantially
different from tissues and blood. Therefore, microbubble contrast
agents comprising fluorinated compounds will cause changes in the
local magnetic fields present in tissues and blood during MRI. As
such, the aforementioned microbubble contrast agents may also be
used for magnetic resonance visualization. Other exemplary MRI
agents, which may be used with the present invention comprise
paramagnetic and supraparamagnetic macromolecular compounds or
particulates that may be associated with microbubbles (i.e. on the
membrane) or mixed with a microbubble contrast agent. Examples of
such imaging agents are to be found in U.S. Pat. Nos. 4,675,173 and
4,849,210, each of which is incorporated herein by reference. With
respect to paramagnetic compounds, gadolinium
diethylenetriaminepentaacetic acid (Gd-DTPA), and transition metal
ions of iron and manganese may be used in conjunction with the
disclosed invention, particularly when attached to a larger
molecule such as human serum albumin, dextran or polylysine.
Regarding supraparamagnetic imaging agents, those comprising iron
oxides may be used to provide perfusion data with the disclosed
methods.
[0056] It will be appreciated that those of ordinary skill in the
art can readily determine other compounds that would perform
suitably that do not meet both the solubility and vapor pressure
criteria, described above. Rather, it will be understood that
certain compounds can be considered outside the preferred range in
either solubility or vapor pressure, if such compounds compensate
for the aberration in the other category and provide a superior
insolubility in water or high vapor pressure or affinity to
dissolve in the surfactant used.
[0057] It will further be understood that other components can be
associated with useful contrast agent formulations. For example,
osmotic agents and stabilizers (described herein), chelators,
surfactants, buffers, viscosity modulators, air solubility
modifiers, salts, sugars, and the like, can be added to fine tune
the contrast agent suspensions for maximum life and contrast
enhancement effectiveness. Such considerations as sterility,
isotonicity, and biocompatibility may govern the use of such
conventional additives to injectable compositions. The use of such
agents will be understood to those of ordinary skill in the art and
the specific quantities, ratios, and types of agents can be
determined empirically without undue experimentation. Such
components associated with useful contrast agents can be in
addition to, or instead of the materials (lipids, polymer, protein,
gas, vapor, liquid, and the like, described herein) which comprise
the contrast agent. In addition, these components can be associated
inside, on or outside the contrast agent. Thus, with respect to
microbubbles, such components can be within the void, part of the
shell or membrane, or part of the solution which surrounds the
microbubble.
[0058] In a preferred embodiment of the present invention, the
lymphatic tissues retain contrast agents of the invention for an
extended period of time. Long retention time may allow the use of
these contrast agents for therapy of the lymphatic tissues,
particularly when using the diagnostic and biologically active or
therapeutic substances of this invention releasing a therapeutic
agent in the site of accumulation. Therefore, if desired, the
contrast agent described herein may further comprise a targeting
agent to alter the biodistribution of an agent, to increase its
concentration in a desirable lymphatic structure, and/or to
decrease its concentration in non-target sites, thereby suppressing
side effects and/or diffusion. To provide accumulation of a
contrast agent in a lymph site, molecules possessing a high
affinity to target tissue are preferably used as target agents.
Antibodies or their fragments, and receptor ligands (e.g.,
hormones, cytokines or their analogues) are common examples of high
affinity molecules employable as target agents.
[0059] In accordance with a preferred embodiment of the present
invention, the contrast agent comprises targeting agents or
modifications to increase uptake and accretion of the contrast
agent by the first lymph node (sentinel lymph node) where they will
be removed by endothelial or phagocytic cells. For example, target
agents which can be included in useful contrast agents include a
radiocolloid-type agent which is scavenged by the
reticuloendothelial system (RES) and accretes the contrast agent in
lymphatic structures beyond the sentinel lymph node, e.g.,
chemically modifying the contrast agent to promote macrophage
uptake by attachment of a macrophage receptor substrate. The
targeting agent may also be a radiolabeled lipid or agent such as
gallium citrate, labeled bleomycin, or the like, which accretes in
lymphatics. In preferred embodiments, these agents will be
associated with a microbubble agent. In addition, and
advantageously for certain cases, it may be a new type of imaging
agent developed especially for this invention, namely, a
radiolabeled antibody which specifically binds to normal lymphatic
tissues or cells, but not to tumors or lesions located therein or
proximal to and draining into the structure, so that it is also
diffusely distributed in the lymph nodes and reveals the internal
structure thereof.
[0060] Various combinations of the materials comprising or
surrounding the contrast agents may be used to modify the
relaxation behavior of the medium or to alter properties such as
the viscosity, osmolarity, stability, sterility, isotonicity,
biocompatibility, imageability, brightness, and the like. For
example, the gas and vapor filled contrast agents used in the
present invention may be controlled according to size, solubility
and heat stability by choosing from among the various additional or
auxiliary stabilizing materials described herein. These materials
can affect these parameters of the contrast agents, especially
contrast agents formulated from lipids, not only by their physical
interaction with the membranes, but also by their ability to modify
the viscosity and surface tension of the surface of the gas and
gaseous precursor filled vesicle.
[0061] Accordingly, the gas and gaseous precursor filled contrast
agents contemplated for use in the present invention may be
favorably modified and further stabilized, for example, by the
addition of one or more of a wide variety of (a) viscosity
modifiers, including, for example, carbohydrates and their
phosphorylated and sulfonated derivatives; polyethers, preferably
with molecular weight ranges between about 400 and about 100,000;
di- and trihydroxy alkanes and their polymers, preferably with
molecular weight ranges between about 200 and about 50,000; and the
like; (b) emulsifying and/or solubilizing agents including, for
example, acacia, cholesterol, diethanolamine, glyceryl
monostearate, lanolin alcohols, lecithin, monoand di-glycerides,
mono-ethanolamine, oleic acid, oleyl alcohol, poloxamer, for
example, poloxamer 188, poloxamer 184, and poloxamer 181,
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, emulsifying wax, and the like; (c)
suspending and/or viscosity-increasing agents, including, for
example, acacia, agar, alginic acid, aluminum mono-stearate,
bentonite, magma, carbomer 934 P, carboxymethylcellulose, calcium
and sodium (and/or sodium 12), carrageenan, cellulose, dextran,
gelatin, guar gum, locust bean gum, veegum, hydroxyethyl cellulose,
hydroxypropyl methylcellulose, magnesium-aluminum-silicate,
Zeolites(r), methylcellulose, pectin, polyethylene oxide, povidone,
propylene glycol alginate, silicon dioxide, sodium alginate,
tragacanth, xanthan gum, alpha -d-gluconolactone, glycerol,
mannitol, and the like; (d) synthetic suspending agents, such as
polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyvinyl
alcohol (PVA), polypropylene glycol (PPG), polysorbate, and the
like; ( e) tonicity raising agents which stabilize and add
tonicity, including, for example, sorbitol, mannitol, trehalose,
sucrose, propylene glycol, glycerol, and the like; and other
similar materials.
[0062] As previously indicated, the contrast agent can be used
alone, or in combination with additional diagnostic, therapeutic or
other agents. Such other agents include excipients such as coloring
materials. As employed herein, the term "diagnostic agents" refers
to detectable agents, in addition to or other than the contrast
agents described herein, useful in diagnostic methods, e.g., MR
agents, CT agents, ultrasound agents, optical imaging agents, dyes,
and the like. As employed herein, the term "bioactive agents"
refers to biologically active agents, e.g., therapeutic compounds.
In this respect it will be appreciated that compatible bioactive
agents may be selected from the group consisting of analgesics,
antibiotics, leukotriene inhibitors or antagonists, antihistamines,
antiinflammatories, antineoplastics, anticholinergics, anesthetics,
enzymes, steroids, genetic material, viral vectors, antisense
agents, proteins, peptides, and the like, and combinations
thereof.
[0063] For optical imaging, optically active gases, such as argon
or neon, may be incorporated in the present compositions. In
addition, optically active materials, for example, fluorescent
materials, including porphyrin derivatives, may also be used.
Elastography is an imaging technique which generally employs much
lower frequency sound, for example, about 60 KHz, as compared to
ultrasound (which can involve frequencies of over about 1 MHz). In
elastography, the sound energy is generally applied to the tissue
and the elasticity of the tissue may then be determined. The lipid
based vesicles described herein are preferably highly elastic, and
they may increase the local elasticity of tissue to which they are
directed. This increased elasticity may then be detected with
elastography. If desired, elastography can be used in conjunction
with other imaging techniques, such as MRI and ultrasound.
[0064] In a preferred embodiment of the present invention, the
contrast agent will comprise a labeling substance, such as a blue
dye or a minute amount of radioactively labeled tracer substance.
Preferably, the labeling substance is carried within the contrast
agent, such as a microbubble, and released when the contrast agent
reaches the sentinel lymph node, i.e., by disruption of the
contrast agent employing ultrasound. The blue dye and the
radioactive tracer are used because the "sentinel node" is not
always easy to find. Through a small incision in the axilla the
surgeon can pick out the node (sometimes there are 2 or 3) that
turns blue with the dye and/or emits a radioactive particle which
is then detected with a probe like a Geiger counter.
Contrast Agent Formation
[0065] Methods for the preparation of contrast agents contemplated
for use in the present invention will be readily apparent to those
skilled in the art, once armed with the present disclosure,
especially when the present disclosure is coupled with information
known in the art, such as that described in Unger, U.S. Pat. No.
5,846,517, the disclosure of which is hereby incorporated herein by
reference in its entirety.
[0066] There are a variety of procedures which can be used to
prepare contrast agents for use with the disclosed methods,
including, for example, shaking, microemulsification, vortexing,
extrusion, filtration, sonication, homogenization, repeated
freezing and thawing cycles, extrusion under pressure through pores
of defined size, and similar methods known to those skilled in the
art. For example, a microbubble preparation may be formed by
reducing the pressure on a flourocarbon-in-water emulsion.
Rehydration of spray dried hollow microspheres to form microbubbles
is preferred. Sonication is also a preferred method for the
formation of contrast agents, i.e., through an ultrasound
transmitting septum or by penetrating a septum with an ultrasound
probe including an ultrasonically vibrating hypodermic needle.
However, it will be appreciated that a variety of other techniques
exist for contrast agent formation. For example, gas injection
techniques can be used, such as venturi gas injection.
[0067] Other methods for forming contrast agents include formation
of particulate microspheres through the ultrasonication of albumin
or other protein as described in European Patent Application
0,359,246; the use of tensides and viscosity increasing agents as
described in U.S. Pat. No. 4,446,442; the use of lipid coated,
nonliposomal, contrast agents as described in U.S. Pat. No.
4,684,479; the use of liposomes having entrapped gases as described
in U.S. Pat. Nos. 5,088,499 and 5,123,414; and the use of denatured
albumin particulate microspheres as described in U.S. Pat. No.
4,718,433. Each of these contrast agent compositions is suitable
for use with the methods of the present invention and, accordingly,
the foregoing patents and applications are hereby incorporated by
reference herein in their entirety.
Administration of Contrast Agents
[0068] Any of the above described contrast agent for imaging
lymphatic structures of interest may be administered to a
vertebrate subject, such as a bird or a mammal. As employed herein,
the term "lymphatic structure" refers to cells, tissues or organs
which comprise or are associated with the lymph system, including
lymph nodes, lymph vessels, lymph canals, lymph cells, macrophages,
injection situs, and the like. Preferably, the vertebrate is a
human, and the lymph structure of interest, such as the lymph
nodes, lymph vessels, and the like, can be imaged with any of the
techniques described herein. This can be useful, e.g., for
detecting the lymph nodes, tumors, necrotic regions, and infected
regions.
[0069] In accordance with a preferred embodiment of the present
invention, there are provided methods for identifying the sentinel
lymph node associated with tissues or organs which are neoplastic
(or presumed neoplastic), infectious, and the like. The present
invention also allows for the diagnosis or detection of defects or
irregularities in lymph nodes. Major areas of interest for imaging
of the sentinel lymph node include regional spread of neoplastic
and infectious lesions of the breast, colon and rectum, prostate,
ovary, testes, skin cancer, and the like. Major lymph nodes
involved in these various lesions include axillary and internal
mammary nodes in the chest, and the pararectal, anterior pelvic
(obturator), internal iliac (hypogastric), presacral, external and
common iliac, and para-aortic nodes. Thus, applications where
lymphographic imaging would be useful include, but are not limited
to, pathological lesions affecting the major organs of the chest,
abdomen and pelvis, as well as the skin, from which the regional
and, subsequently, more distant lymphatics can be involved.
[0070] It will be appreciated that lymphatic structures of interest
can be imaged employing different modes of visualization, including
direct lymphangiography, indirect lymphography, lymph scintigraphy,
MR lymphography, and the like. The different modes of visualization
are known in the art, as well as suitable modes of administration
of contrast agents, are discussed, for example, in Vogl et al.
(Acta Radiol. (1997) Supp 412:47-50), the disclosure of which is
hereby incorporated by reference in its entirety.
[0071] As those skilled in the art would recognize, administration
of the contrast agents described herein, as well as the auxiliary
materials, can be carried out in various fashions which are not
intravascular, including parenteral. Parenteral administration,
which is preferred, includes administration by the following
routes: intramuscular, percutaneous, directly in the lymphatic
vessel or the lymph node, intraepidermal, intramedullary,
intramural or intraparenchymal, interstitially, intraperitoneal,
intrathecal, subcutaneous, intrasynovial, transepithelial
(including transdermal), dermal, in the tumor or pathologic process
itself, and the like. Preferably, the contrast agent is
administered by interstitial injection (or other interstitial
administration) in the vicinity of the lymph node to be imaged,
including subcutaneous (under or in the skin) and intraparenchymal
(into an organ) injection, but not intraperitoneal injection (into
a body cavity). In the case of cancer patients, the contrast agent
is preferably injected in proximity to the cancer. The contrast
agent can also be injected by a combination of two or more
parenteral modes, for example intramuscular, subcutaneous, and in
the pathologic process, insuring its accretion in the lymphatic
structure of interest. Upon administration, the contrast agent is
preferably taken up by the lymphatic system, generally localizing
in lymph nodes (preferably the sentinel lymph node) afferent to the
uptake site. Thus, preferably, the contrast agent would follow the
same route as a metastatic tumor cell would be likely to follow
within the lymphatic system.
[0072] Suspensions or formulations comprising contrast agents are
administered in a manner compatible with the route of
administration, the dosage formulation, and in a diagnostic
effective amount. It is anticipated that dosages between about 0.1
to about 30 ml of the agent (about 10 micrograms up to about 1
milligram per kilogram of body weight) per day will be used for
diagnostic applications. In accordance with a preferred embodiment
of the present invention, a small quantity of the contrast agent
(e.g., about 0.1 ml/Kg based on the body weight of the vertebrate)
is introduced into the animal per injection site, but this can vary
depending on the site and the number of injections. Other
quantities of the contrast agent, such as from about 0.005 ml/Kg to
about 1.0 ml/Kg, are also contemplated for use in the practice of
the present invention. Volumes of the contrast agent will normally
vary somewhat depending upon the site of injection, the
concentration and activity of the preparation, the number of
injections to be used, the particular contrast agent employed, the
characteristics of the lymphatic structure desired, the degree and
duration of effect desired, the judgment of the practitioner, as
well as properties peculiar to each individual. Moreover, suitable
dosage ranges for systemic application depend on the route of
administration. In addition, it will be appreciated that the image
of the lymphatic structure will vary depending on the contrast
agent employed (e.g., depending upon their half-lives, their
sensitivity to the ultrasound or other energy employed, their
imaging characteristics, i.e., energy ranges, emission intensities,
scatter, and the like, the stability of the contrast agent,
especially antibody conjugates, their rate of transport to the
lymph nodes, their distribution and clearance, the time at which
imaging is to be done, and the like), the auxiliary or stabilizing
material and/or suitable carrier, the lymph structure, the
injection site, and the like. Adjustment of these parameters will
be conventional for the ordinary skilled clinician. Suitable
regimes for initial administration are variable, but are typified
by an initial administration followed by repeated doses at one or
more intervals.
[0073] In addition, the contrast agent may be in the form of a
sterile injectable suspension or formulation comprising contrast
agents combined with suitable carriers. Suitable carriers include
non-toxic parenterally-acceptable sterile aqueous or nonaqueous
solutions, suspensions, or emulsions, including the auxiliary or
stabilizing materials, surfactants, and the like (each which have
been described herein). This suspension may be formulated according
to known methods using suitable dispersing or wetting agents and
suspending agents, including the auxiliary or stabilizing materials
described herein. They can also be manufactured in the form of
sterile water, or some other sterile injectable medium immediately
before use. Sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides, fatty
acids (including oleic acid), naturally occurring vegetable oils
like sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or
synthetic fatty vehicles like ethyl oleate, or the like. They may
be sterilized, for example, by filtration through a
bacteria-retaining filter, by incorporating sterilizing agents into
the formulations, by irradiating the formulations, by heating the
formulations, and the like. Sterile injectable suspensions may also
contain adjuvants such as preserving, wetting, emulsifying, and
dispersing agents. Buffers, preservatives, antioxidants, and the
like can also be incorporated as required.
[0074] The imaging agent will normally be administered at a site
and by means that insure that it is mobilized and taken up into the
lymphatic circulation. This will vary with the system to be imaged.
Multiple injection sites may be preferable in order to permit
proper drainage to the regional lymph nodes under investigation. In
some cases, injections around the circumference of a tumor or
biopsy site is desired. In other cases, injection into a particular
sheath or fossa is preferred. Injection into the webs of the
fingers or toes is a common mode used to study peripheral
lymphatics. The contrast agent can be administered to the subject
either pre-operatively and/or intra-operatively to localize the
sentinel lymph node. It is recognized that the present invention,
preferably, allows immediate and real-time identification of the
lymph vessel and the sentinel draining node following percutaneous
injection of the contrast agent in a region of interest.
Administration of the contrast agent does not require significant
lead time to reach the nodes. In addition, additional methodology
can be employed to modify or alter the transport of the contrast
agent to the lymph structure, including massaging the injection
site or stimulating flow by exercise to facilitate transport of the
contrast agent to the lymphatic structure of interest. Preferably,
the site of injection of the contrast contrast agent will be
massaged to promote uptake of the contrast agent by the lymph
vessel to spontaneously and on demand fill the lymph vessel and the
draining lymph node.
[0075] The invention method can be used to visualize iliopelvic
lymphatics in genitourinary cancers. For example, to visualize the
sentinel lymph node associated with genitourinary cancers or
lesions, bilateral deep perianal injection of the contrast agent
into the ischiorectal fossa is effective. For example, the patient
can be placed in the lithotomy position and about 1 ml of the
contrast agent is introduced bilaterally into the ischiorectal
fossa, e.g., with a 22 gauge needle, to a depth of about 1.5 inches
just lateral to the anal margin, at the 9 and 3 o'clock positions.
The patient may also lie on the side if achieving the lithotomy
position is not possible. Subcutaneous dorsal pedal injection of
about 0.5 ml of the contrast agent may also be made, e.g., using a
23 gauge half-inch needle in the first interdigital spaces
bilaterally.
[0076] In certain cases, such as testicular or prostatic cancer or
some cases of rectal carcinoma, intratumoral or peritumoral
injection of imaging agents can be effective.
[0077] The present method also has applicability in locating the
sentinel node associated with breast tumor. Images of axillary,
subclavian and supraclavicular nodes may be obtained by injecting
the contrast agent into and around the tumor and below the skin
adjacent to the tumor or the medial surface of the upper arms
(ipsilateral and contralateral) of patients with breast cancer. A
unilateral injection can be made in the subcostal site ipsilateral
to the tumor, and then repeated later on the contralateral side to
observe cross drainage between the ipsilateral and contralateral
nodes. Alternatively, for example, for visualization of the
internal mammary lymphatics in breast cancer, the contrast agent is
injected into the posterior rectus sheath at the insertion of the
diaphragm in the subcostal site, using about 1 ml of the contrast
agent. By injecting a contrast agent in the vicinity of the tumor,
the practitioner will know that the lymph duct involved and leading
to the sentinel node will be directed toward the axillary, internal
mammary, or supraclavicular chain wherein imaging is effected at
appropriate times after each injection.
[0078] Another approach is to inject about 0.5 to 1 ml of contrast
agent around the areola tissue of the breasts bilaterally, and then
imaging the axillary, internal mammary, or supraclavicular chains.
In addition to periareolar injection, interdigital administration
of the contrast agent may be used for visualization of axillary
lymphatics (see, DeLand et al., (1980), Cancer Res. 40:2997-3001).
Combined interdigital and periareolar administration of the
contrast agent can provide increased accuracy to demonstrate
increased uptake in affected axillary nodes. Intratumoral injection
of the contrast agent can also be performed in patients with breast
cancer or melanoma and is a useful mode of administration for
certain cases.
[0079] Preferably, the contrast agents persist for a sufficient
amount of time following administration to allow measurements of
the rate of increase in contrast agent levels in the target region
and the determination of maximum signal strength. In this respect
preferred imaging agents have a half life of at least about 1
minute following administration. More preferably the imaging agents
have a half life of at least about 2, 3, 5, 10 or 30 minutes or
more following administration. However, those skilled in the art
will appreciate that the disclosed invention may be practiced by
continuous administration of an imaging agent having any half life
including those with half lives on the order of seconds or tens of
seconds.
[0080] In accordance with yet another embodiment of the present
invention, the sentinel node is then removed for evaluation as to
the presence or absence of neoplastic or infectious diseases or
disorders, including metastasis. Thus, the diagnostic procedure is
minimally invasive, as other non-affected regional axillary nodes
are not disturbed. When compared with the conventional surgical
protocols of removing essentially all regional lymph nodes at the
axilla, the minimally invasive aspect of the present methodology
immediately becomes apparent.
Imaging Techniques
[0081] In accordance with the present invention, any imaging
techniques which allow for the monitoring of the infusion of
contrast agent into the target region are compatible with the
teachings herein. In this regard, all forms of imaging techniques
are contemplated in the present invention, including, for example,
imaging by X-ray, computed tomography (CT) imaging, including CT
angiography (CTA) imaging, magnetic resonance (MR) imaging,
magnetic resonance angiography (MRA), nuclear medicine, ultrasound
(US) imaging, optical imaging or spectroscopy, elastography,
infrared imaging, microwave imaging, and the like. Moreover, the
imaging may be combined to provide multiple exposures of the
contrast agent following administration. The imaging techniques
that are employed are known in the art, and these techniques are
described generally in Kopans, D. B. Md., Breast Imaging
(Lippincott-Raven Publishers 1998), the disclosure of which is
incorporated by reference herein in its entirety.
[0082] Computed tomography (CT) is a valuable diagnostic imaging
technique for studying various areas of the body. This technique
measures the radiodensity (electron density) of matter. CT imaging
techniques which are employed are conventional and are described,
for example, in Computed Body Tomography, Lee, J. K. T., Sagel, S.
S., and Stanley, R. J., eds., 1983, Ravens Press, New York, N.Y.,
especially the first two chapters thereof entitled "Physical
Principles and Instrumentation", and Scroggin, Lippincotts Computer
Tomography Review (Lippincott-Raven Publishers 1995), the
disclosures of which are incorporated by reference herein in their
entirety.
[0083] Magnetic resonance imaging (MRI) is another diagnostic
imaging technique which may be used for producing images of the
body in a variety of scanning planes such as, for example, axial,
coronal, sagittal or orthogonal. MRI employs a magnetic field,
radio frequency energy and magnetic field gradients to make images
of the body. Similar to CTs, the magnetic resonance imaging
techniques which are employed are conventional and are described,
for example, in D. M. Kean and M. A. Smith, Magnetic Resonance
Imaging: Principles and Applications, (William and Wilkins,
Baltimore 1986), and in Rajan, S. S., MRI : A Conceptual Overview
(Springer Verlag 1997), the disclosures of which are incorporated
by reference herein in their entirety. Contemplated MRI techniques
include, but are not limited to, nuclear magnetic resonance (NMR),
electronic spin resonance (ESR), and the like. The presently
preferred imaging modality is NMR imaging.
[0084] With respect to ultrasound, ultrasonic imaging techniques
contemplated for use in the present invention are well known in the
art, and are described, for example, in McGahan and Goldberg,
Diagnostic Ultrasound: A Logical Approach (Lippincott-Raven
Publishers 1998), and in Frederick and Kremkau, Diagnostic
Ultrasound: Principles and Instruments, (W B Saunders Co. 1998),
the disclosures of which are hereby incorporated herein by
reference in their entirety. Specific ultrasound imaging modes
useful with the disclosed invention include harmonic or non-linear
imaging, grey scale (B-mode), Doppler (including pulsed wave,
Power, flow, color, amplitude, spectral and harmonic), 3-D imaging,
gated imaging, and the like. With respect to harmonic imaging, it
will be appreciated that the present invention is compatible with
wideband harmonic imaging and pulse inversion harmonic imaging.
Those skilled in the art will further appreciate that any of these
imaging modes may be used to provide the signal levels which, upon
processing, can afford the desired values for fluid flow rates and
fluid content.
[0085] If one desires to use harmonic imaging (an optional
embodiment of the present invention), and the ultrasound imaging
machine is set to image at a particular frequency, the outgoing
waveform supplied to the sonic transducer can be a numerical
fraction of the imaging frequency (e.g., 1/2, 2/3, 1/3, and the
like) or a whole number or fractional multiple of the imaging
frequency (e.g., 2, {fraction (3/2)}, 3, 4, and the like). With any
particular combination of contrast agents and excitation frequency,
certain harmonics will be dominant. The second harmonic is a common
example. Those strongest harmonics are preferred for obvious
reasons, although other harmonics or frequencies may be selected
for reasons such as preparation of multiple images or elimination
of background. Moreover several frequencies, including harmonic and
non-harmonic frequencies or some combination thereof, may be
simultaneously detected to provide the desired image. That is, in
preferred embodiments any frequency other than the interrogation
frequency may be used to provide the desired data. Of course, those
skilled in the art will appreciate that dominant harmonics can be
determined by simple empirical testing of the contrast agent
preparation.
[0086] To detect the reradiated ultrasound energy generated by the
contrast agents, a modified conventional ultrasound scanner system
or commercially available harmonic imaging systems can be used.
These systems are able to detect or select one or more or all of
the new frequencies, or harmonics, radiated by the contrast agents
for production of the ultrasound image. In other words, it detects
a frequency different from the emitted frequency. Equipment
suitable for harmonic ultrasound imaging is disclosed in Williams
et al., WO 91/15999. Many conventional ultrasound imaging devices,
however, utilize transducers capable of broad bandwidth operation,
and the outgoing waveform sent to the transducer is software
controlled. For this reason, reprogramming to emit a waveform
different from the one detected is well within the level of skill
in the art.
[0087] Although harmonic ultrasound imaging is particularly
preferred for use in the disclosed methods and systems, other types
of ultrasound imaging such as B-mode (gray scale imaging), F-mode
(color flow or Doppler imaging) and D-mode (spectral Doppler) are
also compatible and within the purview of the instant
invention.
[0088] In B-mode imaging, the ultrasound system typically transmits
a series of beams, along scan lines, steered to scan a desired
field of view. The ultrasound system typically steers "receive
beams" in a manner that corresponds to the transmit beams. Data
returned from each receive beam is communicated to an image display
subsystem which reconstructs a two-dimensional gray scale image
from the B-mode data and displays it on a console. Such series of
pulses down a single line may be identical or may be of equal or
unequal frequency or have a near 180 degree phase shift (inverted
pulse) to promote the distinction of the contrast agent from the
surrounding tissues.
[0089] F-mode imaging is accomplished in a manner similar to B-mode
imaging, in that the ultrasound system fires and receives a series
of beams to scan a field of view. However, since F-mode imaging
requires calculation of the velocity of targets, each line is fired
and received several times. As with B-mode imaging, the data
returned from each firing of each line is used to reconstruct an
image on a console.
[0090] F-mode imaging is often used concurrently with B-mode
imaging. For example, the gray scale image reconstructed from a
B-mode scan can be superimposed with an F-mode image reconstructed
from an F-mode scan of the same field of view or of a lesser
included field of view. The F-mode information can be displayed
using colors, with different colors indicating different positive
or negative flow velocities or turbulence at the part of the B-mode
image on which the pixel is superimposed. Because F-mode imaging is
intended to provide only qualitative insight into target motion in
the patient's body, the ultrasound system's processing of F-mode
signals need not have high spatial or velocity resolution either in
amplitude or in pixel resolution. However, since an important value
of F-mode imaging is to detect flows relative to anatomical
structures in the body, it is usually important that the F-mode
image be properly registered with the B-mode image on-screen. Since
this technique relies on the correlation of signal obtained from
one pulse versus the subsequent pulse, and since vesicles can be
destroyed by the first pulse, an F-signal is generated that is not
related to motion. This loss of correlation can be shown in a
variety of display formats but is typically displayed in color.
[0091] In D-mode (spectral Doppler) acquisition, the ultrasound
system fires a beam and processes the return signal for a single
target. Spectral Doppler information can be obtained by
transmitting and receiving either continuous wave (CW) or pulsed
wave (PW) ultrasonic energy. In CW Doppler acquisition, for
example, Power Doppler (Doppler angiography), the ultrasound
receiver continuously receives echoes from all objects within the
receiver's area of sensitivity in the body, and cannot isolate
information received from any specific range interval. CW Doppler
is most useful where the instrument's area of sensitivity can be
adjusted, either by physical placement of the probe or by
beamforming, or both, to include only the desired target. In PW
Doppler acquisition, the ultrasound instrument receives echoes from
individual pulses, the timing of which implies a range interval
within the body of the object which produced the echo. A clinician
typically selects a range interval within which the target is
expected to be located.
[0092] In D-mode acquisition, it is desirable to be able to produce
detailed quantitative measurements over a very large range of
signal levels (dynamic range). D-mode information is processed by
the ultrasound system to display either the velocity spectrum of
the target, plotted against time, or to provide an audio output
carrying similar information. Spectral Doppler acquisition is
described in Liv Hatle, M.D. & Bjorn Angelsen, Dr. Techn.,
"Doppler Ultrasound in Cardiology" (1st ed. 1982) and (2d ed.
1984), incorporated herein by reference in its entirety.
[0093] In addition to B-, F- and D-mode acquisition, a fourth mode
also exists, known as M-mode, but this is merely a different
display modality for data acquired in a manner similar to B- or
F-mode acquisition. The requirements for M-mode acquisition are not
significantly different from those for B- or F-mode acquisition.
Alternatively, or in addition, 3-dimensional ultrasound is also
contemplated, wherein 3-D scans require special probes and software
to accumulate and render the images.
[0094] Additional diagnostic techniques contemplated for use in the
present invention are well known in the art, and are described, for
example, in Gamsu et al., Diagnostic Imaging Review (W B Saunders
Co 1998), the disclosure of which are incorporated by reference
herein in its entirety.
[0095] In the case of diagnostic applications (such as ultrasound,
CT, MRI, and the like), energy, such as ultrasonic energy, may be
applied to at least a portion of the patient to image the target
tissue. A visible image of an internal region of the patient,
preferably the lymphatic structure, may be then obtained, such that
the identification of the sentinel lymph node can be ascertained.
Of course, it will be appreciated that the same images could be
used to detect or diagnose defects or irregularities in the
lymphatic structure.
[0096] All U.S. and Foreign Patent publications, textbooks, and
journal publications referred to herein are hereby expressly
incorporated by reference in their entirety. The invention will now
be described in greater detail by reference to the following
nonlimiting examples.
EXAMPLES
[0097] The invention was put in practice using a rabbit model with
a Vx2 tumor implanted by bolus injection in the calf. The popliteal
fossa was imaged with a Siemens Sonoline Elegra scanner equiped
with a 7MHz trasnducer and the enlarged lymph nodes identified. 0.5
mL of AF0150 (Alliance Pharmaceutical Corp.) was injected at the
margin of the leg tumor nearest the popliteal fossa and another 0.5
ml injected in the foot pad of the affected leg using a 23 G
needle. The popliteal lymph nodes were imaged with standard,
wideband, and burst wideband sonography immediately after injection
and during massage of the foot pad. The lymph vessel leading to the
popliteal node could be recognized as an echogenic line leading to
the node. This process could be repeated several times. The lymph
node fed by this vessel was enhanced and complete filling was
achieved with intermittent imaging with a delay of 5 to 10 seconds.
The delay time required to fill the node increased as the
experiment progressed over the following minutes. The enhancement
of the node was best appreciated with wideband harmonic
imaging.
[0098] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
* * * * *