U.S. patent application number 10/530094 was filed with the patent office on 2006-08-03 for method, compounds and preparations for the identification of sentinel lymph nodes.
Invention is credited to Jonny Ostensen, Henrik Rasmussen, Audun Tornes.
Application Number | 20060173339 10/530094 |
Document ID | / |
Family ID | 19914060 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060173339 |
Kind Code |
A1 |
Tornes; Audun ; et
al. |
August 3, 2006 |
Method, compounds and preparations for the identification of
sentinel lymph nodes
Abstract
The present invention relates methods for the identification of
a sentinel lymph node and to compounds and preparations used in
said methods.
Inventors: |
Tornes; Audun; (Oslo,
NO) ; Ostensen; Jonny; (Oslo, NO) ; Rasmussen;
Henrik; (Oslo, NO) |
Correspondence
Address: |
GE HEALTHCARE, INC.
IP DEPARTMENT
101 CARNEGIE CENTER
PRINCETON
NJ
08540-6231
US
|
Family ID: |
19914060 |
Appl. No.: |
10/530094 |
Filed: |
October 1, 2003 |
PCT Filed: |
October 1, 2003 |
PCT NO: |
PCT/NO03/00328 |
371 Date: |
January 26, 2006 |
Current U.S.
Class: |
600/458 |
Current CPC
Class: |
A61K 9/5015 20130101;
A61K 9/0009 20130101; A61K 49/223 20130101 |
Class at
Publication: |
600/458 |
International
Class: |
A61B 8/14 20060101
A61B008/14 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 2002 |
NO |
20024755 |
Claims
1. Method for the identification of a sentinel lymph node in a
subject comprising a) administering to said subject a preparation
comprising microbubbles comprising a shell and a gas or gas
precursor, said microbubbles having a mean particle size of about
0.25-15 .mu.m in diameter and a pressure stability of at least 50%
at a pressure of 120 mm Hg, b) allowing said microbubbles to
accumulate in said sentinel lymph node and c) detecting said
microbubbles in said sentinel lymph node using ultrasound.
2. Method according to claim 1 wherein the preparation is
interstitially, preferably percutaneously, administered.
3. Method according to claim 1 and 2, wherein the shell has an
overall negative or positive net charge, preferably an overall
negative net charge.
4. Method according to claim 1, wherein the shell comprises lipids,
preferably phospholipids.
5. Method according to claim 1, wherein the shell comprises
negatively charged phospholipids in an amount of from 50% to
100%.
6. Method according to claim 1, wherein the microbubbles are stable
for pressure variations associated with ultrasound imaging of a
mechanical index of at least 0.2.
7. Method according to claim 1, wherein the preparation further
comprises a macrophage stimulating compound.
8. Microbubbles for the identification of a sentinel lymph node
comprising a shell and a gas or gas precursor and having a mean
particle size of about 0.25-15 .mu.m in diameter wherein said
microbubbles have a pressure stability of at least 50% at a
pressure of 120 mm Hg.
9. Microbubbles according to claim 8, wherein the shell has an
overall negative net charge.
10. Microbubbles according to claim 8, wherein the shell comprises
lipids, preferably phospholipids.
11. Microbubbles according to claim 8, wherein the shell comprises
negatively charged phospholipids in an amount of from 50% to
100%.
12. Preparations for the identification of a sentinel lymph node
comprising microbubbles according claim 8.
13. Preparations according to claim 12 further comprising a
macrophage stimulating compound.
14. Use of microbubbles according to claim 8 for the manufacture of
a preparation for the identification of a sentinel lymph node.
Description
[0001] The present invention relates to methods for the
identification of a sentinel lymph node and to compounds and
preparations used in said methods.
[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 fluid which enters the
lymph vessels carries with it substances and materials from the
tissue, e.g. antigens, particles and cells. The lymph nodes process
the lymph fluid by sieving it and macrophages inside the nodes
remove particulate and cell material carried by the lymph fluid via
phagocytosis.
[0003] When cancer occurs in tissues or organs, its loose matrix
may allow the dislodging of cells that gain access to the lymphatic
system, become trapped in the lymph node and grow. In 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 and/or can spread downstream to the next node. The
lymph nodes that drain the tissue or organ of interest (i.e., the
cancerous tissue) are called the regional lymph nodes and the first
node that traps the cancer is called the sentinel lymph node.
[0004] Patterns in the spread of tumours are complicated, as
metastasis of neoplastic cells does not simply result in the spread
of the neoplastic cells to the next physically nearest node. These
nodes are more likely to contain the sentinel lymph node; however,
the sentinel lymph node may be in a more distant nodal group. This
can occur due to tumours, infections, injuries or previous
treatment can block the lymph vessels that directly drain the
tissue or organ of interest, promoting the development of aberrant
pathways.
[0005] In the past, it has been normal practice in some situations
to remove all lymph nodes potentially harbouring neoplastic cells
metastasised from a tumour. A high morbidity rate is associated
with this practice. Thus, several methods were developed to
identify and biopsy the sentinel lymph node. If the sentinel lymph
node is free of neoplastic cells, then further lymph node biopsies
and (further) lymph node dissections can be avoided. Sentinel lymph
nodes have been identified by injecting a marking agent into the
tumour-bearing tissue and tracing the pathway of the marking system
through the lymphatic system.
[0006] Visible marking agents such as dyes have been employed to
visually detect the sentinel lymph node with the naked eye (A. E.
Giuliano et al., Ann. Surg. 220, 1994, 391-401). Such a method
requires significant surgical dissection. The nodes are
indistinguishable from the surrounding tissue unless stained and
the dyes unfortunately have an unpredictable and rapid
clearance.
[0007] U.S. Pat. No. 5,732,704 discloses a method to detect
sentinel lymph nodes using radiopharmaceutical compounds and the
localisation of said compounds with the aid of a radiation
detection probe. Although such compounds have a more delayed
transit, patients and medical personnel are exposed to potentially
harmful doses of ionising radiation. Radioactive isotopes also pose
environmental contamination and disposal issues.
[0008] U.S. Pat. No. 5,496,536 describes a method of lymphography
by using particles which are less than 1 .mu.m in diameter and
detecting those particles with different imaging modalities. As
observed by C. Oussoren et al., Biochim Biophys. Acta 1328, 1997,
261-272, small particles are taken up into the lymphatic
capillaries to a high extent; however, they are only poorly
retained by lymph nodes. Such small particles are generally less
effective ultrasound scatters and are hence not well suited for
ultrasound based lymphography. Because of their poor retention by
lymph nodes, they are not selective enough to detect only the
sentinel lymph node, but will proceed on to other lymph nodes.
[0009] The observation made by Oussoren et al. was affirmed by the
disclosures of WO-A-00/45855 and WO-A-00/38579.
[0010] WO-A-00/38579 discloses a method to detect sentinel lymph
nodes using a contrast agent which is capable of migrating to the
lymph node within a certain time frame--preferably within less than
3 hours--and detecting said contrast agent with an adequate
detection modality. To migrate within this time frame, the contrast
agent must comprise particles between 0.05 and 5 .mu.m in
diameter.
[0011] WO-A-00/45855 discloses a method to identify the sentinel
lymph node using particulate contrast agents having a mean particle
size of 1-10 .mu.m and an imaging modality to detect said contrast
agents in the lymph node.
[0012] As outlined in WO-A-00/38579 and WO-A-00/45855, the size of
the contrast agent particles seems to have a significant impact on
the operability of the methods disclosed in said documents.
Unfortunately, the methods described do not work equally well with
respect to the contrast agents and the imaging modalities employed.
Although having about the same size, different contrast agents used
for the same imaging modality behave significantly different, thus,
the use of certain contrast agents in the methods described may
result in insufficient sensitivity.
[0013] Accordingly, there is a need for a reliable method to
identify the sentinel lymph node. Additionally, said method should
be sensitive, safe and easy to carry out.
[0014] It has surprisingly been found that a method for the
identification of a sentinel lymph node in a subject which
comprises [0015] a) administering to said subject a preparation
comprising microbubbles comprising a shell and a gas or gas
precursor, said microbubbles having a mean particle size of about
0.25-15 .mu.m in diameter and a pressure stability of at least 50%
at a pressure of 120 mm Hg, [0016] b) allowing said microbubbles to
accumulate in said sentinel lymph node and [0017] c) detecting said
microbubbles in said sentinel lymph node using ultrasound imaging
fulfils the criteria stated above.
[0018] Thus, the invention provides a method for the identification
of a sentinel lymph node in a subject comprising [0019] a)
administering to said subject a preparation comprising microbubbles
comprising a shell and a gas or gas precursor, said microbubbles
having a mean particle size of about 0.25-15 .mu.m in diameter and
a pressure stability of at least 50% at a pressure of 120 mm Hg,
[0020] b) allowing said microbubbles to accumulate in said sentinel
lymph node and [0021] c) detecting said microbubbles in said
sentinel lymph node using ultrasound imaging.
[0022] In another aspect, the invention is related to a method for
the identification of a sentinel lymph node in a subject comprising
detecting previously administered microbubbles in said sentinel
lymph node of said subject using ultrasound imaging, wherein said
microbubbles comprise a shell and a gas or gas precursor, have a
mean particle size of about 0.25-15 .mu.m in diameter and a
pressure stability of at least 50% at a pressure of 120 mm Hg.
[0023] In yet another aspect, the invention is related to
microbubbles for the identification of a sentinel lymph node which
comprise a shell and a gas or gas precursor, have a mean particle
size of about 0.25-15 .mu.m in diameter and a pressure stability of
at least 50% at a pressure of 120 mm Hg.
[0024] Another aspect of the invention are preparations for the
identification of a sentinel lymph node comprising microbubbles
comprising a shell and a gas or gas precursor, having a mean
particle size of about 0.25-15 .mu.m in diameter and a pressure
stability of at least 50% at a pressure of 120 mm Hg.
[0025] Yet another aspect of the invention is the use of
microbubbles comprising a shell and a gas or gas precursor, having
a mean particle size of about 0.25-15 .mu.m in diameter and a
pressure stability of at least 50% at a pressure of 120 mm Hg for
the manufacture of an agent for the identification of a sentinel
lymph node.
[0026] The microbubbles according to the invention remain intact
upon injection. They are not only easily taken up by the lymphatic
system and the sentinel lymph node but they are also retained in
the sentinel lymph node and remain stable, thus allowing sensitive
and effective ultrasound detection.
[0027] In step a) of the method according to the invention, a
preparation comprising microbubbles comprising a shell and a gas or
gas precursor is administered to a subject. Said microbubbles have
a mean particle size of about 0.25-15 .mu.m in diameter and a
pressure stability of at least 50% at a pressure of 120 mm Hg
[0028] In the context of the present invention, "subject" means a
vertebrate subject like a bird or a mammal and preferably a
human.
[0029] The microbubbles and/or the preparations according to the
invention 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.
[0030] The microbubbles in the context of the invention comprise a
shell and a gas or a gas precursor.
[0031] The term "shell" in the context of the present invention can
be interchangeably used with the term "wall" or "membrane" and
means material surrounding or defining a microbubble. The shell may
be in the form of one or more layers, preferably in the form of a
single monolayer or a bilayer (unilamellar), and the mono- or
bilayer may be used to form one or more mono- or bilayers (oligo-
or multilamellar). In the case of more than one mono- or bilayer,
the mono- or bilayers are preferably concentric. Suitably, the
shell is formulated from lipids, natural or synthetic polymeric
materials, proteinaceous materials, carbohydrates, saccharides, and
the like or combinations thereof. In a preferred embodiment, the
shell has an overall negative or positive net charge. Thus, the
shell may be composed of or comprise polymeric material or
proteinaceous material having an excess of negative or positive
charges or the shell may be composed of or comprise negatively or
positively charged lipids. Alternatively, the shell may be composed
of or comprise neutral polymeric, proteinaceous or lipid materials
combined with incorporation or surface modification using
negatively or positively charged components that give an overall
net charge. Preferably, the shell is composed of or comprises
lipids, more preferably phospholipids, e.g phosphatidylcholines,
preferably dilauroyl phosphatidylcholine, dimyristoyl
phosphatidylcholine, diheptadecanoyl phosphatidylcholine,
dipalmitoyl phospatidylcholine, distearoyl phosphatidylcholine,
diarachidoyl phosphatidylcholine or dibehenoyl phosphatidylcholine,
phosphatidylserines, preferably dipalmitoyl or distearoyl
phosphatidylserine, phosphatidylglycerols, preferably dipalmitoyl
or distearoyl phosphatidylglycerol, phosphatidylethanolamines,
preferably dipalmitoyl or distearoyl phosphatidylethanolamine,
phosphatidylinositols, preferably dipalmitoyl or distearoyl
phosphatidylinositol, phosphatidic acid, preferably dipalmitoyl or
distearoyl phosphatidic acid, cardiolipins or any mixture of the
foregoing named compounds, optionally in a mixture with
cholesterol, cholesterol sulfate, cholesteryl hemisuccinate,
N-palmitoyl homocystein, palmitic acid, oleic acid, stearic acid or
arachidic acid. Alternatively, the shell may also be composed of or
comprise fluorinated analogues of the above-mentioned lipids. In
another preferred embodiment, the shell is composed of or comprises
the above-mentioned lipids which are covalently linked to
hydrophilic polymers such as polyethylene glycol (PEG), preferably
PEG 2000-8000, e.g. dipalmitoyl or distearoyl
phosphatidylethanolamine-polyethyleneglycol 5000. In a particularly
preferred embodiment, the shell is composed of or
comprises--preferably in an amount of from 50 to 100%, more
preferably in an amount of 70 to 90%--negatively charged
phospholipids, more preferably negatively charged phospholipids
based on fatty acids having at least 14 carbon atoms, e.g. myristic
acid, palmitic acid, stearic acid oleic acid or arachidic acid,
e.g. dipalmitoyl phosphatidylglycerol or dipalmitoyl
phosphatidylethanolamine-PEG. In another preferred embodiment, the
shell comprises--preferably in an amount of from 1 to
20%--positively charged synthetic lipids, e.g. cationic lipids
normally used in nucleic acid delivery such as DOTAP
(N-1(-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniumethyl
sulphate), DOTMA
(N-(1-(2,3-dioleoyloxy)-propyl)-N,N,N-trimethylammonium chloride),
DOGS (dioctadecylamidoglycyl spermine) and the like. Alternatively,
the shell is composed of or comprises lipopeptides, for examples
lipopeptides as described in WO-A-99/55383, the content of which is
incorporated herein by reference.
[0032] The term "gas precursor" in the context of the present
invention denotes a material which is a liquid or a solid at
ambient temperature and pressure and changes phase from liquid to
gas at the relevant temperature, e.g. the body temperature of the
subject. When referring to "gas" and "gas precursor", it will be
understood that mixtures of gases and gas precursors fall within
the definition.
[0033] Suitably, microbubbles according to the invention comprise
air, nitrogen and fluorinated compounds, either partially
fluorinated or fully fluorinated (perfluorinated compounds), as
pure compounds or mixtures thereof. In a preferred embodiment, the
microbubbles comprise perfluorinated compounds, e.g.
perfluoropropane, perfluorobutane, perfluoropentane,
perfluorohexane, sulfur hexafluoride and the like, optionally in
admixture with nitrogen.
[0034] The microbubbles according to the invention have a mean
particle size of 0.25-15 .mu.m in diameter, preferably 0.5-7 .mu.m,
particularly preferably 1-5 .mu.m.
[0035] The microbubbles according to the invention have a pressure
stability of at least 50% at a pressure of 120 mm Hg. In the
context of the invention, the term "pressure stability of at least
50%" means that the acoustic attenuation efficacy of the
microbubbles after being exposed to a pressure of 120 mm Hg is at
least 50% of the acoustic attenuation efficacy of said microbubbles
before being exposed to said pressure. Hence, by comparing the
acoustic attenuation efficacy of the microbubbles before and after
exposure to pressure, a measure of the microbubble's ultrasound
imaging efficacy can be obtained. The acoustic attenuation efficacy
can be determined by measuring the dampening (dB/cm) of a sound
beam going through a diluted suspension of the microbubble sample
using one or two broad band transducers with centre frequencies 3.5
and/or 5.0 MHz. Transmission is measured by pulse-echo technique;
short pulses of sound are emitted from the transducer and traversed
through a measuring cell compartment before being reflected from
the back wall of the compartment and received again by the emitting
transducer. The pulses are digitised by an oscilloscope and
frequency spectra are calculated by Fourier transformation. To
compensate for transmission path and transducer characteristics,
the spectra are normalised to spectra of the pure diluent. A
detailed description of the measurement of attenuation spectra and
a suitable system setup for is described in L. Hoff, Acoustic
Characterization of Contrast Agents for Medical Ultrasound Imaging,
Kluwer Academic Publishers, 2001, chapter 4, page 99-109, the
disclosure of which is incorporated herein by reference. The
analysis is normally conducted in the range of 0.degree. C. to
50.degree. C., preferably at ambient room temperature. In a first
step of the analysis, a reference spectrum is taken from the
diluent. Suitable diluents are free of air bubbles and any liquids
in which the microbubbles are stable could be used, preferable
diluents are isotonic saline solution like Isoton II (Coulter
Electronics Ltd. Luton, UK), a 0.9% saline solution comprising a
phosphate buffer and a detergent to reduce surface tension. In a
next step, the microbubble sample is mixed with the diluent and one
or more attenuation spectra are measured at ambient pressure. The
concentration of the microbubble sample is adapted to the size of
the microbubbles. Preferably, the dilution factor is such that the
attenuation from the microbubble sample is between 15 and 20 dB,
i.e. about 3 dB/cm. This typically means that the microbubbles are
diluted by a factor 10.sup.3 to 10.sup.4. In a next step, the
pressure is raised to 120 mm Hg and one or more attenuation spectra
are measured. In order to determine the pressure stability
according to the invention, the acoustic attenuation efficacy of
the microbubble sample before pressure is set to 100% thus allowing
the calculation of the relative acoustic attenuation efficacy of
the microbubble sample after pressure.
[0036] In a preferred embodiment, the microbubbles according to the
invention have a pressure stability of at least 70%, more preferred
of at least 85%, most preferred of at least 95%.
[0037] In a preferred embodiment, the microbubbles according to the
invention are stable for pressure variations associated with
ultrasound imaging of a mechanical index of at least 0.2. A method
to determine the microbubble stability associated with ultrasound
imaging pressures is described in W. T. Shi et al., Ultrasound in
Med. & Biol., Vol. 26, 2000, 1-11.
[0038] Suitably, the microbubbles according to the invention are
echogenic, i.e. they are capable of scattering or reflecting
ultrasound waves. Preferably, the microbubbles are adapted to
return a signal at a frequency different from the transmit
frequency of the ultrasound pulse. That is, the microbubbles are
adapted for harmonic ultrasound imaging as described in U.S. Pat.
No. 5,540,909.
[0039] The microbubbles according to the invention can be prepared
in a variety of ways which are readily apparent to those skilled in
the art, including, e.g. shaking, vortexing, sonication, extrusion,
repeated freezing and thawing cycles, extrusion under pressure
through pores of defined size and spray drying. For example for
lipid comprising microbubbles, the lipid-containing medium may be
subjected to any appropriate emulsion generating technique, e.g.
sonication, high pressure homogenisation, high shear mixing, in the
presence of the selected gas or gas precursor. The gas employed in
the emulsification step need not to be the same as in the final
microbubble. Thus, most of this gas may be removed during a
subsequent lyophilisation step and residual gas may be removed by
evacuation of the dried product, to which an atmosphere or
overpressure of the desired end product gas may then be applied
(see for example WO-A-97/29783, the content of which is
incorporated herein by reference).
[0040] Other methods of forming the microbubbles according to the
invention includes the formation of protein comprising microbubbles
(EP-A-359 246 and U.S. Pat. No. 4,718,433), the formation of lipid
containing microbubbles (U.S. Pat. No. 4,684,479) and the formation
of liposomal microbubbles (U.S. Pat. No. 5,088,499; U.S. Pat. No.
5,123,414 and WO-A-94/28874), the content of which is incorporated
herein by reference.
[0041] According to the method of the invention, the microbubbles
are administered in form of a preparation, preferably in form of a
liquid preparation. In the following, the term "preparation" is
used interchangeably with the term "microbubble preparation".
[0042] Preparations according to the invention comprise the
above-described microbubbles and one or more components selected
form the group consisting of osmotic agents, stabilisers,
surfactants, buffers, viscosity modulators, emulsifiers,
solubilising agents, suspending agents, wetting agents,
antioxidants, viscosity increasing agents, tonicity raising agents,
salts, sugars and the like. Such components are added to ensure
maximum life and effectiveness of the microbubbles. Additionally,
considerations as sterility, isotonicity and biocompatibility may
govern the use of such components.
[0043] Suitable viscosity modulators include, for example,
carbohydrates and their phosphorylated and sulfonated derivatives;
polyethers, preferably with molecular weight ranges between 400 and
100,000 and di- and trihydroxy alkanes and their polymers,
preferably with molecular weight ranges between 200 and 50,000.
[0044] Suitable emulsifying and/or solubilizing agents include, for
example, acacia, cholesterol, glyceryl monostearate, lanolin
alcohols, lecithin, mono- and diglycerides, ethanolamine,
diethanolamine 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.
[0045] Suitable suspending and/or viscosity-increasing agents
include, for example, acacia, agar, alginic acid, aluminum
mono-stearate, bentonite, magma, carbomer 934 P, cellulose,
methylcellulose, carboxymethylcellulose, hydroxyethyl cellulose,
hydroxypropyl methylcellulose, carrageenan, dextran, gelatin, guar
gum, locust bean gum, veegum, magnesium-aluminum-silicate, silicon
dioxide, zeolites, pectin, polyethylene oxide, povidone, propylene
glycol alginate, sodium alginate, tragacanth, xanthan gum,
alpha-d-gluconolactone, glycerol and mannitol.
[0046] Suitable suspending agents are for example polyethylene
glycol (PEG), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA),
polypropylene glycol (PPG), polysorbate and the like.
[0047] Suitable tonicity raising agents which stabilise and add
tonicity are, for example, sorbitol, mannitol, trehalose, sucrose,
propylene glycol and glycerol.
[0048] The preparations according to the invention may further
comprise dyes or biologically active agents, preferably 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 and
peptides.
[0049] In a preferred embodiment, the preparations further comprise
compounds which promote macrophage uptake, e.g. mannans, for
example zymosan, mannose-containing oligo- and polysaccharides, Fc
(fragment crystallizable) portions of immunoglobulin molecules,
complement components, for example C3b or C3bi, ligands for
scavenger receptors, ligands for toll-like receptors, ligands for
LRPs (LDL-receptor-like proteins), bacterial glycopeptides or
lipopolysaccharides for example bleomycin or endotoxin.
[0050] The preparations are preferably sterile injectable
formulations like suspensions or emulsions comprising suitable
carriers including non-toxic parenterally-acceptable aqueous or non
aqueous solutions and the components and/or compounds described
above. Preferred carriers are water or saline.
[0051] The preparations may be formulated according to known
methods. In a preferred embodiment, the preparations are
manufactured immediately before use. Thus, a dry microbubble
product may be mixed with a suitable carrier and one or more of the
above mentioned compounds and components. In another preferred
embodiment, preparations are manufactured immediately before use
and microbubbles are generated in situ during the manufacture of
the preparation, e.g. by adding a suitable carrier to a vial
containing the desired gas and the components comprising the shell
of the microbubbles and agitating this mixture. The preparations
according to the invention are preferably sterilised before
administration and/or are manufactured from sterile starting
materials. Sterilisation of the preparations may be achieved by
filtration through a bacteria-retaining filter, by incorporating
sterilising agents, by irridation and the like.
[0052] Administration according to step a) of the method of the
invention can be carried out in various fashions which are not
intravascular. Methods of parenteral administration are preferred
and include but are not limited to the following routes:
intramuscular, percutaneous, directly in a lymphatic vessel,
interstitially, intraperitoneal, intrathecal, subcutaneous,
intrasynovial, transepithelial (including transdermal) dermal,
intradermal, subdermal, in a tumour or pathologic process itself,
and the like. Preferably, the preparation is interstitially
administered, preferably by interstitial injection including
subcutaneous and intradermal injection. In the case of cancer
patients, the preparation is preferably injected in proximity to
the cancer (peritumoural). The preparation can also be injected by
a combination of two or more parenteral modes, for example
intramuscular, subcutaneous, and in the pathologic process,
ensuring its accretion in the sentinel lymph node.
[0053] The preparation will normally be administered at a site and
by means that ensure that it is mobilised 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 tumour 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 preparation can be administered to the subject
either pre-operatively and/or intra-operatively to localise the
sentinel lymph node. The method according to the invention allows
immediate and real-time identification of the sentinel lymph node
following administration of the preparation in a region of interest
as administration does not require significant lead time to reach
the sentinel lymph node. Moreover, additional methodology can be
employed to modify or alter the transport of the preparation to the
sentinel lymph node, including massaging the injection site or
stimulating flow. Preferably, the site of injection of the
preparation will be massaged.
[0054] The method of the invention has applicability in locating
the sentinel node associated with breast tumour. Images of
axillary, subclavian and supraclavicular nodes may be obtained by
injecting the preparation into and around the tumour and below the
skin adjacent to the tumour. A unilateral injection can be made in
the subcostal site ipsilateral to the tumour, followed by bilateral
lymph node imaging. By injecting the preparation in the vicinity of
the tumour, 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
ultrasound detection is effected at appropriate times after each
injection.
[0055] Another approach is to inject the preparation around the
areola tissue of the breasts bilaterally, and then detecting the
axillary, internal mammary, or supraclavicular chains. In addition
to periareolar injection, interdigital administration of the
preparation 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 preparation can
provide increased accuracy to demonstrate increased uptake in
affected axillary nodes. Intratumoural injection of the contrast
agent can also be performed in patients with breast cancer or
melanoma.
[0056] The preparations will be administered in an effective
amount, i.e. in an amount which allows sufficient detection. It is
anticipated that between 0.1 to 30 ml of the preparations are
administered in liquid form, preferably 0.1 to 3 ml, particularly
preferably 0.5 to 1.5 ml. The particularly preferred volume for
administration corresponds to an administered microbubble gas
volume of about from 5 to 15 .mu.l. In a preferred embodiment,
multiple injections are performed (typically 4), each with a small
quantity of the preparation, e.g. 0.1 to 3 ml are administered to a
subject per injection site. Variations can be due to the number of
injections and the injection site. Other amounts of the
preparations, such as from about 0.005 ml/kg to about 1.0 ml/kg,
are also contemplated according to the method of the invention.
Volumes of the preparations in liquid form will normally vary being
dependent upon, e.g., the site of administration, the concentration
of the preparation, the number of injections, the composition of
the preparation and/or the type of microbubbles present therein and
the properties peculiar to each individual subject.
[0057] In step b) according to the method of the invention, the
microbubbles are allowed to accumulate in the sentinel lymph
node.
[0058] After administration, the microbubbles do not require
significant lead time to reach the sentinel lymph node and
accumulate therein. Thus, immediate and real-time identification of
the sentinel lymph node following administration of the preparation
according to the invention is possible. Generally, the microbubbles
according to the invention are capable of accumulating in the
sentinel lymph node in less than 60 minutes. In a preferred
embodiment, the microbubbles according to the invention accumulate
in the sentinel lymph node in less than 15 minutes and particularly
preferably in less than 5 minutes.
[0059] After administration, the microbubbles according to the
invention have a half life of at least 5 minutes, preferably of at
least 15 minutes and particularly preferably of at least 60
minutes.
[0060] As the accumulation time of the microbubbles in the sentinel
lymph nodes is relatively short, the microbubbles according to
invention allow imaging shortly after administration, improving
logistics and prolonging the detection time.
[0061] In step c) according to the method of the invention, the
microbubbles are detected in the sentinel lymph node using
ultrasound imaging and thereby identifying the sentinel lymph
node.
[0062] With respect to ultrasound, ultrasound 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).
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, colour,
amplitude, spectral and harmonic), 3-D imaging, gated imaging, and
the like. With respect to non-linear imaging, it will be
appreciated that the present invention is compatible with wideband
harmonic imaging and pulse inversion harmonic imaging.
[0063] If one desires to use harmonic imaging 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, ( 3/2, 3, 4, and the like). With any
particular combination of microbubble preparation 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.
[0064] To detect the re-radiated ultrasound energy generated by the
microbubble preparations, a modified conventional ultrasound
scanner system or commercially available non-linear 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
microbubble preparation 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 WO-A-91/15999. Many conventional ultrasound imaging
devices, however, utilise 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.
[0065] Although non-linear ultrasound imaging such as harmonic
ultrasound imaging, second harmonic ultrasound imaging or
preferably pulse inversion is particularly preferred for use in the
disclosed methods, other types of ultrasound conventional imaging
such as B-mode (gray scale imaging), F-mode (colour flow or Doppler
imaging) and D-mode (spectral Doppler) are also compatible and
within the purview of the present invention.
[0066] 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.
[0067] 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.
[0068] 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 colours, with different colours 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 microbubbles 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
colour.
[0069] 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.
[0070] 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 L. Hatle, and B. Angelsen, "Doppler Ultrasound in
Cardiology" (1st ed. 1982) and (2d ed. 1984).
[0071] 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. It will be appreciated that
the emitted ultrasound energy--if sufficiently high--may disrupt
the microbubbles present in the lymphatics. Doppler based imaging
methods as described above may detect this as a "pseudo Doppler"
signal allowing sensitive and specific detection of the
microbubbles that are immobilised or moving very slowly (see for
example U.S. Pat. No. 5,425,366).
[0072] Additional 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).
[0073] Ultrasonic energy may be applied to at least a portion of
the subject to image the target tissue. A visible image of an
internal region of the subject may then be obtained, such that the
identification of the sentinel lymph node can be ascertained.
Another aspect of the invention is a method for the identification
of a sentinel lymph node in a subject preadministered with
microbubbles comprising a shell and a gas or gas precursor, said
microbubbles having a mean particle size of about 0.25-15 .mu.m in
diameter and a pressure stability of at least 50% at a pressure of
120 mm comprising detecting said microbubbles accumulated in said
sentinel lymph node of said subject using ultrasound.
[0074] The method according to the invention it is not only useful
to identify the sentinel lymph node, but also to determine whether
said sentinel lymph node shows defects or irregularities in the
lymphatic structure. Thus, the present invention also provides a
method for the discrimination between benign and malignant sentinel
lymph nodes in a subject comprising [0075] a) administering to said
subject a preparation comprising microbubbles comprising a shell
and a gas or gas precursor, said microbubbles having a mean
particle size of about 0.25-15 .mu.m in diameter and a pressure
stability of at least 50% at a pressure of 120 mm Hg, [0076] b)
allowing said microbubbles to accumulate in said sentinel lymph
node, [0077] c) detecting said microbubbles in said sentinel lymph
node using ultrasound and [0078] d) characterising said sentinel
lymph node as being benign or malignant according to the pattern of
contrast enhancement within the lymph node.
[0079] Mattrey et al. noticed during sentinel lymph node ultrasound
imaging that cancer within the node did not fill with contrast
agent material, thus leaving a filling defect. However, it was
stated that further work is required for confirmation. (R. Mattrey
et al., Academic Radiology 9, 2002, S231-S235). It was now found
that it is possible to classify the sentinel lymph nodes after
having identified them according to the method of the invention
because of their different patterns of contrast enhancement: benign
sentinel lymph nodes appear uniformly echogenic while malignant
sentinel lymph nodes demonstrate a heterogenic enhancement pattern
with both, areas of increased echogenity and areas that do not
enhance. This finding represents a major clinical advance as the
method according to the invention not only allows for noninvasive
and safe detection of sentinel lymph nodes, but also of their
metastatic infiltration.
[0080] The above-mentioned method provides good results just by
visual assessment of acquired ultrasound images. Discrimination
between benign and malignant lymph nodes may be further improved by
application of image processing methods to enhance the difference
in pattern of contrast enhancement between normal and infiltrated
nodes.
[0081] The invention will now be described in greater detail by
reference to the following non-limiting examples.
EXAMPLES
Example 1
Pressure Stability Measurements
[0082] Various ultrasound contrast agents containing microbubbles
were tested for pressure stability:
[0083] Albunex.TM., microbubbles containing air in a shell of
denatured human serum albumine, which can be prepared as disclosed
in EP-A-359 246.
[0084] Sonovue.TM., microbubbles containing SF.sub.6 encapsulated
in a phospholipid membrane Sonazoid.TM., microbubbles containig
perfluorobutan encapsulated in a surfactant membrane
a) Size Distribution and Volume Concentration Determination
[0085] The microbubble concentration and size distribution of all
samples were determined by Coulter counting with a Coulter
Multisizer Mark II (Coulter Electronics Ltd., Luton, England)
fitted with a 50 .mu.m aperture with a nominal measuring range of 1
to 30 .mu.m. Analysis was performed with 64 logarithmically spaced
size channels. A 20 .mu.l sample was diluted in 200 ml Isoton II
(Coulter Electronics) at ambient room temperature and stirred for 4
minutes before analysis. As analytic response, the microbubble
volume concentration as a percentage of suspension volume and the
volume median diameter of the microbubbles were used.
TABLE-US-00001 microbubble volume concentration as a percentage of
suspension volume median diameter volume [%] of the microbubbles
[.mu.m] Albunex .TM. 0.7 10 Sonovue .TM. 0.6 7 Sonazoid .TM. 1.0
3
b) Acoustic Attenuation Measurements
[0086] The acoustic attenuation spectrum of all samples was
measured as described by L. Hoff, Acoustic Characterization of
Contrast Agents for Medical Ultrasound Imaging, Kluwer Academic
Publishers, 2001, chapter 4. The acoustic attenuation of a sound
beam going through a diluted suspension of the samples was measured
using a broadband transducer with a centre frequency of 3.5 MHz. A
suitable sample volume was homogeneously dispersed in 55 ml Isoton
II at ambient room temperature before analysis. Transmission was
measured by a pulsed-echo technique; short pulses of sound were
emitted from the transducer and traversed the measuring cell
compartment before being reflected from the back wall of the
compartment and received again by the emitting transducer. The
pulses were digitised by an oscilloscope and frequency spectra were
calculated by Fourier transformation. To compensate for
transmission path and transducer characteristics, the spectra were
normalised to the spectra of pure Isoton II. Results were
normalised to a 1:1000 dilution. The acoustic attenuation [dB/cm]
at atmospheric pressure was measured at 3.5 MHz and the obtained
measurement reading was set to 100% acoustic attenuation efficacy.
After application of a pressure of 120 mm Hg for 30 seconds, the
attenuation was measured again and the acoustic attenuation
efficacy after pressure was calculated. Pressure stability was
reported as attenuation efficacy after pressure in percent of
attenuation efficacy before pressure. TABLE-US-00002 Pressure
stability at 120 mm Hg [%] Albunex .TM. 0 Sonovue .TM. 79 .+-. 2
Sonazoid .TM. 98 .+-. 1
Example 2
In Vivo Sentinel Lymph Node Detection and Characterization
[0087] Anaesthetised Sinclair pigs with melanoma tumours were used
to investigate sentinel lymph node detection and characterisation
using Sonazoid.TM..
a) In Vivo Sentinel Lymph Node Detection
[0088] 1 ml Sonazoid.TM. was administered intradermally around the
primary tumour. After 5 minutes of gentle massage of the injection
site, ultrasound scanning was performed with a Siemens Elegera
scanner. The sentinel lymph node showed strong contrast enhancement
on pulse inversion gray scale imaging and was further evaluated
with high power colour flow imaging which gave a mosaic enhancement
pattern characteristic of microbubble rupture. The identification
of the sentinel node was further aided by contrast enhancement of
the lymph canal from the injection site to the sentinel node. The
location of the lymph node was marked on the skin. The location of
the sentinel node was confirmed with peritumoural injections of
radiocolloid tracer and blue dye in accordance with current
clinical practice. The contrast enhancement of the sentinel lymph
node was present for up to 3 hours.
[0089] The method described in Example 2a was applied in 6 melanoma
Sinclair pigs. 17 primary tumours with 31 melanomas were assessed
using ultrasound, lymphoscintegraphy and blue dye (the gold
standard). The accuracy of correctly identifying the sentinel nodes
was 90% for ultrasound and 81% for lymphocintegraphy.
b) In Vivo Sentinel Lymph Node Detection and Characterization
[0090] 1 ml Sonazoid.TM. was administered intradermally around each
primary tumour in a Sinclair pig with 3 melanoma tumours. After 5
minutes of gentle massage of the injection site, ultrasound
scanning was performed with a Siemens Elegera scanner and the
sentinel nodes identified as described in Example 2a. One of the
lymph nodes showed a homogenous contrast enhancement pattern, while
the other two showed a spotty, heterogeneous enhancement pattern.
The first node was characterised as normal, while the two latter
were characterised as malignant based on ultrasound imaging.
Microscopic histological examination of the lymph nodes confirmed
absence of tumour in the first node and the presence of tumour in
the two latter nodes. The pathological evaluation produced a
pseudocolor map of the pathology specimens that correlated well
with the contrast enhancement patterns seen on ultrasound with
contrast enhancement of normal lymphatic tissue and absence of
enhancement in tumour tissue.
[0091] The method described in example 2b was applied in 6 melanoma
Sinclair pigs. In total 31 lymph nodes were investigated. The
accuracy of correctly detecting the presence or absence of
metastatic melanoma tumours in the lymph nodes was 86%.
[0092] There were no statistically significant difference between
contrast enhanced ultrasound and pathology.
Example 3
Comparison of Albunex.TM. and Sonazoid.TM.
[0093] 1 ml Albunex.TM. as described in Example 1 was injected
interdigitially in the hind limb of an anesthesised dog. The
injection site was gently massaged for 5 minutes. The draining
lymph node (Inn popliteus) was imaged with an ATL HDI 5000 Scanner
equipped with a L12-5 transducer operating in pulse inversion mode.
No contrast enhancement was seen at imaging 5 and 15 minutes post
injection, respectively.
[0094] 20 minutes after the Albunex.TM. injection, 1 ml
Sonazoid.TM. as described in Example 1 was injected interdigitially
in the same hind limb following identical imaging procedures as
described in Example 3a. A strong contrast enhancement was seen at
imaging 5 minutes post injection and enhancement was maintained at
imaging 15 minutes post injection. Peak contrast enhancement was
more than 7 dB.
* * * * *