Note: Descriptions are shown in the official language in which they were submitted.
CA 02273609 1999-06-04
Radiopharmaceuticals and Methods for Imaging
Field of the Invention
The invention relates to medical diagnostic imaging and in particular imaging
agents, kits
and methods for medical diagnostic imaging.
Background of the Invention
Diagnostic imaging exploits agents that bind or localize to sites selectively
within the body.
Techniques of imaging include positron emission tomography, nuclear magnetic
resonance
imaging, scintigraphy, single photon emission computed tomography, perfusion
contrast
echocardiography, ultrafast X-ray computed tomography, and digital subtraction
angiography.
1o These techniques are used to diagnose many diseases, disorders and abnormal
physical states,
such as cancer, neurological abnormalities, inflammation, infection, and
degenerative diseases.
Lymphoscintigraphy is a useful diagnostic imaging technique. The recognition
of the
importance of lymphoscintigraphy, for identification of the sentinel lymph
nodes) in melanoma
and breast cancer plays a significant role in the clinical management of
patients. The
15 widespread clinical acceptance of this technique and the lack of an
agreement on which
radiopharmaceutical agent has the most ideal properties has resulted in a wide
variety of
agents being used clinically with many other agents being investigation or
developed.
Lymphoscintigraphy has resurged as a valuable technique for the identification
of
lymphatic drainage pathways and the location of the sentinel node(s). The
renewed interest is
20 largely due to a multidisciplinary approach validating the importance of
the pathology of the
sentinel node. A pathological finding in the node is an important factor in a
patient's prognosis,
management and clinical care. The exploration and use of the technique has a
30 year history
over which time numerous radioactive tracers, colored dyes, and combined
approaches have
been investigated to identify lymphatic drainage and the sentinel node(s).
25 Animal studies have demonstrated that the particle size of a
radiopharmaceutical agent
is a critical factor in determining the migration rate from the injection site
and the rate of uptake
in lymph nodes. The particles should be larger than 0.005 nm in size, as
smaller particles may
penetrate or leak into the capillary membranes and therefore become
unavailable to migrate
through the lymphatic channel (1). Particles between 0.005 nm and 5 nm in size
are able to
3o migrate from an intradermal injection site into the lymphatic vessels. The
particles move
CA 02273609 1999-06-04
through the lymphatic system by rhythmical contractions and relaxations of the
smooth muscle
cells in the capillary walls. Muscular activity and respiratory movement
increase the lymph
pressure, thus increasing lymphatic flow. Anesthesia may decrease lymphatic
flow, however
the magnitude of depression can vary considerably depending upon the
anesthetic used (2).
Following migration from the interstitial space and into the lymphatic vessel
the particles are
transported to the lymph nodes where they can be retained by mechanical
trapping or
phagocytosis. The optimal particle size identified from the animal studies for
lymphatic
drainage has been estimated to be about 5 nm (2). Larger particles
approximately 500 nm in
size demonstrate a much slower rate of migration from the injection site and
significantly lower
1o accumulation in the lymph nodes. Larger particles, greater than a few
hundred nanometers in
size are retained primarily at the injection site in the interstitial space.
The size dependence for
particle absorption and movement has been verified in both animal and human
studies. The
number of injected colloid particles has also been reported to influence the
rate of out flow from
the injection site and phagocytosis within the lymph nodes.
15 Radiopharmaceutical Agents
There have been many radiopharmaceuticals that have been evaluated and used
for
lymphoscintigraphy studies. Au-198 colloid was the first agent which was
widely used but was
rapidly replaced by other radionuclides and radiopharmaceuticals. The agents
that are
commonly used are Tc-99m antimony trisulfide colloid, Tc-99m nanocolloid and
Tc-99m sulfur
2o colloid and these agents are available in different parts of the world. In
Europe the predominant
agent used is Tc-99m nanocolloid whereas in North America, Tc-99m sulfur
colloid is the
primary agent of choice.
Colloidal Gold Au-998
One of the first agents widely used for lymphoscintigraphy was colloidal Au-
198.
25 Colloidal gold has a relatively uniform particle size of 3 to 5 nm, which
is optimal (3-5) and was
used clinically for many years to study the lymphatic system. Au-198 has a 2.7
day half life,
emits beta particles matter and a 412 KeV gamma photon. Although it has
favorable particle size
properties, it is no longer widely used as it delivers a high radiation dose
at the site of injection and
has decreased spatial resolution due to its 412 KeV gamma photon. In addition,
tissue necrosis
2
CA 02273609 1999-06-04
at the injection site was sometimes observed due to the large absorbed dose
from electrons
emitted from the beta decay of Au-198 (6,7).
Tc-99m Antimony Trisulfide Colloid
The first particulate Tc-99m agent to be used for lymphoscintigraphy was Tc-
99m
antimony trisulfide colloid. The colloid has a particle size range of 3 to 30
nm and has been
used clinically in the last decade in various locations (6). Tc-99m labeling
of antimony trisulfide
colloid has been proposed to occur on the surface of the particles with the
final particle size
determined by the size of the antimony colloid used (8) (9). This agent was
being developed when
the clinical importance of lymphoscintigraphy studies was not widely
recognized, therefore the
1o radiopharmaceutical agent was never developed commercially worldwide.
Tc-99m Albumin Based Colloid Radiopharmaceuticals
Three types of albumin-based Tc-99m colloid radiopharmaceutical agents have
been
studied; nanocolloid, microaggregated albumin and macroaggregated albumin.
Tc-99m Nanocolloid is available as a kit containing human albumin nanocolloid
particles
1s and stannous chloride dihydrate. Approximately 95% of the colloidal albumin
particles are smaller
than 80 nm in size (10,11 ). Less than 4% of the particles are between 80-100
nm in size. There
is about 1 % of the particles that is larger than 100 nm (10,11 ). The
preparation of Tc-99m
Nanocolloid involves the addition of pertechnetate to a lyophilized vial of
human albumin
nanocolloid particles, stannous chloride, glucose, polyoxamer 238, sodium
phosphate and
2o sodiumphytate. It is critical to exclude oxygen from the vial during the
addition of the
pertechnetate as the oxygen will form a stannous technetium colloid and not
allow the Tc-99m to
bind to the albumin particles. Tc-99m Nanocolloid is being used routinely
clinically in Europe.
Tc-99m microaggregated albumin and Tc-99m macroaggregated albumin have also
been
evaluated for lymphoscintigraphy studies. Tc-99m microaggregated albumin has a
particle size
2s distribution range of 0.2 to 2 ~m in size with 90% less than 1 ~m in size.
Tc-99m
macroaggregated albumin forms larger particles, ranging in size from 10 to 90
~,m. Both of these
agents have been shown to have slow migration from the injection site as would
be expected due
to their larger particle size. Tc-99m microaggregated albumin moves more
rapidly than Tc-99m
macroaggregated albumin and these agents have not been found to be very useful
for
3
CA 02273609 1999-06-04
lymphoscintigraphy (12,13). However, there are some authors which report
reasonably good
studies utilizing Tc-99m microcolloid (14,15).
Tc-99m Sulfur Colloid
Tc-99m sulfur colloid has been evaluated as a potential radiopharmaceutical
agent and
numerous reports have demonstrated its usefulness for lymphoscintigraphic
studies (9,16-22).
Historically, there have been several methods of producing Tc-99m sulfur
colloid particles by
utilizing various starting materials and stabilizing agents. The routine
preparation of Tc-99m sulfur
colloid results in a preparation which has a very wide particle size
distribution, ranging from <0.1
nm to greater than 5 ~m (Table 1 ). The most common kit reagent uses sodium
thiosulfate as the
1o source of sulfur. The ingredients have been used in different amounts to
develop kits which
produce the appropriate particle size and having different degrees of
stability (16,18,23) (17). Tc-
99m sulfur colloid is formed by the reaction of thiosulfate under acidic
conditions. Under these
conditions there are two types of reactions which take place. One reaction
involves the reaction
of thiosulfate to form sulfur and bisulfate which also forms polythionates and
subsequently high
1s molecular weight sulfur and oxygen polymers. The reaction rates, the nature
of the reactions, and
the yields of the various products depends upon the thiosulfate concentration,
acidity, and
temperatures. The second type of reaction is an internal oxidation and
reduction of thiosulfate in
the presence of technetium which forms insoluble sulfides or stable sulfide
complexes. Larson et
al. (8)reported that upon heating there is rapid incorporation of Tc-99m into
the sulfur colloid
2o particles. The nucleation process of the reaction has been studied and it
has been reported that
the Tc colloid particles form more rapidly than the sulfur colloid. Thus, the
sulfur colloid forms at
least in part with Tc-colloid serving as its nucleus. In addition, some sulfur
molecules form
independently. The smaller particles generally contain relatively low amounts
of sulfur and larger
amounts of technetium (8,9).
2s The use of Tc-99m sulfur colloid for lymphoscintigraphy initially used the
standard
commercial formulation, however the success and reliability rate with this
formulation was
extremely variable. Recently, filtering the preparation prior to injection
and/or modifying its method
of production has provided a good radiopharmaceutical agent. Several reports
have been
published which state that filtering a standard sulfur colloid preparation
through various sizes of
3o membrane filters increases the quality of the preparation for
lymphoscintigraphy (17,23). An
unfiltered Tc-99m sulfur colloid kit was prepared and then filtered through a
0.1 ~,m membrane
4
CA 02273609 1999-06-04
filter, after filtration the average particle size distribution had a range of
10 nm with a small (<
0.1 %) secondary population averaging 89-173 nm (23). Other studies have
demonstrated that
the use of a 0.22 um membrane filter also gave a product that could image
lymphatic drainage to
identify the sentinel node(17,18).
An alternative to filtering the standard Tc-99m sulfur colloid preparation
would be to alter
the labeling procedure. Altering the preparation parameter has been reported
to provide an agent
which contains particles small enough to visualize the lymphatic drainage and
particles large
enough for prolonged retention within the lymph node to enhance the utility of
the study. To
achieve this Eshima et al (16) evaluated the particle size distribution and
stability of the Tc-99m
io sulfur colloid kit utilizing different labeling conditions. The particle
size distribution and the stability
of the different Tc-99m sulfur colloid kit preparation parameters were
evaluated over a 6 hour
period utilizing polycarbonate filtration. The optimal labeling conditions
required the addition of
pertechnetate that had the longest ingrowth of Tc-99 pertechnetate, heating
the reaction for 3
minutes, allowing the reaction to cool for 2 minutes and then neutralizing the
reaction.
Tc-99m sulfur colloid has a variable particle size distribution pattern. All
Tc-99m sulfur
colloid preparations have a bimodal distribution pattern, regardless of the
preparation procedure
utilized (16). The use of a reduced heating protocol results in a dramatic
increase in the
percentage of particles smaller than 0.3 p.m in size, regardless of the age of
the generator elution
while prolonged heating significantly decreases the percentage of small
particles. In addition, this
2o preparation does not appear to form a significant amount of particles
smaller than 0.005 nm as
there is no evidence of visualization or localization of activity outside of
the lymphatic system.
This preparation procedure demonstrates rapid movement of the particles and
utilizing rapid
dynamic images it has been able to map and imaging the lymphatic drainage
system (24). In
addition to rapid movement, the preparation has demonstrated prolonged
retention within the
nodes. Furthermore, the particle size distribution of the modified preparation
of the kit formulation
following filtration did not change over a six hour period (25).
Currently, several studies are underway to identify additional methods which
would further
reduce the average particle size formed during the preparation of Tc-99m
sulfur colloid. Particle
size studies utilizing Tc-99m sodium pertechnetate which has been obtained
from generators
3o which have up to 7 days of ingrowth of Tc-99 pertechnetate are being
investigated. Also, studies
in which an additive such as Re has been introduced into the kit demonstrated
that there are more
5
CA 02273609 1999-06-04
nucleation sites for the particles to form. To optimize the percentage of
small particles, a rhenium
sulfide colloid kit formulation has been formulated and developed. TCK-17 is a
kit formulation
which utilizes rhenium sulfide colloid for the preparation of the agent. The
addition of cold
rhenium provides additional nucleation sites which allows for the formation of
smaller size
particles (26). In-vitro particle size distribution studies were conducted
with the Tc-99m TCK-17
kit, these studies demonstrated a more homogeneous particle size distribution
and no bimodal
distribution pattern. In addition, there was a significantly larger percentage
of particles smaller
than 0.03 ~.m (63%). Preliminary animal studies demonstrated that there is a
very rapid migration
from the injection site; however additional studies need to be conducted to
determine if the agent
1o has significant retention in the lymph nodes (27).
Miscellaneous Lymphoscintigraphy Agents
Technetium-99m human serum albumin (Tc-99m HSA) has been evaluated for
lymphoscintigraphy studies and, following an intradermal injection, has been
used to image
lymphatic flow. However, the agent is not particulate and there is minimal
retention of the agent
15 within the lymph nodes. In addition, delayed images may miss the sentinel
node which makes it a
suboptimal agent for use with the intraoperative probe (27,32). There are a
number of other
agents that have been used or are being developed for lymphoscintigraphy
including Hg-197
sulfide colloid, Ga-67 citrate, and monoclonal antibodies labeled with In-111,
I-131 and I-125 (2)
Although several of these agents have been utilized with various degrees of
success the
2o use of Tc-99m agents are more common due to its availability and favorable
radionuclidic
properties. Tc-99m gives comparatively low radiation exposures to patients and
staff, has an
optimal photon energy for scintillation camera imaging and surgical probes
have been
developed to localize the sentinel node during the surgical procedure. Several
other Tc-99m
based radiopharmaceutical agents have been evaluated as potential
lymphoscintigraphy agents
25 including hydroxyethyl starch (Tc-99m HES), dextran (Tc-99m DXT), and Tc-
99m stannous
phytate (1,33,34).
Problems have arisen utilizing a dye alone due to movement of the patient
following
imaging and positioning of the patient during surgery. The development of an
intraoperative
surgical probe has facilitated localization of the node but this procedure is
not ideal. There is a
6
CA 02273609 1999-06-04
need for an imaging method to precisely identify the lymphatic drainage or
nodes so that surgery
to excise a node can be completed quickly and easily.
Summary of Invention
The invention relates to a double tracer technique to locate a node,
preferably the sentinel
node. Visible blue dyes such as isosulphan blue (28) (29), lymphazurin (30),
or patent blue dye
(PBD) (31) have been coinjected along with radiopharmaceutical agents for
lymphoscintigraphy
studies. In most cases, the addition of a blue dye serves as a useful adjunct
to the
radiopharmaceutical in the identification and surgical removal of the sentinel
node. The invention
relates to a radiopharmaceutical agent including a probe (preferably
radioactive) and a dye and
1o methods of using the dye in diagnostic imaging, preferably
lymphoscintigraphy for diagnosis of
cancer. The agents are also useful to identify drug delivery sites and the
could be used to
incorporate a therapeutic agent such as a chemotherapeutic agent or other
useful toxins which
could be released at the site.
The invention relates to a method of imaging a tissue in a mammal, comprising:
a)
15 administering to the mammal an effective amount of an agent including (i) a
probe (ii) a dye,
wherein the agent is capable of interacting with the tissue for detection; b)
detecting the probe;
and c) detecting the dye. In one embodiment, the method of claim 1, wherein
the probe is
radioactive. The probe is preferably connected to the dye. The tissue includes
lymphatic tissue
or a lymphatic node. The dye can be a coloured dye. The dye is preferably
selected from the
2o group consisting of a nonfluorescent dye, a fluorescent dye, an ultraviolet
fluorescent dye, a
visible fluorescent dye, an infared fluorescent dye, a chemiluminescent dye, a
phosphorescent
dye and a bioluminescent dye. The dye may be selected from the group
consisting of:
7
CA 02273609 1999-06-04
Food dyes Methylene Blue Evans Blue a Coumarin-based
Flavoproteins a Porphyrin dye Tolonium Chloridedye
Indocyanine GreenBromosulfophthaleinCongo Red Luciferins
(ICG) (BSP) Hydroxy- Green Fluorescent
Fluorescein and Rose Bengal pyrenetrisulfonateProtein (GFP)
disodium fluoresceinIsosulfan Blue (HPT) an Acridinium-based
Fluorescein a Pyrene-based dye
dye
isothiocyanate Cibacron Blue;
(FITC) and
an Inorganic Dye.
The radioactive probe can be a gamma emitting radionuclide. The radioactive
probe
preferably is a metal or a radioisotopic metal selected from the group
consisting of Tc, Re, Mn,
Fe, Co, Ni, Zn, Cd, Mo, W, Cu, Ag, Au, Ti, Hg, Cr and Rh, a halogen, Br, I,
CI, F, At. The
s imaging is preferably done with a technique selected from the group
consisting of
lymphoscintigraphy, scintigraphy, X-ray contrast, Barium particle imaging,
positron emission
tomography, nuclear magnetic resonance imaging, single photon emission
computed
tomography, perfusion contrast echocardiography, ultrafast X-ray computed
tomography,
digital subtraction angiography, spiral CT, gamma probe detectors, hand held
gamma probe
1o detectors, fMRI (fast Magnetic Resonance Imaging) and standard X-Ray
equipment.
The probe includes a probe selected from the group consisting of hydroxyethyl
starch,
dextran, stannous phytate, sulfide colloid, sulfur colloid, citrate, a
monoclonal antibody, a
polyclonal antibody, human serum albumin antimony trisulfide colloid,
nanocolloid, albumin,
albumin-based colloid, nanocolloid albumin, microaggregated albumin and
macroaggregated
15 albumin. The probe can be selected from the group consisting of
hydroxyethyl starch (Tc-99m
HES), dextran (Tc-99m DXT), Tc-99m stannous phytate, lymphoscintigraphy, Hg-
197 sulfide
colloid, Ga-67 citrate, a monoclonal antibody labeled with In-111, I-131 or I-
125, Tc-99m human
serum albumin (Tc-99m HSA), Tc-99m antimony trisulfide colloid, Tc-99m
nanocolloid, Tc-99m
sulfur colloid, albumin-based Tc-99m colloid, nanocolloid, microaggregated
albumin and
2o macroaggregated albumin.
The agent can be administered by methods including intradermally,
subcutaneously,
bydirect lymphatic injection into a lymphatic channel, by intravenous
injection or by intraarterial
injection. The radioactive probe may be detected with a gamma camera. The dye
may be
detected with a uv light source.
8
CA 02273609 1999-06-04
Another aspect of the invention relates to a method of diagnosing cancer in a
mammal,
by administering to the mammal an effective amount of an agent including (i) a
probe and (ii) a
dye, wherein the agent is capable of migrating through a lymphatic channel
into a lymphatic
node for detection; detecting the probe and thereby detecting the location of
the lymphatic
node; detecting the dye and thereby detecting the location of the lymphatic
node; removing the
node; and determining the presence or absence of cancer in the node. The node
preferably
comprises a sentinel node. The cancer includes breast cancer or melanoma.
Another aspect of the invention relates to a method of imaging and assessing
lymphatic
drainage or a node, comprising: administering to the mammal an effective
amount of an agent
1o including (i) a probe and (ii) a dye, wherein the agent is capable of
migrating through a
lymphatic channel for detection; detecting the probe; and detecting the dye.
The method may
further include removing the node, preferably a sentinel node. The probe is
preferably at least
about 0.05nm in size and about 5nm in size The dye can be any suitable dye
disclosed in this
application or known in the art, such as FITC. The probe can be any suitable
probe disclosed
15 in this application or known in the art and is preferably selected from the
group consisting of a
peptide, a polypeptide, a protein, an antibody, Tc-99m sulfur colloid and Tc-
99m colloidal
albumin. The antibody can be a polyclonal antibody or a monoclonal antibody or
a fragment of
either of the foregoing.
Another aspect of the invention includes a method of medical treatment of
cancer in a
2o mammal, by administering to the mammal an effective amount of an agent
including (i) a probe
which selectively interacts with a cancer cell and (ii) a dye; detecting the
probe and thereby
detecting the location of the cancer cell; detecting the dye and thereby
detecting the location of
the cancer cell; administering an anti-cancer agent or treatment proximate to
the cancer cell.
The probe can include an antibody. The cancer can include liver cancer,
melanoma or breast
25 cancer.
Another aspect of the invention relates to a method of producing an imaging
agent,
comprising connecting a probe with a dye.
Another aspect of the invention is a composition including a carrier and an
agent
including (i) a radioactive probe and (ii) a dye. In a variation, the
composition includes a carrier
3o and an agent including (i) a radioactive probe and (ii) a dye, wherein the
agent is capable of
migrating through a lymphatic channel. The composition preferably includes a
pharmaceutical
composition. The carrier may include albumin particles, inert microspheres,
sulfur colloid
9
CA 02273609 1999-06-04
particles or Re-Sulfur colloidal paricles. The composition may include a probe
and dye for
coinjection, as disclosed in this application.
Another aspect of the invention relates to a kit for imaging, inlcuding an
agent including
(i) a radioactive probe and (ii) a dye. In a variation, the invention includes
a kit for imaging,
including an agent including (i) a radioactive probe and (ii) a dye, wherein
the agent is capable
of migrating through a lymphatic channel. Another aspect of the invention
relates to a kit for
producing an imaging agent, including (i) a radioactive probe compound and
(ii) a dye
compound. The kit may include a probe and dye for coinjection, as disclosed in
this application.
The invention also includes a method of detecting the presence or assessing of
the
1o severity of a disease, disorder or abnormal physical state in a mammal
comprising: (a)
administering an effective amount of a composition of the invention; and (b)
detecting the
presence or assessing the severity of the disease, disorder or abnormal
physical state. The
probe can be radioactive.
The invention also includes a method of imaging a tissue in a mammal, by:
15 administering to the mammal an effective amount of (i) a probe (ii) a dye,
other than a blue dye,
wherein the agent is capable of interacting with the tissue for detection;
detecting the probe; and detecting the dye. The probe can be radioactive.
The dye is preferably selected from the group consisting of a nonfluorescent
dye, a fluorescent
dye, an ultraviolet fluorescent dye, a visible fluorescent dye, an infared
fluorescent dye, a
2o chemiluminescent dye, a phosphorescent dye and a bioluminescent dye.
Brief Description of the Drawings
Preferred embodiments of the invention will be described in relation to the
drawings in
which:
Figure 1 (a) -(c) Node activity as measured by y camera.
25 Figure 2(a)-(d) Node activity as measured by ycamera.
Figure 3 y camera image and corresponding image taken under UV light.
Figure 4 (a) -(c) Node activity as measured by y camera.
Figure 5(a) Activity of sulfur colloid fractions; (b) Activity of fractions of
Tc99m sulfur colloid.
CA 02273609 1999-06-04
Figure 6(a) Activity of fractions of Tc99m sulfur colloid; (b) Fluorescence of
FITC-Tc99m sulfur
colloid vs. fraction; (c) Fluorescence vs. Fraction # for 1 mg fluorescein;
(d) Amount of activity
per fraction of 99mTc-fluorescein-SC; (e) Amount of fluorescence per fraction
of 99mTc-
fluorescein-SC; (f) Node activity as measured by y camera.
Detailed Description of the Invention
The invention relates to a radiopharmaceutical agent including a probe and a
dye and
methods of using the dye in diagnostic imaging, preferably lymphoscintigraphy
for diagnosis of
cancer. The probe is preferably radioactive. The agents are also useful to
identify drug delivery
sites and the could be used to incorporate a therapeutic agent such as a
chemotherapeutic agent
or other useful toxins which could be released at the site.
The probe and dye are preferably connected. The invention also includes
methods of
coinjecting probe and dye for imaging. If we coinject dyes, such as the
fluorescent dyes it allows
one to identify lymphatic flow and lead to the location of the sentinel node.
In addition by co
injection the dye may become trapped along with the radiolabeled probe which
is useful in
is diagnostic imaging.
In addition to the blue dye there are numerous other dyes that can be
incorporated into
lymphoscintigraphy studies to identify lymphatic drainage, the sentinel
node(s), or other nodes.
These dyes may be incorporated either as an adjuvant in solution or
incorporated into a particle
for prolonged retention within the lymph node. Dyes that are encompassed in
this patent
2o application include nonflourescent, fluorescent (ultraviolet, visible and
infrared),
chemiluminescent, phosphorescent and bioluminescent dyes. Possible dyes that
can be used
include:
11
CA 02273609 1999-06-04
1. Food dyes 6. Methylene Blue11. Evans Blue 16. Coumarin-based
2. Flavoproteins 7. Porphyrin dyes12. Tolonium Chloridedyes, i.e., 3-
3. Indocyanine i.e., Pd- 13. Congo Red (carboxymethylest
Green (ICG) uroporphyrin 14. Hydroxy- er)-7-
4. Fluorescein 8. Bromosulfophthalepyrenetrisulfonatejulolidinocoumarin
and
disodium in (BSP) (HPT) 17. Luciferins
fluorescein 9. Rose Bengal 15. Pyrene-based 18. Green Fluorescent
5. Fluorescein 10. Isosulfan dyes i.e., 1,3- Protein (GFP)
Blue
isothiocyanate dihydroxy 6,8- 19. Acridinium-based
(FITC) pyrenedisodiumsuldyes
fonate 20. Cibacron Blue
21. Inorganic
Dyes
Such As Re(I)
metal ligand
complexes
This patent application demonstrates that FITC can be incorporated into Tc-99m
sulfur
colloid or albumin particles and be administered intradermally or
subcutaneously. This
combination approach allows the detection of the lymphatic drainage pathway
and sentinel
s nodes) as well as other nodes easily by gamma camera scintigraphy. These
nodes can be
marked on the skin surface to more easily locate the node during the surgical
procedure.
We have incorporated FITC into Tc-99m sulfur colloid and Tc-99m colloidal
albumin
particles. The methodology for incorporation of FITC are listed below.
1 ) Add Tc-99m in 3ml saline to sulfur colloid kit vial
1o Add syringe A to vial
Boil vial for 1.5 min
Add 15mg FITC/600u1 DMF to vial
Boil vial for 1.5 mins
Cool vial for 2 mins at room temperature
1s Filter through a 0.22 micron filter and use or
Add 0.7 ml of this to 0.3m1 FITC/DMF (25mg/ml) and use
2) Add Tc-99m to Microlite kit vial and Vortex briefly
Add 250u1 of NaHC03 (1 M) pH 8.5
2o Vortex for 10 seconds
Add 500u1 of 10mglml FITC in DMF
12
CA 02273609 1999-06-04
Vortex for 10 seconds
Incubate for 1 hr at 4°C and use or
Filter through a 0.22 micron filter and use
The methodology given above may be readily adapted or modified by one skilled
in the
art.
Following the preparation of the radiolabeled and UV agent the material was
injected
intradermally or subcutaneously into a rat. Dynamic images were obtained and
movement of
the radioactivity was monitored with the gamma camera. At 1.5 hours post
injection, the
animals were sacrificed and an incision was made and a UV light source was
used to readily
localize the node by emitting a yellow to green fluorescent color.
A modified labeling procedure was also used to incorporate fluorescein into
the Microlite
particles and similar results were obtained; gamma camera visualization of the
flow of particles
and after an incision was made the UV light was able to rapidly localize the
sentinel node.
There are other methods that can be utilized to incorporate or attach dyes to
radioactive
particles which will be apparent to those skilled in the art.
Metal
Suitable metals include the transition metals, lanthanide metals, halogens and
actinide
metals. Complex-forming metals useful in preparing agents for radiotherapy or
imaging are
preferably the metals (or radioisotopes of the metals) Tc, Re, Mn, Fe, Co, Ni,
Zn, Cd, Mo, W, Cu,
2o Ag, Au, Ti, Hg, Cr, Rh., At, Gd, Ga, Ho, In, Lu, Sm, Yb and Y.
Non-Metal Agents may also be useful and can include, F, CL, Br and At. These
can be
attached utilizing standard labeling procedures.
The probe does not have to be radioactive for all applications of the
invention. For
example, in MRI, fMRI, X-Ray, CT and Ultrasound it would not have to be
radioactive.The
halide salt, in particular chloride salt, or oxide of these complex-forming
metals are forms capable
of complexing with a desired ligand and are suitable for the present
invention. Radionuclide
labeled imaging agents employ complex-forming metal isotopes that include f3-
emitters such as
rhenium-186 and -188; and y-emitters such as technetium-99m. The complex-
forming metal most
preferred for radiodiagnostic imaging is technetium-99m due to its
advantageous half life of 6
13
CA 02273609 1999-06-04
hours and inexpensive preparation from a molybdenum-99 generator. Technetium
and rhenium
labeling is accomplished by procedures established in the art. Either complex-
forming metal may
be introduced to the ligand in aqueous solution in oxo, dioxo or nitrido form,
for example
pertechnetate (~'"'Tc04 ) or perrhenate, with a suitable reducing agent such
as stannous chloride.
Alternatively, radiodiagnostic agents may be formed by a transchelation
reaction which entails use
of the complex-forming metal in the form of a weak metal complex such as
technetium-gluconate,
heptagluconate, tartrate or citrate to give a desired labeled ligand.
Transchelation reactions are
typically heated to facilitate conversion of technetium from the weak complex
to a complex with
the ligand, for example in a boiling hot water bath.
to
There are two general methodologies which can be utilized to make a combined
probe-
dye conjugate. The first methodology requires the covalent attachment of the
dye directly to the
probe. The probe can then subsequently be radiolabeled utilizing standard
labeling
methodologies. Alternatively, the probe may be radiolabeled followed by the
addition of the dye
15 via a covalent attachment to the radiolabeled probe. The second methodology
involves
incorporation into the probe utilizing a number of different techniques. Some
of which include;
modifications of the labeling procedure for sulfur colloid particles so as to
incorporate the dye
during the heating step while the particles are being formed during the
radiolabeling procedure.
An alternative method would be to allow the dye particles to incubate with the
probe and therefore
2o be allowed to bind to the particles through electrostatically or other non-
covalent properties. The
probe can then be radiolabeled prior to the binding or after the radiolabeling
of the probe.
Pharmaceutical Compositions
The invention also includes compositions, preferably pharmaceutical
compositions for
radiotherapy or imaging, including an agent prepared according to a method of
the invention.
25 Pharmaceutical compositions may be formulated according to known
techniques.
The invention includes a method of detecting the presence or assessing of the
severity of
a disease, disorder or abnormal physical state in a mammal comprising: (a)
administering an
agent or composition of the invention and (b) detecting the presence or
assessing the severity of
the disease, disorder or abnormal physical state. The presence or severity of
the disease,
3o disorder or abnormal physical state is preferably detected or assessed with
lymphoscintigraphy, or
14
CA 02273609 1999-06-04
a technique selected from the group consisting of positron emission tomography
(PET), magnetic
resonance imaging (MRI) scintigraphy, single photon emission computed
tomography, perfusion
contrast echocardiography, ultrafast X-ray computed tomography, digital
subtraction angiography,
spiral CT, hand held gamma probe detectors, fMRI (fast Magnetic Resonance
Imaging) and
standard X-Ray equipment. Suitable methods and materials for imaging are
described in:
Handbook of Nuclear Medicine second ed., 1993, Mosby Press, Frederic I. Datz;
Fundamentals of
Nuclear Pharmacy third ed., 1992 Springer-Verlag, Gopal B. Saha; Principles
and practice of
Nuclear Medicine second ed., 1995, Mosby press, Paul J. Early and D. Bruce
Sodee; which are
incorporated by reference in their entirety.
to The invention also includes a method of radiotherapy of a disease, disorder
or abnormal
physical state in a mammal including administering an agent or composition of
the invention.
Methods of performing radiotherapy are described in, for example, Principles
and Practice of
Nuclear Medicine, 2"d Ed., P.J. Early and D.B. Sodee, Chapter 32, which is
incorporated by
reference in its entirety.
15 The particles could be used therapeutically by incorporating an alpha or
beta radionuclide
into the probe. After visual localization of the node and demonstrating that
the probe is being
retained in the lymph node the therapeutic radionuclide could deliver a large
radiation dose to
destroy the tumor cells. This would be especially useful for those probes
which may not be able
to be surgically removed because of their location or the morbidity which
might result in removal of
2o the node.
The pharmaceutical compositions are used to treat diseases and provide images
in
diseases, disorders or abnormal physical states including cancer. Other
diseases, disorders
and abnormal physical states will be apparent to those skilled in the art
andlor on review of this
application or references cited in this application.
25 Pharmaceutical compositions used for imaging or to treat patients having
diseases,
disorders or abnormal physical states preferably include an agent of the
invention and an
acceptable vehicle or excipient (Remington's Pharmaceutical Sciences 18~" ed,
(1990, Mack
Publishing Company) and subsequent editions). Vehicles include saline and D5W
(5%
dextrose and water) or other acceptable injection vehicles. Excipients include
additives such as
3o a buffer, solubilizer, suspending agent, emulsifying agent, viscosity
controlling agent, lactose
CA 02273609 1999-06-04
filler, antioxidant, preservative or disintegrants. The compositions may
further include a
reducing agent, a bulking agent or a pH stabilising agent. There are preferred
excipients for
stabilizing peptides for parenteral and other administration. The excipients
preferably include
serum albumin, glutamic or aspartic acid, phospholipids and fatty acids.
Intradermal,
subcutaneous, direct lymphatic injection or parenteral (injectable)
administration can be utilized.
The methods for the preparation of pharmaceutically acceptable compositions
which can be
administered to patients are known in the art.
The pharmaceutical compositions can be administered to humans or animals
(preferably
mammals). Dosages to be administered depend on individual patient condition,
indication of
to the drug, physical and chemical stability of the drug, toxicity, the
desired effect and on the
chosen route of administration (Robert Rakel, ed., Conn's Current Therapy
(1995, W.B.
Saunders Company, USA)). Preferably the amount of complex-forming metal
labeled agent
administered to the mammal is approximately 100 to 1000 microcuries of
activity for an
intradermal injection injected around the primary site for melanoma studies,
in the case of
i5 identification of lymph nodes in breast cancer 1 to 5 mci may be injected
around the primary site.
about 0.01 mcglkg/minute to 1,000 mcglkglminute and more preferably about 0.01
to 50
mcglkglminutes.
Kits
The invention also includes kits for imaging, and preferably
lymphoscintigraphy. Since the
2o radioisotopic metals often have a very short half life, it is advantageous
to omit them from the
kit.
The metal is preferably one or more of the metals and radioisotopic metal
forms of Tc, Re,
Mn, Fe, Co, Ni, Zn, Cd, Mo, W, Cu, Ag, Au, Ti, Hg, Cr and Rh.
Preferred embodiments of the invention are described below in examples which
are not
25 intended to in any way limit the scope of the invention
Example 1
We demonstrated that the FITC-TKPR co-injected with Tc-99m sulfur colloid
could be
detected in the sentinel node of the rat at 30 minutes, 1 hour, 1.5 hours and
2 hours post
injection. The accompanying gamma camera images are in Figures 1 (a)-(c). The
FITC-TKPR
16
CA 02273609 1999-06-04
Tc-99m sulfur colloid was generated using the protocol outlined below and 100
~I was injected
into each rat hindpad.
Add Tc-99m in 3ml saline to sulfur colloid kit vial
Add syringe A to vial
Boil vial for 3 minutes
Cool vial for 2 minutes at room temperature
Add syringe B
Filter Tc-99m sulfur colloid through 0.22-micron filter
Add 2mg FITC-TKPRI250u1 to vial and use
1o Example 2
We demonstrated that the FITC-conjugated Tc-99m sulfur colloid could be
detected in
the sentinel node of the rat at 30 minutes, 1 hour, 1.5 hours and 2 hours post
injection. This
data supports the accompanying gamma camera images that are located in Figures
2(a)-2(d)
and Figure 3. The FITC Tc-99m sulfur colloid was generated using the protocol
outlined below
and 100 ~I was injected into each rat hindpad.
Add Tc-99m in 3ml saline to sulfur colloid kit vial
Add syringe A to vial
Boil vial for 1.5 min
Add 15mg FITCI600u1 DMF to vial
2o Boil vial for 1.5 minutes
Cool vial for 2 minutes at room temperature
Filter through a 0.22 micron filter and use or
Add 0.7 ml of this to 0.3m1 FITCIDMF (25mglml) and use
Example 3
We demonstrated that the FITC-conjugated Tc-99m Microlyte could be detected in
the
sentinel node of the rat at 30 minutes, 1 hour, 1.5 hours and 2 hours post
injection. This data
support the accompanying gamma camera images in Figures 4(a)-(c). The FITC-
conjugated Tc-
99m Microlyte was generated using the protocol outlined below and 100 pl was
injected into each
rat hindpad.
17
CA 02273609 1999-06-04
9m to Microlyte kit vial
Vortex briefly
Add 250u1 of NaHC03 (1 M) pH 8.5
Vortex for 10 seconds
Add 500u1 of 10mglml FITC in DMF
Vortex for 10 seconds
Incubate for 1 hr at 4°C and use or
Filter through a 0.22 micron filter and use
Example 4
io We demonstrated that To-99m sulfur colloid particles can be separated using
a Pharmacia
PD-10 column (G-25 column). A sample of To-99m sulfur colloid (2.5 ml) was
loaded upon the
column and then eluted off in 1 ml fractions. This data is presented in graph
format in Figures
5(a) and (b). This data show that the Tc-99m sulfur colloid particles can be
separated using a PD-
column. The To-99m sulfur colloid was prepared using the following protocol.
Add Tc-99m in 3ml saline to sulfur colloid kit vial
Add syringe A to vial
Boil vial for 3 minutes
Cool vial for 2 minutes at room temperature
Add syringe B
2o Filter Tc-99m sulfur colloid through 0.22-micron filter and use
Example 5
We determined whether fluorescein could be incorporated into Tc-99m sulfur
colloid
particles during their formation (Figure 6(a),(b)). The fluorescein Tc-99m
sulfur colloid was
generated using the protocol outlined below. This data characterizes the
elution pattern of the Tc-
99m sulfur colloid off the PD-10 column. '
- _ This data characterizes
the elution pattern of the fluorescein off the PD-10 column. The results from
this experiment
showed that fluorescein was not incorporated into the Tc-99m sulfur colloid
particles using the
labeling protocol outlined below. Imaging was achieved by coinjecting the
compounds.
18
CA 02273609 1999-06-04
Add Tc-99m in 3ml saline to the sulfur colloid kit vial
Add syringe A to vial
Boil vial for 2 minutes
Add 100u1 of 50mgI0.1 ml fluorescein
Boil vial for 1 minute
Cool vial for 2 minutes at room temperature
Add syringe B to vial
Filter though a 0.22 micron filter and use
to We determined the elution pattern of fluorescein on a Pharmacia PD-10
column (G-25
column) as shown by Figure 6(c), .
Example 6
We determined whether fluorescein could be incorporated into Tc-99m sulfur
colloid
particles during their formation (Figures 6(d)-(f)). The fluorescein Tc-99m
sulfur colloid was
generated using the protocol outlined below. This data characterizes the
elution pattern of the
Tc-99m sulfur colloid off the PD-10 column. The data in the chart on page 50
and 51 of lab
r
~n
. The
fluorescein Tc-99m sulfur colloid was generated using the protocol outlined
below and 100 ul
was injected into each rat hindpad.
The results from this experiment showed that fluorescein was not incorporated
into the
Tc-99m sulfur colloid particles using the labeling protocol outlined below,
however, the addition
of fluorescein to the Tc-99m sulfur colloid kit did not interfere with the
generation of functional
particles.
Add Tc-99m in 3ml saline to the sulfur colloid kit vial
Add syringe A to vial
3o Boil vial for 1.5 minutes
19
CA 02273609 1999-06-04
Add 200u1 of 50mg10.1 ml tluorescein
Boil vial for 1.5 minutes
Cool vial for 2 minutes at room temperature
Add syringe B to vial
Filter though a 0.22 micron filter and use
The present invention has been described in detail and with particular
reference to the preferred
embodiments; however, it will be understood by one having ordinary skill in
the art that changes can be
made thereto without departing from the spirit and scope of the invention.
1o All publications, patents and patent applications are incorporated by
reference in their
entirety to the same extent as if each individual publication, patent or
patent application was
specifically and individually indicated to be incorporated by reference in its
entirety.
CA 02273609 1999-06-04
References
1. Henze E, Schelbert HR, Collins JD, et al. Lymphoscintigraphy with Tc-99m-
labeled dextran.
J. Nucl. Med. 23:923-929, 1982
2. Strand SE, Bergqvist L. Radiolabeled colloids and macromolecules in the
lymphatic system.
Crit. Rev. Ther. Drug Carrier. Sysf. 6:211-238, 1989
3. Kirk J, Gray WM, Watson ER. Cumulative radiation effect. 3. Continuous
radiation therapy--
short- lived sources. CIin.RadioL 24:1-11, 1973
4. Kirk J, Gray WM, Watson ER. Cumulative radiation effect. part IV.
Normalisation of
1o fractionated and continuous therapy - area and volume correction factors.
Clin.Radiol.
26:77-88, 1975
5. Strand SE, Persson BR. Quantitative lymphoscintigraphy I: Basic concepts
for optimal
uptake of radiocolloids in the parasternal lymph nodes of rabbits. J.NucLMed.
20:1038-
1046, 1979
6. Bergqvist L, Strand SE, Persson B, et al. Dosimetry in lymphoscintigraphy
of Tc-99m
antimony sulfide colloid. J.Nucl.Med. 23:698-705, 1982
7. Meyer CM, Lecklitner ML, Logic JR, et al. Technetium-99m sulfur-colloid
cutaneous
lymphoscintigraphy in the management of truncal melanoma. Radiology 131:205-
209, 1979
8. Larson SM, Nelp WB. Radiopharmacology of a simplifield technetium-99m-
colloid
2o preparation for photoscanning. J.NucLMed. 7:817-826, 1966
9. Steigman J, Eckelman W.C.: Chapter IV. Technetium (VII) Compounds, in
Steigman J,
Eckelman W.C. (eds): The Chemistry of Technetium in Medicine. Washinton D.C.,
National
Academy Press, 1992, pp 10-15
10. Solco NANOCOLL (kit for the preparation of technetium Tc-99m nanocolloid
albumin
summary of the product characteristics. 12-96. Eidhoven, The Netherlands,
Amersham
Cygne. (GENERIC)
Ref Type: Pamphlet
21
CA 02273609 1999-06-04
11. Wilhelm AJ, Mijnhout GS, Franssen EJ. Radiopharmaceuticals in sentinel
lymph-node
detection - an overview. Eur.J.Nucl.Med. 26 Suppl 1:S36-S421999
12. ALBU-Res (Kit for the preparation of technetium Tc-99m microcolloid
albumin) summary
of the product characteristics. 1996. Eindhoven, The Netherlands, Amersham
Cygne.
(GENERIC)
Ref Type: Serial (Book,Monograph)
13. Sadek S, Owunwanne A, Abdel-Dayem HM, et al. Preparation and evaluation of
Tc-99m
hydroxyethyl starch as a potential radiopharmaceutical for lymphoscintigraphy:
comparison
with Tc-99m human serum albumin, Tc-99m dextran, and Tc-99m sulfur
microcolloid.
to Lymphology 22:157-166, 1989
14. Paganelli G, De CC, Cremonesi M, et al. Optimized sentinel node
scintigraphy in breast
cancer. Q.J.Nucl.Med. 42:49-53, 1998
15. Pijpers R, Meijer S, Hoekstra OS, et al: Sentinel Node Identification with
Tc-99m
Colloidal Albumin Lymphoscintigraphy and Gamma Probe Guidance in Breast
Cancer.
J.NucLMed.37:2591996(Abstract)
16. Eshima D, Eshima LA, Gotti NM, et al. Technetium-99m-sulfur colloid for
lymphoscintigraphy: effects of preparation parameters. J.NucLMed. 37:1575-
1578, 1996
17. Goldfarb LR, Alazraki NP, Eshima D, et al. Lymphoscintigraphic
identification of sentinel
lymph nodes: clinical evaluation of 0.22-micron filtration of Tc-99m sulfur
colloid. Radiology
208:505-509, 1998
18. Hung JC, Wiseman GA, Wahner HW, et al. Filtered technetium-99m-sulfur
colloid
evaluated for lymphoscintigraphy. J.Nucl.Med. 36:1895-1901, 1995
19. Kelly WN, Ice RD. Pharmaceutical quality of technetium-99m sulfur colloid.
Am. J. Hosp. Pharm. 30:817-820, 1973
20. Krogsgaard OW. Technetium-99m-sulfur colloid. In vitro studies of various
commercial
kits. Eur. J. Nucl. Med. 1:31-35, 1976 '
21. Steigman J, Solomon NA, Hwang LL. Technetium-sulfur colloid.
Int.J.Rad.Appl.lnstrum. jA.J 37:223-229, 1986
22
CA 02273609 1999-06-04
22. ~Ihelm AJ, Mijnhout GS, Franssen EJ. Radiopharmaceuticals in sentinel
lymph-node
detection - an overview [In Process Citation). Eur.J.NucLMed. 26:S36-S421999
23. Dragotakes S.C., Callahan R.J., LaPointe L.C., et al. Particle Size
Characterization of a
Filtered Tc-99m Sulfur Colloid Preparation for Lymphoscintigraphy. J.Nucl.Med.
36:80P1995
24. Taylor AJ, Murray D, Herda S, et al. Dynamic lymphoscintigraphy to
identify the sentinel
and satellite nodes. CIin.Nucl Med 21:755-758, 1996
25. Corrigan P, Eshima L, Eshima D: Stability of Filtered Tc-99m Sulfur
Colloid for
Lymphoscintigraphy Studies. J.NucLMed.Technol 25:1531997(Abstract)
26. Eshima L, Algozine C, Taylor AT, et al: Particle Size Evaluation of Tc-99m
TCK-17 a
Potential New Radiopharmaceutical for Lymphoscintigraphy Studies. J Nucl Med
38:112P1997(Abstract)
27. Bergqvist L, Strand SE, Persson BR. Particle sizing and biokinetics of
interstitial
lymphoscintigraphic agents. Semin.NucLMed. 13:9-19, 1983
28. Bostick P, Essner R, Sarantou T, et al. Intraoperative lymphatic mapping
for early-stage
melanoma of the head and neck. Am.J.Surg. 174:536-539, 1997
29. Bostick P, Essner R, Glass E, et al. Comparison of blue dye and probe-
assisted
intraoperative lymphatic mapping in melanoma to identify sentinel nodes in 100
lymphatic
basins. Arch.Surg. 134:43-49, 1999
30. Echt ML, Finan MA, Hoffman MS, et al. Detection of sentinel lymph nodes
with
lymphazurin in cervical, uterine, and vulvar malignancies. South.Med J 92:204-
208, 1999
31. Kapteijn BA, Nieweg OE, Liem I, et al. Localizing the sentinel node in
cutaneous
melanoma: gamma probe detection versus blue dye. Ann.Surg.OncoL 4:156-160,
1997
32. Ohtake E, Matsui K, Kobayashi Y, et al. Dynamic lymphoscintigraphy with Tc-
99m
human serum albumin. Radiat.Med. 1:132-136, 1983
33. Henze E, Robinson GD, Kuhl DE, et al. Tc-99m dextran: a new blood-pool-
labeling
agent for radionuclide angiocardiography. J.NucLMed. 23:348-353, 1982
23
CA 02273609 1999-06-04
34. Yueh TC, Pui MH, Zeng SQ. Intestinal lymphangiectasia: value of Tc-99m
dextran
lymphoscintigraphy. CIin.Nucl.Med. 22:695-696, 1997
24
