Note: Descriptions are shown in the official language in which they were submitted.
~32~ ~
METHOD FOR LOCALIZATION AND TREATMENT OF TUMORS AND
COMPLEXES THEREFOR
The present invention relates to methods for the
location and treatment of tumors and complexes for use in
such methods.
The localization of tumors, such as astrocytomas in
the brain in vivo and the determination of the margin
between normal tissue and tumor can be useful for
surgical, radiotherapeutic and chemotherapeutic
approaches to the tumor. Although gliomas generally do
not metastasize, they do recur locally after surgical
resection and carry a grave prognosis (1). The grave
prognosis results in part from the inability to delineate
clearly the boundary between tumor and normal brain
tissue, and from the restricted permeability of the blood
brain barrier to im~ging and therapeutic agents. The
successful delivery of magnetic resonance contrast agents
or of radionuclides for positron or gamma imaging might
contribute to the more precise localization of tumor
margins.
Monoclonal antibodies prepared against the tumor
have been proposed for use in the past as effective
carrier molecules for the delivery of contrast and
radionuclide agents (2,3). However, the use of such
monoclonal antibodies is accompanied by disadvantages.
Antibodies are very large molecules that also can carry
cross-reactive antiqenic determinants that could cause
-2- c~
problems. In addition, the monoclonal antibodies seldom
bind more than 70~ of cells, even in clonogenic tumors.
In addition to monoclonal antibodies, various
synthetic polypeptides, such as polylysine which
S selectively binds to tumor cells as compared to normal
~rain cells, have been considered for use as carrier
agents for therapeutic agents.
Notwithstanding prior efforts, a need still exists
for reliable, safe methods for the localization,
targeting and treatment of tumors and for complexes that
can be used in such methods.
The objects of the present invention include
disclosing novel methods for the localization and the
treatment of tumors.
The objects also include the disclosure of novel
complexes which can be used in such methods and a kit
containing a complex.
In the practice of the present invention, a safe and
effective amount of a novel complex of a polylysine, a
linking molecule and an imaging agent or a chemothera-
peutic agent having a high net positive charge is
injected into the arterial system of an animal and the
complex is carried to and selectively binds to tumors
having a greater net negative charge than non-tumor
cells. The method is especially useful for the imaging
of polyanionic charged tumors such as ~ilms tumors, brain
tumors, small cell carcinoma of the lung and melanomas.
The novel complexes of the present invention
comprise polylysine, a linking molecule and an imaging
agent or a chemotherapeutic agent. In the complexes, the
linking molecule is bound to less than all of the lysyls
of the polylysine so that the complexes have a high net
positive charge and will bind selectively to tumors
having a higher net negative charge than non-tumor
cells. The ratio of lysyl residues to linking agent will
range from about 5 to l to about 20 to 1.
~ 3 2 ~ A~ 3
The advantages o~ using polylysine in the complexes
are: 1) the chemical homogeneity of the synthetic
polymer, 2) the ease of preparation of polylysines having
different molecular sizes, 3) the ease of modification of
the polymer-linking agent complex with tri- or
tetravalent radionuclides, such as gadolinium and
zirconium, 4) the ease of labelling polylysine with
iodide radionuclides using the Bolton Hunter reagent.
The ability to select polylysine polymers of low
molecular weight can facilitate the delivery of the
imaging or therapeutic agent through the areas where
blood-brain barrier is intact without recourse to
permeabilization with mannitol (2).
The ease with which polylysines may be modified with
multiple nuclides provides an approach to determine
dosage and concentration of polymer that is required for
successful imaging in a patient population and for
simultaneous imaging and therapy (e.g. with Gd and 90Y.)
Finally, the polylysine is not immunogenic as
compared to immunoglobulins; therefore, the novel
polylysine containing complexes are selective for any
tumors having greater net negative charges than non-tumor
cells and may be used for multiple imaging or multiple
therapeutic procedures.
The primary advantage o~ immunoglobulins, such as
antibodies, resides in the specificity of these proteins
for particular tumors and in the relative lack of
toxicity as compared with polylysine which exhibits
toxicity at concentrations above 1.0 mg per 100 gm body
weight (8,10,11). However, the unexpected sensitivity of
the methods of the present invention permits the use of
- complexes containing polylysine concentrations that are
not toxic.
Other objects and advantages of the present
invention will be apparent from the description.
In preparing the complexes of the present invention,
the polylysine is coupled covalently to a linking
~ s?J~ f,
molecule, diethylene triamine pentaacetic acid
dianhydride (DTPA), with a ratio of lysine residue/DTPA
in the range of 5-20 lysyls per DTPA. This ratio assures
that the complexes will have an adequate net positive
charge to bind to tumors having a greater net negative
charge than non-tumor cells.
The preferred degree of polymerization of the
polylysine chain length is in the range of 50-100. The
polylysine-DTPA complex is preferrably separated from low
molecular weight reagents by gel filtration
chromatography on Sephadex G-25.
The imaging and chemotherapeutic agents, such as
metal ions that are paramagnetic (e.g. gadolinium,
man9anese)~ positron emitters (e.g. 89Zr), gamma emitters
(e.g. 's3Gd) or beta emitters (e.g. 90Y), are then added
to the polylysine-DTPA complex by ci~rate exchange and
the low molecular weight materials present are separated
from the polylysine-DTPA-metal ion complex by gel
filtration. A paramagnetic component in the complex
permits MR imaging, a positron emitter permits PET
imaging and the presence of a beta emitter in the complex
provides a radiotherapeutic agent.
In the preferred practice of the methods of the
present invention, a selected complex is injected into
the arterial system in the area of the suspected tumor so
that the concentrations of polylysine are less than 100
~g per 100 gms body weight. When the method is an
imaging method, the MRI or PET imaging is then done in a
conventional manner after a suitable time delay (24 to 96
hours) to permit maximum contrast between tumor and
surrounding tissue.
Novel complexes of polylysine-DTPA and the metallic
ions Is3Gd and 89Zr have been found to bind to C6 astro-
cytoma and U87 MG glioblastoma cells six to eight times
more readily than they bind to endothelial cells from the
brain or the aorta and to provide superior imaging.
~fi~ 2 ~ ~ 3
Many tumors have a greater net negative charge than
non-tumor cells. Polylysine in the complexes contributes
a positive charge that increases their tendency to bind
to tumor cells. Therefore, the high net positive charge
on the polylysine-DTPA-metal ion complexes, prepared as
described, results in selective binding of the complex to
the tumor cells.
The ingredients for preparing the novel complexes of
the present invention may be provided in kit form for the
convenience of users. In addition, the novel complexes
of the present invention may also be supplied with other
ingredients for use in test kits for the in vitro
analysis of tumor cells, tumor cell fragments or tumor
specific proteins in spinal fluid or plasma.
In the description that follows, the efficacy of
compositions of polylysine, DTPA and Is3Gd, or a9zr or
l2sI to image C6 astrocytoma in vivo in the rat brain are
described.
Materials and Methods
Fluorescein-labelled polylysine hydrobromide (DP 88,
weight average by light scattering), unmodified
polylysine tDP 299 by light scattering, DP 267 by
viscosity) and DTPA dianhydride were purchased from Sigma
~St. Louis, MO). The Is3Gd and the l2sI Bolton-Hunter
reagents (12) were purchased fro~ DuPont-New England
Nuclear (Boston, MA); the agzr was generated from an
yttrium target by the reaction 89Y(P,n)89Zr in the 11 MeV
proton beam of the University of Wisconsin, Medical
Physics cyclotron (CTI, Inc.). The carrier free a9zr was
purified by the method of Scadden and Ballou (13). The
agzr was coupled to the polylysyl DTPA on the day it was
generated from the yttrium. At 24 hours after
preparation, 1 picomole of carrier free agzr contains 300
~Ci,
The polylysine hydrobromide was dissolved in
bicarbonate buffer (0.1 mol/l, pH 9.0); then, DTPA,
dissolved in anhydrous dimethyl sulfoxide (DMSO), was
6 ~ y
added immediately to the polylysine. Polylysine-DTPA
complexes were prepared with one DTPA per 16 lysyl
residues. To prepare these complexes, a stoichiometric
ratio of one DTPA dianhydride in DMSO was added per 10
lysyl residues. The reaction was permitted to proceed
for 60 min. at 25C; then, the reactants were passed
through G-25 Sephadex columns (PD-I0 columns from
Pharmacia, Piscataway, NJ) that were preequilibrated with
citrate buffer (0.1 mol/l, pH 5.5). The polylysine-DTPA
product emerged at the void volume of the column. In a
similar manner, but with different stoichiometric ratios,
polylysine-DTPA complexes containing other DTPA/lysine
ratios were prepared.
The DTP~-modified polylysine in citrate buffer was
reacted with gadolinium chloride (Aldrich Chem.,
Milwaukee, WI) dissolved in the same citrate buffer. The
polylysine-DTPA-Gd chelates were then passed through G-25
Sephadex gel filtration columns, which were
preequilibrated with 0.15 mol/l of sodium chloride, to
separate the free gadolinium from the polylysine-DTPA-Gd
complex. The complexes containing zirconium and other
metal ions can be prepared in a similar manner.
The C6 astrocytoma cells were cultured in Ham's F-10
medium, supplemented with 2.5% fetal calf serum (Hyclone
Lab, Logan UT), 15~ horse serum (Gibco), 100 ~g
streptomycin and 100 unites penicillin per ml, and 1.2 gm
bicarbonate buffer per liter.
Male Wistar Furth rats (over 300 gms each) were
purchased from Harlan Sprague Dawley (Indianapolis,
IN). The rats were anesthetized with chloral hydrate and
a 0.5 mm burr hole was placed over the left frontal
cortex of each rat 3 mm to the left of the midline. Each
of 6 rat~ was then injected with 2X106 C6 astrocytoma
cells in 10~1 of F-10 Ham's medium containing 0.5%
agarose (Sigma). Two other rats were injected with the
same medium, but without tumor cells. Eight days later
the rats were anesthetized with chloral hydrate and the
-7- ~; 5~ ~ 'S ~
brains were imaged by MRI to visualize the tumor cell
injection site. The animals were catheterized through
the femoral artery to the ascending aorta. Then 500 ul
of saline solution containing 100 ug of 125I polylysine
(Bolton Hunter), or 100 ug of ~s3Gd-and ~s7Gd-labelled
polylysine or 100 ug of ~9Zr labelled polylysine was
injected into the aorta. Each rat received 0.23 mCi
89Zr. The animals were permitted to recover for three
additional days to permit the background radiation level
to fall. On the 11th day after tumor implantation the
animals were anestheized with chloral hydrate and imaged
by the Signa MRI using the GE extremity coil t17 cm
diameter~. The Tl weighted images were obtained at
either 3 or 4 mm thickness (TR=600; TE=20).
Blood samples were taken from the rats while they
were under anesthesia and the samples were retained for
analysis of radionuclides; the rats were sacrificed by
exsanguination, placed in the PET imager in groups of
four and counted for 14 hours. The PET images were
generated by a CTI Inc Model 933/04-12. It provides a 4
ring, 7 slice positron tomograph with 5 mm full width,
half maximum (FWHM) spatial resolution (transverse) and 6
mm FWHM resolution axially.
The brains and kidneys of each animal were removed
after imaging. The liver, lungs, spleen, thyroid,
testes, bone, heart and pancreas of animal three (a tumor
recipient) were removed. All three nuclides were
measured in the tissue samples by analysis with a Ge(Li)-
type counter (15% efficiency germanium gamma
spectrometer).
Tissue histology was performed on all brain samples
to validate the location of the implanted C6 cells.
Frozen sections (10 um) of formalin fixed brains were
cut, stained with thionine and covered with DePex
embedding material (Gurr Microscopy Ltd) and a cover
slip. The sections were examined in a Leitz-DADS
microscope and photographed.
Results
Poly-L-lysine hydrobromide (DP88) was modified cova-
lently with either the chelator, DTPA or with '2sI-Bolton
Hunter reagent. The polylysine-DTPA was then reacted
either with the positron emitter 89Zr, or paramagnetic
stable gadolinium and the gamma emitter ls3Gd. The 89Zr
was produced in the 11 meV cyclotron by the reaction
89Y(p,n)a9Zr and purified by fractional solubilization
techniques utilizing acid and organic solvents. The
polylysine-DTPA-nuclide and the polylysine-iodide
complexes were separated from the unbound nuclide or
metal ion by gel chromatography. Wistar Furth rats were
implanted intracranially with C6 astrocytoma and 8 days
later they were injected, through a catheter placed in
the aorta, with the polylysine nuclide complexes~ On the
eleventh day after tumor implantation the rats were
imaged by magnetic resonance imaging (MRI), and by
positron emission tomography (PET). The organs were
removed from the rats and the amount of each nuclide was
determined by Ge(Li) counting. Frozen sections of the
brains were prepared and stained with thionine to
validate the tumor growth and the margin between tumor
and normal brain. The signal intensity (SI) of the Tl
weighted MR images revealed enhancement by the
polylysine-DTPA-Gd; the central region of the tumor had a
low SI with a high SI at the periphery in all cases.
Ge(Li) counting revealed a 3-8 fold higher level of 89Zr
in the tumor containing hemisphere than in the non-tumor
hemisphere in 4 of 5 rats surviving 11 days with the
implanted tumor. The PET revealed the whole body
distribution of the polylysine-DTPA-~9Zr: the major
organs labelled were the tumor, kidney, spleen, thymus,
heart, bone, testes and liver and the radioactivity
recorded on a counts per second per gram normalized
basis. Cytological studies of the thionine stained
sections revealed good correlation with the tumor
morphology as demonstrated by MR imaging. These
_9_
observations suggest that polylysine- ~ ~ ~. ~
polylysine-DTPA-~9Zr complexes may have utility in
detecting the margin between astrocytoma and normal brain
by MRI and possibly by PET. Polylysine-DTPA-beta
emitting metal nuclide complexes may have utility in
radiotherapy of such tumors in situ.
In the description that follows, the magnetic
resonance images of the brains of the rats injected wi'h
the C6 astrocytoma will be described first. The MR
images obtained pre- and post-injection of the modified
polylysine will be shown. The MR images obtained from
tumor free rats that were injected with the modified
polylysines will also be shown. Then the positron
emission tomographs obtained from the rats will be dis-
cussed. The distribution of the polylysine-DTPA-nuclides
derivatives in the brain and other organs, as determined
by Ge(Li) counting, will then be described. Finally the
histology of the tumor in situ will be discussed.
Magnetic Resonance Imaging of the Rat Brain
The rat brains were imaged on the eighth day after
tumor implantation. The images of the other 3 tumor
bearing rats were similar. These images were obtained
prior to injection of the gadolinium, zirconium and
iodide labeled polylysines (DP88). It may be observed
that some degree of asymmetry is detectable at this stage
(animal 2 and animal 3) and one animal had a low signal
intensity (SI) on Tl weighted images (TWI) in the region
of the implanted tumor (animal 6). The control animals
injected with cell free agarose, by contrast, revealed no
unusual features at the same plane of section (animal
8). The images were taken at three mm thickness.
On the 11th day after tumor implantation and the
third day after injection of the radionuclide-labelled
polylysines (DP88), a circular central region of low
signal intensity (SI) on the Tl weighted imases (TWI),
and a circumference of high SI in the same region of
animal 2 was observed. The peripherally increased SI is
--10 ~ ~ ~ f `~
also seen in the adjacent 3 mm MRI image from this
animal. The MRI of animal 3 also shows low central SI on
Tl and high SI in the circumference. The tumor contain-
ing region o~ animal 6 also reveals a low SI on TWI in
the central region and a high SI in the circumference.
The right hemisphere however, also has a large central
region of law SI. The latter point is relevant since the
organ counts of the 89Zr-DTPA-polylysine reported below
for rat 6 indicate a higher distribution of the
polylysine-DTPA-89Zr in the right hemisphere than in the
left. A similar study of control rat brain (animal 8)
,revealed no areas of tumor-like appearance. Of the 6
rats that were injected with tumors, 5 survived to day 11
after tumor implantation; all 5 had tumor growth identi-
fied and the MRI of these brains revealed the tumorlocation and morphology. The high SI and TWI at the
tumor margin is consistent with an enhancement of the
relaxation of water protons caused by the localization of
the Gd-DTPA polylysine complex in the tumor. The central
zone of the C6 astrocytoma in the rat brain is frequently
necrotic, an observation consistent with the low SI
images obtained. The tumor region is clearly resolved in
the 3 mm thick sections when the extremity coil is
used. The tumor histology section below indicates that
there was no evidence of hemorrhage in the tumor area or
surrounding brain.
PET of the Tumor Containing and Control Rats
The positron emission tomographs of the rats were
obtained in 7 planes, from the dorsal to the ventral
surface of the rats. Four rats were imaged simul-
taneously and ring sources were used to correct for
position in the apparatus. Each set of animals was
imaged for 10 hours or longer to obtain the data for
image reconstruction. These images ~eveal that the
majority of the ~9Zr-DTPA-polylysine was localized in the
kidneys and a second major area of positron source was
the snout. It was of interest in this regard that the
animals, all of whom received 300 ug of polylysine, had
blood in the urine and 2 evidenced nose bleeds. Further
examination of the tomographs indicated that the brain
does contain substantial positron emission activity. The
localization of the Zr source to the right or left hemi-
sphere by PET is difficult to ascertain. The apparent
advantage of the PET over MRI is a rapid evaluation of
the distribution of the Zr source in the whole body and
the low concentration of polylysine required for
imaging. The MRI clearly resolves the tumor area and
permits demarcation of the tumor zone from the normal
brain.
Organ Distribution of the Nuclide Labelled Polylysine
The brain was separated into the right and left
hemisphere for determination of the counts per second of
each nuclide in the organ. The tumor cells in all cases
were implanted in the left frontal region of the brain.
Table I below reports the counts per sec per gram tissue
normalized to the whole rat body for each animal. The
nuclide distribution was determined with a Ge(Li)
counter; the ~9Zr was measured from the 909 keV peak, the
~s3Gd from the 105 keV peak and the l2sI from the 27keV
peak. An aliquot of the injected polylysine nuclide
material was used for calculatinq the organ distribution
of the radionuclides.
From Table I, it may be observed that the
distribution of the Zr was three to eight times higher in
the left hemisphere (containing the tumor) than in the
right in four of the 5 surviving rats that were injected
with tumors. In one rat that contained C6 tumor, the
right hemisphere had more radioactivity. The gadolinium
reflected a similar increased localization in the left
hemisphere but the proportion of the left in the compared
with the right hemisphere was smaller than that observed
for the Zr. There is some disproportionation of the Gd
and Zr distribution even though both were chelated with
the DTPA on polylysines of identical polymer size, This
12 ~ 5 ~ `J ~".
suggests that either the DTPA-Gd is released at a
different rate from the polylysine chain than the DTPA-Zr
or that the polylysyl-DTPA-Gd metabolite of polylysine
localizes differently from lysyl-DTPA-a9Zr. Because the
S polylysyl-DTPA-Gd complex contained more metal ion (cold
Gd was added) than the Zr complex, it is possible that
the excess metal in the Gd-DTPA complex affected nuclide
distribution. The iodide label was equivalent in both
hemispheres. This is consistent with the recognized loss
of iodide from iodide labeled proteins in the presence of
serum and other tissue fluids. The Zr is the label of
choice from these observations and the iodide is least
preferred of the three nuclides.
Table I
Distribution of each of three Polylysine-DTPA-
Nuclide Derivatives in the Left and Right 3rain
Hemispheres
39Zr l53Gd lZsI
Rat Left Right Left Right Left Right
2 0.212 0.063 0.030 0.021 0.004 0.004
3 0.851 0.108 0.051 0.03~ 0.008 0.006
4 0.300 0.~87 0.038 0.020 0.007 0.~03
0.365 0.057 0.060 0.031 0.007 0.009
6 00713 1.521 0.031 0.262 0.005 0.006
7 0.075 0.062 0.047 0.054 0.006 0.007
8 0.078 0.068 0.063 0.027 0.015 0.010
The distribution of the nuclides in other organs of
the body is illustrated in Table II using rat 3 as an
example. This table reveals that the organs with highest
Zr contents are the kidney, spleen, heart, thymus, bone
and testes. The high nuclide content of the spleen,
heart, thymus and testes is anticipated since the poly-
lysine was injected directly into the aorta. Positron
emission tomographs of the rats reflect the Ge(Li) counts
as anticipated and the PET may be used to follow tempo-
rally the polylysine-DTPA-89Zr organ distribution. When
normalized to kidney, the ratio of the Zr to the Gd dif-
-13- ~5~p~,S ~1
fers in several organs indicating that Zr uptake is high
in bone and low in liver whereas Gd is high in liver.
Table II
Distribution of Polylysine-DTPA-Nuclide in
5Organs of Rat 3
Counts per Sec per gm tissue normalized to
the whole body
39Zr Is3Gd l2sI
Kidney 12.472 1.739 1.276
bone 2.500 0.323 0.004
heart 3.208 0.950 0.016
liver 0.922 2.844 0.092
lung 1.414 0.243 0.005
testes 2.604 0.040 0.000
thymus 3.055 0.617 0.012
spleen 5.520 9.193 1.351
pancreas 1.427 0.678 0.027
Histology of the C6 Astrocytoma in the Brain
The thionine stained sections of the formalin fixed
rat brains clearly revealed nests of tumor cells. The
tumor cells were located at discrete sites in the left
hemisphere, including the frontal and the parietal
cortex. The tumors in the brain, that were revealed by
histological stain, correlated with the sites revealed by
MRI. From the histological examination it may be seen
that the tumor infiltrated the normal brain tissue around
the tumor. The tumor proper contains small round cells
and larger round cells with pale nuclei and condensed
chromatin. The histology confirms that the C6 tumors
grew in the adult Wistar Furth rats, that the cell type
and structure is consistent with the properties of the C6
tumor line, and that the MRI images obtained in vivo
correlate with the histological appearance of the
tissue. There was no evidence of hemorrhagic changes in
the tumor or surrounding brain even though necrotic
central zone could be seen in some tumors.
1 4
From the foregoing it is clear that polylysine
derivatives, containing DTPA chelated to paramagnetic
ions, such as Gd, enhances the MRI of intracerebral
tumors. The distribution of the polylysine-DTPA-39Zr, as
determined by Ge(Li) counting, is higher in the tumor
containing hemisphere by a factor of 3-8, than in the
contralateral side. These data represent the first
successful use of a tumor selective carrier vehicle,
polylysine, to deliver paramagnetic Gd and positron
emitting 89Zr in vivo to a syngenic model rat glioma for
the purposes of neuroradiological imaging. This delivery
system enhanced the relaxation of water in the area of
the C6 astrocytoma. The proof of principle that polyly-
cine-DTPA-89Zr may be used for the PET imaging of intra-
cranial tumors has also been demonstrated. These resultsare consistent with an earlier study from our
laboratories3 which indicated that C6 astrocytoma cells
and U87 MG glioblastoma cells, in vitro, bind 6-8 fold
times more polylysine-DTPA-39Zr than does endothelial
cells from brain or aorta.
Other investigators have previously imaged C6 tumors
by MR, in vivo, with the use of a 10 Cm internal diameter
coil (17). Rats with intracerebral C6 tumors were
injected with DTPA-Gd (17) and their brains were
imaged. The DTPA-Gd treatment enhanced the relaxation of
water protons and provided MRI contrast. DTPA-Gd pene-
trates the tumor region in the brain transiently, as a
result of a compromised blood brain barrier. The influx
of the DTPA-Gd and efflux is relatively rapid because the
DTPA is not bound by the tumor. The advantage of the
polylysine-DTPA-&d complex compared with DTPA-Gd alone
resides in the selective binding of the polylysine to the
tumor cell surface. Because the polylysine is bound by
the tumor, the imaging may be performed after the blood
level of the contrast material (i.e. polylysine) has
fallen to very low levels. The signal to noise ratio is
thus enhanced by the use of polylysine.
-15- ?~ ~jh'~ ~ ,n
The polylysine can also be used to deliver several
nuclides or chemotherapeutic agents simultaneously,
because of the abundance of epsilon amino groups on the
polymer. This permits an analysis of drug delivery or Oe
radiation dosage effects by a comparison of the PET image
with the MRI image. Polylysine does exhibit toxic
properties at concentrations exceeding 1.5 mg per lO0 gm
body weight in the rat (10,11). However the picomole
concentrations of a9Zr, Gd and 90Y required for PET
imaging, for MRI or for radiotherapy is several
magnitudes below toxic concentrations. Polylysine has
been used a complexing agent for poly I:poly C in the
chemotherapy of tumors (18), and for the delivery of
methotrexate to ovarian cells (19). The toxicity problem
is therefore amenable to solution.
The C6 astrocytoma is a good model for human gliomas
because the tumor produces S-lO0 (20), ~lutathione S
transferase (21) and glial maturation factor (22). The
successful imaging of this tumor in vivo has direct
applications to the imaging of human brain gliomas in
vivo. In the human subject the polylysine-DTPA-nuclide
complex will have greater utility than in the rat. The
placement of the catheter into the carotid artery in the
area of the tumor is readily achieved as is discernment
by PET of the tumor mass. The size of the rat brain
(0.8-l.0 cm dorsal ventral dimension) approximates that
of a single PET slice (5 mm). This resolution in a human
brain provides the information necessary to determine the
tumor margin and the suitability of surgery.
The polylysine polymers preferred for use in the
complexes of the present invention are those lysine
polypeptide or homopolymers having a molecular weight of
about 5,000 to about 20,000 dalton. They can be made by
the process described in U. S. Patent No. 3,215,684. Any
polylysine which covalently bonds to the chelating agent
and possesses an adequate net positive charge to be
attracted to and bind to tumor cells and a favorable
toxicity ratio can be used.
-16 ~ d . ~
The preferred chelating agent for use in the present
invention is DTPA which is also known as pentetic acid and
diethylenetriamine pentaacetic acid. The purpose of the
chelating agent is to covalently bind to the polylysine
and the metallic ions which are imaging or therapeutic
agents. DTPA can be prepared as described in U. S. Patent
No. 2,384,816. Other chelating agents that might be used
include ethylene diamine tetraacetic acid and DOTA.
Representative of metallic agents that can be used
as MR imaging agents in the novel complexes of the
present invention are paramagnetic ions such as
gadolinium, manganese, and cobalt.
Representative of the metallic ions which can be
used as PET imaging agents are 39Zr, and ~s2Mn or ssCo.
Representative of the ions that can be used as y-
camera imaging agents is ~In.
Representatives of metallic ions that can be used as
therapeutic agents in the complexes of the present
invention are 90Y and 2llA+ (astatine).
Two other technologies that have clinical utility in
tumor studies are possible because of the binding of
polylysine containing complexes or probes to tumor
cells: a) Spinal fluid samples may be centrifuged at low
speed, 1000g for 10 minutes, to recover any cell or cell
fragments present. Spinal fluid normally does not
contain cells but may contain cells or cell fragments in
the case of central nervous system tumors. The resultant
pellet is resuspended in bicarbonate buffer containing
saline and the radiolabelled polylysine is added to the
suspension. The cell suspension is recentrifuged at the
same force indicated above, the pellet recovered and
washed three times with the buffer. The pellet is then
counted to determine the number of cells per volume of
spinal fluid. b) Spinal fluid samples or samples of
blood plasma can be incubated with polylysine probes
(polylysine alone, polylysine-DTPA-metal ions,
polylysine-fluorescein [or other fluorescent probe]).
The mixture is centrifuged at low speed, <2000g, for 10
-17- ~6~fi^i
minutes. The pellets are resuspended in bicarbonate
buffer and applied to pure nitrocellulose membranes.
The polylysine probes bind avidly to the nitrocellu-
lose membrane, even when the polylysine is complexed with
other materials. The tumor cell-polylysine complexes
also will adhere to the membrane. The cells on the
membrane may then be incubated with immunoglobulins that
are specific for particular tumor cell types. These
immunoglobulins are available commercially or can be
prepared. Then traditional western blot procedures can
be employed to yield spot tests which identify the tumor
cell fragments adherent to the nitrocellulose membrane.
Specifically, the initial anti-tumor immunoglobulin may
be of varied origin, i.e. from the patient, mice, sheep,
goat etc. Depending upon the source of the initial
immunoglobulin, a second immunoglobulin (i.e. anti-human,
anti-mouse, anti-sheep, anti-goat IgG and IgM) coupled to
a reporter molecule such as peroxidase or phosphatase, is
incubated with the nitrocellulose membrane. ~he mem-
branes are washed after each step. Finally the membranesare incubated with an appropriate substrate which yields
a new signal (e.g. color, electric output).
The complexes of the present invention when used ln
vivo as diagnostic agents or therapeutic agents are
preferably combined with conventional diagnostic or
pharmaceutical diluents, such as Sterile Water for
Injection U.S.P., lactose, salts and the like and
packaged as sterile preparations. The preparations will
normally contain a safe and effective amount of the metal
ions, which are either known imaging or diagnostic
agen's, for their intended use.
It will be apparent to those skilled in the art that
a n~mbe of modifications and changes may be made without
departing from the spirit and scope of the present inven-
^S ~ion. Therefore, it is intended that scope of inventionnot ~e limited by the foregoing specific description but
only by the claims.
-18- ~ ~; c~
'J
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