U.S. patent application number 13/552414 was filed with the patent office on 2013-06-27 for bisaminoethanethiol-targeting ligand conjugates and compositions.
This patent application is currently assigned to BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM. The applicant listed for this patent is E. Edmund Kim, Chun W. Liu, David J. Yang, Dong-Fang Yu. Invention is credited to E. Edmund Kim, Chun W. Liu, David J. Yang, Dong-Fang Yu.
Application Number | 20130165631 13/552414 |
Document ID | / |
Family ID | 27080078 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130165631 |
Kind Code |
A1 |
Yang; David J. ; et
al. |
June 27, 2013 |
BISAMINOETHANETHIOL-TARGETING LIGAND CONJUGATES AND
COMPOSITIONS
Abstract
The invention provides, in a general sense, a new labeling
strategy employing .sup.99mTc chelated with ethylenedicysteine
(EC). EC is conjugated with a variety of ligands and chelated to
.sup.99mTc for use as an imaging agent for tissue-specific
diseases. The drug conjugates of the invention may also be used as
a prognostic tool or as a tool to deliver therapeutics to specific
sites within a mammalian body. Kits for use in tissue-specific
disease imaging are also provided.
Inventors: |
Yang; David J.; (Sugarland,
TX) ; Liu; Chun W.; (Sugarland, TX) ; Yu;
Dong-Fang; (Houston, TX) ; Kim; E. Edmund;
(Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yang; David J.
Liu; Chun W.
Yu; Dong-Fang
Kim; E. Edmund |
Sugarland
Sugarland
Houston
Houston |
TX
TX
TX
TX |
US
US
US
US |
|
|
Assignee: |
BOARD OF REGENTS, THE UNIVERSITY OF
TEXAS SYSTEM
Austin
TX
|
Family ID: |
27080078 |
Appl. No.: |
13/552414 |
Filed: |
July 18, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12563724 |
Sep 21, 2009 |
8236279 |
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13552414 |
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11760456 |
Jun 8, 2007 |
7615208 |
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12563724 |
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11627299 |
Jan 25, 2007 |
7582281 |
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11760456 |
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10672763 |
Sep 26, 2003 |
7223380 |
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11627299 |
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09599152 |
Jun 21, 2000 |
7067111 |
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10672763 |
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09587583 |
Jun 2, 2000 |
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09599152 |
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09434313 |
Oct 25, 1999 |
6692724 |
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09587583 |
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Current U.S.
Class: |
530/330 ;
530/408; 534/14 |
Current CPC
Class: |
A61K 51/0459 20130101;
A61P 43/00 20180101; A61K 51/0453 20130101; A61K 51/088 20130101;
A61K 51/0491 20130101; A61K 51/0497 20130101; A61K 51/087
20130101 |
Class at
Publication: |
530/330 ; 534/14;
530/408 |
International
Class: |
A61K 51/04 20060101
A61K051/04; A61K 51/08 20060101 A61K051/08 |
Claims
1. A composition for imaging comprising: a) a radionuclide; b)
ethylenedicysteine; and c) a tissue specific ligand conjugated to
said ethylenedicysteine; wherein said ethylenedicysteine forms an
N.sub.2S.sub.2 chelate with said radionuclide label.
2.-41. (canceled)
42. A kit for preparing a radiopharmaceutical preparation, said kit
comprising a sealed container including a predetermined quantity of
an ethylenedicysteine-tissue specific ligand conjugate composition
and a sufficient amount of reducing agent to label the conjugate
with .sup.99mTc.
43.-51. (canceled)
Description
BACKGROUND OF TILE INVENTION
[0001] The government does not own rights in the present
invention.
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of
labeling, radioimaging and chemical synthesis. More particularly,
it concerns a strategy for radiolabeling target ligands. It further
concerns methods of using those radiolabeled ligands in tumor
imaging and tissue-specific disease imaging.
[0004] 2. Description of Related Art
[0005] Improvement of scintigraphic tumor imaging is extensively
determined by development of more tumor specific
radiopharmaceuticals. Due to greater tumor specificity,
radiolabeled ligands as well as radiolabeled antibodies have opened
a new era in scintigraphic detection of tumors and undergone
extensive preclinical development and evaluation. (Mathias et al.,
1996, 1997a, 1997b). Radionuclide imaging modalities (positron
emission tomography, PET; single photon emission computed
tomography, SPECT) are diagnostic cross-sectional imaging
techniques that map the location and concentration of
radionuclide-labeled radiotracers. Although CT and MRI provide
considerable anatomic information about the location and the extent
of tumors, these imaging modalities cannot adequately differentiate
invasive lesions from edema, radiation necrosis, grading or
gliosis. PET and SPECT can be used to localize and characterize
tumors by measuring metabolic activity.
[0006] The development of new tumor hypoxia agents is clinically
desirable for detecting primary and metastatic lesions as well as
predicting radioresponsiveness and time to recurrence. None of the
contemporary imaging modalities accurately measures hypoxia since
the diagnosis of tumor hypoxia requires pathologic examination. It
is often difficult to predict the outcome of a therapy for hypoxic
tumor without knowing at least the baseline of hypoxia in each
tumor treated. Although the Eppendorf polarographic oxygen
microelectrode can measure the oxygen tension in a tumor, this
technique is invasive and needs a skillful operator. Additionally,
this technique can only be used on accessible tumors (e.g., head
and neck, cervical) and multiple readings are needed. Therefore, an
accurate and easy method of measuring tumor hypoxia will be useful
for patient selection. However, tumor to normal tissue uptake
ratios vary depending upon the radiopharmaceuticals used.
Therefore, it would be rational to correlate tumor to normal tissue
uptake ratio with the gold standard Eppendorf electrode measures of
hypoxia when new radiopharmaceuticals are introduced to clinical
practice.
[0007] [.sup.18F]FMISO has been used to diagnose head and neck
tumors, myocardial infarction, inflammation, and brain ischemia
(Martin et al. 1992; Yeh et al. 1994; Yeh et al., 1996; Liu et al.,
1994). Tumor to normal tissue uptake ratio was used as a baseline
to assess tumor hypoxia (Yet et al. 1996). Although tumor hypoxia
using [.sup.18F]FMISO was clearly demonstrated, introducing new
imaging agents into clinical practice depends on some other factors
such as easy availability and isotope cost. Although tumor
metabolic imaging using [.sup.18F]FDG was clearly demonstrated,
introducing molecular imaging agents into clinical practice depends
on some other factors such as easy availability and isotope cost.
[.sup.18F]fluorodeoxyglucose (FDG) has been used to diagnose
tumors, myocardial infarction, and neurological disease. In
addition, PET radiosynthesis must be rapid because of short
half-life of the positron isotopes. .sup.18F chemistry is also
complex. The .sup.18F chemistry is not reproducible in different
molecules. Thus, it would be ideal to develop a chelator which
could conjugate to various drugs. The preferred isotope would be
.sup.99mTc due to low cost ($0.21/mCi vs. $50/mCi for .sup.18F) and
low energy (140 Key vs. 571 Key for .sup.18F). .sup.99mTc is easily
obtained from a .sup.99Mo generator. Due to favorable physical
characteristics as well as extremely low price, .sup.99mTc has been
preferred to label radiopharmaceuticals.
[0008] Several compounds have been labeled with .sup.99mTc using
nitrogen and sulfur chelates (Blondeau et al., 1967; Davison et
al., 1980). Bis-aminoethanethiol tetradentate ligands, also called
diaminodithol compounds, are known to form very stable Tc(V)O
complexes on the basis of efficient binding of the oxotechnetium
group to two thiolsulfur and two amine nitrogen atoms.
.sup.99mTc-L,L-ethylenedicysteine (.sup.99mTc-EC) is a recent and
successful example of N.sub.2S.sub.2 chelates. EC can be labeled
with .sup.99mTc easily and efficiently with high radiochemical
purity and stability, and is excreted through the kidney by active
tubular transport (Surma et al., 1994; Van Nerom et al., 1990,
1993; Verbruggen et al., 1990, 1992). Other applications of EC
would be chelated with galium-68 (a positron emitter, t1/2=68 min)
for PET and gadolinium, iron or manganese for magnetic resonance
imaging (MRI). .sup.99mTc-EC-neomycin and
.sup.99mTc-EC-deoxyglucose were developed and their potential use
in tumor characterization was evaluated.
SUMMARY OF THE INVENTION
[0009] The present invention overcomes these and other drawbacks of
the prior art by providing a new radiolabeling strategy to target
tissues for imaging. The invention provides radiolabeled
tissue-specific ligands, as well as methods for making the
radiolabeled ligands and for using them to image tissue-specific
diseases.
[0010] The present invention provides compositions for tissue
specific disease imaging. The imaging compositions of the invention
generally include a radionuclide label chelated with
ethylenedicysteine and a tissue specific ligand conjugated to the
ethylenedicysteine on one or both of its acid arms. The
ethylenedicysteine forms an N.sub.2S.sub.2 chelate with the
radionuclide label. Of course, the chelated compound will include
an ionic bond between the radionuclide and the chelating compound.
The terms "EC-tissue specific ligand conjugate," "EC-derivative"
and "EC-drug conjugate" are used interchangeably herein to refer to
the unlabeled ethylenedicysteine-tissue specific ligand compound.
As used herein, the term "conjugate" refers to a covalently bonded
compound.
[0011] Ethylenedicysteine is a bis-aminoethanethiol (BAT)
tetradentate ligand, also known as diaminodithiol (DADT) compounds.
Such compounds are known to form very stable Tc(V)O-complexes on
the basis of efficient binding of the oxotechnetium group to two
thiol-sulphur and two amine-nitrogen atoms. The .sup.99mTc labeled
diethylester (.sup.99mTc-L,L-ECD) is known as a brain agent.
.sup.99mTc-L,L-ethylenedicysteine (.sup.99mTc-L,L-EC) is its most
polar metabolite and was discovered to be excreted rapidly and
efficiently in the urine. Thus, .sup.99mTc-L,L-EC has been used as
a renal function agent (Verbruggen et al. 1992).
[0012] A tissue specific ligand is a compound that, when introduced
into the body of a mammal or patient, will specifically bind to a
specific type of tissue. It is envisioned that the compositions of
the invention may include virtually any known tissue specific
compound. Preferably, the tissue specific ligand used in
conjunction with the present invention will be an anticancer agent,
DNA topoisomerase inhibitor, antimetabolite, tumor marker, folate
receptor targeting ligand, tumor apoptotic cell targeting ligand,
tumor hypoxia targeting ligand, DNA intercalator, receptor marker,
peptide, nucleotide, organ specific ligand, antimicrobial agent,
such as an antibiotic or an antifungal, glutamate pentapeptide or
an agent that mimics glucose. The agents that mimic glucose may
also be referred to as "sugars."
[0013] Preferred anticancer agents include methotrexate,
doxorubicin, tamoxifen, paclitaxel, topotecan, LHRH, mitomycin C,
etoposide, tomudex, podophyllotoxin, mitoxantrone, captothecin,
coldhicine, endostatin, fludarabin and gemcitabine. Preferred tumor
markers include PSA, ER, PR, AFP, CA-125, CA-199, CEA, interferons,
BRCA1, cytoxan, p53, VEGF, integrins, endostatin, HER-2/neu,
antisense markers or a monoclonal antibody. It is envisioned that
any other known tumor marker or any monoclonal antibody will be
effective for use in conjunction with the invention. Preferred
folate receptor targeting ligands include folate, methotrexate and
tomudex. Preferred tumor apoptotic cell or tumor hypoxia targeting
ligands include annexin V, colchicine, nitroimidazole, mitomycin or
metronidazole. Preferred antimicrobials include ampicillin,
amoxicillin, penicillin, cephalosporin, clidamycin, gentamycin,
kanamycin, neomycin, natamycin, nafcillin, rifampin, tetracyclin,
vancomycin, bleomycin, and doxycyclin for gram positive and
negative bacteria and amphotericin B, amantadine, nystatin,
ketoconazole, polymycin, acyclovir, and ganciclovir for fungi.
Preferred agents that mimic glucose, or sugars, include neomycin,
kanamycin, gentamycin, paromycin, amikacin, tobramycin, netilmicin,
ribostamycin, sisomicin, micromicin, lividomycin, dibekacin,
isepamicin, astromicin, aminoglycosides, glucose or
glucosamine.
[0014] In certain embodiments, it will be necessary to include a
linker between the ethylenedicysteine and the tissue specific
ligand. A linker is typically used to increase drug solubility in
aqueous solutions as well as to minimize alteration in the affinity
of drugs. While virtually any linker which will increase the
aqueous solubility of the composition is envisioned for use in
conjunction with the present invention, the linkers will generally
be either a poly-amino acid, a water soluble peptide, or a single
amino acid. For example, when the functional group on the tissue
specific ligand, or drug, is aliphatic or phenolic-OH, such as for
estradiol, topotecan, paclitaxel, or raloxifen etoposide, the
linker may be poly-glutamic acid (MW about 750 to about 15,000),
poly-aspartic acid (MW about 2,000 to about 15,000), bromo
ethylacetate, glutamic acid or aspartic acid. When the drug
functional group is aliphatic or aromatic-NH.sub.2 or peptide, such
as in doxorubicin, mitomycin C, endostatin, annexin V, LHRH,
octreotide, and VIP, the linker may be poly-glutamic acid (MW about
750 to about 15,000), poly-aspartic acid (MW about 2,000 to about
15,000), glutamic acid or aspartic acid. When the drug functional
group is carboxylic acid or peptide, such as in methotrexate or
folic acid, the linker may be ethylenediamine, or lysine.
[0015] While the preferred radionuclide for imaging is .sup.99mTc,
it is envisioned that other radionuclides may be chelated to the
EC-tissue specific ligand conjugates, or EC-drug conjugates of the
invention, especially for use as therapeutics. For example, other
useful radionuclides are .sup.188Re, .sup.186Re, .sup.153Sm,
.sup.166Ho, .sup.90Y, .sup.89Sr, .sup.67Ga, .sup.68Ga, .sup.111In,
.sup.153Gd, and .sup.59Fe. These compositions are useful to deliver
the therapeutic radionuclides to a specific lesion in the body,
such as breast cancer, ovarian cancer, prostate cancer (using for
example, .sup.186/188Re-EC-folate) and head and neck cancer (using
for example, .sup.186/188Re-EC-nitroimidazole).
[0016] Specific embodiments of the present invention include
.sup.99mTc-EC-annexin V, .sup.99mTc-EC-colchicine,
.sup.99mTc-EC-nitroimidazole, .sup.99mTc-EC-glutamate pentapeptide,
.sup.99mTc-EC-metronidazole, .sup.99mTc-EC-folate,
.sup.99mTc-EC-methotrexate, .sup.99mTc-EC-tomudex,
.sup.99mTc-EC-neomycin, .sup.99mTc-EC-kanamycin,
.sup.99mTc-EC-aminoglycosides, (glucosamine, EC-deoxyglucose),
.sup.99mTc-EC-gentamycin, and .sup.99mTc-EC-tobramycin.
[0017] The present invention further provides a method of
synthesizing a radiolabeled ethylenedicysteine drug conjugate or
derivative for imaging or therapeutic use. The method includes
obtaining a tissue specific ligand, admixing the ligand with
ethylenedicysteine (EC) to obtain an EC-tissue specific ligand
derivative, and admixing the EC-tissue specific ligand derivative
with a radionuclide and a reducing agent to obtain a radionuclide
labeled EC-tissue specific ligand derivative. The radionuclide is
chelated to the EC via an N.sub.2S.sub.2 chelate. The tissue
specific ligand is conjugated to one or both acid arms of the EC
either directly or through a linker as described above. The
reducing agent is preferably a dithionite ion, a stannous ion or a
ferrous ion.
[0018] The present invention further provides a method for labeling
a tissue specific ligand for imaging, therapeutic use or for
diagnostic or prognostic use. The labeling method includes the
steps of obtaining a tissue specific ligand, admixing the tissue
specific ligand with ethylenedicysteine (EC) to obtain an EC-ligand
drug conjugate, and reacting the drug conjugate with .sup.99mIc in
the presence of a reducing agent to form an N.sub.2S.sub.2 chelate
between the ethylenedicysteine and the .sup.99mTc.
[0019] For purposes of this embodiment, the tissue specific ligand
may be any of the ligands described above or discussed herein. The
reducing agent may be any known reducing agent, but will preferably
be a dithionite ion, a stannous ion or a ferrous ion.
[0020] In another embodiment, the present invention provides a
method of imaging a site within a mammalian body. The imaging
method includes the steps of administering an effective diagnostic
amount of a composition comprising a .sup.99mTc labeled
ethylenedicysteine-tissue specific ligand conjugate and detecting a
radioactive signal from the .sup.99mTc localized at the site. The
detecting step will typically be performed from about 10 minutes to
about 4 hours after introduction of the composition into the
mammalian body. Most preferably, the detecting step will be
performed about 1 hour after injection of the composition into the
mammalian body.
[0021] In certain preferred embodiments, the site will be an
infection, tumor, heart, lung, brain, liver, spleen, pancreas,
intestine or any other organ. The tumor or infection may be located
anywhere within the mammalian body but will generally be in the
breast, ovary, prostate, endometrium, lung, brain, or liver. The
site may also be a folate-positive cancer or estrogen-positive
cancer.
[0022] The invention also provides a kit for preparing a
radiopharmaceutical preparation. The kit generally includes a
sealed via or bag, or any other kind of appropriate container,
containing a predetermined quantity of an ethylenedicysteine-tissue
specific ligand conjugate composition and a sufficient amount of
reducing agent to label the conjugate with .sup.99mTc. In certain
cases, the ethylenedicysteine-tissue specific ligand conjugate
composition will also include a linker between the
ethylenedicysteine and the tissue specific ligand. The tissue
specific ligand may be any ligand that specifically binds to any
specific tissue type, such as those discussed herein. When a linker
is included in the composition, it may be any linker as described
herein.
[0023] The components of the kit may be in any appropriate form,
such as in liquid, frozen or dry form. In a preferred embodiment,
the kit components are provided in lyophilized form. The kit may
also include an antioxidant and/or a scavenger. The antioxidant may
be any known antioxidant but is preferably vitamin C Scavengers may
also be present to bind leftover radionuclide. Most commercially
available kits contain glucoheptonate as the scavenger. However,
glucoheptonate does not completely react with typical kit
components, leaving approximately 10-15% left over. This leftover
glucoheptonate will go to a tumor and skew imaging results.
Therefore, the inventors prefer to use EDTA as the scavenger as it
is cheaper and reacts more completely.
[0024] Another aspect of the invention is a prognostic method for
determining the potential usefulness of a candidate compound for
treatment of specific tumors. Currently, most tumors are treated
with the "usual drug of choice" in chemotherapy without any
indication whether the drug is actually effective against that
particular tumor until months, and many thousands of dollars,
later. The imaging compositions of the invention are useful in
delivering a particular drug to the site of the tumor in the form
of a labeled EC-drug conjugate and then imaging the site within
hours to determine whether a particular drug.
[0025] In that regard, the prognostic method of the invention
includes the steps of determining the site of a tumor within a
mammalian body, obtaining an imaging composition which includes a
radionuclide chelated to EC which is conjugated to a tumor specific
cancer chemotherapy drug candidate, administering the composition
to the mammalian body and imaging the site to determine the
effectiveness of the candidate drug against the tumor. Typically,
the imaging step will be performed within about 10 minutes to about
4 hours after injection of the composition into the mammalian body.
Preferably, the imaging step will be performed within about 1 hour
after injection of the composition into the mammalian body.
[0026] The cancer chemotherapy drug candidate to be conjugated to
EC in the prognostic compositions may be chosen from known cancer
chemotherapy drugs. Such drugs appear in Table 2. There are many
anticancer agents known to be specific for certain types of
cancers. However, not every anticancer agent for a specific type of
cancer is effective in every patient. Therefore, the present
invention provides for the first time a method of determining
possible effectiveness of a candidate drug before expending a lot
of time and money on treatment.
[0027] Yet another embodiment of the present invention is a reagent
for preparing a scintigraphic imaging agent. The reagent of the
invention includes a tissue specific ligand, having an affinity for
targeted sites in vivo sufficient to produce a
scintigraphically-detectable image, covalently linked to a
.sup.99mTc binding moiety. The .sup.99mTc binding moiety is either
directly attached to the tissue specific ligand or is attached to
the ligand through a linker as described above. The .sup.99mTc
binding moiety is preferably an N.sub.2S.sub.2 chelate between
.sup.99mTc in the +4 oxidation state and ethylenedicysteine (EC).
The tissue specific ligand will be covalently linked to one or both
acid arms of the EC, either directly or through a linker as
described above. The tissue specific ligand may be any of the
ligands as described above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0029] FIG. 1. Synthetic scheme of .sup.99mTc-EC-folate.
[0030] FIG. 2. Synthetic scheme of .sup.99mTc-EC-MTX
(methotrexate).
[0031] FIG. 3. Synthetic scheme of .sup.99mTc-EC-TDX (tomudex).
[0032] FIG. 4. Biodistribution studies for .sup.99mTc-EC and
.sup.99mTc-EC-folate.
[0033] FIG. 5. Blocking studies for tumor/muscle and tumor/blood
count ratios with .sup.99mTc-EC-folate.
[0034] FIGS. 6A and 6B. Scintigraphic images of tumor in
.sup.99mTc-EC-folate injected group as compared to .sup.99mTc-EC
injected group.
[0035] FIG. 7. Synthetic scheme of EC-MN (metronidazole)
[0036] FIG. 8A and FIG. 8B. For EC-NIM, FIG. 8A shows the synthetic
scheme and FIG. 8B illustrates the .sup.1H-NMR confirmation of the
structure.
[0037] FIG. 9. Biodistribution studies (tumor/blood ratios) for
.sup.99mTc-EC-MN, [.sup.18F]FMISO and [.sup.131I]IMISO.
[0038] FIG. 10. Biodistribution studies (tumor/muscle ratios) for
.sup.99mTc-EC, [.sup.18F]FMISO and [.sup.131I]IMISO.
[0039] FIGS. 11A and 11B. Scintigraphic images of tumor in
.sup.99mTc-EC-MN (FIG. 11A) and .sup.99mTc-EC (FIG. 11B) injected
groups.
[0040] FIG. 12. Autoradiograms performed at 1 hour after injection
with .sup.99mTc-EC-MN.
[0041] FIG. 13. Illustrates stability of .sup.99mTc-EC-NIM in dog
serum samples.
[0042] FIG. 14A and FIG. 14B. Illustrates breast tumor uptake of
.sup.99mTc-EC-NIM vs. .sup.99mTc-EC in rats (FIG. 14A) and in rats
treated with paclitaxel compared to controls (FIG. 14B).
[0043] FIG. 15A, FIG. 15B, FIG. 15C, and FIG. 15D. Illustrates
ovarian tumor uptake of .sup.99mTc-EC-NIM vs. .sup.99mTc-EC in rats
(FIG. 15A) The tumor uptake in rats treated with paclitaxel (FIG.
15B) was less than tumor uptake in rats not treated with paclitaxel
(FIG. 15A). Also illustrated is tumor uptake of .sup.99mTc-EC-NIM
in rats having sarcomas. FIG. 15C shows tumor uptake in sarcoma
bearing rats treated with paclitaxel while FIG. 15D shows tumor
uptake in rats not treated with paclitaxel. There was a decreased
uptake of .sup.99mTc-EC-NIM after treatment with paclitaxel.
[0044] FIG. 16. Synthetic scheme of EC-GAP (pentaglutamate).
[0045] FIG. 17. Scintigraphic images of breast tumors in
.sup.99mTc-EC-GAP injected group.
[0046] FIG. 18. Scintigraphic images of breast tumors in
.sup.99mTc-EC-ANNEX V injected group at different time
intervals.
[0047] FIG. 19A and FIG. 19B. Comparison of uptake difference of
.sup.99mTc-EC-ANNEX V between pre- (FIG. 19A) and post- (FIG. 19B)
paclitaxel treatment in ovarian tumor bearing group.
[0048] FIG. 20A and FIG. 20B. Comparison of uptake difference of
.sup.99mTc-EC-ANNEX V between pre- (FIG. 20A) and post- (FIG. 20B)
paclitaxel treatment in sarcoma tumor bearing group.
[0049] FIG. 21. Synthetic scheme of EC-COL (colchicine).
[0050] FIG. 22. Illustration that no degradation products observed
in EC-COL synthesis.
[0051] FIG. 23. Ratios of tumor to muscle and tumor to blood as
function of time for .sup.99mTc-EC-COL.
[0052] FIG. 24. Ratios of tumor to muscle and tumor to blood as
function of time for .sup.99mTc-EC.
[0053] FIG. 25. In vivo imaging studies in breast tumor bearing
rats with .sup.99mTc-EC-COL.
[0054] FIG. 26. In vivo imaging studies in breast tumor bearing
rats with .sup.99mTc-EC.
[0055] FIG. 27. Computer outlined region of interest after
injection of .sup.99mTc-EC-COL vs. .sup.99mTc-EC.
[0056] FIG. 28. SPECT with .sup.99mTc-EC-MN of 59 year old male
patient who suffered stroke. Images taken one hour
post-injection.
[0057] FIG. 29. MRI T1 weighted image of same patient as FIG.
28.
[0058] FIG. 30. SPECT with .sup.99mTc-EC-MN of 73 year old male
patient one day after stroke at one hour post-injection.
[0059] FIG. 31. SPECT with .sup.99mTc-EC-MN of same 73 year old
patient as imaged in FIG. 30 twelve days after stroke at one hour
post-injection.
[0060] FIG. 32. CT of same 73 year old male stroke patient as
imaged in FIG. 30, one day after stroke.
[0061] FIG. 33. CT of same 73 year old male stroke patient as
imaged in FIG. 32, twelve days after stroke. Note, no marked
difference between days one and twelve using CT for imaging.
[0062] FIG. 34. SPECT with 99mTc-EC-MN of 72 year old male patient
who suffered a stroke at one hour post-injection.
[0063] FIG. 35. CT of same 72 year old stroke patient as imaged in
FIG. 34. Note how CT image exaggerates the lesion size.
[0064] FIG. 36. Synthetic scheme of 99mTc-EC-neomycin.
[0065] FIG. 37A. Scintigraphic image of breast tumor-bearing rats
after administration of .sup.99mTc-EC and .sup.99mTc-EC-neomycin
(100 .mu.Ci/rat, iv.) showed that the tumor could be well
visualized from 0.5-4 hours postinjection.
[0066] FIG. 37B. Scintimammography with .sup.99mTc-EC-neomycin (30
mCi, iv.) of a breast cancer patient. Images taken two hours
post-injection.
[0067] FIG. 38A. .sup.1H-NMR of EC.
[0068] FIG. 38B. .sup.1H-NMR of neomycin.
[0069] FIG. 38C. .sup.1H-NMR of EC-neomycin.
[0070] FIG. 39A and FIG. 39B. Mass spectrometry of EC-neomycin
(M+1112.55).
[0071] FIG. 40A. UV wavelength scan of EC.
[0072] FIG. 40B. UV wavelength scan of neomycin.
[0073] FIG. 40C. UV wavelength scan of EC-neomycin.
[0074] FIG. 41. Radio-TLC analysis of .sup.99mTc-EC-neomycin.
[0075] FIG. 42. HPLC analysis of .sup.99mTc-EC-neomycin
(radioactive detector).
[0076] FIG. 43. HPLC analysis of .sup.99mTc-EC-neomycin (UV 254
nm).
[0077] FIG. 44. HPLC analysis of .sup.18F-FDG (radioactive
detector).
[0078] FIG. 45. HPLC analysis of .sup.18F-FDG (UV 254 nm).
[0079] FIG. 46. In vitro cellular uptake assay of a series of
.sup.99mTc-EC-drug conjugates in lung cancer cell line.
.sup.99mTc-EC-neomycin showed highest uptake in the agents
tested.
[0080] FIG. 47. Effect of glucose on cellular (A549) uptake of
.sup.99mTc-EC-neomycin and .sup.18F-FDG.
[0081] FIG. 48A and FIG. 48B. Effect of glucose on cellular (H1299)
uptake of .sup.99mTc-EC-neomycin and .sup.18F-FDG illustrated as
percent of drug uptake (FIG. 48A) and as percent of change with
glucose loading (FIG. 48B).
[0082] FIG. 49. Synthetic scheme of .sup.99mTc-EC-Glucosamine
[0083] FIG. 50. Hexokinase assay of glucose.
[0084] FIG. 51. Hexokinase assay of glucosamine.
[0085] FIG. 52. Hexokinase assay of EC-glucosamine.
[0086] FIG. 53. Hexokinase assay of EC-GAP-glucosamine.
[0087] FIG. 54. Synthetic scheme of
.sup.99mTc-EC-GAP-glucosamine.
[0088] FIG. 55A, FIG. 55B, FIG. 55C. In vitro cellular uptake assay
of .sup.99mTc-EC (FIG. 56A), .sup.99mTc-EC-deoxyglucose-GAP (FIG.
56B), and .sup.18F-PDG (FIG. 56C) in lung cancer cell line (A549).
.sup.99mTc-EC-DG showed similar uptake compared to
.sup.18F-FDG.
[0089] FIG. 56. Tumor-to-tissue count density ratios of
.sup.99mTc-EC-GAP in breast tumor-bearing rats.
[0090] FIG. 57 In vitro cellular uptake of .sup.18PDG with glucose
loading at 2 hours post-injection in breast cancer cell line
(13762).
[0091] FIG. 58. In vivo tissue uptake of .sup.99mTc-EC-neomycin in
breast tumor-bearing mice.
[0092] FIG. 59. Synthetic scheme of .sup.99mTc-EC-deoxyglucose.
[0093] FIG. 60. Mass spectrometry of EC-deoxyglucose.
[0094] FIG. 61. .sup.1H-NMR of EC-deoxyglucose (EC-DG).
[0095] FIG. 62. .sup.1H-NMR of glucosamine.
[0096] FIG. 63. Radio-TLC analysis of .sup.99mTc-EC-DG.
[0097] FIG. 64. HPLC analysis of .sup.99mTc-EC-deoxyglucose and
.sup.99mTc-EC-(radioactive detector).
[0098] FIG. 65. HPLC analysis of .sup.99mTc-EC-deoxyglucose and
.sup.99mTc-EC (radioactive detector, mixed).
[0099] FIG. 66. Hexokinase assay of glucose.
[0100] FIG. 67. Hexokinase assay of FDG.
[0101] FIG. 68. Hexokinase assay of EC-DG.
[0102] FIG. 69. In vitro cellular uptake assay of
.sup.99mTc-EC-deoxyglucose, .sup.99mTc-EC and .sup.18F-FDG in lung
cancer cell line (A549). .sup.99mTc-EC-DG showed similar uptake
compared to .sup.18F-FDG.
[0103] FIG. 70. Effect of d- and 1-glucose on breast cellular
(13762 cell line) uptake of .sup.99mTc-EC-DG.
[0104] FIG. 71. Effect of d- and I-glucose on breast cellular
(13762 cell line) uptake of .sup.18F-FDG.
[0105] FIG. 72. Effect of d- and l-glucose on breast cellular (A549
cell line) uptake .sup.18F-FDG.
[0106] FIG. 73. Effect of d- and I-glucose on breast cellular (A549
cell line) uptake of .sup.99mTc-EC-DG.
[0107] FIG. 74. Effect of in vivo blood glucose level induced by
glucosamine and EC-DG (1.2 mmol/kg, i.v.).
[0108] FIG. 75. Effect of in vivo blood glucose level induced by
FDG (1.2 and 1.9 mmol/kg, i.v.) and insulin.
[0109] FIG. 76. Tumor-to-tissue count density ratios of
.sup.99mTc-EC-deoxyglucose in breast tumor-bearing rats.
[0110] FIG. 77. In vivo biodistribution of
.sup.99mTc-EC-deoxyglucose in breast tumor-bearing rats.
[0111] FIG. 78. In vivo tissue uptake of .sup.99mTc-EC-deoxyglucose
in lung tumor-bearing mice.
[0112] FIG. 79. In vivo tissue uptake of .sup.99mTc-EC-neomycin in
lung tumor-bearing mice.
[0113] FIG. 80. In vivo tissue uptake of .sup.18F-FDG in lung
tumor-bearing mice.
[0114] FIG. 81. Planar image of breast tumor-bearing rats after
administration of .sup.99mTc-EC and .sup.99mTc-EC-deoxyglucose (100
.mu.Ci/rat, iv.) showed that the tumor could be well visualized
from 0.5-4 hours postinjection.
[0115] FIG. 82A. MRI of a patient with malignant astrocytoma.
[0116] FIG. 82B. SPECT with .sup.99mTc-EC-DG of a patient with
malignant astrocytoma.
[0117] FIG. 83A. MRI of a patient with hemorrhagic astrocytoma.
[0118] FIG. 83B. SPECT with .sup.99mTc-EC-DG of a patient with
malignant astrocytoma.
[0119] FIG. 84A. MRI of a patient with benign meningioma.
[0120] FIG. 84B. SPECT with .sup.99mTc-EC-DG of a patient with
benign meningioma showed no focal intensed uptake.
[0121] FIG. 85A. CT of a patient with TB in lung.
[0122] FIG. 85B. SPECT with .sup.99mTc-EC-DG of a patient with TB
showed no focal intensed uptake.
[0123] FIG. 86A. CT of patient with lung cancer.
[0124] FIG. 86B. Whole body images of .sup.99mTc-EC-DG of a patient
with lung cancer.
[0125] FIG. 86C. SPECT with .sup.99mTc-EC-DG of a patient with lung
cancer, the tumor showed focal intensed uptake.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0126] In the field of nuclear medicine, certain pathological
conditions are localized, or their extent is assessed, by detecting
the distribution of small quantities of internally-administered
radioactively labeled tracer compounds (called radiotracers or
radiopharmaceuticals). Methods for detecting these
radiopharmaceuticals are known generally as imaging or radioimaging
methods.
[0127] In radioimaging, the radiolabel is a gamma-radiation
emitting radionuclide and the radiotracer is located using a
gamma-radiation detecting camera (this process is often referred to
as gamma scintigraphy). The imaged site is detectable because the
radiotracer is chosen either to localize at a pathological site
(termed positive contrast) or, alternatively, the radiotracer is
chosen specifically not to localize at such pathological sites
(termed negative contrast).
[0128] A variety of radionuclides are known to be useful for
radioimaging, including .sup.67Ga, .sup.99mTc, .sup.111In,
.sup.123I, .sup.125I, .sup.169Yb or .sup.186Re. Due to better
imaging characteristics and lower price, attempts have been made to
replace the .sup.123I, .sup.131I, .sup.67Ga and .sup.111In labeled
compounds with corresponding .sup.99mTc labeled compounds when
possible. Due to favorable physical characteristics as well as
extremely low price ($0.21/mCi), .sup.99mTc has been preferred to
label radiopharmaceuticals. Although it has been reported that
DTPA-drug conjugate could be labeled with .sup.99mTc effectively
(Mathias et al., 1997), DTPA moiety does not chelate with
.sup.99mTc as stable as with .sup.111In. (Goldsmith, 1997).
[0129] A number of factors must be considered for optimal
radioimaging in humans. To maximize the efficiency of detection, a
radionuclide that emits gamma energy in the 100 to 200 keV range is
preferred. To minimize the absorbed radiation dose to the patient,
the physical half-life of the radionuclide should be as short as
the imaging procedure will allow. To allow for examinations to be
performed on any day and at any time of the day, it is advantageous
to have a source of the radionuclide always available at the
clinical site. .sup.99mTc is a preferred radionuclide because it
emits gamma radiation at 140 keV, it has a physical half-life of 6
hours, and it is readily available on-site using a
molybdenum-99/technetium-99m generator.
[0130] Bis-aminoethanethiol tetradentate ligands, also called
diaminodithiol compounds, are known to form very stable
Tc(V)O-complexes on the basis of efficient binding of the
oxotechnetium group to two thiolsulfur and two amine nitrogen
atoms. (Davison et al., 1980; 1981; Verbruggen et al., 1992).
.sup.99mTc-L,L-ethylenedicysteine (.sup.99mTc-EC) is the most
recent and successful example of N.sub.2S.sub.2 chelates.
(Verbruggen et al., 1992; Van Nerom et al., 1993; Surma et al.,
1994). EC, a new renal imaging agent, can be labeled with
.sup.99mTc easily and efficiently with high radiochemical purity
and stability and is excreted through kidney by active tubular
transport. (Verbruggen et al., 1992; Van Nerom et al., 1993; Surma
et al., 1994; Verbruggen et al., 1990; Van Nerom et al., 1990;
Jamar et al., 1993). Other applications of EC would be chelated
with galium-68 (a positron emitter, t1/2=68 minutes) for PET and
gadolinium, iron or manganese for magnetic resonance imaging
(MRI).
[0131] The present invention utilizes .sup.99mTc-EC as a labeling
agent to target ligands to specific tissue types for imaging. The
advantage of conjugating the EC with tissue targeting ligands is
that the specific binding properties of the tissue targeting ligand
concentrates the radioactive signal over the area of interest.
While it is envisioned that the use of .sup.99mTc-EC as a labeling
strategy can be effective with virtually any type of compound, some
suggested preferred ligands are provided herein for illustration
purposes. It is contemplated that the .sup.99mTc-EC-drug conjugates
of the invention may be useful to image not only tumors, but also
other tissue-specific conditions, such as infection, hypoxic tissue
(stroke), myocardial infarction, apoptotic cells, Alzheimer's
disease and endometriosis.
[0132] Radiolabeled proteins and peptides have been reported in the
prior art. (Ege et al., U.S. Pat. No. 4,832,940, Abrams et al.,
1990; Bakker et al., 1990; Goldsmith et al., 1995, 1997; Olexa et
al. 1982; Ranby et al. 1988; Hadley et al. 1988; Lees et al. 1989;
Sobel et al. 1989; Stuttle, 1990; Maraganore et al. 1991; Rodwell
et al. 1991; Tubis et al. 1968; Sandrehagen 1983). However,
.sup.99mTc-EC has not been used in conjunction with any ligands,
other than as the diethylester (Kabasakal, 2000), prior to the
present invention. The diethylester of EC was used as a cerebral
blood flow agent (Kikukawa, et al., 2000).
[0133] Although optimal for radioimaging, the chemistry of
.sup.99mTc has not been as thoroughly studied as the chemistry of
other elements and for this reason methods of radiolabeling with
.sup.99mTc are not abundant. .sup.99mTc is normally obtained as
.sup.99mTc pertechnetate (TcO.sub.4.sup.-; technetium in the +7
oxidation state), usually from a molybdenum-99/technetium-99m
generator. However, pertechnetate does not bind well with other
compounds. Therefore, in order to radiolabel a compound, .sup.99mTc
pertechnetate must be converted to another form. Since technetium
does not form a stable ion in aqueous solution, it must be held in
such solutions in the form of a coordination complex that has
sufficient kinetic and thermodynamic stability to prevent
decomposition and resulting conversion of .sup.99mTc either to
insoluble technetium dioxide or back to pertechnetate.
[0134] For the purpose of radiolabeling, it is particularly
advantageous for the .sup.99mTc complex to be formed as a chelate
in which all of the donor groups surrounding the technetium ion are
provided by a single chelating ligand--in this case,
ethylenedicysteine. This allows the chelated .sup.99mTc to be
covalently bound to a tissue specific ligand either directly or
through a single linker between the ethylenedicysteine and the
ligand.
[0135] Technetium has a number of oxidation states: +1, +2, +4, +5,
+6 and +7. When it is in the +1 oxidation state, it is called Tc
MIBI. Tc MIBI must be produced with a heat reaction. (Seabold et
al. 1999). For purposes of the present invention, it is important
that the Tc be in the +4 oxidation state. This oxidation state is
ideal for forming the N.sub.2S.sub.2 chelate with EC. Thus, in
forming a complex of radioactive technetium with the drug
conjugates of the invention, the technetium complex, preferably a
salt of .sup.99mT.sub.c pertechnetate, is reacted with the drug
conjugates of the invention in the presence of a reducing
agent.
[0136] The preferred reducing agent for use in the present
invention is stannous ion in the form of stannous chloride
(SnCl.sub.2) to reduce the Tc to its +4 oxidation state. However,
it is contemplated that other reducing agents, such as dithionate
ion or ferrous ion may be useful in conjunction with the present
invention. It is also contemplated that the reducing agent may be a
solid phase reducing agent. The amount of reducing agent can be
important as it is necessary to avoid the formation of a colloid.
It is preferable, for example, to use from about 10 to about 100
.mu.g SnCl.sub.2 per about 100 to about 300 mCi of Tc
pertechnetate. The most preferred amount is about 0.1 mg SnCl.sub.2
per about 200 mCi of Tc pertechnetate and about 2 ml saline. This
typically produces enough Tc-EC-tissue specific ligand conjugate
for use in 5 patients.
[0137] It is often also important to include an antioxidant in the
composition to prevent oxidation of the ethylenedicysteine. The
preferred antioxidant for use in conjunction with the present
invention is vitamin C (ascorbic acid). However, it is contemplated
that other antioxidants, such as tocopherol, pyridoxine, thiamine
or rutin, may also be useful.
[0138] In general, the ligands for use in conjunction with the
present invention will possess either amino or hydroxy groups that
are able to conjugate to EC on either one or both acid arms. If
amino or hydroxy groups are not available (e.g., acid functional
group), a desired ligand may still be conjugated to EC and labeled
with .sup.99mTc using the methods of the invention by adding a
linker, such as ethylenediamine, amino propanol,
diethylenetriamine, aspartic acid, polyaspartic acid, glutamic
acid, polyglutamic acid, or lysine. Ligands contemplated for use in
the present invention include, but are not limited to,
angiogenesis/antiangiogenesis ligands, DNA topoisomerase
inhibitors, glycolysis markers, antimetabolite ligands,
apoptosis/hypoxia ligands, DNA intercalators, receptor markers,
peptides, nucleotides, antimicrobials such as antibiotics or
antifungals, organ specific ligands and sugars or agents that mimic
glucose.
[0139] EC itself is water soluble. It is necessary that the EC-drug
conjugate of the invention also be water soluble. Many of the
ligands used in conjunction with the present invention will be
water soluble, or will form a water soluble compound when
conjugated to EC. If the tissue specific ligand is not water
soluble, however, a linker which will increase the solubility of
the ligand may be used. Linkers may attach to an aliphatic or
aromatic alcohol, amine or peptide or to a carboxylic and or
peptide. Linkers may be either poly amino acid (peptide) or amino
acid such as glutamic acid, aspartic acid or lysine. Table 1
illustrates desired linkers for specific drug functional
groups.
TABLE-US-00001 TABLE 1 Drug Functional Group Linker Example
Aliphatic or phenolio-OH EC-Poly (glutamic acid) A (MW. 750-15,000)
or EC. poly(aspertic acid) (MW. 2000-15,000) or bromo ethylacetate
or EC-glutamic acid or EC-aspertic acid. Aliphatic or
aromatic-NH.sub.2 EC-poly(glutamic acid) B or peptide (MW.
750-15,000) or EC- poly(aspertic acid) (MW. 2000-15,000) or EC-
glutamic acid (mono- or diester) or EC-aspartic acid. Carboxylic
acid or peptide Ethylene diamine, lysine C Examples: A. estradiol,
topotecan, paclitaxel, raloxlfen etoposide B. doxorubicin,
mitomycin C, endostatin, annexin V. LHRH, octreotide, VIP C.
methotrexate, folic acid
[0140] It is also envisioned that the EC-tissue specific ligand
drug conjugates of the invention may be chelated to other
radionuclides and used for radionuclide therapy. Generally, it is
believed that virtually any .alpha., .beta.-emitter,
.gamma.-emitter, or .beta., .gamma.-emitter can be used in
conjunction with the invention. Preferred .beta., .gamma.-emitters
include .sup.166Ho, .sup.188Re, .sup.186Re, .sup.153Sm, and
.sup.89Sr. Preferred .beta.-emitters include .sup.90Y and
.sup.225Ac. Preferred .gamma.-emitters include .sup.67Ga,
.sup.68Ga, .sup.64Cu, .sup.62Cu and .sup.111In. Preferred
.alpha.-emitters include .sup.211At and .sup.212Bi. It is also
envisioned that para-magnetic substances, such as Gd, Mn and Fe can
be chelated with EC for use in conjunction with the present
invention.
[0141] Complexes and means for preparing such complexes are
conveniently provided in a kit form including a sealed vial
containing a predetermined quantity of an EC-tissue specific ligand
conjugate of the invention to be labeled and a sufficient amount of
reducing agent to label the conjugate with .sup.99mTc. .sup.99mTc
labeled scintigraphic imaging agents according to the present
invention can be prepared by the addition of an appropriate amount
of .sup.99mTc or .sup.99mTc complex into a vial containing the
EC-tissue specific ligand conjugate and reducing agent and reaction
under conditions described in Example 1 hereinbelow. The kit may
also contain conventional pharmaceutical adjunct materials such as,
for example, pharmaceutically acceptable salts to adjust the
osmotic pressure, buffers, preservatives, antioxidants, and the
like. The components of the kit may be in liquid, frozen or dry
form. In a preferred embodiment, kit components are provided in
lyophilized form.
[0142] Radioactively labeled reagents or conjugates provided by the
present invention are provided having a suitable amount of
radioactivity. In forming .sup.99mTc radioactive complexes, it is
generally preferred to form radioactive complexes in solutions
containing radioactivity at concentrations of from about 0.01
millicurie (mCi) to about 300 mCi per mL.
[0143] .sup.99mTc labeled scintigraphic imaging agents provided by
the present invention can be used for visualizing sites in a
mammalian body. In accordance with this invention, the .sup.99mTc
labeled scintigraphic imaging agents are administered in a single
unit injectable dose. Any of the common carriers known to those
with skill in the art, such as sterile saline solution or plasma,
can be utilized after radiolabeling for preparing the injectable
solution to diagnostically image various organs, tumors and the
like in accordance with this invention. Generally, the unit dose to
be administered has a radioactivity of about 0.01 mCi to about 300
mCi, preferably 10 mCi to about 200 mCi. The solution to be
injected at unit dosage is from about 0.01 mL to about 10 mL. After
intravenous administration, imaging of the organ or tumor in vivo
can take place, if desired, in hours or even longer, after the
radiolabeled reagent is introduced into a patient. In most
instances, a sufficient amount of the administered dose will
accumulate in the area to be imaged within about 0.1 of an hour to
permit the taking of scintiphotos. Any conventional method of
scintigraphic imaging for diagnostic or prognostic purposes can be
utilized in accordance with this invention.
[0144] The .sup.99mTc-EC labeling strategy of the invention may
also be used for prognostic purposes. It is envisioned that EC may
be conjugated to known drugs of choice for cancer chemotherapy,
such as those listed in Table 2. These EC-drug conjugates may then
be radio labeled with .sup.99mTc and administered to a patent
having a tumor. The labeled EC-drug conjugates will specifically
bind to the tumor. Imaging may be performed to determine the
effectiveness of the cancer chemotherapy drug against that
particular patient's particular tumor. In this way, physicians can
quickly determine which mode of treatment to pursue, which
chemotherapy drug will be most effective. This represents a
dramatic improvement over current methods which include choosing a
drug and administering a round of chemotherapy. This involves
months of the patient's time and many thousands of dollars before
the effectiveness of the drug can be determined.
[0145] The .sup.99mTc labeled EC-tissue specific ligand conjugates
and complexes provided by the invention may be administered
intravenously in any conventional medium for intravenous injection
such as an aqueous saline medium, or in blood plasma medium. Such
medium may also contain conventional pharmaceutical adjunct
materials such as, for example, pharmaceutically acceptable salts
to adjust the osmostic pressure, buffers, preservatives,
antioxidants and the like. Among the preferred media are normal
saline and plasma.
[0146] Specific, preferred targeting strategies are discussed in
more detail below.
Tumor Folate Receptor Targeting
[0147] The radiolabeled ligands, such as pentetreotide and
vasoactive intestinal peptide, bind to cell receptors, some of
which are overexpressed on tumor cells (Britton and Granowska,
1996; Krenning et al., 1995; Reubi et al., 1992; Goldsmith et al.,
1995; Virgolini et al., 1994). Since these ligands are not
immunogenic and are cleared quickly from the plasma, receptor
imaging would seem to be more promising compared to antibody
imaging.
[0148] Folic acid as well as antifolates such as methotrexate enter
into cells via high affinity folate receptors
(glycosylphosphatidylinositol-linked membrane folate-binding
protein) in addition to classical reduced-folate carrier system
(Westerhof et al., 1991; On et al., 1995; Hsuch and Dolnick, 1993).
Folate receptors (FRs) are overexposed on many neoplastic cell
types (e.g., lung, breast, ovarian, cervical, colorectal,
nasopharyngeal, renal adenocarcinomas, malign melanoma and
ependymomas), but primarily expressed only several normal
differentiated tissues (e.g., choroid plexus, placenta, thyroid and
kidney) (Orr et al., 1995; Weitman et al., 1992a; Campbell et al.,
1991; Weitman et al., 1992b; Holm et al., 1994; Ross et al., 1994;
Franklin et al., 1994; Weitman et al., 1994). FRs have been used to
deliver folate-conjugated protein toxins, drug/antisense
oligonucleotides and liposomes into tumor cells overexpressing the
folate receptors (Ginobbi et al., 1997; Leamon and Low, 1991;
Leamon and Low, 1992; Leamon et al., 1993; Lee and Low, 1994).
Furthermore, bispecific antibodies that contain anti-FR antibodies
linked to anti-T cell receptor antibodies have been used to target
T cells to FR-positive tumor cells and are currently in clinical
trials for ovarian carcinomas (Canevari et al., 1993; Bolhuis et
al., 1992; Patrick et al., 1997; Coney et al., 1994; Kranz et al.,
1995). Similarly, this property has been inspired to develop
radiolabeled folate-conjugates, such as
.sup.67Ga-deferoxamine-folate and .sup.111In-DTPA-folate for
imaging of folate receptor positive tumors (Mathias et al., 1996;
Wang et al., 1997; Wang et al., 1996; Mathias et al., 1997b).
Results of limited in vitro and in vivo studies with these agents
suggest that folate receptors could be a potential target for tumor
imaging. In this invention, the inventors developed a series of new
folate receptor ligands. These ligands are .sup.99mTc-EC-folate,
.sup.99mTc-EC-methotrexate (.sup.99mTc-EC-MTX),
.sup.99mTc-EC-tomudex (.sup.99mTc-EC-TDX).
Tumor Hypoxia Targeting
[0149] Tumor cells are more sensitive to conventional radiation in
the presence of oxygen than in its absence; even a small percentage
of hypoxic cells within a tumor could limit the response to
radiation (Hall, 1988; Bush et al., 1978; Gray et al., 1953).
Hypoxic radioresistance has been demonstrated in many animal tumors
but only in few tumor types in humans (Dische, 1991; Gatenby et
al., 1988; Nordsmark et al., 1996). The occurrence of hypoxia in
human tumors, in most cases, has been inferred from histology
findings and from animal tumor studies. In vivo demonstration of
hypoxia requires tissue measurements with oxygen electrodes and the
invasiveness of these techniques has limited their clinical
application.
[0150] Misonidazole (MISO) is a hypoxic cell sensitizer, and
labeling MISO with different radioisotopes (e.g., .sup.18F,
.sup.123I, .sup.99mTc) may be useful for differentiating a hypoxic
but metabolically active tumor from a well-oxygenated active tumor
by PET or planar scintigraphy. [.sup.18F]Fluoromisonidazole (FMISO)
has been used with PET to evaluate tumors hypoxia. Recent studies
have shown that PET, with its ability to monitor cell oxygen
content through [.sup.18F]FMISO, has a high potential to predict
tumor response to radiation (Koh et al., 1992; Valk et al., 1992;
Martin et al., 1989; Rasey et al., 1989; Rasey et al., 1990; Yang
et al., 1995). PET gives higher resolution without collimation,
however, the cost of using PET isotopes in a clinical setting is
prohibitive. Although labeling MISO with iodine was the choice,
high uptake in thyroid tissue was observed. Therefore, it is
desirable to develop compounds for planar scintigraphy that the
isotope is less expensive and easily available in most major
medical facilities. In this invention, the inventors present the
synthesis of .sup.99mTc-EC-2-nitroimidazole and
.sup.99mTc-EC-metronidazole and demonstrate their potential use as
tumor hypoxia markers.
Peptide Imaging of Cancer
[0151] Peptides and amino acids have been successfully used in
imaging of various types of tumors (Wester et al., 1999; Coenen and
Stocklin, 1988; Raderer et al., 1996; Lambert et al., 1990; Bakker
et al., 1990; Stella and Mathew, 1990; Butterfield et al., 1998;
Piper et al., 1983; Mochizuki et al., Dickinson and Hiltner, 1981).
Glutamic acid based peptide has been used as a drug carrier for
cancer treatment (Stella and Mathew, 1990; Butterfield et al.,
1998; Piper et al., 1983; Mochizuki et al., 1985; Dickinson and
Hiltner, 1981). It is known that glutamate moiety of folate
degraded and formed polyglutamate in vivo. The polyglutamate is
then re-conjugated to folate to form folyl polyglutamate, which is
involved in glucose metabolism. Labeling glutamic acid peptide may
be useful in differentiating the malignancy of the tumors. In this
invention, the inventors report the synthesis of EC-glutamic acid
pentapeptide and evaluate its potential use in imaging tumors.
Imaging Tumor Apoptotic Cells
[0152] Apoptosis occurs during the treatment of cancer with
chemotherapy and radiation (Lennon et al., 1991; Abrams et al.,
1990; Blakenberg et al., 1998; Blakenberg et al., 1999; Tait and
Smith, 1991) Annexin V is known to bind to phosphotidylserin, which
is overexpressed by tumor apoptotic cells (Blakenberg et al., 1999;
Tait and Smith, 1991). Assessment of apoptosis by annexin V would
be useful to evaluate the efficacy of therapy such as disease
progression or regression. In this invention, the inventors
synthesize .sup.99mTc-EC-annexin V (EC-ANNEX) and evaluate its
potential use in imaging tumors.
Imaging Tumor Angiogenesis
[0153] Angiogenesis is in part responsible for tumor growth and the
development of metastasis. Antimitotic compounds are antiangiogenic
and are known for their potential use as anticancer drugs. These
compounds inhibit cell division during the mitotic phase of the
cell cycle. During the biochemical process of cellular functions,
such as cell division, cell motility, secretion, ciliary and
flagellar movement, intracellular transport and the maintenance of
cell shape, microtubules are involved. It is known that antimitotic
compounds bind with high affinity to microtubule proteins
(tubulin), disrupting microtubule assembly and causing mitotic
arrest of the proliferating cells. Thus, antimitotic compounds are
considered as microtubule inhibitors or as spindle poisons (Lu,
1995).
[0154] Many classes of antimitotic compounds control microtubule
assembly-disassembly by binding to tubulin (Lu, 1995; Goh et al.,
1998; Wang et al., 1998; Rowinsky et al., 1990; Imbert, 1998).
Compounds such as colchicinoids interact with tubulin on the
colchicine-binding sites and inhibit microtubule assembly (Lu,
1995; Goh et al., 1998; Wang et al., 1998). Among colchicinoids,
colchicine is an effective anti-inflammatory drug used to treat
prophylaxis of acute gout. Colchicine also is used in chronic
myelocytic leukemia. Although colchicinoids are potent against
certain types of tumor growth, the clinical therapeutic potential
is limited due to inability to separate the therapeutic and toxic
effects (Lu, 1995). However, colchicine may be useful as a
biochemical tool to assess cellular functions. In this invention,
the inventors developed .sup.99mTc-EC-colchicine (EC-COL) for the
assessment of biochemical process on tubulin functions.
Imaging Tumor Apoptotic Cells
[0155] Apoptosis occurs during the treatment of cancer with
chemotherapy and radiation. Annexin V is known to bind to
phosphotidylserin, which is overexpressed by tumor apoptotic cells.
Assessment of apoptosis by annexin V would be useful to evaluate
the efficacy of therapy such as disease progression or regression.
Thus, .sup.99mTc-EC-annexin V (EC-ANNEX) was developed.
Imaging Tumor Hypoxia
[0156] The assessment of tumor hypoxia by an imaging modality prior
to radiation therapy would provide rational means of selecting
patients for treatment with radiosensitizers or bioreductive drugs
(e.g., tirapazamine, mitomycin C). Such selection of patients would
permit more accurate treatment patients with hypoxic tumors. In
addition, tumor suppressor gene (P53) is associated with multiple
drug resistance. To correlate the imaging findings with the
overexpression of P53 by histopathology before and after
chemotherapy would be useful in following-up tumor treatment
response. .sup.99mTc-EC-2-nitroimidazole and
.sup.99mTc-EC-metronidazole were developed.
Imaging Tumor Angiogenesis
[0157] Angiogenesis is in part responsible for tumor growth and the
development of metastasis. Antimitotic compounds are antiangiogenic
and are known for their potential use as anticancer drugs. These
compounds inhibit cell division during the mitotic phase of the
cell cycle. During the biochemical process of cellular functions,
such as cell division, cell motility, secretion, ciliary and
flagellar movement, intracellular transport and the maintenance of
cell shape, microtubules are involved. It is known that antimitotic
compounds bind with high affinity to microtubule proteins
(tubulin), disrupting microtubule assembly and causing mitotic
arrest of the proliferating cells. Thus, antimitotic compounds are
considered as microtubule inhibitors or as spindle poisons.
Colchicine, a potent antiangiogenic agent, is known to inhibit
microtubule polymerization and cell arrest at metaphase. Colchicine
(COL) may be useful as a biochemical tool to assess cellular
functions. .sup.99mTc-EC-COL was then developed.
Imaging Hypoxia Due to Stroke
[0158] Although tumor cells are more or less hypoxic, it requires
an oxygen probe to measure the tensions. In order to mimic hypoxic
conditions, the inventors imaged 11 patients who had experienced
stroke using .sup.99mTc-EC-metronidazole (.sup.99mTc-EC-MN).
Metronidazole is a tumor hypoxia marker. Tissue in the area of a
stroke becomes hypoxic due to lack of oxygen. The SPECT images were
conducted at 1 and 3 hours post injection with .sup.99mTc-EC-MN.
All of these imaging studies positively localized the lesions. CT
does not show the lesions very well or accurately. MRI and CT in
some cases exaggerate the lesion size. The following are selected
cases from three patients.
[0159] Case 1. A 59 year old male patient suffered a stroke in the
left basal ganglia. SPECT .sup.99mTc-EC-MN identified the lesions
at one hour post-injection (FIG. 28), which corresponds to MRI T1
weighted image (FIG. 29).
[0160] Case 2. A 73 year old male patient suffered a stroke in the
left medium cerebral artery (MCA) territory. SPECT .sup.99mTc-EC-MN
was obtained at day 1 and day 12 (FIGS. 30 and 31) at one hour
post-injection. The lesions showed significant increased uptake at
day 12. CT showed extensive cerebral hemorrhage in the lesions. No
marked difference was observed between days 1 and 12 (FIGS. 32 and
33). The findings indicate that the patient symptoms improved due
to the tissue viability (from anoxia to hypoxia). SPECT
.sup.99mTc-EC-MN provides functional information which is better
than CT images.
[0161] Case 3. A 72 year old male patient suffered a stroke in the
right MCA and PCA area. SPECT .sup.99mTc-EC-MN identified the
lesions at one hour post-injection (FIG. 34). CT exaggerates the
lesion size. (FIG. 35).
Tumor Glycolysis Targeting
[0162] The radiolabeled ligands, such as polysaccharide (neomycin,
kanamycin, tobramycin) and monosaccharide (glucosamine) bind to
cell glucose transporter, followed by phosphorylation which are
overexpressed on tumor cells (Rogers et al., 1968; Fanciulli et
al., 1994; Popovici et al., 1971; Jones et al., 1973; Hermann et
al., 2000). Polysaccharide (neomycin, kanamycin, tobramycin) and
monosaccharide (glucosamine) induced glucose level could be
suppressed by insulin (Harada et al., 1995; Moller et al., 1991;
Offield et al., 1996; Shankar et al., 1998; Yoshino et al., 1999;
Villevalois-Cam et al., 2000) Since these ligands are not
immunogenic and are cleared quickly from the plasma, metabolic
imaging would seem to be more promising compared to antibody
imaging.
[0163] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Tumor Folate Receptor Targeting
Synthesis of EC
[0164] EC was prepared in a two-step synthesis according to the
previously described methods (Ratner and Clarke, 1937; Blondeau et
al., 1967; each incorporated herein by reference). The precursor,
L-thiazolidine-4-carboxylic acid, was synthesized (m.p.
195.degree., reported 196-197.degree.). EC was then prepared (m.p.
237.degree., reported 251-253.degree.). The structure was confirmed
by .sup.1H-NMR and fast-atom bombardment mass spectroscopy
(FAB-MS).
Synthesis of Aminoethylamido Analogue of Methotrexate
(MTX-NH.sub.2)
[0165] MIX (227 ma, 0.5 mmol) was dissolved in 1 ml of HCI solution
(2N). The pH value was <3. To this stirred solution, 2 ml of
water and 4 ml of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
(EEDQ, 6.609% in methanol, 1 mmol) were added at room temperature.
Ethylenediamine (EDA, 0.6 ml, 10 mmol) was added slowly. The
reaction mixture was stirred overnight and the solvent was
evaporated in vacuo. The raw solid material was washed with diethyl
ether (10 ml), acetonitrile (10 ml) and 95% ethyl alcohol (50 ml)
to remove the unreacted EEDQ and EDA. The product was then dried by
lyophilization and used without further purification. The product
weighed 210 mg (84.7%) as a yellow powder. m.p. of product:
195-198.degree. C. (dec, MIX); .sup.1H-NMR (D.sub.2O) .delta.
2.98-3.04 (d, 8H, --(CH.sub.2).sub.2CONH(CH.sub.0).sub.2NH.sub.2),
4.16-4.71 (m, 6H, --CH.sub.2- pteridinyl, aromatic-NCH.sub.3,
NH--CH--COOH glutamate), 6.63-6.64 (d, 2H, aromatic-CO), 7.51-753
(d, 2H. aromatic-N), 8.36 (s, 1H, pteridinyl). FAB MS m/z calcd for
C.sub.22H.sub.28, N.sub.10O.sub.4(M).sup.+ 496.515, found
496.835.
Synthesis of Aminoethylamido Analogue of Folate
(Folate-NH.sub.2)
[0166] Folic acid dihydrate (1 g, 2.0 mmol) was added in 10 ml of
water. The pH value was adjusted to 2 using HCI (2 N). To this
stirred solution, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
(EEDQ, 1 g in 10 ml methanol, 4.0 mmol) and ethylenediamine (EDA,
1.3 ml, 18 mmol) were added slowly. The reaction mixture was
stirred overnight at room temperature. The solvent was evaporated
in vacuo. The product was precipitated in methanol (50 ml) and
further washed with acetone (100 ml) to remove the unreacted EEDQ
and EDIT. The product was then freeze-dried and used without
further purification. Ninhydrin (2% in methanol) spray indicated
the positivity of amino group. The product weighed 0.6 g (yield
60%) as a yellow powder. m.p. of product: 250.degree. (dec).
.sup.1H-NMR (D.sub.2O) .delta.1.97-2.27 (m, 2H, --CH.sub.2
glutamate of folate), 3.05-3.40 (d, 6H,
--CH.sub.2CONH(CH.sub.2).sub.2NH.sub.2), 4.27-4.84 (m, 3H,
--CH.sub.2-pteridinyl, NH--CH--COOH glutamate), 6.68-6.70 (d, 2H,
aromatic-CO), 7.60-7.62 (d, 2H, aromatic-N), 8.44 (s, 1H,
pteridinyl). FAB MS m/z calcd for
C.sub.21H.sub.25N.sub.9O.sub.5(M).sup.+ 483, found 483.21.
Synthesis of Ethylenedicysteine-Folate (EC-Folate)
[0167] To dissolve EC, NaOH (2N, 0.1 ml) was added to a stirred
solution of EC (114 ma, 0.425 mmol) in water (1.5 ml). To this
colorless solution, sulfo-NHS (92.3 mg, 0.425 mmol) and EDC (81.5
mg, 0.425 mmol) were added. Folate-NH.sub.2 (205 mg, 0.425 mmol)
was then added. The mixture was stirred at room temperature for 24
hours. The mixture was dialyzed for 48 hours using Spectra/POR
molecular porous membrane with molecule cut-off at 500 (Spectrum
Medical Industries Inc., Houston, Tex.). After dialysis, the
product was freeze dried. The product weighed 116 mg (yield 35%).
m.p. 195.degree. (dec). .sup.1H-NMR (D.sub.2O) .delta.1.98-2.28 (m,
21-1, --CH.sub.2 glutamate of folate), 2.60-2.95 (m, 4H and
--CH.sub.2--SH of EC). 3.24-3.34 (m, 10H, --CH.sub.2--CO,
ethylenediamine of folate and ethylenediamine of EC), 4.27-4.77 (m,
511, --CH-pteridinyl, NH--CH--COOH glutamate of folate and
NH--CH--COOH of EC), 6.60-6.62 (d, 2H, aromatic-CO), 7.58-7.59 (d,
2H. aromatic-N), 8.59 (s, 1H, pteridinyl). Anal. calcd for
C.sub.29H.sub.37N.sub.iiS.sub.2O.sub.8Na.sub.2(8H.sub.2O), FAB MS
m/z (M).sup.+ 777.3 (free of water). C, 37.79; H. 5.75; N, 16.72;
S, 6.95. Found: m/z (M).sup.+ 777.7 (20), 489.4 (100). C, 37.40; H,
5.42; N. 15.43; S, 7.58.
Radiolabeling of EC-Folate and EC with .sup.99mTc
[0168] Radiosynthesis of .sup.99mTc-EC-folate was achieved by
adding required amount of .sup.99mTc-pertechnetate into home-made
kit containing the lyophilized residue of EC-folate (3 mg),
SnCl.sub.2 (100 .mu.g), Na.sub.2HPO.sub.4 (13.5 mg), ascorbic acid
(0.5 mg) and NaEDTA (0.5 mg). Final pH of preparation was 7.4.
.sup.99mTc-EC was also obtained by using home-made kit containing
the lyophilized residue of EC (3 mg), SnCl.sub.2 (100 .mu.g),
Na.sub.2, IPO.sub.4 (13.5 mg), ascorbic acid (0.5 mg) and NaEDTA
(0.5 mg) at pH 10. Final pH of preparation was then adjusted to
7.4. Radiochemical purity was determined by TLC (ITLC SG, Gelman
Sciences, Ann Arbor, Mich.) eluted with, respectively, acetone
(system A) and ammonium acetate (1M in water):methanol (4:1)
(system B). From radio-TLC (Bioscan, Washington, D.C.) analysis,
the radiochemical purity was >95% for both radiopharmaceuticals.
Radio-TLC data are summarized in Table 2. Synthesis of
.sup.99mTc-EC-folate is shown in FIG. 1.
TABLE-US-00002 TABLE 2 DRUGS OF CHOICE FOR CANCER CHEMOTHERAPY The
tables that follow list drugs used for treatment of cancer in the
USA and Canada and their major adverse effects. The Drugs of Choice
listing based on the opinions of Medical Letter consultants. Some
drugs are listed for indications for which they have not been
approved by the US Food and Drug Administration. Anticancer drugs
and their adverse effects follow. For purposes of the present
invention, these lists are meant to be exemplary and not
exhaustive. DRUGS OF CHOICE Cancer Drugs of Choice Some
alternatives Adrenocortical** Mitotane Doxorubicin, streptozocin,
Cisplatin etoposide Bladder* Local: Instillation of BCG
Instillation of mitomycin, Systemic: Methotrexate + vinblastine +
doxorubicin or thiotape doxorubicin + claplatin (MVAC) Pecitaxel,
substitution of Claplatin + Methotrexate + vinblastine carboplatin
for claplatin in (CMV) combinations Brain Anaplastic astrocytoma*
Procarbazine + lamuatine + vincristine Carmustine, Claplatin
Anaplastic oligodendro- Procarbazine + lamustine + vincristine
Carmustine, Claplatin Giloma* Gilabiastome** Carmustine or
lamustine Procarbazine, claplatin Medulloblastoma Vincristine +
carmustine .+-. mechiorethamine .+-. Etoposide methotrexate
Mechiorethamine + vincristine + procarbazine + prednisone (MOPP)
Vincristine + claplatin .+-. cyclophosphamide Primary central
nervous Methotrexate (high dose Intravenous and/or system lymphoma
Intrathecal) .+-. cytarabine (Intravenous and/or Intrathecal)
Cyclophosphamide + Doxorubicin + vincristine + prednisone (CHOP)
Breast Adjuvant.sup.1: Cyclophosphamide + methotrexate +
fluorouracil (CMF); Cyclophosphamide + Doxorubicin .+-.
fluorouracil (AC or CAF); Tamoxifen Metastatic: Cyclophosphamide +
Paclitaxel; thiotepa + methotrexate + fluorouracil (CMF) or
Doxorubicin + vinblastine; Cyclophosphamide + duxorubicin .+-.
mitomycin + vinblastine; fluorouracil (AC or CAF) for receptor-
mitomycin + methotrexate + negative and/or hormone-refractory;
mitoxantrone; fluorouracil by Tamoxifen for receptor-positive
and/or continuous infusion; Bone hormone-sensitive.sup.2 marrow
transplant.sup.3 Cervix** Claplatin Chlorambucil, vincristine,
Ifosfamide with means fluorouracil, Doxorubicin, Bleomycin +
ifosfamide with means + methotrexate, altretamine claplatin
Chorlocarcinoma Methotrexate .+-. leucovorin Methotrexate +
dactinomycin + Dactinomycin cyclophosphamide (MAC) Etoposide +
methotrexate + dactinomycin + cyclophosphamide + vincristine
Colorectal* Adjuvant colon.sup.4: Fluorouracil + levamisole;
Hepatic metastases: fluorouracil + leucovorin Intrahepatic-arterial
floxuridine Metastatic: fluorouracil + leucovorin Mitomycin
Embryonal rhabdomyosarcoma.sup.5 Vincristine + dectinomycin .+-.
Same + Doxorubicin cyclophasphamide Vincristine + ifosfamide with
means + etoposide Endometrial** Megastrol or another progestin
fluorouracil, tamoxifen, Doxorubicin + claplatin .+-. altretamine
cyclophosphamide Esophageal* Claplatin + fluorouracil Doxorubicin,
methotraxate, mitomycin Ewing's sarcoma.sup.5 Cyclophosphamide (or
ifosfamide with CAV + etoposide means) + Doxorubicin + vincristine
(CAV) .+-. dactinomycin Gastric** Fluorouracil .+-. leucavorin
Claplatin Doxorubicin, etoposide, methotrexate + leucovorin,
mitomycin Head and neck squambus cell*.sup.6 Claplatin +
fluorouracil Blomycin, carboplatin, paclitaxel Methotrexate Islet
cell** Streptozocin + Doxorubicin Streptozocin + fluorouracil;
chlorozotocin.sup..dagger.; octreotide Kaposi's sarcoma*
(Aids-related) Etoposide or interferon alfa or vinblastine
Vincristine, Doxorubicin, Doxorubicin + bleomycin + vincristine or
bleomycin vinblastine (ABV) Leukemia Acute lymphocytic leukemia
Induction: Vincristine + prednisone + Induction: same .+-.
high-dose (ALL).sup.7 asparaginase .+-. daunorubicin methotrexate
.+-. cyterabine; CNS prophylaxis: Intrathecal methotrexate .+-.
pegaspargase instead of systemic high-dose methotrexate with
asparaginese leutovorin .+-. Intrathecal cytarabine .+-. Teniposide
or etoposide Intrathecal hydrocortisone High-dose cytarabine
Maintenance: Methotrexate + Maintenance: same + periodic
mercaptopurine vincristine + prednisone Bone marrow
transplant..sup.3 .sup.8 Acute myeloid leukemia (AML).sup.9
Induction: Cytsrabine + either daunorubicin Cytarabine +
mitoxentrone or idarubicin High-dose cyterabine Post Induction:
High-dose cytarabine .+-. other drugs such as etoposide Bone marrow
transplant.sup.3. Chronic lymphocytic leukemia Chlorambucil .+-.
prednisone Cladribine, cyclophosphamide, (CLL) Fludarabin
pentostatin, vincristine, Doxorubicin Chronic myeloid leukemia
(CML).sup.10 Chronic phase Bone marrow transplant.sup.3 Busulfan
Interferon alfa Hydroxyures Accelerated.sup.11 Bone marrow
transplant.sup.3 Hydroxyures, busulfen Blast crisis.sup.11
Lymphoid: Vincristine + prednisone + L- Tretinoln.sup..dagger.
separaginess + intrathecal methotrexate Amsecrine,
.sup..dagger.azacitidine (.+-.maintenance with methotrexate + 8-
Vincristine .+-. plicamycin marcaptopurine) Hairy cell Leukemia
Pentostatin or cladribine Interferon alfa, chlorambucil, fludarabin
Liver** Doxorubicin Intrahepatic-arterial floxuridine Fluorouracil
or claplatin Lung, small cell (cat cell) Claplatin + etoposide (PE)
Ifosfamide with means + Cyclophosphamide + doxorubicin +
carboplatin + etoposide (ICE) vincristine (CAV) Daily oral
etoposide PE alternated with CAV Etoposide + ifosfamide with
Cyclophosphamide + etoposide + claplatin means + claplatin (VIP
(CEP) Paclitaxel Duxorubicin + cyclophosphamide + etoposide (ACE)
Lung Claplatin + etoposide Claplatin + fluorouracil + (non-small
cell)** Claplatin + Vinblastine .+-. mitomycin leucovorin Claplatin
+ vincrisine Carboplatin + paclitaxel Lymphomas Hodgkin's.sup.12
Doxorubicin + bleomycin + vinblastine + Mechlorethamine +
vincristine + dacarbazine (ABVD) procarbazine + prednisone ABVD
alternated with MOPP (MOPP) Mechlorethamine + vincristine +
Chlorambusil + vinblastine + procarbazine (.+-.prednisone) +
doxorubicin + procarbazine + prednisone .+-. bleomycin +
vinblastine (MOP[P]-ABV) carmustine Etoposide + vinblastine +
doxorubicin Bone marrow transplant.sup.3 Non-Hodgkin's Burkitt's
lymphoma Cyclophosphamide + vincristine + Ifosfamide with means
methotrexate Cyclophosphamide + Cyclophosphamide + high-dose
cytarabine .+-. doxorubicin + vincrletine + methotrexate with
leutovorin prednisone (CHOP) Intrathecal methotrexate or cytarabine
Difuse large-cell lymphoma Cyclophosphamide + doxorubicin +
Dexamethasone sometimes vincristine + prednisone (CHOP) substituted
for prednisone Other combination regimens, which may include
methotrexate, etoposide, cytarabine, bleomycin, procarbazine,
ifosfamide and mitoxantrone Bone marrow transplant.sup.3 Follicular
lymphoma Cyclophosphamide or chlorambusil Same .+-. vincristine and
prednisone, .+-. etoposide Interferon alfa, cladribine, fludarabin
Bone marrow transplant.sup.3 Cyclophosphamide + doxorubicin +
vincristine + prednisone (CHOP) Melanoma** Interferon Alfa
Carmustine, lomustine, cisplatin Dacarbazine Dacarbazine +
clapletin + carmustine + tamoxifen Aldesleukin Mycosis fungoides*
PUVA (psoralen + ultraviolet A) Isotretinoin, topical carmustine,
Mechlorethamine (topical) pentosistin, fludarabin, Interferon alfa
cladribine, photopheresis (extra- Electron beam radiotherapy
corporeal photochemitherapy), Methotrexate chemotherapy as in non-
Hodgkin's lymphoma Mysloma* Melphelan (or cyclophosphamide) +
Interferon alfa prednisons Bone marrow transplant.sup.3 Melphalan
.+-. carmustine + High-dose dexamethasons cyclophosphamide +
prednisons + vincristine Dexamethasone + doxorubicin + vincristine
(VAD) Vincristine + carmustine + doxorubicin + prednisons (VBAP)
Neuroblestoma* Doxorubicin + cyclophosphamide + Carboplatin,
etoposide claplatin + teniposide or etoposide Bone marrow
transplant.sup.3 doxorubicin + cyclophosphamide Claplatin +
cyclophosphamide Osteogenic sarcoma.sup.5 Doxorubicin + claplatin
.+-. etopside .+-. Ifosfamide with means, ifosfamide etoposide,
carboplatin, high- dose methotrexate with leucovorin
Cyclophosphamide + etoposide Ovary Claplatin (or carboplatin) +
paclitaxel Ifosfamide with means, Claplatin (or carboplatin) +
paclitaxel, tamoxifen, cyclophosphamide (CP) .+-. doxorubicin
melphalan, altretamine (CAP) Pancreatic** Fluoroutacil .+-.
leucovorin Gemoltabinet Prostate Leuprolide (or goserelln) .+-.
flutamide Estramustine .+-. vinblastine, aminoglutethimide +
hydrocortleone, estramustine + etoposide, diethylstllbestrol,
nilutamide Renal** Aldesleukin Vinblastine, floxuridine Inteferon
alfa Retinoblestoma.sup.5* Doxorubicin + cyclophosphamide .+-.
Carboplatin, etoposide, claplatin .+-. etoposide .+-. vincristina
Ifosfamide with means Sarcomas, soft tissue, adult* Doxorubicin
.+-. decarbazine .+-. Mitornyeln + doxorubicin + cyclophosphamide
.+-. Ifosfamide with claplatin means Vincristina, etoposide
Testicular Claplatin + etoposide .+-. bleomycin Vinblestine (or
etoposide) + (PEB) Ifosfamide with means + claplatin (VIP) Bone
marrow transplant.sup.3 Wilms' tumor.sup.5 Dectinomycln +
vincriatine .+-. Ifosfamide with means, doxorubicin .+-.
cyclophosphamide etoposide, carboplatin *Chemotherapy has only
moderate activity. **Chemotherapy has only minor activity.
.sup.1Tamoxifen with or without chemotherapy is generally
recommended for postmenopausal estrogen-receptor-positive,
mode-positive patients and chemotherapy with or without tamoxlfen
for premenopausal mode-positive patients. Adjuvant treatment with
chemotherapy and/or tamoxifen is recommended for mode-negative
patients with larger tumors or other adverse prognostic indicators.
.sup.2Megastrol and other hormonal agents may be effective in some
patients with tamoxifen fails. .sup.3After high-dose chemotherapy
(Medical Letter, 34: 79, 1982). .sup.4For rectal cancer,
postoperative adjuvant treatment with fluoroutacil plus radiation,
preceded and followed by treatment
with fluorouracil alone. .sup.5Drugs have major activity only when
combined with surgical resection, radiotherapy or both. .sup.6The
vitamin A analog lactratinoln (Acgutana) can control pre-neoplastic
lesions (leukoplakla) and decreases the rate of second primary
tumors (S E Banner et al, J Natl Cancer Inst, 88: 140 1994).
.sup..dagger.Available in the USA only for investigational use.
.sup.7High-risk patients (e.g., high counts, cytogenetic
abnormalities, adults) may require additional drugs for induction,
maintenance and "Intensificiation" (use of additional drugs after
achievement of remission). Additional drugs include
cyclophosphamida, mitoxantrone and thloguanine. The results of one
large controlled trial in the United Kingdom suggest that
Intensificiation may improve survival in all children with ALL (J M
Chasselle et al, Lancet, 34B: 143, Jan. 21, 1995). .sup.8 Patients
with a poor prognosis initially or those who relapse after
remission. .sup.9Some patients with acute promyelocytic leukemia
have had complete responses to tratinoin. Such treatment can cause
a toxic syndrome characterized primarily by fever and respiratory
distress (R P Warrell, Jr et al, N Engl J Med. 328: 177, 1993).
.sup.10Allogeheic HLA-identical sibling bone marrow transplantation
can cure 40% to 70% of patients with CML in chronic phase, 18% to
28% of patients with accelerated phase CML, and <15% patients in
blast crisis. Disease-free survival after bone marrow
transplantations adversely influenced by age >50 years, duration
of disease >3 years from diagnosis, and use of one-antigen-
mismatched or matched-unrelated donor marrow. Interferon also may
be curative in patients with chronic phase CML who achieve a
complete cytogenetic response (about 10%); it is the treatment of
choice for patents >80 years old with newly diagnosed chronic
phase CML and for all patients who are not candidates for an
allgensic bone marrow transplant. Chemotherapy alone is palliative.
.sup.11If a second chronic phase is achieved with any of these
combinations, allogeneic bone marrow transplant should be
considered. Bone marrow transplant in second chronic phase may be
curative for 30% to 35% of patients with CML. .sup.12Limited-stage
Hodgkin's disease (stages 1 and 2) is curable by radiotherapy.
Disseminated disease (stages 3b and 4) require chemotherapy. Some
intermediate stages and selected clinical situations may benefit
from both. + Available in the USA only for investigational use.
ANTICANCER DRUGS AND HORMONES Drug Acute Toxicity .dagger-dbl.
Delayed toxicity .dagger-dbl. Aldesleukin (Interleukin-2; Fever;
fluid retention; hypertension; Neuropsychiatric disorders;
Proleukin - Cetus respiratory distress; rash; anemia;
hypothyrldiam; nephrotic Oncology) thrombocytophenia; nausea and
syndrome; possibly acute vomiting; diarrhea; capillary leak
leukoencaphalopathy; syndrome; naphrotoxlolty; myocardial brachial
plexopathy; bowel toxicity; hepatotoxicity; erytherna perforation
nodosum; neutrophil chemotactic defects Altretamine Nausea and
vomiting Bone marrow depression; (hexamethylmelamine; Hexalen - CNS
depression; peripheral U Bioscience) neuropathy; visual
hallucinations; stexis; tremors, alopecia; rash Aminogiutethimide
(Cytadren - Drowsiness; nausea; dizziness; rash Hypothryroidism
(rare); bone Ciba) marrow depression; fever; hypotension;
mascullinization .dagger.Amsacrine (m-AMSA; Nausea and vomiting;
diarrhea; pain or Bone marrow depression; amaidine; AMSP P-D-
phlebitis on infuelon; anaphylaxia hepactic injury; convulsions;
Parke-Davis, Amsidyl- stomatitle; ventricular Warner-Lambert)
fibrillation; alopecia; congestive heart failure; renal dysfunction
Asparaginase (Elspar-merck; Nausea and vomiting; fever; chills; CNS
depression or Kidrolase in Canada) headache; hypersensitivity,
anaphylexia; hyperexcitability; acute abdominal pain; hyperglycemia
leading hemorrhagic pancreatitis; to coma coagulation defects;
thromboals; renal damage; hepactic damage Cervix ** Claplatin
Ifosfamide with means Chlorambucil, vincristine, Bleomycin patin
fluoroutacil, doxorubicin, Ifosfamide with means methotrexete,
altretamine Chorlocarcinoma Methotrexete .+-. leucovorin
Methotrexete + dectinomycin + Dactinomyclin cyclophosphamide (MAC)
Etoposide + methotrexate + dactinomycin + cyclophosphamide +
vincrlatine Colorectal * Adjuvant colon.sup.4: Fluoroutacil +
Hepatic metastases: lavamleole; fluoroutacil + leucovarin
Intrahepactic-arterial Metastatic: Fluoroutacil + leucvarin
floxuridine Mitomyclin Embryonal Vincriatine + dectinomycin .+-.
Same + doxorubicin rhebdomyosarcoma.sup.6 cyclophosphamide
Vincristine + Ifosfamide with means + etoposide Endometrial **
Megastrol or another progeetin Fluoroutacil, tamoxifen, Doxorubicin
+ claplatin .+-. altretamine cyclophosphamide Esophageal *
Claplatin + Fluoroutacil Doxorubicin, methotrexete, Ewing's
sarcoma.sup.5 Cyclophosphamide (or ifosfamide with mitomycin means)
+ doxorubicin + vincrietine CAV + etoposide (CAV) .+-. dectinomycin
Gastric ** Fluoroutacil .+-. leucovoin Claplatin, doxorubicin,
etoposide, methotrexete + leucovorin, mitomycin Head and neck
squamous Claplatin + fluoroutacil Blaonycin, carboplatin, cell
*.sup.5 Methotrexete paciltaxel Islet call ** Streptozocin +
doxorubicin Streptozocln + fluoroutacil; chlorozotocin; actreatide
Kaposal's sercoma * Etoposide or Interferon alfa or Vincristine,
doxorubicin, (AIDS-related) vinbleomycin stine bleomycln
Doxorubicin + bleomycin + vincristine or vinbleomycin stine (ABV)
Leukemias Induction: Vincristine + prednisone + Industion: same
.+-. high-dose Acute lymphocytic leukemia asparaginase .+-.
daunorubieln methotrexete .+-. cyterabine; (ALL).sup.7 CNS
prophylaxia; Intrathecal pegaspargase instead of methotrexete .+-.
systemic high-dose aspareginese methotrexete with leucovorin .+-.
Teniposide or etoposide Intrethecal cytarabine .+-. Intrathecal
High-dose cytarabine hydrocortisone Maintenance: same +
Maintenance: methotrexete .+-. periodic vincristine +
mercaptopurine prednisone Bone marrow transplant.sup.3 Acute
myeloid leukemia Induction: Cytarabine + either Cytarabine +
mitoxantrone (AML).sup.9 daunbrublein or idarubieln High-dose
cytarabine Post Induction: High-dose cytarabine .+-. other drugs
such as etoposide Bone marrow transplant.sup.3 Chronic lymophocytic
Chlorambuell .+-. prednisone Claplatin, cyclophosphamide, leukemia
(CLL) Fludarabin pentostatin, vinorlstine, doxorubicin
.dagger.Available in the USA only for investigational use.
.dagger-dbl. Dose-limiting effects are in bold type. Cutaneous
reactions (sometimes severe), hyperpigmentation, and ocular
toxicity have been reported with virtually all nonhormonal
anticancer drugs. For adverse interactions with other drugs, see
the Medical Letter Handbook of Adverse Drug Interactions, 1995.
.sup.1Available in the USA only for investigational use.
.sup.2Megestrol and other hormonal agents may be effective in some
pateients when tamoxifen fails. .sup.3After high-dose chemotherapy
(Medical Letter, 34: 78, 1992). .sup.4For rectal cancer,
postoperative adjuvant treatment with fluoroutacil plus radiation,
preceded and followed by treatment with fluoroutacil alone.
.sup.5Drugs have major activity only when combined with surgical
resection, radiotherapy or both. .sup.6The vitamin A analog
isotretinoin (Accutane) can control pre-neoplastic isions
(leukoplaka) and decreases the rats of second primary tumors (S E
Senner et al., J Natl Cancer Inst. 88: 140, 1994). .sup.7High-risk
patients (e.g., high counts, cytogenetic abnormalities, adults) may
require additional drugs for Induction, maintenance and
"Intensification" (use of additional drugs after achievement of
remission). Additional drugs include cyclophosphamide, mitoxantrone
and thioguamine. The results of one large controlled trial in the
United Kingdom suggest that intensilibation may improve survival in
all children with ALL (j m Chassella et al., Lancet, 348: 143, Jan.
21, 1998). .sup.8Patients with a poor prognosis initially or those
who relapse after remission .sup.9Some patients with acute
promyclocytic leukemia have had complete responses to tretinoin.
Such treatment can cuase a toxic syndrome characterized primarily
by fever and respiratory distress (R P Warrell, Jr et al. N Eng J.
Med, 329: 177, 1993). .sup.10Allogenaic HLA Identical sibling bone
marrow transplantation can cure 40% to 70% of patients with CML in
chroni phase, 15% to 25% of patients with accelerated phase CML,
and <15% patients in blast crisis. Disease-free survival after
bone marrow transplantation is adversely influenced by age >50
years, duration of disease >3 years from diagnosis, and use of
one antigen mismatched or matched-unrelated donor marrow. Inteferon
alfa may be curative in patients with chronic phase CML who achieve
a complete cytogenetic resonse (about 10%); It is the treatment of
choices for patients >50 years old with newly diagnosed chronic
phase CML and for all patients who are not candidates for an
allogenic bone marrow transplant. Chemotherapy alone is
palliative.
Radiolabeling of EC-MTX and EC-TDX with .sup.99mTc
[0169] Use the same method described for the synthesis of
EC-folate, EC-MTX and EC-TDX were prepared. The labeling procedure
is the same as described for the preparation of
.sup.99mTc-EC-folate except EC-MTX and EC-TDX were used. Synthesis
of .sup.99mTc-EC-MTX and .sup.99mTc-EC-TDX is shown in FIG. 2 and
FIG. 3.
Stability Assay of .sup.99mTc-EC-Folate, .sup.99mTc-EC-MTX and
.sup.99mTc-EC-TDX
[0170] Stability of .sup.99mTc-EC-Folate, .sup.99mTc-EC-MTX and
.sup.99mTc-EC-TDX was tested in serum samples. Briefly, 740 KBq of
1 mg .sup.99mTc-EC-Folate, .sup.99mTc-EC-MIX and .sup.99mTc-EC-TDX
was incubated in dog serum (200 .mu.l) at 37.degree. C. for 4
hours. The serum samples was diluted with 50% methanol in water and
radio-TLC repeated at 0.5, 2 and 4 hours as described above.
Tissue Distribution Studies
[0171] Female Fischer 344 rats (150.+-.25 g) (Harlan
Sprague-Dawley, Indianapolis, Ind.) were inoculated subcutaneously
with 0.1 ml of mammary tumor cells from the 13762 tumor cell line
suspension (10.sup.6 cells/rat, a tumor cell line specific to
Fischer rats) into the hind legs using 25-gauge needles. Studies
performed 14 to 17 days after implantation when tumors reached
approximately 1 cm diameter. Animals were anesthetized with
ketamine (10-15 mg/rat, intraperitoneally) before each
procedure.
[0172] In tissue distribution studies, each animal injected
intravenously with 370-550 KBq of .sup.99mTc-EC-folate or
.sup.99mTc-EC (n=3/time point). The injected mass of each ligand
was 10 .mu.g per rat. At 20 min, 1, 2 and 4 h following
administration of the radiopharmaceuticals, the anesthetized
animals were sacrificed and the tumor and selected tissues were
excised, weighed and counted for radioactivity by a gamma counter
(Packard Instruments, Downers Grove, Ill.). The biodistribution of
tracer in each sample was calculated as percentage of the injected
dose per gram of tissue wet weight (% ID/g). Counts from a diluted
sample of the original injectate were used for reference.
Tumor/nontarget tissue count density ratios were calculated from
the corresponding % ID/g values. Student-t test was used to assess
the significance of differences between two groups.
[0173] In a separate study, blocking studies were performed to
determine receptor-mediated process. In blocking studies, for
.sup.99mTc-EC-folate was co-administrated (i.v.) with 50 and 150
.mu.mol/kg folic acid to tumor bearing rats (n=3/group). Animals
were killed 1 h post-injection and data was collected.
Scintigraphic Imaging and Autoradiography Studies
[0174] Scintigraphic images, using a gamma camera (Siemens Medical
Systems, Inc., Hoffman Estates, Ill.) equipped with low-energy,
parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v.
injection of 18.5 MBq of .sup.99mTc-labeled radiotracer.
[0175] Whole-body autoradiogram were obtained by a quantitative
image analyzer (Cyclone Storage Phosphor System, Packard, Meridian,
CI.). Following i.v. injection of 37 MBq of .sup.99mTc-EC-folate,
animal killed at 1 h and body was fixed in carboxymethyl cellulose
(4%). The frozen body was mounted onto a cryostat (LKB 2250
cryomicrotome) and cut into 100 .mu.m coronal sections. Each
section was thawed and mounted on a slide. The slide was then
placed in contact with multipurpose phosphor storage screen (MP,
7001480) and exposed for 15 h .sup.99mTc-labeled). The phosphor
screen was excited by a red laser and resulting blue light that is
proportional with previously absorbed energy was recorded.
Results
[0176] Chemistry and Stability of .sup.99mTc-EC-Folate
[0177] A simple, fast and high yield aminoethylamido and EC
analogues of folate, MTX and TDX were developed. The structures of
these analogues were confirmed by NMR and mass spectroscopic
analysis. Radiosynthesis of EC-folate with .sup.99mTc was achieved
with high (>95%) radiochemical purity. .sup.99mTc-EC-folate was
found to be stable at 20 min. 1, 2 and 4 hours in dog serum
samples.
[0178] Biodistribution of .sup.99mTc-EC-Folate
[0179] Biodistribution studies showed that tumor/blood count
density ratios at 20 min-4 h gradually increased for
.sup.99mTc-EC-folate, whereas these values decreased for
.sup.99mTc-EC in the same time period (FIG. 4). % ID/g uptake
values, tumor/blood and tumor/muscle ratios for
.sup.99mTc-EC-folate and .sup.99mTc-EC were given in Tables 3 and
4, respectively.
TABLE-US-00003 TABLE 3 Biodistribution of .sup.99mTc-EC-folate in
Breast Tumor-Bearing Rats % of injected .sup.99mTc-EC-folate dose
per organ or tissue 20 min 1 h 2 h 4 h Blood 0.370 .+-. 0.049 0.165
.+-. 0.028 0.086 .+-. 0.005 0.058 .+-. 0.002 Lung 0.294 .+-. 0.017
0.164 .+-. 0.024 0.092 .+-. 0.002 0.063 .+-. 0.003 Liver 0.274 .+-.
0.027 0.185 .+-. 0.037 0.148 .+-. 0.042 0.105 .+-. 0.002 Stomach
0.130 .+-. 0.002 0.557 .+-. 0.389 0.118 .+-. 0.093 0.073 .+-. 0.065
Kidney 4.328 .+-. 0.896 4.052 .+-. 0.488 5.102 .+-. 0.276 4.673
.+-. 0.399 Thyroid 0.311 .+-. 0.030 0.149 .+-. 0.033 0.095 .+-.
0.011 0.066 .+-. 0.011 Muscle 0.058 .+-. 0.004 0.0257 .+-. 0.005
0.016 .+-. 0.007 0.008 .+-. 0.0005 Intestine 0.131 .+-. 0.013 0.101
.+-. 0.071 0.031 .+-. 0.006 0.108 .+-. 0.072 Urine 12.637 .+-.
2.271 10.473 .+-. 3.083 8.543 .+-. 2.763 2.447 .+-. 0.376 Tumor
0.298 .+-. 0.033 0.147 .+-. 0.026 0.106 .+-. 0.029 0.071 .+-. 0.006
Tumor/Blood 0.812 .+-. 0.098 0.894 .+-. 0.069 1.229 .+-. 0.325
1.227 .+-. 0.129 Tumor/Muscle 5.157 .+-. 0.690 5.739 .+-. 0.347
6.876 .+-. 2.277 8.515 .+-. 0.307 Values shown represent the mean
.+-. standard deviation of data from 3 animals
Scintigraphic Imaging and Autoradiography Studies
[0180] Scintigraphic images obtained at different time points
showed visualization of tumor in .sup.99mTc-EC-folate injected
group. Contrary, there was no apparent tumor uptake in
.sup.99mTc-EC injected group (FIG. 6). Both radiotracer showed
evident kidney uptake in all images. Autoradiograms performed at 1
h after injection of .sup.99mTc-EC-folate clearly demonstrated
tumor activity.
Example 2
Tumor Hypdxia Targeting
Synthesis of 2-(2-methyl-5-nitro-.sup.1H imidazolyl)ethylamine
(amino analogue of metronidazole, MN--NH.sub.2)
[0181] Amino analogue of metronidazole was synthesized according to
the previously described methods (Hay et al., 1994). Briefly,
metronidazole was converted to a mesylated analogue (m.p.
149-150.degree. C., reported 153-154.degree. C., TLC:ethyl acetate,
Rf=0.45), yielded 75%. Mesylated metronidazole was then reacted
with sodium azide to afford azido analogue (TLC:ethyl acetate,
Rf=0.52), yielded 80%. The azido analogue was reduced by triphenyl
phosphine and yielded (60%) the desired amino analogue (m.p.
190-192.degree. C., reported 194-195.degree. C., TLC:ethyl acetate,
Rf=0.15). Ninhydrin (2% in methanol) spray indicated the positivity
of amino group of MN--NH.sub.2. The structure was confirmed by
.sup.1H-NMR and mass spectroscopy (FAB-MS) ink 171 (M.sup.+H,
100).
Synthesis of Ethylenedicysteine-Metronidazole (EC-MN)
[0182] Sodium hydroxide (2N, 0.2 ml) was added to a stirred
solution of EC (134 ma, 0.50 mmol) in water (5 ml). To this
colorless solution, sulfo-NHS (217 mg, 1.0 mmol) and 1.about.)C
(0.192 ma. 1.0 mmol) were added. MN-NH: dihydrochloride salt (340
mg, 2.0 mmol) was then added. The mature was stirred at room
temperature for 24 hours. The mixture was dialyzed for 48 hrs using
Spectra/POR molecular porous membrane with cut-off at 500 (Spectrum
Medical Industries Inc., Houston, Tex.). After dialysis, the
product was frozen dried using lyophilizer (Labconco, Kansas City,
Mo.). The product weighed 315 mg (yield 55%). .sup.1H-NMR
(D.sub.2O) .delta. 2.93 (s, 6H, nitroimidazole-CH.sub.3), 2.60-2.95
(m, 4H and --CH.sub.2--SH of EC), 3.30-3.66 (m, 8H, ethylenediamine
of EC and nitromidazole-CH.sub.2--CH.sub.2--NH.sub.2), 3.70-3.99
(t, 2H, NH--CH--CO of EC), 5.05 (t, 4H,
metronidazole-CH.sub.2--CH.sub.2--NH.sub.2) (s, 2H, nitroimidazole
C.dbd.CH). FAB MS rniz 572 (M.sup.+, 20). The synthetic scheme of
EC-MN is shown in FIG. 7.
Synthesis of 3-(2-nitro-.sup.1H-imidazolyl)propylamine (amino
analogue of nitroimidazole, NIM-NH.sub.2)
[0183] To a stirred mixture containing 2-nitroimidazole (1 g, 8.34
mmol) and Cs.sub.2, CO.sub.3 (2.9 g, 8.90 mmol) in dimethylformaide
(DMF, 50 ml), 1,3-ditosylpropane (3.84 g, 9.99 mmol) was added. The
reaction was heated at 80.degree. C. for 3 hours. The solvent was
evaporated under vacuum and the residue was suspended in
ethylacetate. The solid was filtered, the solvent was concentrated,
loaded on a silica gel-packed column and eluted with
hexane:ethylacetate (1:1). The product,
3-tosylpropyl-(2-nitroimidazole), was isolated (1.67 g, 57.5%) with
m.p. 108-111.degree. C. .sup.1H-NMR (CDCl.sub.3) .delta. 2.23 (m,
2H), 2.48 (S. 3H), 4.06 (t, 2H, J=5.7 Hz), 4.52 (t, 2H, J=6.8 Hz),
7.09 (S. 1H), 7.24 (S. 1H), 7.40 (d, 2H, J=8.2 Hz). 7.77 (d, 2H,
J=8.2 Hz).
[0184] Tosylated 2-nitroimidazole (1.33 g, 4.08 mmol) was then
reacted with sodium azide (Q29 g, 4.49 mmol) in DMF (10 ml) at
100.degree. C. for 3 hours. After cooling, water (20 ml) was added
and the product was extracted from ethylacetate (3.times.20 ml).
The solvent was dried over MgSO.sub.4 and evaporated to dryness to
afford azido analogue (0.6 g, 75%, TLC: hexane:ethyl acetate; 1:1,
Rf=0.42). .sup.1H-NMR (CDCl.sub.3) .delta. 2.14 (m, 2), 3.41 (t,
2H, J=6.2 Hz), 4.54 (t, 2H, J=6.9 Hz), 7.17 (S. 2H).
[0185] The azido analogue (0.57 g, 2.90 mmol) was reduced by
taphenyl phosphine (1.14 g, 4.35 mmol) in tetrahydrofuran (PHI;) at
room temperature for 4 hours. Concentrate HCI (12 ml) was added and
heated for additional 5 hours. The product was extracted from
ethylacetate and water mixture. The ethylacetate was dried over
MgSO.sub.4 and evaporated to dryness to afford amine hydrochloride
analogue (360 ma, 60%). Ninhydrin (2% in methanol) spray indicated
the positivity of amino group of NIM-NH. .sup.1H-NMR (D.sub.2O)
.delta. 2.29 (m, 2H), 3.13 (t, 2H, J=7.8 Hz), 3.60 (br, 2H), 4.35
(t, 2H, J=7.4 Hz), 7.50 (d, 1H, J=2.1 Hz), 7.63 (d, 1H, J=2.1
Hz).
Synthesis of Ethylenedicysteine-Nitroimidazole (EC-NIM)
[0186] Sodium hydroxide (2N, 0.6 ml) was added to a stirred
solution of EC (134 ma, 0.50 mmol) in water (2 ml). To this
colorless solution, sulfo-NHS (260.6 mg, 1.2 mmol), EDC (230 ma,
1.2 mmol) and sodium hydroxide (2N, 1 ml) were added. NIM-NH.sub.2
hydrochloride salt (206.6 mg, 1.0 mmol) was then added. The mixture
was stirred at room temperature for 24 hours. The mixture was
dialyzed for 48 hrs using Spectra/POR molecular porous membrane
with cut-off at 500 (Spectrum Medical Industries Inc., Houston,
Tex.). After dialysis, the product was frozen dried using
lyophilizer (Labconco, Kansas City, Mo.). The product weighed 594.8
mg (yield 98%). The synthetic scheme of EC-NIM is shown in FIG. 8A.
The structure is confirmed by .sup.1H-NMR (D.sub.2O) (FIG. 8B).
Radiolabeling of EC-MN and EC-NIM with .sup.99mTc
[0187] Radiosynthesis of .sup.99mTc-EC-MN and .sup.99mTc-EC-NEVI
were achieved by adding required amount of pertechnetate into
home-made kit containing the lyophilized residue of EC-MN or EC-NIM
(3 mg), SnCl.sub.2, (100 .mu.g), Na.sub.2HPO.sub.4 (13.5 mg),
ascorbic acid (0.5 mg) and NaEDTA (0.5 mg). Final pH of preparation
was 7.4. Radiochemical purity was determined by TLC (ITLAC SG,
Gelman Sciences, Ann Arbor, Mich.) eluted with acetone (system A)
and ammonium acetate (1M in water):methanol (4:1) (system B),
respectively. From radio-TLC (Bioscan, Washington, D.C.) analysis,
the radiochemical purity was >96% for both radiotracers.
Synthesis of [.sup.18F]FMISO and .sup.131I]IMISO
[0188] [Should this be .sup.18?][Fl]uoride was produced by the
cyclotron using proton irradiation of enriched .sup.18O-water in a
small-volume silver target. The tosyl MIS0 (Hay et al., 1994) (20
mg) was dissolved in acetonitrile (1.5 ml), added to the
kryptofix-fluoride complex. After heating, hydrolysis and column
purification, A yield of 25-40% (decay corrected) of pure product
was isolated with the end of bombardment (EOB) at 60 min. HPLC was
performed on a C-18 ODS-20T column, 4.6.times.25 mm (Waters Corp.,
Milford, Mass.), with water/acetonitrile, (80/20), using a flow
rate of 1 ml/min. The no-carrier-added product corresponded to the
retention time (6.12 min) of the unlabeled FMISO under similar
conditions. The radiochemical purity was greater than 99%. Under
the UV detector (310 tun), there were no other impurities. The
specific activity of [.sup.18F]FMISO determined was 1 Ci/.mu.mol
based upon UV and radioactivity detection of a sample of known mass
and radioactivity.
[0189] [.sup.13I]IMISO was prepared using the same precursor (Chemf
et al., 1994), briefly, 5 mg of tosyl MISO was dissolved in
acetonitrile (1 ml), and Na.sup.131I (1 mCi in 0.1 ml 1N NaOH)
(Dupont New England Nuclear, Boston. MA) was added. After heating
and purification, the product (60-70% yield) was obtained.
Radio-TLC indicated the Rf values of 0.01 for the final product
using chloroform methanol (7:3) as an eluant.
Stability Assay of .sup.99mTc-EC-MN and .sup.99mTc-EC-NIM
[0190] Stability of labeled .sup.99mTc-EC-MN and .sup.99mTc-EC-NIM
were tested in serum samples. Briefly, 740 KBq of 1 mg
.sup.99mTc-EC-MN and .sup.99mTc-EC-NIM were incubated in dog serum
(200 .mu.l) at 37.degree. C. for 4 hours. The serum samples were
diluted with 50% methanol in water and radio-TLC repeated at 0.5, 2
and 4 hours as described above.
Tissue Distribution Studies of .sup.99mTc-EC-MN
[0191] Female Fischer 344 rats (150.+-.25 g) (Harlan
Sprague-Dawley, Indianapolis, Ind.) were inoculated subcutaneously
with 0.1 ml of mammary tumor cells from the 13762 tumor cell line
suspension (10.sup.6 cells/rat, a tumor cell line specific to
Fischer rats) into the hind legs using 25-gauge needles. Studies
performed 14 to 17 days after implantation when tumors reached
approximately 1 cm diameter. Rats were anesthetized with ketamine
(10-15 mg/rat, intraperitoneally) before each procedure.
[0192] In tissue distribution studies, each animal was injected
intravenously with 370-550 KBq of .sup.99mTc-EC-MN or .sup.99mTc-EC
(n=3/time point). The injected mass of .sup.99mTc-EC-MN was 10
.mu.g per rat. At 0.5, 2 and 4 hrs following administration of the
radiotracers, the rats were sacrificed and the selected tissues
were excised, weighed and counted for radioactivity. The
biodistribution of tracer in each sample was calculated as
percentage of the injected dose per gram of tissue wet weight (%
ID/g). Tumor/nontarget tissue count density radios were calculated
from the corresponding % ID/g values. The data was compared to
[.sup.18F]FMISO and [.sup.131I]IMISO using the same animal model.
Student t-test was used to assess the significance of differences
between groups.
Scintigraphic Imaging and Autoradiography Studies
[0193] Scintigraphic images, using a gamma camera (Siemens Medical
Systems, Inc., Hoffman Estates, Ill.) equipped with low-energy,
parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v.
injection of 18.5 MBq of each radiotracer.
[0194] Whole-body autoradiogram was obtained by a quantitative
image analyzer (Cyclone Storage Phosphor System, Packard, Meridian,
Conn.). Following i.v. injection of 37 MBq of .sup.99mTc-EC-MN, the
animals were killed at 1 h and the body were fixed in carboxymethyl
cellulose (4%) as previously described (Yang et al., 1995). The
frozen body was mounted onto a cryostat (LKB 2250 cryomicrotome)
and cut into 100 .mu.m coronal sections. Each section was thawed
and mounted on a slide. The slide was then placed in contact with
multipurpose phosphor storage screen (MP, 7001480) and exposed for
15 hrs.
[0195] To ascertain whether .sup.99mTc-EC-NIM could monitor tumor
response to chemotherapy, a group of rats with tumor volume 1.5 cm
and ovarian tumor-bearing mice were treated with paclitaxel (40
mg/kg/rat, 80 mg/kg/mouse, i.v.) at one single dose. The image was
taken on day 4 after paclitaxel treatment. Percent of injected dose
per gram of tumor weight with or without treatment was
determined.
Polarographic Oxygen Microelectrode pO.sub.2 Measurements
[0196] To confirm tumor hypoxia, intratumoral pO.sub.2 measurements
were performed using the Eppendorf computerized histographic
system. Twenty to twenty-five pO.sub.2 measurements along each of
two to three linear tracks were performed at 0.4 mm intervals on
each tumor (40-75 measurements total). Tumor pO measurements were
made on three tumor-bearing rats. Using an on-line computer system,
the pot measurements of each track were expressed as absolute
values relative to the location of the measuring point along the
track, and as the relative frequencies within a pO.sub.2 histogram
between 0 and 100 mmHg with a class width of 2.5 mm.
Results
[0197] Radiosynthesis and Stability of .sup.99mTc-EC-MN and
.sup.99mTc-EC-NIM
[0198] Radiosynthesis of EC-MN and EC-NIM with .sup.99mTc were
achieved with high (>95%) radiochemical purity Radiochemical
yield was 100%. .sup.99mTc-EC-MN and .sup.99mTc-EC-NIM (FIG. 13)
were found to be stable at 0.5, 2 and 4 hrs in dog serum samples.
There was no degradation products observed. Radiofluorination and
radioiodination of MISO were achieved easily using the same
precursor. In both labeled MISO analogues, the radiochemical purity
was greater than 99%.
In Vivo Tissue Distribution Studies
[0199] The tissue distribution of .sup.99mTc-EC-MN and
.sup.99mTc-EC in the tumor-bearing rats is shown in Tables 4 and 5.
Due to high affinity for ionic .sup.99mTc, there was no significant
and consistent thyroid uptake, suggesting the in vivo stability of
.sup.99mTc-EC-MN (Table 5).
TABLE-US-00004 TABLE 4 Biodistribution of .sup.99mTc-EC in Breast
Tumor-Bearing Rats % of injected .sup.99mTc-EC dose per organ or
tissue 20 min 1 h 2 h 4 h Blood 0.435 .+-. 0.029 0.273 .+-. 0.039
0.211 .+-. 0.001 0.149 .+-. 0.008 Lung 0.272 .+-. 0.019 0.187 .+-.
0.029 0.144 .+-. 0.002 0.120 .+-. 0.012 Liver 0.508 .+-. 0.062
0.367 .+-. 0.006 0.286 .+-. 0.073 0.234 .+-. 0.016 Stomach 0.136
.+-. 0.060 0.127 .+-. 0.106 0.037 .+-. 0.027 0.043 .+-. 0.014
Kidney 7.914 .+-. 0.896 8.991 .+-. 0.268 9.116 .+-. 0.053 7.834
.+-. 1.018 Thyroid 0.219 .+-. 0.036 0.229 .+-. 0.118 0.106 .+-.
0.003 0.083 .+-. 0.005 Muscle 0.060 .+-. 0.006 0.043 .+-. 0.002
0.028 .+-. 0.009 0.019 .+-. 0.001 Intestine 0.173 .+-. 0.029 0.787
.+-. 0.106 0.401 .+-. 0.093 0.103 .+-. 0.009 Urine 9.124 .+-. 0.808
11.045 .+-. 6.158 13.192 .+-. 4.505 8.693 .+-. 2.981 Tumor 0.342
.+-. 0.163 0.149 .+-. 0.020 0.115 .+-. 0.002 0.096 .+-. 0.005
Tumor/Blood 0.776 .+-. 0.322 0.544 .+-. 0.004 0.546 .+-. 0.010
0.649 .+-. 0.005 Tumor/Muscle 5.841 .+-. 3.253 3.414 .+-. 0.325
4.425 .+-. 1.397 5.093 .+-. 0.223 Values shown represent the mean
.+-. standard deviation of data from 3 animals
[0200] In blocking studies, tumor/muscle and tumor/blood count
density ratios were significantly decreased (p<0.01) with folic
acid co-administrations (FIG. 5).
TABLE-US-00005 TABLE 5 Biodistribution of
.sup.99mTc-EC-metronidazole conjugate in breast tumor bearing
rats.sup.1 30 Min. 2 Hour 4 Hour Blood 1.46 .+-. 0.73 1.19 .+-.
0.34 0.76 .+-. 0.14 Lung 0.79 .+-. 0.39 0.73 .+-. 0.02 0.52 .+-.
0.07 Liver 0.83 .+-. 0.36 0.91 .+-. 0.11 0.87 .+-. 0.09 Spleen 0.37
.+-. 0.17 0.41 .+-. 0.04 0.37 .+-. 0.07 Kidney 4.30 .+-. 1.07 5.84
.+-. 0.43 6.39 .+-. 0.48 Muscle 0.08 .+-. 0.03 0.09 .+-. 0.01 0.07
.+-. 0.01 Intestine 0.27 .+-. 0.12 0.39 .+-. 0.24 0.22 .+-. 0.05
Thyroid 0.051 .+-. 0.16 0.51 .+-. 0.09 0.41 .+-. 0.02 Tumor 0.034
.+-. 0.13 0.49 .+-. 0.02 0.50 .+-. 0.09 .sup.1Each rat received 99m
Tc-EC-metronidazole (10 .mu.Ci, iv). Each value is percent of
injected dose per gram weight (n = 3)/time interval. Each data
represents mean of three measurements with standard deviation.
[0201] Biodistribudon studies showed that tumor/blood and
tumor/muscle count density ratios at 0.54 hr gradually increased
for .sup.99mTc-EC-MN, [.sup.18F]FMISO and [.sup.13I]IMISO, whereas
these values did not alter for .sup.99mTc-EC in the same time
period (FIG. 9 and FIG. 10). [.sup.18F]FMISO showed the highest
tumor-to-blood uptake ratio than those with [.sup.131I]IMso and
.sup.99mTc-EC-MN at 30 min, 2 and 4 hrs post-injection. Tumor/blood
and tumor/muscle ratios for .sup.99mTc-EC-MN and [.sup.131I]IMISO
at 2 and 4 hrs postinjection were not significantly different
(p<0.05).
Scintigraphic Imaging and Autoradiographic Studies
[0202] Scintigraphic images obtained at different time points
showed visualization of tumor in .sup.99mTc-EC-MN and
.sup.99mTc-EC-NIM groups. Contrary, there was no apparent tumor
uptake in .sup.99mTc-EC injected group (FIG. 11). Autoradiograms
performed at 1 hr after injection of .sup.99mTc-EC-MN clearly
demonstrated tumor activity (FIG. 12). Compare to .sup.99mTc-EC-NM,
.sup.99mTc-EC-NIM appeared to provide better scintigraphic images
due to higher tumor-to-background ratios. In breast tumor-bearing
rats, tumor uptake was markedly higher in .sup.99mTc-EC-NIM group
compared to .sup.99mTc-EC (FIG. 14A). Data obtained from percent of
injected dose of .sup.99mTc-EC-NIM per gram of tumor weight
indicated that a 25% decreased uptake in the rats treated with
paclitaxel when compared to control group (FIG. 14B).
[0203] In ovarian tumor-bearing mice, there was a decreased tumor
uptake in mice treated with paclitaxel (FIG. 15A and FIG. 15B).
Similar results were observed in sarcoma-bearing (FIG. 15C and FIG.
15D). Thus, .sup.99mTc-EC-NIM could be used to assess tumor
response to paclitaxel treatment.
Polarographic Oxygen Microelectrode pO.sub.2 Measurements
[0204] Intratumoral PO.sub.2 measurements of tumors indicated the
tumor oxygen tension ranged 4.6.+-.1.4 mmHg as compared to normal
muscle of 35.+-.10 mmHg. The data indicate that the tumors are
hypoxic.
Example 3
Peptide Imaging of Cancer
Synthesis of Ethylenedieysteine-Pentaglutamate (EC-GAP)
[0205] Sodium hydroxide (1N, 1 ml) was added to a stirred solution
of EC (200 mg, 0.75 mmol) in water (10 ml). To this colorless
solution, sulfo-NHS (162 mg, 0.75 mmol) and EDC (143 mg, 0.75 mmol)
were added. Pentaglutamate sodium salt (M.W. 750-1500, Sigma
Chemical Company) (500 mg, 0.67 mmol) was then added. The mixture
was stirred at room temperature for 24 hours. The mixture was
dialyzed for 48 hrs using Spectra/POR molecular porous membrane
with cut-off at 500 (Spectrum Medical Industries Inc., Houston,
Tex.). After dialysis, the product was frozen dried using
lyophilizer (Labconco, Kansas City, Mo.). The product in the salt
form weighed 0.95 g. The synthetic scheme of EC-GAP is shown in
FIG. 16.
Stability Assay of .sup.99mTc-EC-GAP
[0206] Radiolabeling of EC-GAP with .sup.99mTc was achieved using
the same procedure described previously. The radiochemical purity
was 100%. Stability of labeled .sup.99mTc-EC-GAP was tested in
serum samples. Briefly, 740 KBq of 1 mg .sup.99mTc-EC-GAP was
incubated in dog serum (200 .mu.l) at 37.degree. C. for 4 hours.
The serum samples were diluted with 50% methanol in water and
radio-TLC repeated at 0.5, 2 and 4 hours as described above.
Scintigraphic Imaging Studies
[0207] Scintigraphic images, using a gamma camera equipped with
low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4
hrs after i.v. injection of 18.5 MBq of each radiotracer.
Results
[0208] Stability Assay of .sup.99mTc-EC-GAP
[0209] .sup.99mTc-EC-GAP found to be stable at 0.5, 2 and 4 hrs in
dog serum samples. There was no degradation products observed.
Scintigraphic Imaging Studies
[0210] Scintigraphic images obtained at different time points
showed visualization of tumor in .sup.99mTc-EC-GAP group. The
optimum uptake is at 30 min to 1 hour post-administration (FIG.
17).
Example 4
Imaging Tumor Apoptotic Cells
Synthesis of Ethylenedicysteine-Annexin V (EC-ANNEX)
[0211] Sodium bicarbonate (1N, 1 ml) was added to a stirred
solution of EC (5 mg, 0.019 mmol). To this colorless solution,
sulfo-NHS (4 mg, 0.019 mmol) and EDC (4 mg, 0.019 mmol) were added.
Annexin V (M.W. 33 kD, human, Sigma Chemical Company) (0.3 mg) was
then added. The mixture was stirred at room temperature for 24
hours. The mixture was dialyzed for 48 hrs using Spectra/POR
molecular porous membrane with cut-off at 10,000 (Spectrum Medical
Industries Inc., Houston, Tex.). After dialysis, the product was
frozen dried using lyophilizer (Labconco, Kansas City, Mo.). The
product in the salt form weighed 12 mg.
Stability Assay of .sup.99mTc-EC-ANNEX
[0212] Radiolabeling of EC-ANNEX with .sup.99mTc was achieved using
the same procedure described in EC-GAP. The radiochemical purity
was 100%. Stability of labeled .sup.99mTc-EC-ANNEX was tested in
serum samples. Briefly, 740 KBq of 1 mg .sup.99mTc-EC-ANNEX was
incubated in dog serum (200 .mu.l) at 37.degree. C. for 4 hours.
The serum samples were diluted with 50% methanol in water and
radio-TLC repeated at 0.5, 2 and 4 hours as described above.
Scintigraphic Imaging Studies
[0213] Scintigraphic images, using a gamma camera equipped with
low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4
hrs after i.v. injection of 18.5 MBq of the radiotracer. The animal
models used were breast, ovarian and sarcoma. Both breast and
ovarian-tumor bearing rats are known to overexpress high apoptotic
cells. The imaging studies were conducted on day 14 after tumor
cell inoculation. To ascertain the tumor treatment response, the
pre-imaged mice were administered paclitaxel (80 mg/Kg, iv, day 14)
and the images were taken on day 18.
Results
[0214] Stability Assay of .sup.99mTc-EC-ANNEX
[0215] .sup.99mTc-EC-ANNEX found to be stable at 0.5, 2 and 4 hrs
in dog serum samples. There was no degradation products
observed.
Scintigraphic Imaging Studies
[0216] Scintigraphic images obtained at different time points
showed visualization of tumor in .sup.99mTc-EC-ANNEX group (FIGS.
18-20). The images indicated that highly apoptotic cells have more
uptake of .sup.99mTc-EC-ANNEX. There was no marked difference of
tumor uptake between pre- and post-[aclitaxel treatment in the high
apoptosis (ovarian tumor-bearing) group (FIG. 19A and FIG. 19B) and
in the low apoptosis (sarcoma tumor-bearing) group (FIG. 20A and
FIG. 20B).
Example 5
Imaging Tumor Angiogenesis
Synthesis of (Amino Analogue of Colchcine, COL-NH.sub.2)
[0217] Demethylated amino and hydroxy analogue of colchcine was
synthesized according to the previously described methods (On et
al., 1995). Briefly, colchicine (4 g) was dissolved in 100 ml of
water containing 25% sulfuric acid. The reaction mixture was heated
for 5 hours at 100.degree. C. The mixture was neutralized with
sodium carbonate. The product was filtered and dried over freeze
dryer, yielded 2.4 g (70%) of the desired amino analogue (m.p.
153-155.degree. C., reported 155-157.degree. C.). Ninhydrin (2% in
methanol) spray indicated the positivity of amino group of
COL-NH.sub.2. The structure was confirmed by .sup.1H-NMR and mass
spectroscopy (FAB-MS). .sup.1H-NMR (CDCl.sub.3) .delta. 8.09 (S,
1H), 7.51 (d, 1H, J=12 Hz), 7.30 (d, 1H, J=12 Hz), 6.56 (S, 1H),
3.91 (S, 6H), 3.85 (m, 1H), 3.67 (S, 3H), 2.25-2.52 (m, 4H). m/z
308.2 (M.sup.+, 20), 307.2 (100).
Synthesis of Ethylenedicysteine-Colchcine (EC-COL)
[0218] Sodium hydroxide (2N, 0.2 ml) was added to a stirred
solution of EC (134 mg, 0.50 mmol) in water (5 ml). To this
colotiess solution, sulfo-NHS (217 mg, 1.0 mmol) and EDC (192 mg,
1.0 mmol) were added. COL-NH.sub.2 (340 mg, 2.0 mmol) was then
added. The mixture was stirred at room temperature for 24 hours.
The mixture was dialyzed for 48 hrs using Spectra/POR molecular
porous membrane with cut-off at 500 (Spectrum Medical Industries
Inc., Houston, Tex.). After dialysis, the product was frozen dried
using lyophilizer (Labconco, Kansas City, Mo.). The product weighed
315 mg (yield 55%). .sup.1H-NMR (D.sub.2O) .delta. 7.39 (S, 1H),
7.20 (d, 1H, J=12 Hz), 7.03 (d, 1H, J=12 Hz), 6.78 (S, 1H),
4.25-4.40 (m, 1H), 3.87 (S, 3H, --OCH.sub.3), 3.84 (S, 3H,
--OCH.sub.3), 3.53 (S, 3H, --OCH.sub.3), 3.42-3.52 (m, 2H),
3.05-3.26 (m, 4H), 2.63-2.82 (m, 4H), 2.19-2.25 (m, 411). FAB MS
m/z 580 (sodium salt, 20). The synthetic scheme of EC-COL is shown
in FIG. 21.
Radiolabeling of EC-COL and EC with .sup.99mTc
[0219] Radiosynthesis of .sup.99mTc-EC-COL was achieved by adding
required amount of .sup.99mTc-pertechnetate into home-made kit
containing the lyophilized residue of EC-COL (5 mg), SnCl.sub.2
(100 .mu.g), Na.sub.2HPO.sub.4 (13.5 mg), ascorbic acid (0.5 mg)
and NaEDTA (0.5 mg). Final pH of preparation was 7.4. .sup.99mTc-EC
was also obtained by using home-made kit containing the lyophilized
residue of EC (5 mg), SnCl.sub.2 (100 .mu.g), Na.sub.2HPO.sub.4
(13.5 mg), ascorbic acid (0.5 mg) and NaFDTA (0.5 mg) at pH 10.
Final pH of preparation was then adjusted to 7.4. Radiochemical
purity was determined by TLC (ITLC SG, Gelman Sciences, Ann Arbor,
Mich.) eluted with ammonium acetate (1M in water):methanol (4:1).
Radio-thin layer chromatography (TLC, Bioscan, Washington, D.C.)
was used to analyze the radiochemical purity for both
radiotracers.
Stability Assay of .sup.99mTc-EC-COL
[0220] Stability of labeled .sup.99mTc-EC-COL was tested in serum
samples. Briefly, 740 KBq of 5 mg .sup.99mTc-EC-COL was incubated
in the rabbinate serum (500 .mu.l) at 37.degree. C. for 4 hours.
The serum samples was diluted with 50% methanol in water and
radio-TLC repeated at 0.5, 2 and 4 hours as described above.
Tissue Distribution Studies
[0221] Female Fischer 344 rats (150.+-.25 g) (Harlan
Sprague-Dawley, Indianapolis, Ind.) were inoculated subcutaneously
with 0.1 ml of mammary tumor cells from the 13762 tumor cell line
suspension (10 cells/rat, a tumor cell line specific to Fischer
rats) into the hind legs using 25-gauge needles. Studies performed
14 to 17 days after implantation when tumors reached approximately
1 cm diameter. Rats were anesthetized with ketamine (10-15 mg/rat,
intraperitoneally) before each procedure.
[0222] In tissue distribution studies, each animal was injected
intravenously with 370-550 KBq of .sup.99mTc-EC-COL or
.sup.99mTc-EC (n=3/time point). The injected mass of
.sup.99mTc-EC-COL was 10 .mu.g per rat. At 0.5, 2 and 4 hrs
following administration of the radiotracers, the rats were
sacrificed and the selected tissues were excised, weighed and
counted for radioactivity. The biodistribution of tracer in each
sample was calculated as percentage of the injected dose per gram
of tissue wet weight (% ID/g). Tumor/nontarget tissue count density
ratios were calculated from the corresponding %1D/g values. Student
t-test was used to assess the significance of differences between
groups.
Scintigraphic Imaging Studies
[0223] Scintigraphic images, using a gamma camera (Siemens Medical
Systems, Inc., Hoffman Estates, Ill.) equipped with low-energy,
parallel-hole collimator, were obtained 0.5, 2 and 4 hrs after i.v.
injection of 300 .mu.Ci of .sup.99mTc-EC-COL and .sup.99mTc-EC.
Computer outlined region of interest (ROI) was used to quantitate
(counts per pixel) the tumor uptake versus normal muscle
uptake.
Results
[0224] Radiosynthesis and Stability of .sup.99mTc-EC-COL
[0225] Radiosynthesis of EC-COL with .sup.99mTc was achieved with
high (>95%) radiochemical purity (FIG. 21). .sup.99mTc-EC-COL
was found to be stable at 0.5, 2 and 4 hrs in rabbit serum samples.
There was no degradation products observed (FIG. 22).
In Vivo Biodistribution
[0226] In vivo biodistribution of .sup.99mTc-EC-COL and
.sup.99mTc-EC in breast-tumor-bearing rats are shown in Tables 4
and 6. Tumor uptake value (% ID/g) of .sup.99mTc-EC-COL at 0.5, 2
and 4 hours was 0.436.+-.0.089, 0.395.+-.0.154 and 0.22110.006
(Table 6), whereas those for .sup.99mTc-EC were 0.342.+-.0.163,
0.115.+-.0.002 and 0.097.+-.0.005, respectively (Table 4).
Increased tumor-to-blood (0.52.+-.0.12 to 0.72.+-.0.07) and
tumor-to-muscle (3.47.+-.0.40 to 7.97.+-.0.93) ratios as a function
of time were observed in .sup.99mTc-EC-COL group (FIG. 23).
Conversely, tumor-to-blood and tumor-to-muscle values showed
time-dependent decrease with .sup.99mTc-EC when compared to
.sup.99mTc-EC-COL group in the same time period (FIG. 24).
TABLE-US-00006 TABLE 6 Biodistribution of .sup.99mTc-EC-Colchicine
in Breast Tumor Bearing Rats 30 Min. 2 Hour 4 Hour Blood 0.837 .+-.
0.072 0.606 .+-. 0.266 0.307 .+-. 0.022 Lung 0.636 .+-. 0.056 0.407
.+-. 0.151 0.194 .+-. 0.009 Liver 1.159 .+-. 0.095 1.051 .+-. 0.213
0.808 .+-. 0.084 Spleen 0.524 .+-. 0.086 0.559 .+-. 0.143 0.358
.+-. 0.032 Kidney 9.705 .+-. 0.608 14.065 .+-. 4.007 11.097 .+-.
0.108 Muscle 0.129 .+-. 0.040 0.071 .+-. 0.032 0.028 .+-. 0.004
Stomach 0.484 .+-. 0.386 0.342 .+-. 0.150 0.171 .+-. 0.123 Uterus
0.502 .+-. 0.326 0.343 .+-. 0.370 0.133 .+-. 0.014 Thyroid 3.907
.+-. 0.997 2.297 .+-. 0.711 1.709 .+-. 0.776 Tumor 0.436 .+-. 0.089
0.395 .+-. 0.154 0.221 .+-. 0.006 * Each rat received
.sup.99mTc-EC-Colchicine (10 .mu.Ci, iv.). Each value is the
percent of injected dose per gram tissue weight (n = 3)/time
interval. Each data represents mean of three measurements with
standard deviation.
TABLE-US-00007 TABLE 7 Rf Values Determined by Radio-TLC (ITLC-SG)
Studies System A* System B.dagger. .sup.99mTc-EC-folate 0
1(>95%) .sup.99mTc-EC- 0 1(>95%) Free .sup.99mTc 1 1 Reduced
.sup.99mTc 0 0 *Acetone .dagger.Ammonium Acetate (1M in
water):Methanol (4:1)
Gamma Scintigraphic Imaging of .sup.99mTc-EC-COL in Breast
Tumor-Bearing Rats
[0227] In vivo imaging studies in three breast-tumor-bearing rats
at 1 hour post-administration indicated that the tumor could be
visualized well with .sup.99mTc-EC-COL group (FIG. 25), whereas,
less tumor uptake in the .sup.99mTc-EC group was observed (FIG.
26). Computer outlined region of interest (ROI) showed that
tumor/background ratios in .sup.99mTc-EC-COL group were
significantly higher than .sup.99mTc-EC group (FIG. 27).
Tumor Glycolysis Targeting
Example 6
Development of .sup.99mTc-EC-Neomycin
Synthesis of EC
[0228] EC was prepared in a two-step synthesis according to the
previously described methods (Ratner and Clarke, 1937; Blondeau et
al., 1967). The precursor, L-thiazolidine-4-carboxylic acid, was
synthesized (m.p. 195.degree., reported 196-197.degree.). EC was
then prepared (m.p. 237.degree., reported 251-253.degree.). The
structure was confirmed by .sup.1H-NMR and fast-atom bombardment
mass spectroscopy (FAB-MS).
Synthesis of Ethylenedicysteine-Neomycin (EC-Neomycin)
[0229] Sodium hydroxide (2N, 0.2 ml) was added to a stirred
solution of EC (134 mg, 0.50 mmol) in water (5 ml). To this
colorless solution, sulfo-NHS (217 mg, 1.0 mmol) and EDC (192 mg,
1.0 mmol) were added. Neomycin trisulfate salt (909 mg, 1.0 mmol)
was then added. The mixture was stirred at room temperature for 24
hours. The mixture was dialyzed for 48 hours using Spectra/POR
molecular porous membrane with cut-off at 500 (Spectrum Medical
Industries Inc., Houston, Tex.). After dialysis, the product was
frozen dried using lyophilizer (Labconco, Kansas City, Mo.). The
product weighed 720 mg (yield 83%). The synthetic scheme of
EC-neomycin is shown in FIG. 36. The structure is confirmed by
.sup.1H-NMR (FIGS. 38A-B), mass spectrometry (FIGS. 39A-B) and
elemental analysis (Galbraith Laboratories, Inc. Knoxyille, Tenn.).
Elemental analysis
C.sub.39H.sub.75N.sub.10S.sub.4O.sub.19.15H.sub.2O(C,H,N,S), Calc.
C, 33.77; H, 7.58; N, 10.11, S:9.23; found C, 32.44; H, 5.90; N,
10.47, S:10.58. UV wavelength of EC-neomycin was shifted to 270.5
nm when compared to EC and neomycin (FIGS. 40A-C)
Radiolabeling of EC-MN and EC-Neomycin with .sup.99mTc
[0230] Radiosynthesis of .sup.99mTc-EC and .sup.99mTc-EC-neomycin
were achieved by adding required amount of .sup.99mTc-pertechnetate
into home-made kit containing the lyophilized residue of EC or
EC-neomycin (10 mg), SnCl.sub.2 (100 .mu.g), Na.sub.2HPO.sub.4
(13.5 mg) and ascorbic acid (0.5 mg). NaEDTA (0.5 mg) in 0.1 ml of
water was then added. Final pH of preparation was 7.4.
Radiochemical purity was determined by TLC (ITLC SG, Gelman
Sciences, Ann Arbor, Mich.) eluted with ammonium acetate (1M in
water):methanol (4:1). From radio-TLC (Bioscan, Washington, D.C.)
analysis (FIG. 41) and HPLC analysis (FIGS. 42-45), the
radiochemical purity was >95% for both radiotracers.
Stability Assay of .sup.99m Tc-EC and .sup.99mTc-EC-neomycin
[0231] Stability of labeled .sup.99mTc-EC and
.sup.99mTc-EC-neomycin were tested in dog serum samples. Briefly,
740 KBq of 1 mg .sup.99mTc-EC and .sup.99mTc-EC-neomycin were
incubated in dog serum (200 .mu.l) at 37.degree. C. for 4 hours.
The serum samples were diluted with 50% methanol in water and
radio-TLC repeated at 0.5, 2 and 4 hours as described above.
Tissue Distribution Studies of .sup.99mTc-EC-Neomycin
[0232] Female Fischer 344 rats (150+25 g) (Harlan Sprague-Dawley,
Indianapolis, Ind.) were innoculated subcutaneously with 0.1 ml of
mammary tumor cells from the 13762 tumor cell line suspension
(10.sup.6 cells/rat, a tumor cell line specific to Fischer rats)
into the hind legs using 25-gauge needles. Studies performed 14 to
17 days after implantation when tumors reached approximately 1 cm
diameter. Rats were anesthetized with ketamine (10-15 mg/rat,
intraperitoneally) before each procedure.
[0233] In tissue distribution studies, each animal was injected
intravenously with 10-20 .mu.Ci of .sup.99mTc-EC or
.sup.99mTc-EC-neomycin (n=3/time point). The injected mass of
.sup.99mTc-EC-neomycin was 200 .mu.g per rat. At 0.5, 2 and 4 hours
following administration of the radiotracers, the rats were
sacrificed and the selected tissues were excised, weighed and
counted for radioactivity. The biodistribution of tracer in each
sample was calculated as percentage of the injected dose per gram
of tissue wet weight (% ID/g). Tumor/nontarget tissue count density
ratios were calculated from the corresponding % ID/g values. When
compared to .sup.99mTc-EC (Table 4) and free technetium (Table 9),
tumor-to tissue ratios increased as a function of time in .sup.99m
Tc-EC-neomycin group (Table 8).
Scintigraphic Imaging Studies
[0234] Scintigraphic images, using a gamma camera (Siemens Medical
Systems, Inc., Hoffman Estates, Ill.) equipped with low-energy,
parallel-hole collimator, were obtained 0.5, 2 and 4 hours after
i.v. injection of 100 .mu.Ci of each radiotracer. Compare to
.sup.99mTc-EC, high uptake in the tumors was observed (FIG. 37A).
Preliminary clinical imaging studies were conducted in a patient
with breast cancer. The tumor was visualized well at 2 hours
post-administration of .sup.99mTc-EC-neomycin (FIG. 37B).
TABLE-US-00008 TABLE 8 Biodistribution of .sup.99mTc-EC-neomycin in
Breast Tumor Bearing Rats 30 Min. 1 Hour 2 Hour 4 Hour Blood 0.463
.+-. 0.007 0.262 .+-. 0.040 0.139 .+-. 0.016 0.085 .+-. 0.004 Lung
0.344 .+-. 0.011 0.202 .+-. 0.030 0.114 .+-. 0.014 0.080 .+-. 0.003
Liver 0.337 .+-. 0.012 0.269 .+-. 0.013 0.221 .+-. 0.020 0.195 .+-.
0.012 Stomach 0.279 .+-. 0.039 0.147 .+-. 0.001 0.061 .+-. 0.008
0.054 .+-. 0.008 Spleen 0.159 .+-. 0.008 0.114 .+-. 0.013 0.095
.+-. 0.007 0.089 .+-. 0.003 Kidney 8.391 .+-. 0.395 8.804 .+-.
0.817 8.356 .+-. 0.408 8.638 .+-. 0.251 Thyroid 0.349 .+-. 0.008
0.202 .+-. 0.028 0.114 .+-. 0.007 0.086 .+-. 0.001 Muscle 0.093
.+-. 0.001 0.049 .+-. 0.010 0.021 .+-. 0.006 0.010 .+-. 0.001
Intestine 0.159 .+-. 0.004 0.093 .+-. 0.014 0.061 .+-. 0.004 0.266
.+-. 0.200 Urine 25.402 .+-. 8.621 21.786 .+-. 2.690 0.224 .+-.
0.000 2.609 .+-. 2.377 Tumor 0.419 .+-. 0.023 0.279 .+-. 0.042
0.166 .+-. 0.023 0.131 .+-. 0.002 Brain 0.022 .+-. 0.001 0.014 .+-.
0.003 0.010 .+-. 0.001 0.007 .+-. 0.001 Heart 0.147 .+-. 0.009
0.081 .+-. 0.012 0.040 .+-. 0.004 0.029 .+-. 0.002 Tumor/Blood
0.906 .+-. 0.039 1.070 .+-. 0.028 1.196 .+-. 0.061 1.536 .+-. 0.029
Tumor/Muscle 4.512 .+-. 0.220 5.855 .+-. 0.458 8.364 .+-. 1.469
12.706 .+-. 0.783 Tumor/Brain 19.495 .+-. 1.823 20.001 .+-. 0.890
17.515 .+-. 2.035 20.255 .+-. 1.693 Values shown represent the mean
.+-. standard deviation of data from 3 animals.
TABLE-US-00009 TABLE 9 Biodistribution of .sup.99mTc Pertechnetate
in Breast Tumor Bearing Rats 30 Min. 2 Hour 4 Hour Blood 1.218 .+-.
0.328 0.666 .+-. 0.066 0.715 .+-. 0.052 Lung 0.646 .+-. 0.291 0.632
.+-. 0.026 0.387 .+-. 0.024 Liver 0.541 .+-. 0.232 0.304 .+-. 0.026
0.501 .+-. 0.081 Spleen 0.331 .+-. 0.108 0.187 .+-. 0.014 0.225
.+-. 0.017 Kidney 0.638 .+-. 0.197 0.489 .+-. 0.000 0.932 .+-.
0.029 Thyroid 24.821 .+-. 5.181 11.907 .+-. 15.412 17.232 .+-.
5.002 Muscle 0.130 .+-. 0.079 0.076 .+-. 0.002 0.063 .+-. 0.003
Intestine 0.153 .+-. 0.068 0.186 .+-. 0.007 0.344 .+-. 0.027 Tumor
0.591 .+-. 0.268 0.328 .+-. 0.016 0.423 .+-. 0.091 Brain 0.038 .+-.
0.014 0.022 .+-. 0.002 0.031 .+-. 0.009 Heart 0.275 .+-. 0.089
0.145 .+-. 0.015 0.166 .+-. 0.012 Tumor/Blood 0.472 .+-. 0.093
0.497 .+-. 0.073 0.597 .+-. 0.144 Tumor/Muscle 4.788 .+-. 0.833
4.302 .+-. 0.093 6.689 .+-. 1.458 Tumor/Liver 1.084 .+-. 0.023
1.084 .+-. 0.115 0.865 .+-. 0.270 Values shown represent the mean
.+-. standard deviation of data from 3 animals.
In Vitro Cellular Uptake of .sup.99mTc-EC-Drug Conjugates
[0235] To evaluate the cellular uptake of .sup.99mTc-EC-drug
conjugates, each well containing 80,000 cells (A549 lung cancer
cell line) was added with 2 .mu.Ci of .sup.99mTc-EC-neomycin and
.sup.18F-FDG. After incubation at 0.5-4 hours, the cells were
washed with phosphate buffered saline 3 times and followed by
trypsin to lose the cells. The cells were then counted by a gamma
counter. .sup.99mTc-EC-neomycin showed highest uptake among those
agents tested in human lung cancer cell line (FIG. 46).
Effect of Glucose on Cellular Uptake of .sup.99mTc-EC-Neomycin and
.sup.18F-FDG
[0236] Neomycin is known to influence glucose absorption (Rogers et
al., 1968; Fanciulli et al., 1994). Previous experiments have shown
that .sup.99mTc-EC-neomycin has higher uptake than .sup.18F-IDG in
human lung cancer cell line (A549). To determine if uptake of
.sup.99mTc-EC-neomycin is mediated via glucose-related mechanism,
glucose (0.1 mg-2.0 mg) was added to each well containing either
50,000 (breast) cells or 80,000 cells (lung) along with 2 .mu.Ci of
.sup.99mTc-EC-neomycin and .sup.18F-FDG. After incubation, the
cells were washed with phosphate buffered saline 3 times and
followed by trypsin to lose the cells. The cells were then counted
by a gamma counter.
[0237] By adding glucose at the concentration of 0.1-2.0 mg/well,
decreased uptake of .sup.99mTc-EC-neomycin in two lung cancer cell
lines and one breast cell line was observed. Similar results were
observed in .sup.18F-PDG groups. .sup.99mTc-EC (control) showed no
uptake. The findings suggest that the cellular uptake of
.sup.99mTc-EC-neomycin may be mediated via glucose-related
mechanism (FIGS. 47, 48A and 48B).
Example 7
Tumor Metabolic Imaging with .sup.99mTc-EC-Deoxyglucose
Synthesis of EC-Deoxyglucose (EC-DG)
[0238] Sodium hydroxide (1N, 1 ml) was added to a stirred solution
of EC (110 mg, 0.41 mmol) in water (5 ml). To this colorless
solution, sulfo-NHS (241.6 mg, 1.12 mmol) and EDC (218.8 mg, 1.15
mmol) were added. D-Glucosamine hydrochloride salt (356.8 mg, 1.65
mmol) was then added. The mixture was stirred at room temperature
for 24 hours. The mixture was dialyzed for 48 hours using
Spectra/POR molecular porous membrane with cut-off at 500 (Spectrum
Medical Industries Inc., Houston, Tex.). After dialysis, the
product was frozen dried using lyophilizer (Labconco, Kansas City,
Mo.). The product in the salt form weighed 568.8 mg. The synthetic
scheme is shown in FIG. 59. The structure was confirmed by mass
spectrometry (FIG. 60) and proton NMR (FIGS. 61 and 62).
Radiochemical purity of .sup.99mTc-EC-DG was 100% as determined by
radio-TLC (FIG. 63) and HPLC (FIGS. 64 and 65) analysis.
Hexokinase Assay
[0239] To determine if EC-DG mimics glucose phosphorylation, a
hexokinase assay was conducted. Using a ready made kit (Sigma
Chemical Company), EC-DG, glucosamine and glucose (standard) were
assayed at UV wavelength 340 nm. Glucose, EC-DG and glucosamine
showed positive hexokinase assay (FIGS. 66-68).
In Vitro Cellular Uptake Assay
[0240] In vitro cellular uptake assay was conducted by using a
human lung cancer cell line (A549). Two .mu.Ci of .sup.99mTc-EC-DG
and .sup.18F-FDG were added to wells containing 80,000 cells each.
After incubation at 0.5-4 hours, the cells were washed with
phosphate buffered saline 3 times and followed by trypsin to lose
the cells. The cells were then counted by a gamma counter. The
uptake of .sup.99mTc-EC-DG was comparable to FDG (FIG. 69).
Effect of d- and l-Glucose on Cellular Uptake of
.sup.99mTc-EC-Deoxyglucose and .sup.18F-FDG
[0241] To evaluate if the uptake of .sup.99mTc-EC-deoxyglucose is
mediated via d-glucose mechanism, d- and 1-glucose (1 mg and 2.0
mg) were added to, each well containing either breast or lung
cancer cells (50,000/0.5 ml/well), along with 2 .mu.Ci of
.sup.99mTc-EC-deoxyglucose and .sup.18F-FDG. After 2 hours
incubation, the cells were washed with phosphate buffered saline 3
times and followed by trypsin to lose the cells. The cells were
counted by a gamma counter.
[0242] By adding glucose at the concentration of 1-2.0 mg/well, a
decreased uptake of .sup.99mTc-EC-deoxyglucose and .sup.18F-FDG by
d-glucose in breast and lung cancer cells was observed. However,
there was no influence on both agents by 1-glucose (FIG. 70-73).
The findings suggest that the cellular uptake of
.sup.99mTc-EC-deoxyglucose is mediated via d-glucose mechanism.
Effect of EC-Deoxyglucose Loading on Blood Glucose Level in Normal
Rats
[0243] Previous experiments have shown that cellular uptake of
.sup.99mTc-EC-deoxyglucose is similar to FDG. For instance, the
hexokinase assay (glucose phosphorylation) was positive. The uptake
of .sup.99mTc-EC-deoxyglucose is mediated via d-glucose mechanism.
This study is to determine whether blood glucose level could be
induced by either FDG or EC-deoxyglucose and suppressed by
insulin.
[0244] Normal healthy Fischer 344 rats (weight 145-155 g) were
fasting overnight prior to the experiments. The concentration of
glucosamine hydrochloride, FDG and EC-deoxyglucose prepared was 60%
and 164% (mg/ml). The blood glucose level (mg/dl) was determined by
a glucose meter (Glucometer DEX, Bayer Corporation, Elkhart, Ind.).
Prior to the study, the baseline of blood glucose level was
obtained. Each rat (n=3/group) was administered 1.2 mmol/kg of
glucosamine, FDG and EC-deoxyglucose. In a separate experiment, a
group of rats was administered EC-deoxyglucose and FDG. Insulin (5
units) was administered after 30 minutes. Blood samples were
collected from the tail vein every 30 minutes up to 6 hours
post-administration.
[0245] Blood glucose level was induced by bolus intravenous
administration of glucosamine, FDG and EC-deoxyglucose. This
increased blood glucose level could be suppressed by
co-administration of EC-deoxyglucose or FDG and insulin (FIGS. 74
and 75).
Tissue Distribution Studies of .sup.99mTc-EC-DG
[0246] For breast tumor-bearing animal model, female Fischer 344
rats (150+25 g) (Harlan Sprague-Dawley, Indianapolis, Ind.) were
innoculated subcutaneously with 0.1 ml of mammary tumor cells from
the 13762 tumor cell line suspension (10.sup.6 cells/rat, a tumor
cell line specific to Fischer rats) into the hind legs using
25-gauge needles. Studies were performed 14 to 17 days after
implantation when tumors reached approximately 1 cm diameter. Rats
were anesthetized with ketamine (10-15 mg/rat, intraperitoneally)
before each procedure.
[0247] For lung tumor-bearing animal model, each athymic nude mouse
(20-25 g) was innoculated subcutaneously with 0.1 ml of human lung
tumor cells from the A549 tumor cell line suspension (10.sup.6
cells/mouse) into the hind legs using 25-gauge needles. Studies
were performed 17 to 21 days after implantation when tumors reached
approximately 0.6 cm diameter.
[0248] In tissue distribution studies, each animal was injected
intravenously with 10-20 .mu.Ci (per rat) or 1-2 .mu.Ci (per mouse)
of .sup.99mTc-EC or .sup.99mTc-EC-DG (n=3/time point). The injected
mass of .sup.99mTc-EC-DG was 1 mg per rat. At 0.5, 2 and 4 hours
following administration of the radiotracers, the rodents were
sacrificed and the selected tissues were excised, weighed and
counted for radioactivity. The biodistribution of tracer in each
sample was calculated as percentage of the injected dose per gram
of tissue wet weight (% ID/g). Tumor/nontarget tissue count density
ratios were calculated from the corresponding % ID/g values. When
compared to .sup.99mTc-EC (Table 4) and free technetium (Table 9),
tumor-to tissue ratios increased as a function of time in
.sup.99mTc-EC-DG group (FIGS. 76-80).
Scintigraphic Imaging Studies
[0249] Scintigraphic images, using a gamma camera equipped with
low-energy, parallel-hole collimator, were obtained 0.5, 2 and 4
hours after i.v. injection of 100 .mu.Ci of the radiotracer. The
animal model used was breast tumor-bearing rats. Tumor could be
visualized well when compared to .sup.99mTc-EC (control group)
(FIG. 81). Preliminary clinical studies were conducted in 5
patients (3 brain tumors and 2 lung diseases). The images were
obtained at 1-2 hours post-administration. .sup.99mTc-EC-DG was
able to differentiate benign versus malignant tumors. For instance,
malignant astrocytoma showed high uptake (FIGS. 82A, 82B, 83A and
83B). Benign meningioma showed poor uptake compared to malignant
meningioma (FIGS. 84A and B). Poor uptake was observed in patient
with TB (FIG. 85A and FIG. 85B), but high uptake was observed in
lung tumor (FIG. 86A, FIG. 86B, and FIG. 86C).
[0250] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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