Note: Descriptions are shown in the official language in which they were submitted.
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1,4,8,11-TETRAAZACYCLOTETRADECANE DERIVATIVES AS
RADIODIAGNOSTIC AGENTS AND THEIR USE
IN DETERMINING HYPOXIA AND RADIORESISTANCE OF TUMORS
Pursuant to 35 U.S.C. ~202(c), it is hereby acknowledged that the
U.S. Government has certain rights in the invention described herein, which
was
made in part with funds from the National Institutes of Health (Grant No. CA
06927)
BACKGROUND OF THE INVENTION
This invention relates to derivatives of 1,4,8,11-
tetraazacyclotetradecane (cyclam) which demonstrate hypoxic cell selectivity
and
retention and which are useful as radiodiagnostic agents for assessing tissue
oxygenation status non-invasively, as well as to methods for preparing such
derivatives and radionuclide metal-containing complexes thereof and their use
for
imaging and non-invasively determining tissue hypoxia and radioresistance of
tumors.
Hypoxic cells are known to exist in both animal and human
tumors and this cell population has been shown to be 2.5 to 3 times more
resistant
than normally oxygenated cells to therapeutic radiation. The presence of these
radioresistant populations in tumors presents a serious obstacle to the
curative
potential of clinical radiotherapy. Radiobiologic hypoxic fraction (HF), the
fraction of clonogenic tumor cells that exhibit maximum radioresistance, has
been shown to be an important parameter for predicting tumor treatment
resistance and for the selection of aggressive and metastatic cell phenotypes.
If
this tumor property were known by clinicians at the time of diagnosis, it
would be
a useful predictor of tumor treatment response and would define subsets of
patients for whom targeted therapies would produce improved cure rates. Thus,
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radiodiagnostic agents that can accurately assess the presence and extent of
hypoxic tissues in tumors would be of invaluable assistance in designing the
appropriate therapeutic regimen and in following the response of tumor tissue
to
therapy.
Although various agents and methods have been proposed for the
detection and measurement of hypoxic cells in tumors, and direct p02
measurements have been obtained from accessible tumors using microelectrodes,
there is no practical, clinically useful diagnostic agent or method currently
available. Some experimental procedures to assess tumor hypoxia are invasive,
requiring multiple biopsy specimens that cannot always be obtained.
A promising approach to the assessment of hypoxic tissue is
suggested by the ability of certain classes of compounds to selectively
localize in
these tissues after intravenous administration. The radiosensitizing drug,
misonidazole (MISO), was shown to become selectively bound to the
macromolecular fraction of EMT-6 murine tumor and V-79 hamster lung cells in
hypoxic in vitro incubation studies (J.D. Chapman et al., Cancer Res., 43:
1523-
1528 (1983)) and to EMT-6 tumors in BALB/C mice (B.M. Garrecht et al., Brit.
J. Radiol., 56: 745-753 (1983)). Selective binding of a y-emitting analogue of
a
suitable compound would allow imaging and measurement of hypoxic tissue by
conventional nuclear medicine techniques. Based on this approach, a number of
analogues of 2-nitroimidazole have been investigated as potential non-
invasive,
hypoxic tissue specific, nuclear medicine imaging agents. The selective
toxicity
to hypoxic cells by 2-nitroimidazole has been shown to correlate with the
accumulation of cellular reduction products of these compounds (J.D. Chapman,
Cancer, 54: 2441-2449 (1984)).
The exact mechanism of the binding to the macromolecular
fraction of cells remains under investigation, but is believed to rely on
reduction
of the nitroheterocycle through a series of one-electron transfers that
produce
nitroso, hydroxylamino and amino products (J.E. Biaglow et al., Biochem.
Pharmacol., 35: 77-90 (1986)). This process is reliant on flavoproteins known
as
nitroreductases. The enzymes are functional only in viable cells and the
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progression of the reduction past the one-electron stage (nitro radical anion)
is
strongly inhibited by oxygen, since this species can accept the electron from
the
nitro radical anion, thereby regenerating the nitroheterocycle. Further
reductive
metabolism and subsequent binding will, therefore, only be expected to take
place
in poorly oxygenated yet viable (hence hypoxic) cells.
A number of experimental hypoxic tissue imaging agents
incorporating y-emitting radionuclides have been investigated. These include 4-
bromomisonidazole (D.C. Jette et al., Int. J. Nucl. Med. Biol., 10: 205-210
(1983); J.S. Rasey et al., Radiat. Rse., 91: 542-554 (1982)), 1-(2-
iodophenoxy)-
ethyl)-2-nitroimidazole (L.I. Wiebe, Nucelarmedizine, 23: 63-67 (1984)), a
series
of iodinated acetophenone derivatives of 2-nitroimidazole (J.R. Mercer et al.,
J.
Lab. Comp. Radiopharm., 25: 107-108 (1988)), fluoromisonidazole (F-MISO)
(P.A. Jerabek et al., Appl. Radiat. Isot., 37: 599-605 (1986)) the 2-
nitroimidazole
nucleoside analogue, iodoazomycin riboside (IAZR) (D.C. Jette et al., Radiat.
Res., 105: 169-179 (1986); L.I. Wiebe et al., In Nuclear Medicine in Clinical
Oncology, Heidelberg, Springer-Verlag, pp. 402-407 (1986)) and certain
azomycin nucleosides, namely iodoazomycin arabinoside (IAZA), iodoazomycin
galactopyranoside (IAZGP) and iodoazomycin xylopyranoside (IAZXP), which
are the subject of U.S. Patent 5,401,490.
At millimolar concentrations, IAZGP and IAZA have been found
to be selectively toxic to hypoxic EMT-6 tumor cells and also sensitize these
cells
to the lethal effects of ionizing radiation. At micromolar concentrations,
IAZA is
taken up preferentially in EMT-6 tumor tissue at a level useful for non-
invasive
imaging. An imaging study using'ZSI-IAZA showed EMT-6 tumor tissue to be
clearly delineated from surrounding tissue. More recently, pilot clinical
studies
using'z3I-IAZA with single photon emission computed tomography (SPELT) and
'8F-FMISO with positron emission tomography (PET) have been reported. These
data were recently reviewed and it appears unlikely that either marker is
optimal
for routinely determining the HF of individual human tumors (J.D. Chapman et
al., Radiother. Oncol., 46: 229-37 (1998)). Among the azomycin-nucleoside
markers, IAZXP and IAZGP have been shown to exhibit the most favorable
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properties for marking the HF of solid rodent tumors in vivo (J.D. Chapman et
al.
(1988), supra).
Although the azomycin nucleosides, and particularly IAZGP, have
been found to have useful hypoxic marking properties, an extensive research
effort is ongoing to identify hypoxic markers which exhibit good
bioavailability
to all tissues, low lipophilicity to promote rapid renal excretion, are
amenable to
detection at optimal times after administration and can be convenientily
labelled
with the preferred nuclear medicine isotopes.
SUMMARY OF THE INVENTION
In accordance with one aspect of the present invention, cyclam
derivatives are provided having the formula:
20
(I)
wherein at least one of said R,, R2, R3 and R4 contains 2-nitro-imidazol-
1-yl moiety of the formula:
n
N ~ N -
N02
which is chemically linked to the cyclam nitrogen through any of various
divalent
linking groups. The remaining Rs are hydrogen.
Preferably, the linking group comprises a chain of 2 to 7
atoms selected from the group of carbon or nitrogen, wherein the moiety
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connecting the cyclam nitrogen and the 1-imidazole nitrogen to the remainder
of
the linking group is a -CHZ- moiety, the carbons in the remainder of the
linking
moiety being optionally substituted with a hydroxyl group, and two adjacent
carbons in the linking groups being optionally replaceable by an amide group.
Stereoisomers, including both enantiomers, meso forms and
diastereomers of the cyclam derivatives of the above general formula, as well
as
the pharmaceutically acceptable salts of such cyclam derivatives and their
isomers, are included within the present invention.
The present invention also provides radiodiagnostic complexes
comprising a cyclam derivative, as described above, and various complex-
forming radionuclide metals.
According to a further aspect of the invention, a process is
provided for preparation of the cyclam derivatives of the invention. Briefly,
this
method involves the nucleophilic displacement by cyclam of a suitable leaving
group on a moiety containing azomycin.
According to yet another aspect, the present invention provides
radiodiagnostic compositions comprising the above-mentioned radionuclide
metal-containing complexes and a biologically compatible carrier medium for
use
in determining tumor hypoxia and radioresistance, as well as in imaging body
tissue of a living test subject.
There is also provided, in accordance with this invention, a
method of preparing a radionuclide metal-containing complex, as described
above, by causing a salt or chelate of a radionuclide metal to undergo a
complex-
forming reaction with a cyclam derivative of the above formula, or a metal
adduct
thereof. A kit is also provided for carrying out such method. The kit contains
(i) a cyclam derivative, or a metal adduct thereof, and (ii) instructions for
reacting
the cyclam derivative or metal adduct thereof, with a radionuclide metal
selected
from the group of the radioactive isotopes of Tc, Cu, Ru, Co, Pt, Fe, Os, Ir,
W,
Re, Cr, Mo, Mn, Ni, Rh, Pd, Nb, Pb, Ga, As, In and Ta to produce the complex.
According to yet a further aspect of the invention, a novel non-
invasive method is provided for the detection and measurement of tissue
hypoxia
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in a mammal comprising the steps of:
(a) administering a radiodiagnostic complex, as described
above, such that that complex is taken up selectively and retained by the
hypoxic
tissue; and
(b) quantifying the extent of retained complex by the y-
emission from said complex.
According to still a further aspect of this invention, there is
provided a method for imaging body tissue of a living test subject by
administering to the test subject a radionuclide metal-containing complex, as
described herein, and detecting the localization of the complex in the body
tissue
using a suitable radiation detector. Of course, different detectors may be
employed depending on the nature of the emission produced by the radioactive
metal present in the complex. Suitable detectors include, without limitation,
SPECT and PET cameras.
The cyclam derivatives of the present invention are considered
superior to previously reported radiodiagnostic agents for measuring hypoxia
and
predicting tumor radioresistance because of their high specific activity and
the
large differential labelling of experimental tumors having a an HF of 15-20%,
as
compared to tumor tissue that exhibits no detectable HF.
Furthermore, in that radiation resistance and some
chemotherapeutic drug resistance correlate with tumor hypoxia, the ability to
define one type of tumor resistance in advance of therapy is important, since
various modalities of targeted cancer treatment, such as chemical
radiosensitizers,
chemopotentiation agents and bioreductive drugs, can now be directed towards
treatment-resistant hypoxic cells.
In addition, a radiodiagnostic agent for oxygenation status will be
useful in detecting and defining other disease states, including myocardial
infarct
and cerebrovascular hemorrhage, in which ischemia and/or infarct play a role
and
in infections which involve anaerobic foci.
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BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in
color. Copies of this patent with color drawings) will be provided by the
Patent
and Trademark Office upon payment of the necessary fee.
FIGURE 1 is a graphical representation of typical marker
biodistribution data obtained by measuring percentage of injected dose of
marker/gram of assessed tissue (% 1D/G) as a function of time for eight
different
tissues from five EMT6 tumor-bearing scid mice, up to 10 hours after
intravenous
(i.v.) administration of a Tc-99m containing mono-[2-hydroxy-3-(2-
nitroimidazol-1-yl)propyl-substituted cyclam complex. The eight tissues
evaluated are represented as follows: ~ = blood; ~ = liver; o = kidney; v =
spleen; ~ = lungs;, = muscle; O = brain; ~ = tumor.
FIGURES 2A-D show collimated, gamma-camera planar images
of two pair of R 3327-AT (left; hypoxic) and R 3327-H (right; non-hypoxic)
tumor-bearing rats six hours after i.v. administration of Tc-99m-labelled
Ceretec0 (Figures 2A and 2B) and a Cu-67-containing tetra-[2-hydroxy-3-(2-
nitroimidazol-1-yl)-propyl]-substituted cyclam (Figures 2C and 2D). The tumor
site is indicated by an arrow in each image.
FIGURE 3 is a graphical representation of data showing the
binding rate (dpm/106 cells/hr) of a Cu-64 containing tetra-[2-hydroxy-3-(2-
nitroimidazol-1-yl)propyl]-substituted cyclam to tumor cells in vitro, as a
function of oxygen concentration.
FIGURES 4A and B show collimated, gamma camera planar
images of a pair of 83327-AT (left; hypoxic) and 83327-H (right; non-hypoxic)
tumor bearing rats 5-7 hours after administration of a Cu-64 containing tetra-
[2-
hydroxy-3-(2-nitroimidazol-1-yl)propyl]-substituted cyclam.
DETAILED DESCRIPTION OF THE INVENTION
The cyclam derivatives of formula I, above, can be prepared from
known starting materials and the syntheses of specific embodiments of such
derivatives that are within the scope of the invention are exemplified below.
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Certain of these derivatives have been used for radiodiagnostic imaging of
animal
tumor models and for evaluating their ability to determine tissue hypoxia and
assess their hypoxic marking properties, as reported below.
Suitable linking groups for linking the 2-nitro-imidazolyl moiety
to the cyclam nucleus are those of the formula
-(CHRa)n X-(CHRb)m-Y-(CHR~)P , wherein Ra and R~ represent hydrogen or alkyl
(C1-C4) and Rb represents hydrogen, alkyl (C1-C4), hydroxyl, X and Y may be
the
same or different and represent a structural group, such as an amide
(-C(=O)NH-), urea (-NH-C(=O)-NH-), thiourea (-NH-C(=S)-NH-), carbamic
(-NH-C(=O)-O-), amine (-NH-), carbonyl (-C(=O)-), and amidine
(-NH-C=(NH~)-); disulfide (-S-S-), ether (-O-), thioether (-S-) or sulfonamide
(-S(=O)~-NH-) group, n is an integer from 1 to 6, m is an integer from 1 to 6
and
p is an integer from 1 to 6.
Preferably, at least one of the cyclam ring nitrogens is linked to a
nitroimidazolyl moiety of the above formula through an unsubstituted or
hydroxyl-substituted divalent alkylene linking moiety of 2 to 7 carbon atoms,
which may optionally be interrupted by at least one group containing the amide
moiety. Representative examples of suitable linking moieties include -CH,-
CH(OH)-CHZ ; -(CHZ)2-NH-C(=O)-CHZ ; -CH~-C(=O)-NH-(CH~)2-;
(CHz)a-C(=O)-~-(CHz)s-~ -(CHZ)3-.
Particularly preferred embodiments of the present invention are
the mono-, di- and tetra-2-nitroimidazol-1-yl-substituted cyclam derivatives
in
which the nitroimidazolyl moiety is linked to the ring nitrogen of cyclam via
a
linkage having the structure -CHI-CH(OH)-CHI-. These particular derivatives
have an asymmetric center in the linking moiety. This makes the mono-
substituted compound resolvable into a pair of optical isomers. On the other
hand, the di- and tetra-substituted derivatives are separable into d, 1
(racemic) and
meso forms, which contain a plane of symmetry and, therefore, are resolvable
into optical isomers.
The cyclam derivatives of this invention can form soluble salts
with pharmaceutically acceptable acids, such as hydrochloric, phosphoric,
tartaric
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and citric acids, and these salts are also within the scope of the present
invention.
The pharmaceutically acceptable salts of the cyclam derivatives described
herein
can be prepared following procedures that are familiar to those skilled in the
art.
The cyclam derivatives described herein form a complex with
various radionuclide metals selected from the group of the radioactive
isotopes of
Tc, Cu, Ru, Co, Pt, Fe, Os, Ir, W, Re, Cr, Mo, Mn, Ni, Rh, Pd, Nb, Pb, Ga, As,
In
and Ta, for example, Tc-99m, Cu-67, Cu-64, Cu-62, Pb-203, Ga-67, Ga-68, As-
72, In-111, In-113m, Ru-97, Fe-52, Mn-52m, Cn-51 and Co-57. Particularly
preferred complex-forming radionuclide metals for use in the present invention
are Tc-99m, Cu-67, Cu-64 and In-111. The complexes have a net positive charge
and are associated with counter anions, which may be BF4-, CI04-, Br-,
CF3C00-, CI-, or the like, depending on the manner in which they are prepared.
The radionuclide metal-containing complexes, as described above,
are prepared by causing a salt or chelate of a suitable radionuclide metal to
undergo a complex-forming reaction with a cyclam derivative of the invention.
Complex formation is effected by contacting the cyclam derivative with the
desired metal in the form of a salt or in the form of a chelate wherein the
metal is
bound to a relatively weak chelator. In using a metal chelate as starting
material
for the complex formation, the desired complex is formed via the principle of
ligand exchange.
A reducing agent may be required for the above reaction, as in
cases where technetium metal is used. Suitable reducing agents for this
purpose
include Sn(II) compounds, dithionites, sodium borohydride, and the like.
Given the relatively short half-life of the radionuclide metal used
in the practice of this invention, it is frequently impossible to put the
ready-for-
use complexes at the disposal of those wanting to use them. In such cases, the
user will carry out the labeling reaction with the radionuclide in the
clinical or
laboratory setting. To that end, the various reaction components may be
conveniently provided in kit form. Because the radiodiagnostic composition of
the present invention can be prepared in a sample manner, this process may be
readily carried out by experienced researches or clinicians. Such a kit will
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typically contain (i) a cyclam derivative of the general formula I, shown
above, to
which an inert biologically acceptable carrier medium and/or formulating
agent(s), e.g., reducing agent(s), and/or auxiliary substances) may optionally
be
added, and (ii) instructions for carrying out a complex-forming reaction using
the
components present in the kit. As mentioned above, for such complex-forming
reaction the desired radionuclide may be contacted with cyclam derivative in
the
form of a chelate, bound to a relatively weak chelator, such as a
glucoheptonate,
pyrophosphate, a polyphosphate, a phosphonate or polyphosphonate, an oxinate,
a carboxylate, a hydroxycarboxylate, an aminocarboxylate, an enolate or a
mixture thereof, such reaction being carried out under moderate conditions.
The kit embodiment of this invention may optionally comprise a
solution of the radionuclide metal. Because such solutions have a limited
shelf
life, however, they may be made available separately.
The components of the above-described kit may be supplied as a
solution, for example, in the form of a physiological saline solution, or in a
buffer
solution. If desired, the above-mentioned components may be stabilized in a
usual way with suitable stabilizers such as ascorbic acid, gentisic acid or
salts of
these acids.
Radiodiagnostic compositions comprising a radionuclide metal-
containing complex, as described above, may be conveniently formulated for
administration with a biologically acceptable carrier medium. According to a
preferred embodiment, the carrier medium is sterile, pyrogen-free phosphate
buffered sale (PBS). The radiodiagnostic agents of the invention should be
delivered to tissue in trace amounts, on the order of IO-'ZM. Nuclear medicine
markers are usually administered according to their biodistribution, with
isotope
dose up to 40 mCi in the case of Tc-99m, for example. In all cases, any
substance used in formulating a radiodiagnostic composition in accordance with
this invention should be virus-free, pharmaceutically pure and substantially
non-
toxic in the amount used. If necessary or desirable, the risk of contaminating
microorganisms may be prevented by various antibacterial and antifungal
agents,
such as parabens, chlorobutanol, phenol, surbic acid, thimerosal or the like.
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may also be beneficial to include in the formulation isotonic agents such as
glucose.
As used herein, the term "biologically acceptable carrier medium"
is intended to include any and all solvents, dispersion media and the like
which
may be appropriate for the desired route of administration of the composition.
The use of such media for hypoxic markers is known in the art. Except insofar
as
any conventional carrier medium is incompatible with the particular
radiodiagnostic agents described herein, its use in the compositions of the
invention is contemplated.
The radiodiagnostic agents of the invention are administered
parenterally, intravenous administration being the preferred route.
After administration of the radiodiagnostic composition to a test
subject, an image is obtained to detect accumulated radioactivity and thus to
determine the location thereof in the body tissue of the test subject.
The radionuclide metal-containing complexes of the invention
may be utilized in a number of different clinical and research imaging
techniques,
including without limitation, SPECT and PET.
PET has been gaining increasing acceptance in the field of
oncology. One of the principal advantages of positron imaging currently is its
high detection efficiency and higher resolution, as compared to prior gamma-
detectors. This technique is particularly well suited to the detection of
tumor
oxygen levels, providing results that are consistent with those obtained
through
invasive polarographic electrode measurements.
Cu-64 is the preferred radionuclide for PET applications. It can be
obtained from the Mallinckrodt Institute of Radiology, or may be prepared as
described in U.S. Patent 6,001,825. PET imaging systems are available from
commercial sources. See also, U.S. Patent 5,451,789 to Wong et al. ("High
Performance Positron Camera").
The following examples are provided to describe the invention in
futher detail. These examples, which set forth the best mode presently
contemplated for carrying out the invention, are intended to illustrate and to
not
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limit the invention. Examples 1-4 describe the synthesis and purification of
representative cyclam derivatives of the invention, as well as the preparation
of
complexes of such derivatives with suitable y-emitting radionuclides. In the
examples, all temperatures are given in degrees Centigrade, unless otherwise
indicated.
EXAMPLE 1
Preparation of mono-[2-hydroxy-3-(2-nitroimidazol-1-yl)
propyl]-substituted cyclam
1,4,8,11-Tetraazacyclotetradecane (98°Io), 1.88 g (9.4 mmol) was
dissolved in 12 ml of methanol. A solution of 1-(2,3-epoxypropyl)-2-
nitroimidazole, 366.8 mg (2.17 mmol) in 3.0 ml of methanol, was added
dropwise to the cyclam solution with stirring. After 20 minutes at 25°,
a trace of
white solid appeared. The flask was wrapped in aluminum foil and allowed to
stand five days.
Thin-layer chromatography (TLC) on an E. Merck silica gel 60F
plate, developed in chloroform 3: abs. ethanol 6: conc. NH40H 1, showed no
evidence of the starting epoxide. A 5.00 ml aliquot of the reaction mixture
was
evaporated on a film evaporator and the residue dried over CaCl2 in vacuo. The
solid was refluxed with 10 ml of methylene chloride for 35 minutes and the
insoluble material separated by filtration. The clear yellow solution was
evaporated to give 0.4393 g of residue that was dissolved in 1.0 ml of abs
ethanol
and applied to a 30 x 153 mm column of E. Merck silica gel 60 (230-400 mesh).
The column was developed with a mixture of chloroform 400 ml; abs. ethanol
500 ml; and conc. NH40H 100 ml; 20 ml fractions collected.
Fractions 7-14 gave a clear yellow oil that was dissolved in 4.0 ml
of 1 N HCI. The flask was placed in an evacuated desiccator over CaClz. After
4
days, some small white flocs were removed from the yellow solution by
filtration
through sintered glass and the filtrate concentrated in a stream of NZ at
60°. The
flask was placed in a beaker with 25 ml of abs. ethanol; the whole placed in a
dessicator. After 7 days, the product consisted of a conical mass of pale
yellow
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crystal at the bottom of the flask and large clusters of fine white crystals
over the
surface of the liquid and on the upper walls of the flask. After removing the
supernatant and drying overnight over CaCl2 in vacuo, the types of crystals
were
separated mechanically. The yellow crystals weighed 190.1 mg. A mixture of
white and yellow crystals that could not be readily separated weighed 86.1 mg.
This material was dissolved in 300 ~,l of 1 N HCI. The flask was placed in a
beaker containing 25 ml of abs. ethanol in a dessicator. After 2 days, a large
portion of the white crystals was removed and the remaining crystals combined
with the 190 mg fraction. The combined products were dissolved in 2 ml of
water, the solution rendered basic with 3.0 ml of 1 N NaOH and extracted with
5
x 2 ml of methylene chloride. The solvent was evaporated from the combined
extracts and the residue dried to give 184.6 mg of partially crystalline
yellow
material. This product was dissolved in 300 ~.L of abs. ethanol and applied to
a
x 153 mm column of E. Merck silica gel 60 (230-400 mesh). The column was
15 developed with: chloroform 175 ml; abs. ethanol 275 ml; conc. NH40H 50 ml.
Fractions (10 ml) 7-19 were collected, concentrated and the oily yellow
residue
dissolved in methanol. This solution was filtered through a 0.45 ~. PTFE
syringe
filter and the methanol evaporated. The residue was dried over CaCh in vacuo.
This product weighed 114 mg. TLC showed two slow moving components. This
20 material was rechromatographed in the same flash chromatography system.
Fractious 8-11 gave 60.5 mg of a yellow syrup from which a few needle-like
crystals separated. This product was dissolved in 820 ~.L of 1 N HCI. The
flask
was placed in a beaker of abs. ethanol in a dessicator at atmospheric pressure
for
4 days. At this time, there was a small area of crystaline material in the
neck.
There were numerous small globules of clear yellow liquid on the walls. The
slightly cloudy mother liquor was transferred to another flask that was placed
in a
beaker of alcohol for 4 days. Cream colored crystals were deposited. After
drying over CaCl2 followed by P205 in vacuo, this product, the
tetrahydrochloride
of 1-[2-hydroxy-3-(2-nitroimidazol-1-yl)propyl]-,1,4,8,11-
tetraazacyclotetradecane weighed 44.1 mg.
Electrospray mass spectrometry gave (M+1) = 370.2; calculated
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value 370.
EXAMPLE 2
Di-[2-hydroxy-3-(2-nitroimadazol-1-yl)-
propyl]-substituted cyclam
Fractions 3 and 4 from the first column chromatography in the
preparation of the mono-substituted compound (Example 1) gave 16.6 mg of
material that was dissolved in 200 ~L of 1 N HCI. The flask was placed in a
beaker with abs. ethanol and kept in a dessicator at atmospheric pressure for
21
days. The yield of small bright yellow crystals was 12.5 mg.
A small portion was converted to the base, extracted into
chloroform and subjected to TLC in the system described in Example 1. A major
component of Rf 0.36 was by far the largest and most intense. Smaller spots
were seen at Rf 0.44, 0.54 and 0.68.
In another experiment, a very small amount of a bright yellow
crystalline salt was obtained that on electrospray mass spectrometry gave
(M+1) = 539.4. Calculated value 539.
EXAMPLE 3
Tetra-[2-hydroxy-3-(2-nitroimidazol-1-yl)-
propyl]-substituted cyclam
1-(2,3-Epoxypropyl)-2-nitroimidazole, 152.3 mg (0.900 mmol)
was dissolved in 500 ~,L of acetonitrile. 1,4,8,11-Tetraazacyclotetradecane,
41.0
mg (0.205 mmol) was added, followed by 100 ~.L of acetonitrile and 10 ~.L of
water. The cyclam did not dissolve. Methanol, 300 ~,L, was added and the
mixture stirred at 32°. All but a trace of the cyclam dissolved. After
standing 2
days, the thick slurry, containing a light yellow, very finely divided solid,
was
heated to 56-61° with stirring under reflux for 2-1/4 hours. Methanol,
500 ~.L,
was added after the first hour.
After standing 3 weeks, the solvent was removed in a stream of NZ
while heating in a bath at 55°. The residue was broken up under 3.0 ml
of
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tetrahydrofuran. Di-tert-Butyl dicarbonate (99%) 301.5 mg, dissolved in 1.0 ml
of tetrahydrofuran was added and the mixture stirred at room temperature for 4
days. The finely divided pale yellow solid was collected and dried overnight.
This solid weighed 92.9 mg. It was stirred with 1.5 ml of 1.0 N HCI. The
material that did not dissolve was stirred with 1.5 ml of cold trifluoroacetic
acid.
On warming to 250, a clear solution resulted. After filtration to remove a few
white crystals, the bulk of the trifluoroacetic acid was evaporated in a
stream of
NZ and the residue dried over NaOH pellets in vacuo. The clear light yellow
glass
was dissolved in 1.00 ml of water and passed over a canon exchange column (9
mm dia x 11 mm long) containing 3.0 ml of IR-120 (plus) resin (capacity 1.9
meq/ml). Water, 4.0 mL was used as chaser. The effluent turned cloudy and a
fine white precipitate separated. The solution was stirred while 6.0 N HCl was
added dropwise until it was clear. After standing overnight, small pale yellow
crystals of the tetrahydrochloride salt had deposited on the walls of the
flask.
These were collected and dried over CaClz in a vacuum dessicator.
Electrospray mass spectrometry gave (M+1)= 877. Calculated
value 877.
EXAMPLE 4
Radiolabelling of Cyclam Derivatives
a) N-[2-hydroxy)-3-(2-nitroimidazole-1-yl)propyl]-1,4,8,11-
tetraazacyclotetradecanato-technetium V.
Into an 4 ml screw-cap vial were placed: 3.0 mg (5.8157 x 10-6
moles) cyclam derivative prepared in Example 1, above; 3.0 ml deareated
distilled water; 0.332 ml deareated 0.1 N sodium hydroxide solution; 0.2 ml
(13.55 mCl) NH4Tc04 generator eluate; and 20 ~.l saturated stannous tartarate
solution prepared fresh using deareated distilled water. The mixture was
incubated at room temperature for 0.5 hr.
The solution was passed through a 0.8 X 4 cm BioRad Poly-Prep
Chromatography Column (cat #731-1550) containing 0.31 g BioRad Ag 1-X8 ion
exchange resin, chloride form, 200-400 mesh (cat # 140-1451). The column was
CA 02361115 2001-07-26
WO 00/43004 PCT/US00/01754
rinsed with 1.0 ml deareated distilled water. At this point, the radiolabeled
ligand
in the eluant was ready as a radiodiagnostic agent.
The relative levels of Tc-99m delivered using the complex of this
example in animal tissues, as shown in Figure 1, did not change significantly
over
the first ten hours after administration. [Add explanation of symbols in Fig.
1].
b) N, N', N", N"'-Tetra[(2-hydroxy)-3-(2-nitroimidazole-1-
yl) propyl]-1,4,8,11-tetraazacyclotetradecanato-copper II.
To the 10 ml serum vials (each of which contained 5.1 m Cu
6'CuCl2 in 0.240 ml 0.1 N HCl) were added: 3.00 ml distilled water; 3.8 mg
(3.7155 x 10-6 moles) of the cyclam derivative prepared in Example 3, above;
and
0.240 ml 0.1 N sodium hydroxide solution. The pH was determined and adjusted
to 7.52 with additional 1N or 0.1 N sodium hydroxide solution as required.
The solution was allowed to incubate at room temperature for
one- half hour.
The solution was filtered through a 0.22 ~, filter, which renders it
ready for use as a radiodiagnostic agent.
Figure 2 shows two pairs of Fischer X Copenhagen rats in which
83327-AT and 83327-H tumors of approximately equal volume were growing
and to which Ceretec0, radiolabeled with Tc-99m, and the complex of this
example were administered i.v.. Ceretec~ is a commercial tumor perfusion
marker. Planar images using a collimated gamma camera were acquired six to
seven hours after marker administration. After the imaging procedure, animals
were sacrificed and the specific activity of hypoxic marker in blood, muscle
and
tumor tissue was measured. The tumor specific activity of these markers is
presented in Example 5, below.
c.) A Cu-64-containing N, N', N", N"'-tetra(2-hydroxy)-3-(2-
nitroimidazole-1-yl)propyl]-substituted cyclam was prepared following the same
general procedure described in Example 4b, above.
The absolute binding rate of this radiodiagnostic complex to DU-
145 prostate cancer cells (ATCC) in vitro, as a function of oxygen
concentration
is shown in Figure 3. In this experiment, background binding to aerobic cells
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WO 00/43004 PCT/US00/01754
(21 % OZ) was observed, which was probably due to ionic trapping of isotope on
the filters.
Figure 4 shows the uptake of the radiodiagnostic complex of this
example in a pair of Fischer X Copenhagen rats in which 83327-AT (Fig. 4A)
and 83327-H (Fig. 4B) tumors of approximately equal volume were growing.
The tumors, of 8-10 gm. weight, are indicated by a black circle in each
figure.
Tumor specific activity was determined to be about 2.2.
EXAMPLE 5
The following table sets forth data concerning hypoxic marking
properties of radiolabelled cyclam derivatives of Examples 1 and 3, above.
Ex. HSF''Kmh TB' T/M' % 1Z7/g % )D/g ATBe
No. AT'~SE H''SE
4a ~ 0.3%7-8 30-40 ND ND ND
6
4b ND ND ND ND 0.2630.0350.0890.0072.96
TC-99m
labelledND ND ND ND 0.820.0020.1390.0160.59
Ceretec~
HSF - hypoxia-specific factor refers to the amount of marker bound to (or
retained in) hypoxic cells relative to aerobic cells in vitro. See Chapman
et al., Radiotherapy and Oncology, 46: 229-37 (1998)
Km-concentration of 02 at which binding rate of marker is at 50% of
maximum (normally defined in vitro)
TB-maximal tumor/blood ratio of tissue-specific radioactivity
T/M-maximal tumor/muscle ratio of tissue-specific radioactivity
d %>D/g AT - percentage of injected dose of marker/g in anaplastic tumor
tissue assessed 6 hours following injection. Specific activities were
corrected to a standard rat weight of 400 g. Anaplastic tumors formed
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following injection of cells from the Dunning rat prostate carcinoma,
83 327-AT.
%ID/g H - percentage of injected dose of marker/g in well-differentiated
tumor tissue assessed 6 hours following injection. Specific activities were
corrected to a standard rat weight of 400 g. Well-differentiated tumors
formed following injection of cells from the Dunning rat prostate
carcinoma, 83327-H.
AT/H - ratio of specific activities in anaplastic (hypoxic) versus well-
differentiated (non-hypoxic) tumor tissues.
ND = Not determined
As the data show in the above table, the radiodiagnostic agent of
Example 4b, above, was recovered from the hypoxic tumors at a level of about
three times higher than the non-hypoxic, well oxygenated tumors. It is also
noteworthy that up to six-fold higher levels were observed in individual pairs
of
animals. By comparison, Tc-99m labelled Ceretec~ distributed to the well
oxygenated tumors at a level of about 1.7 times higher than hypoxia tumors. In
this connection, see Moore et al., Brit. J. Cancer, 65: 491-97 (1992).
The following example describes PET imaging, using Cu-64-
labelled cyclam derivatives of the present invention to detect and quantify
tumor
hypoxic microenvironments in prostate carcinomas of laboratory animal test
subjects.
EXAMPLE 6
A micro-PET imaging system using luetium oxyorthosilicate
detector elements (Concord Microsystems, Knoxville, TN) with an 8-cm field of
view, a spatial resolution of 2 mm at the center of the field of view and a
volumetric resolution of about 8 ~,1 may be used to image and quantitate rat
tumor
metabolism and viable hypoxic cells. The animals, bearing implanted hypoxic
and non-hypoxic prostate carcinomas, are anesthetized with Avertin (Aldridge,
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WO 00/43004 PCT/US00/01754
Milwaukee, WI) before the imaging procedure. The long access of the rats is
parallel to the long access of the scanner. On the day of the procedure, each
rat is
injected i.v. with 2 mCi of labelled fluorodeoxyglucose ('BFDG) (1.0-1.5 ml)
followed by a line flush with 500 ~.l of physiological saline. Three
sequential
whole body studies are performed on each rat using 5 minute acquisitions for
each bed position, between 3-8, depending on the rat length. The following
day,
each rat is reanesthesized and repositioned so as to acquire PET images from
the
tumor region. The rat is then injected with 2 mCi of optimal Cu-64-labelled
cyclam derivative. Sequential scans are obtained from each rat using 5 minute
aquisitions at 6 different times up to 8 hours after marker administration.
Attenuation correction is determined with a 1 mCi Ge-68 ring transmission
source and rat images are reconstructed using the 3-dimensional filtered back
projection algorithm with the appropriate ramp filtered cutoff. Time activity
curves are established for each organ in the field, including tumor, and the
relative distribution of FDG and labelled cyclam derivative are determined.
The
standardized unit value (SLTV) of metabolic activity and HF are calculated for
each tumor at various times and statistical analysis of data are performed to
assess
differences in metabolism and hypoxic fraction of the two tumor types.
Although the present invention has been described and
exemplified in terms of certain preferred embodiments, other embodiments will
be apparent to those skilled in the art. The invention is, therefore, not
limited to
the particular embodiments described and exemplified, but is capable of
modification or variation without departing from the spirit of the invention,
the
full scope of which is delineated by the appended claims.
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