U.S. patent application number 14/675364 was filed with the patent office on 2016-10-06 for labeled evans blue dye derivative for in vivo serum albumin labeling.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Xiaoyuan Chen, Lixin Lang, Gang Niu.
Application Number | 20160287730 14/675364 |
Document ID | / |
Family ID | 57015517 |
Filed Date | 2016-10-06 |
United States Patent
Application |
20160287730 |
Kind Code |
A1 |
Chen; Xiaoyuan ; et
al. |
October 6, 2016 |
LABELED EVANS BLUE DYE DERIVATIVE FOR IN VIVO SERUM ALBUMIN
LABELING
Abstract
Disclosed is a compound of formula (I): ##STR00001## wherein L,
R.sup.1-R.sup.5, A, B, M, and n are as defined in the
specification, as well as a method of preparing the compound. Also
disclosed are a method of blood-pool imaging in a mammal and a
method of imaging a lymph node in a mammal, comprising use of the
compound.
Inventors: |
Chen; Xiaoyuan; (Potomac,
MD) ; Lang; Lixin; (North Potomac, MD) ; Niu;
Gang; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
57015517 |
Appl. No.: |
14/675364 |
Filed: |
March 31, 2015 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 51/0482 20130101;
C07B 59/004 20130101; C09B 29/30 20130101; C09B 45/00 20130101 |
International
Class: |
A61K 51/04 20060101
A61K051/04; C09B 45/00 20060101 C09B045/00 |
Claims
1. A compound of formula (I): ##STR00024## wherein L is a linker
group selected from aryl, biaryl, heteroaryl, and biheteroaryl,
wherein the aryl, biaryl, heteroaryl, or biheteroaryl is optionally
substituted with one or more groups selected from alkyl, halo,
hydroxy, and alkyloxy, wherein A is selected from a bond, C.dbd.O,
and C.sub.1-C.sub.6 alkyl, wherein B is a chelating group selected
from 1, 4, 7-triazacyclononane-N,N',N''-triacetic acid, 1, 4,
7,10-tetrazacyclononane-N,N',N''-triacetic acid,
triethylenetetramine, diethylenetetramine pentaacetic acid, and
hydrazinonicotinamide, wherein R.sup.1-R.sup.5 are independently
selected from hydrogen, OH, NH.sub.2, and SO.sub.3H, wherein n is 0
or 1, and wherein M is selected from .sup.18F-AlF, .sup.60Cu,
.sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.68Ga, .sup.86Y,
.sup.89Zr, .sup.111In, .sup.99mTc, .sup.186Re, .sup.188Re,
Gd.sup.3+, and Mn.sup.2+.
2. The compound of claim 1, wherein L is biphenyl, optionally
substituted with one or more groups selected from alkyl, halo,
hydroxy, and alkyloxy.
3. The compound of claim 2, wherein A is a bond.
4. The compound of claim 3, wherein B is 1, 4,
7-triazacyclononane-N,N',N''-triacetic acid.
5. The compound of claim 4, wherein n is 0 and the compound is:
##STR00025##
6. The compound of claim 1, wherein n is 1 and M is .sup.18F-AlF,
.sup.64Cu, or .sup.68Ga.
7. An imaging composition comprising a compound of formula (I) and
a pharmaceutically acceptable carrier.
8. The imaging composition of claim 7, wherein the pharmaceutical
composition further comprises Evans blue dye.
9. A method of blood-pool imaging in a mammal, comprising
administering to the mammal a compound of claim 1, wherein n is 1,
M is selected from .sup.18F-AlF, .sup.60Cu, .sup.61Cu, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.68Ga, .sup.86Y, .sup.89Zr, .sup.111In,
.sup.99mTc, .sup.186Re, .sup.188Re, Gd.sup.3+, and Mn.sup.2+, and
PET imaging the mammal.
10. The method of claim 9, which involves imaging a heart of the
mammal.
11. The method of claim 10, wherein the imaging is performed at
least twice.
12. The method of claim 11, which involves measuring a ventricular
ejection volume.
13. The method of claim 9, which involves imaging a vascularized
muscular tissue.
14. The method of claim 13, which involves detecting a vascular
leakage.
15. The method of claim 9, which involves imaging a hepatic
hemangioma.
16. A method of imaging a lymph node in a mammal, comprising
administering to the mammal a compound of claim 1, wherein n is 1,
M is selected from .sup.18F-AlF .sup.60Cu, .sup.61Cu, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.68Ga, .sup.86Y, .sup.89Zr, .sup.111In,
.sup.99mTc, .sup.186Re, .sup.188Re, Gd.sup.3+, and Mn.sup.2+, and
imaging the mammal.
17. The method of claim 16, wherein the imaging is PET imaging.
18. The method of claim 17, wherein the lymph node is a sentinel
lymph node.
19. The method of claim 16, comprising coadministering to the
mammal Evans blue dye.
20. The method of claim 19, wherein the imaging is performed
visually.
21. A method of preparing a compound of formula (IV): ##STR00026##
wherein L is a linker group selected from aryl, biaryl, heteroaryl,
and biheteroaryl, wherein the aryl, biaryl, heteroaryl, or
biheteroaryl is optionally substituted with one or more groups
selected from alkyl, halo, hydroxy, and alkyloxy, wherein M is
selected from .sup.18F-AlF, .sup.64Cu, or .sup.68Ga, and wherein n
is 0 or 1, comprising the steps of: (i) reacting a his amino
compound of the formula: H.sub.2N-L-NH.sub.2 wherein L is aryl,
biaryl, heteroaryl, and biheteroaryl, wherein the aryl, biaryl,
heteroaryl, or biheteroaryl is optionally substituted with one or
more groups selected from alkyl, halo, hydroxy, and alkyloxy, with
1, 4, 7-triazacyclononane-N,N',N''-triacetic acid to form a
compound of formula (II): ##STR00027## (iii) reacting the compound
of formula (II) with a diazotization reagent to form a compound of
formula (III): ##STR00028## and (iv) reacting the compound of
formula (III) with 4-amino-5-hydroxynaphthalene-1,3-disulfonic acid
to form the compound of formula (IV).
22. The method of claim 21, further comprising a step of reacting
the compound of formula (IV) with MX.sub.2, wherein M is selected
from .sup.18F-AlF, .sup.64Cu, or .sup.68Ga and wherein X is a
halogen, to provide a compound of formula (V): ##STR00029##
23. The method of claim 22, wherein the compound of formula (V) is:
##STR00030##
Description
BACKGROUND OF THE INVENTION
[0001] As the most abundant plasma protein, serum albumin has
emerged as a versatile carrier for therapeutic agents, primarily
for treating diabetes, cancer, rheumatoid arthritis and infectious
diseases (Elsadek B et al., Kratz F. J Control Release 2012;
157(1): 4-28). Serum albumin was also used directly as an imaging
probe after labeling with fluorescent dyes for optical imaging
(Klohs J. et al., J Neurosci Methods. 2009; 180(1): 126-132),
radioisotopes for scintillation scanning or positron emission
tomography (PET) (McAfee J G et al., J Nucl Med 1964; 5:936-946;
Hoffend J. et al., Nucl Med Biol 2005; 32(3): 287-292), or
Gd.sup.3+ for magnetic resonance imaging (MRI) (Lauffer R B et al.,
Radiology. 1998; 207(2): 529-538). The major applications of
labeled serum albumin mentioned above include blood pool imaging
and angiography.
[0002] In clinical nuclear medicine, kit preparations for indirect
and direct .sup.99mTc-radiolabeling of red blood cells (RBCs) are
still the dominant methods for blood pool imaging. Compared with
single-photon emission computed tomography (SPECT), PET is more
sensitive and has higher spatial resolution with clinical
instruments. However, to date, only very few blood-pool tracers
have been introduced for PET. For example, carbon monoxide (CO)
containing either .sup.11C or .sup.15O has been used to label RBCs
for PET. However, due to their short half-lives (20.4 min for
.sup.11C and 2.05 min for .sup.15O), these tracers can only be used
in centers with an in-house cyclotron. Moreover, the gaseous form
of CO and the need for administration by inhalation necessitates
sophisticated equipment for either human or animal studies.
[0003] Commercial availability of the species specific isoforms of
albumin including human serum albumin (HSA) makes blood drawing
unnecessary. In fact, .sup.131I-labeled HSA is the only FDA
approved radiologic agent for measuring blood volume. For imaging
purpose, albumin has been labeled with various radioisotopes for
PET imaging including .sup.68Ga (Hoffend J. et al., Nucl Med Biol.
2005; 32(3): 287-292), .sup.62Cu (Okazawa H. et al., J Nucl Med
1996; 37(7): 1080-1085), and .sup.64Cu (Anderson C J et al., Nucl
Med Biol. 1993; 20(4): 461-467). Compared with these radiometals,
.sup.18F has the advantages of being a pure positron emitter and
having an ideal half-life. It is the dominant radioisotope used for
PET imaging for both clinical applications and preclinical
investigations.
[0004] As a protein, albumin can be labeled with .sup.18F through
reaction of N-succinimidyl 4-.sup.18F-fluorobenzoate (SFB) with an
amine group or N-[2-(4-.sup.18F-fluorobenzamido)ethyl]maleimide
(FBEM) on the thiol group. In one study, Wangler et al. prepared
4-(di-tert-butyl-.sup.18F-fluorosilyl)benzenethiol
(.sup.18F-SiFASH) and coupled it directly to rat serum albumin
(RSA) (Wangler B. et al., Bioconjug Chem 2009; 20(2): 317-321).
However, high liver uptake was observed on the .sup.18F-SiFA-RSA
blood pool scan, indicating that the albumin structure may have
been disrupted to some extent during labeling. One alternative is
in vivo labeling of endogenous albumin with a pre-labeled albumin
binder. Ideally, the binder will not affect the in vivo behavior of
the serum albumin such as circulation, extravascularization, and
turn-over; thus the imaging results will reflect the distribution
and metabolism of serum albumin accurately. Currently available
albumin binders include small molecules, peptides that possess an
albumin binding domain, and antibodies.
[0005] Identification of liver lesions is of critical importance
due to the increasing incidence of primary hepatic malignancies
worldwide and an increase in detection of benign liver lesions by
the widespread use of abdomen cross-sectional imaging modalities.
Although many typical lesions can be detected by traditional
imaging tests such as ultrasound, CT, and MRI, there remains a
challenge to diagnose atypical lesions. For example, hypervascular
neuroendocrine tumors often share the same appearance as
hemangiomas on MRI. Some atypical hepatic cysts may also show
overlapping features with hepatic metastasis from ovarian
malignancies.
[0006] The lymphatic system plays a key role in maintaining tissue
interstitial pressure by collecting protein-rich fluid that is
extracted from capillaries. The lymphatic system is also a critical
component of the immune system. Many types of malignant tumors such
as breast cancer, melanoma, and prostate cancer are prone to
metastasize to regional lymph nodes (LNs), possibly through tumor
associated lymphatic channels. The status of these sentinel LNs
(SLNs) not only provides a marker for tumor staging but also serves
as an indicator of prognosis. Consequently, detection and mapping
of SLNs is a key step in therapeutic decision-making (Veronesi U,
et al., Lancet 1997, 349(9069): 1864-1867).
[0007] One common method used in the clinic is a two-step procedure
which consists of local administration of radionuclide-labeled
colloids, mostly with technetium-99m, several hours before the
injection of a vital dye such as Patent blue (isosulfan blue). SLNs
can be visualized either by gamma scintigraphy or SPECT (single
photon emission computed tomography). The SLNs during surgery can
be located with a hand-held gamma ray counter and visual contrast
of the blue dye. However, this method requires separate
administration of two agents because of different rates of local
migration of the colloidal particles and blue dye molecules. Due to
the relatively low sensitivity and poor spatial resolution of
scintigraphy and SPECT, it is highly desirable to develop new
imaging probes for other imaging modalities. The objective is to
improve the detection of SLNs either for noninvasive visualization
or intrasurgical guidance.
[0008] Recently, imaging guided surgery, especially with
fluorescent probes, has been intensively studied due to its low
cost, simplicity, and adaptability. The limited tissue penetration
of light is less critical because of open field of view during
surgery. For example, NIR fluorescence dyes, such as indocyanine
green (ICG), have been investigated for sentinel node navigation
during surgery either alone or in combination with nanoformulations
(Hirano A, et al., Ann Surg Oncol 2012, 19(13):4112-4116; Koo J, et
al., Phys Med Biol 2012, 57(23):7853-7862). Owing to the
nanometer-scale size, stability and strong fluorescence, various
nanoparticles and nanoformulations have been applied for SLN
imaging and showed promising results in preclinical models.
However, most of these probes are composed of heavy metals making
their clinical translation difficult due to the acute and chronic
toxicity. In addition, scattering and tissue attenuation cause poor
results for pre-surgical evaluation of SLNs using optical
imaging.
[0009] Thus, there remains a need in the art for improved methods
for imaging of blood pools and the lymphatic system.
BRIEF SUMMARY OF THE INVENTION
[0010] The invention provides a compound of formula (I):
##STR00002##
[0011] wherein L is a linker group selected from aryl, biaryl,
heteroaryl, and biheteroaryl, wherein the aryl, biaryl, heteroaryl,
or biheteroaryl is optionally substituted with one or more groups
selected from alkyl, halo, hydroxy, and alkyloxy,
[0012] wherein A is selected from a bond, C.dbd.O, and
C.sub.1-C.sub.6 alkyl,
[0013] wherein B is a chelating group selected from 1, 4,
7-triazacyclononane-N,N',N''-triacetic acid, 1, 4,
7,10-tetrazacyclononane-N,N',N''-triacetic acid,
triethylenetetramine, diethylenetetramine pentaacetic acid, and
hydrazinonicotinamide,
[0014] wherein R.sup.1-R.sup.5 are independently selected from
hydrogen, OH, NH.sub.2, and SO.sub.3H,
[0015] wherein n is 0 or 1, and
[0016] wherein M is selected from .sup.18F-AlF, 60Cu, .sup.61Cu,
.sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.68Ga, .sup.86Y, .sup.89Zr,
.sup.111In .sup.99mTc, .sup.186Re, .sup.188Re, Gd.sup.3+, and
Mn.sup.2+.
[0017] The invention also provides a method of blood-pool imaging
in a mammal, comprising administering to the mammal a compound of
formula (I):
##STR00003##
[0018] wherein L is a linker group selected from aryl, biaryl,
heteroaryl, and biheteroaryl, wherein the aryl, biaryl, heteroaryl,
or biheteroaryl is optionally substituted with one or more groups
selected from alkyl, halo, hydroxy, and alkyloxy,
[0019] wherein A is selected from a bond, C.dbd.O, and
C.sub.1-C.sub.6 alkyl,
[0020] wherein B is a chelating group selected from 1, 4,
7-triazacyclononane-N,N',N''-triacetic acid, 1, 4,
7,10-tetrazacyclononane-N,N',N''-triacetic acid,
triethylenetetramine, diethylenetetramine pentaacetic acid, and
hydrazinonicotinamide,
[0021] wherein R.sup.1-R.sup.5 are independently selected from
hydrogen, OH, NH.sub.2, and SO.sub.3H,
[0022] wherein n is 0 or 1, and
[0023] wherein M is selected from .sup.18F-AlF, .sup.60Cu,
.sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.68Ga, .sup.86Y,
.sup.89Zr, .sup.111In, .sup.99mTc, .sup.186Re, .sup.188Re,
Gd.sup.-3, and Mn.sup.2+, and PET imaging the mammal.
[0024] The invention further provides a method of imaging a lymph
node in a mammal, comprising administering to the mammal a compound
of formula (I):
##STR00004##
[0025] wherein L is a linker group selected from aryl, biaryl,
heteroaryl, and biheteroaryl, wherein the aryl, biaryl, heteroaryl,
or biheteroaryl is optionally substituted with one or more groups
selected from alkyl, halo, hydroxy, and alkyloxy,
[0026] wherein A is selected from a bond, C.dbd.O, and
C.sub.1-C.sub.6 alkyl,
[0027] wherein B is a chelating group selected from 1, 4,
7-triazacyclononane-N,N',N''-triacetic acid, 1, 4,
7,10-tetrazacyclononane-N,N',N''-triacetic acid,
triethylenetetramine, diethylenetetramine pentaacetic acid, and
hydrazinonicotinamide,
[0028] wherein R.sup.1-R.sup.5 are independently selected from
hydrogen, OH, NH.sub.2, and SO.sub.3H,
[0029] wherein n is 0 or 1, and
[0030] wherein M is selected from .sup.18F-AlF, .sup.60Cu,
.sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.68Ga, .sup.86Y,
.sup.89Zr, .sup.111In, .sup.99mTc, .sup.186Re, .sup.188Re,
Gd.sup.3+, and Mn.sup.2+, and PET imaging the mammal.
[0031] The invention additionally provides a method of preparing a
compound of formula (IV):
##STR00005##
wherein L is a linker group selected from aryl, biaryl, heteroaryl,
and biheteroaryl, wherein the aryl, biaryl, heteroaryl, or
biheteroaryl is optionally substituted with one or more groups
selected from alkyl, halo, hydroxy, and alkyloxy, wherein M is
selected from .sup.18F-AlF, .sup.64Cu, or .sup.68Ga, and wherein n
is 0 or 1, comprising the steps of:
[0032] (i) reacting a bis amino compound of the formula:
H.sub.2N-L-NH.sub.2 wherein L is aryl, biaryl, heteroaryl, and
biheteroaryl, wherein the aryl, biaryl, heteroaryl, or biheteroaryl
is optionally substituted with one or more groups selected from
alkyl, halo, hydroxy, and alkyloxy, with 1, 4,
7-triazacyclononane-N,N',N''-triacetic acid to form a compound of
formula (II):
##STR00006##
[0033] (iii) reacting the compound of formula (II) with a
diazotization reagent to form a compound of formula (III):
##STR00007##
and
[0034] (iv) reacting the compound of formula (III) with
4-amino-5-hydroxynaphthalene-1,3-disulfonic acid to form the
compound of formula (IV).
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0035] FIGS. 1A and 1B show the absorbance and fluorescence
emission of Evans blue with and without albumin. Evans blue showed
a strong absorbance peak at 620 nm with or without albumin. Evans
blue is almost not fluorescent without albumin. However, with
albumin, Evans blue showed a strong fluorescence emission peak at
680 nm.
[0036] FIG. 2A shows representative reconstructed coronal PET
images of inflamed popliteal (upper) and sciatic (lower) LNs in the
turpentine oil-induced hind limb inflammation model. LNs were
pointed out by arrows.
[0037] FIG. 2B show the T.sub.2-weighted NRI of an enlarged
inflamed popliteal LN, as indicated by the arrow.
[0038] FIG. 2C shows the overlay of PET with a 2D X-ray image. The
LN is indicated by an arrow and the injection sites by
arrowheads.
[0039] FIG. 2D shows quantitative analysis based on the PET images
of FIGS. 2A-2C. There is significantly higher total tracer uptake
in inflamed popliteal LNs than that of contralateral normal LNs at
0.5, 1, 2, and 3 h after tracer injection (*P<0.05)
[0040] FIG. 2E shows quantitative analysis of tracer uptake in
sciatic LNs. No statistical significance was found between LNs in
the left and right side.
[0041] FIGS. 3A-3C show representative .sup.18F-AlF-NEB PET images
of an axillary LN in the orthotropic breast cancer model. FIG. 3A
is a transaxial image, FIG. 3B is a sagittal image, and Figure C is
a coronal image. PET scans were performed at 30 min after tracer
injection. Arrows indicate tumor-draining axillary LNs and
arrowheads indicate primary tumors. A dotted line was added to
indicate animal contour.
[0042] FIGS. 3D and 3E show confirmation of tracer uptake of an
ipsilateral axillary LN after intratumoral injection of
.sup.18F-AlF-NEB.
[0043] FIG. 3F shows a coronal image illustrating a cervical LN.
Arrows indicate tumor-draining axillary LNs and arrowheads indicate
primary tumors.
[0044] FIG. 4 shows a series of maximum-intensity-projection PET
images in normal mice after intravenous injection of either
.sup.18F-AlF-NEB or .sup.18F-FB-MSA. Each mouse received around 3.7
MBq of radioactivity. Images were reconstructed from a 60-min
dynamic scan.
[0045] FIG. 5A shows time-activity curves of ROSs outlined over
muscle, heart, liver, and bladder regions of .sup.18F-AlF-NEB
images.
[0046] FIG. 5B shows time-activity curves of ROSs outlined over
muscle, heart, liver, and bladder regions of .sup.18F-FB-MSA
images.
[0047] FIG. 6 shows ECG-gated blood-pool imaging of control and MI
mice. Transaxial images were reconstructed to display 8 intervals
of 1 cardiac cycle.
[0048] FIG. 7A show left ventricular volume curve calculated from
PET.
[0049] FIG. 7B shows left ventricular ejection fraction calculated
from ECG-gated PET.
[0050] FIG. 8 shows hematoxylin and eosin staining of inflammatory
muscles at 2 h after local injection of turpentine oil. Left panel:
control. Right panel: inflammation.
[0051] FIG. 9 shows transaxial PET images of mice that received
turpentine oil infection. Ten-min static PET scans were obtained
after intravenous injection of 3.7 MBq of .sup.19F-AlF-NEB.
Apparent radioactivity accumulation was observed in inflamed
muscles at both 1 h (upper panel) and 2.5 h (lower panel) after
tracer injection, as indicated by arrows.
[0052] FIG. 10 shows time-activity curves over inflamed and
contralateral healthy muscles based on PET images from a 60-min
dynamic scan using .sup.18F-AlF-NEB.
[0053] FIG. 11 shows quantitative analysis of .sup.18F-AlF-NEB
uptake in both inflamed and contralateral healthy muscles at 1 and
2.5 h after tracer injection.
[0054] FIG. 12 shows a series of maximum-intensity-projection PET
images of UM-tumor-bearing mice after intravenous injection of
.sup.64Cu-NEB. Tumors are indicated by arrows.
[0055] FIG. 13 shows time-activity curves of ROIs over heart and
tumor regions.
[0056] FIG. 14A shows representative .sup.18F-AlF-NEB PET images of
axillary LNs in the orthotropic breast cancer model (left panel:
transaxial; middle panel: sagittal; right panel: coronal image).
Arrowheads indicate primary tumors.
[0057] FIG. 14B shows representative .sup.18F-AlF-NEB PET images of
a cervical LH in the orthotropic breast cancer model (left panel:
transaxial; middle panel: sagittal; right panel: coronal image).
Arrowheads indicate primary tumors.
[0058] FIG. 15A shows representative BLI imaging of a metastatic
popliteal LN (arrow) located near the primary tumor
(arrowhead).
[0059] FIG. 15B shows axial T.sub.2-weighted MRI of an enlarged
metastatic popliteal LN as indicated by the arrow.
[0060] FIG. 15C shows confirmation of the existence of metastatis
in the popliteal LN by imunofluorescence staining.
[0061] FIG. 16 shows that the average long-axis diameter of the
left LN measured by MRI is significantly larger than that of the
right one in the popliteal LN depicted in FIG. 15A.
[0062] FIG. 17A shows representative coronal PET images of
metastatic popliteal LNs (arrows) at different time points after
local injection of .sup.18F-AlF-NEB. Arrowheads indicate the
injection site.
[0063] FIG. 17B shows autoradiography of the popliteal LN,
confirming the metastasis (cold area in the LN).
[0064] FIG. 17C shows the quantitative analysis of the total tracer
uptake in tumor-draining LN (TLN) and right side normal LN (RLN).
The value was corrected by the weights of LNs (*P<0.05).
[0065] FIG. 18A depicts representative PET images which show high
tracer uptake in sciatic LN. Left panel: trasaxial; middle panel:
coronal; right panel: sagittal.
[0066] FIG. 18B depicts representative PET images which show high
tracer uptake in inguinal LN. Left panel: trasaxial; middle panel:
coronal; right panel: sagittal.
[0067] FIG. 18C shows the H&E stain of a healthy popliteal
LN.
[0068] FIG. 18D shows the H&E stain of a metastatic popliteal
LN. The dashed line delineates metastasis foci at the subscapular
sinus area.
[0069] FIGS. 18E and 18F show that H&E staining found
micrometastasis foci inside two of the tumor-draining LNs at 4
weeks after inoculation of Fluc.sup.+ 4T1 cells via hock
injection.
[0070] FIG. 19A shows LN mapping with Evans blue dye in a
turpentine oil-induced hind limb inflammation model. The lower two
arrows indicate popliteal LNs and the upper arrow shows the left
sciatic LN.
[0071] FIG. 19B shows a photograph of excised LNs. The upper two
are popliteal LNs, and the lower two are sciatic LNs. LNs on the
left side are harvested from the inflamed hind limb, whereas those
on the right side are from a normal limb.
[0072] FIG. 20 shows a quantitative analysis of LN size based on
its weight (*P<0.05).
[0073] FIG. 21 depicts the measurement of UV showing the difference
of Evans blue dye in different LNs (*P<0.05).
[0074] FIG. 22 shows longitudinal fluorescence imaging of the
lymphatic system after hock injection of .sup.18F-AlFNEB/EB. LNs
and lymphatic vessels can be clearly seen with the migration of the
tracer along with time.
[0075] FIG. 23A shows ex vivo optical imaging of LNs without
skin.
[0076] FIG. 23B shows a photograph of the same mice to show the
blue color (shown in black) of the LNs.
[0077] FIG. 24A shows coregistration of optical image (left panel)
and PET image (middle panel) with the overlay shown in the right
panel. Popliteal LNs are indicated by the arrow.
[0078] FIG. 24B shows coregistration of optical image (left panel)
and PET image (middle panel) with the overlay shown in the right
panel. Sciatic LNs are indicated by the arrow.
[0079] FIG. 25 shows a representative coronal maximum intensity
projection of PET images of 60 dynamic acquisitions. For dynamic
PET scans, four BALB/C mice was injected intravenously with 1.85
MBq (50 .mu.Ci) of .sup.68Ga-NEB under isoflurane anesthesia. A 60
min list mode acquisition was performed with an Inveon PET scanner.
Image reconstruction was done by the 2-dimensional ordered subsets
expectation maximum (OSEM) algorithm without attenuation or scatter
correction.
[0080] FIG. 26 shows decay corrected biodistribution of
.sup.68Ga-NEB in normal Balb/c mice (n=5/group).
[0081] FIG. 27A shows coronal maximum intensity projection (MIP) in
a male volunteer. The PET image was acquired at 30 min after
intravenous administration of 3.75 mCi of .sup.68Ga-NEB. Principal
organs and regions of uptake are labeled: superior sagittal sinus
(1), arch of aorta (2), cardiac ventricles (3), liver (4), spleen
(5), abdominal aorta (6), limb vessels (7), kidneys and (8),
bladder (9).
[0082] FIG. 27B shows corresponding axial PET and PET/CT fusion
images at key levels to reflect arch of aorta, cardiac ventricles,
liver and spleen, kidneys and bladder.
[0083] FIG. 28 shows multiple time point whole-body maximum
intensity projection PET images of a healthy 52-year-old woman
volunteer at 5, 10, 15, 30, 45, 60, 75, and 90 min after
intravenous administration of .sup.68Ga-NEB.
[0084] FIGS. 29A-29H show .sup.68Ga-NEB PET of a patient with
multiple hemangiomas. The lesion located in the left lobe was
discerned on the whole body MIP of PET (29A). Different lesions of
hemangioma were pointed out by arrows while the abdominal aorta was
pointed out by triangle (29B-29H).
[0085] FIG. 30 shows images of hepatic hemangioma:v .sup.68Ga-NEB
PET/CT (A) shows strong local accumulation of radioactivity with
the hepatic nodule, while .sup.18F-FDG PET/CT (B) shows relatively
low local uptake. vThe nodule is also identified by CT (C) without
much signal contrast. v(D-F) Images of hepatic carcinoma.v The
lesion shows negative contrast compared with surrounding normal
hepatic tissue on .sup.68Ga-NEB PET/CT (D) while increased FDG
uptake is observed with .sup.18F-FDG PET/CT (E).v (G-I) A case of
neuroendocrine tumor (NET) liver metastases.v Multiple liver
nodules are detected on CT scan (I).v .sup.68Ga-NEB PET/CT (G)
shows low uptake in all the hepatic nodules, compared with mild to
moderate uptake in .sup.18F-FDG PET/CT (H).
[0086] FIG. 31A-31C show .sup.68Ga-NEB PET of a patient
hepatocellular carcinoma. A case of hepatocellular carcinoma
presented by whole body MIP of PET (A), transaxial CT (B) and
.sup.68Ga-NEB PET/CT (C). .sup.68Ga-NEB PET showed decreased
accumulation of radioactivity within the hepatic nodule.
DETAILED DESCRIPTION OF THE INVENTION
[0087] The invention provides a compound of formula (I):
##STR00008##
[0088] wherein L is a linker group selected from aryl, biaryl,
heteroaryl, and biheteroaryl, wherein the aryl, biaryl, heteroaryl,
or biheteroaryl is optionally substituted with one or more groups
selected from alkyl, halo, hydroxy, and alkyloxy,
[0089] wherein A is selected from a bond, C.dbd.O, and
C.sub.1-C.sub.6 alkyl,
[0090] wherein B is a chelating group selected from 1, 4,
7-triazacyclononane-N,N',N''-triacetic acid, 1, 4,
7,10-tetrazacyclononane-N,N',N''-triacetic acid,
triethylenetetramine, diethylenetetramine pentaacetic acid, and
hydrazinonicotinamide,
[0091] wherein R.sup.1-R.sup.5 are independently selected from
hydrogen, OH, NH.sub.2, and SO.sub.3H,
[0092] wherein n is 0 or 1, and
[0093] wherein M is selected from .sup.18F-AlF, .sup.60Cu,
.sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.68Ga, .sup.86Y,
.sup.89Zr, .sup.111In, .sup.99mTc, .sup.186Re, .sup.188Re,
Gd.sup.3+, and Mn.sup.2+.
[0094] In an embodiment, L is biphenyl, optionally substituted with
one or more groups selected from alkyl, halo, hydroxy, and
alkyloxy.
[0095] In an embodiment, A is a bond.
[0096] In an embodiment, B is 1, 4,
7-triazacyclononane-N,N',N''-triacetic acid.
[0097] In a preferred embodiment, the compound of formula (I)
is:
##STR00009##
[0098] In a preferred embodiment, n is 1 and M is .sup.18F-AlF,
.sup.64Cu, or .sup.68Ga.
[0099] In a preferred embodiment, the compound of formula (I)
is:
##STR00010##
[0100] M can be any suitable diagnostic or therapeutic metal.
Non-limiting examples of suitable diagnostic and therapeutic metals
include paramagnetic metal ions, gamma-emitting radioisotopes,
positron-emitting radioisotopes, and x-ray absorbers. Non-limiting
examples of suitable paramagnetic metal ions include Gd(III),
Dy(III), Fe(III), and Mn(II). Non-limiting examples of suitable
gamma-emitting radioisotopes or positron-emitting radioisotopes
include .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu,
.sup.68Ga, .sup.99mTc, .sup.95Tc, .sup.111In, .sup.90Y, .sup.149Pr,
.sup.153Sm, .sup.159Gd, .sup.166Ho, .sup.169Yb, .sup.177Lu,
.sup.186Re, and .sup.213Bi. Non-limiting examples of suitable x-ray
absorbers include Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Yb,
Dy, Cu, Rh, Ag, and Ir. Preferably, M is selected from
.sup.18F-AlF, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu,
.sup.67Cu, .sup.68Ga, .sup.86Y, .sup.89Zr, .sup.111In, .sup.99mTc,
.sup.186Re, .sup.188Re, Gd.sup.3+, and Mn.sup.2+. More preferably,
M is .sup.18F-AlF, .sup.64Cu, or .sup.68Ga.
[0101] The compound of formula (I) can be prepared using any
suitable method. In an embodiment, the compound of formula (I) is
prepared by a method comprising the steps of:
[0102] (i) reacting a bis amino compound of the formula:
H.sub.2N-L-NH.sub.2 wherein L is aryl, biaryl, heteroaryl, and
biheteroaryl, wherein the aryl, biaryl, heteroaryl, or biheteroaryl
is optionally substituted with one or more groups selected from
alkyl, halo, hydroxy, and alkyloxy, with 1, 4,
7-triazacyclononane-N,N',N''-triacetic acid to form a compound of
formula (II):
##STR00011##
[0103] (iii) reacting the compound of formula (II) with a
diazotization reagent to form a compound of formula (III):
##STR00012##
and
[0104] (iv) reacting the compound of formula (III) with
4-amino-5-hydroxynaphthalene-1,3-disulfonic acid to form a compound
of formula (IV):
##STR00013##
[0105] In an embodiment, the method further comprises a step of
reacting the compound of formula (IV) with MX.sub.2, wherein M is
selected from .sup.18F-AlF, .sup.64Cu, or .sup.68Ga and wherein X
is a halogen, to provide a compound of formula (V):
##STR00014##
[0106] In a preferred embodiment, the compound of formula (V)
is:
##STR00015##
[0107] In another preferred embodiment, the compound of formula (V)
is:
##STR00016##
[0108] In another preferred embodiment, the compound of formula (V)
is:
##STR00017##
[0109] In a particular embodiment, the compound of formula (I)
(also referred to herein as "NEB") wherein n is 0 can be
synthesized in two steps starting from o-tolidine as shown in
Scheme 1. 1, 4, 7-triazacyclononane-N,N,N''-triacetic acid-3HCl
("NOTA") was first coupled to o-tolidine using diethyl
cyanophosphonate to give NOTA-o-tolidine in 26% yield after
preparative reversed-phase HPLC. NOTA-o-tolidine was then coupled
to 1-amino-8-naphthol-2,4-disulfonic acid to give NEB through the
formation of diazonium salt with a yield of 46.6%. The purity of
the product was >98% based on HPLC analysis and the identity of
the product was confirmed by LC-MS. This truncated version of Evans
blue retained the binding ability with albumin since stable
NEB/albumin complex was confirmed by LC/MS. The complex formation
was also confirmed by saturation binding assay. The bound NEB could
be separated from the unbound NEB using agarose gel
electrophoresis. Quantitation of the mass of unbound NEB as a
function of its concentration allowed determination of the
dissociation constant (K.sub.d, 48.35.+-.3.81 .mu.M). The
.sup.18F-AlF complex of NEB is also referred to herein as
.sup.18F-AlF-NEB. The .sup.64CU complex of NEB is also referred to
herein as .sup.64Cu-NEB.
[0110] The procedure for synthesis of the compound of formula (I)
and its radiolabeling is shown in Scheme 1. The radiochemical yield
for .sup.18F-AlF-NEB was 58.4.+-.11.3% (n=5) with a total synthesis
and work-up time of 20-30 min. A single peak was detected on TLC
and the radiochemical purity was >95% based on HPLC analysis
(Supplementary FIGS. 3 and 4). Labeling of NEB with .sup.64Cu also
took 20-30 min with a radiochemical yield of 74%. For in vitro
labeling of albumin, .sup.18F-SFB was first prepared and purified
by HPLC and then used for protein labeling. The whole labeling
process took about 2-3 h with a radiochemical purity >95%. Both
.sup.18F-AlF-NEB and .sup.64Cu-NEB showed very good stability in
mouse serum even after 120 min incubation at 37.degree. C.
##STR00018##
[0111] The compound of the invention can be used for imaging using
any suitable imaging method. Non-limiting examples of suitable
imaging methods include magnetic resonance imaging, single photon
emission imaging, and positron emission tomographic imaging (PET).
In a preferred embodiment, the imaging method is PET.
[0112] To meet the requirement for both clinical application and
preclinical research, herein, a fast in vivo albumin labeling
method for PET imaging was investigated. The preparation procedure
is rapid and efficient. PET images using in vivo labeled albumin
through a newly developed truncated Evans blue derivative
.sup.18F-AlF-NEB are comparable to those using in vitro labeled
albumin through .sup.18F-FB-Albumin. The in vivo labeling strategy
can be applied to blood pool imaging to evaluate cardiac function
under both physiological and pathological conditions. This method
can also be used to evaluate vascular permeability in tumors,
inflammatory diseases, and ischemic/infarcted lesions. Due to the
simple synthesis procedure, this PET tracer has great potential for
clinical translation. .sup.18F-AlF-NEB is expected to be an ideal
alternative to radiolabeled RBCs for blood pool imaging since
autologous blood products present significant risks to both the
operator handling the product and the patient receiving it.
[0113] As expected, a majority of the radioactivity was retained in
the circulatory system after intravenous injection of
.sup.18F-AlF-NEB, which justifies the feasibility of using this
tracer as a blood pool imaging agent. It is of note that
immediately after intravenous injection of .sup.18F-AlF-NEB, a
small amount of tracer was rapidly cleared out of circulation
before binding to albumin. The possibility of dissociation of
NEB/albumin complex, cannot be excluded, especially at the early
phase, since the dissociation constant of NEB to albumin is
approximately 50 .mu.M. However, due to the very low amount of NEB
injected and highly abundant reservoir of albumin protein, the
complex formation and its stability in the blood circulation is not
of concern. Indeed, from 15 min to 60 min p.i., the tracer showed a
much slower clearance of radioactivity from the blood. It is
believed that this downward slope was caused mainly by the turnover
of albumin from blood circulation to the interstitial space.
[0114] As the most widely used FDA approved PET imaging probe,
.sup.18F-FDG has been used for ECG gated PET imaging to evaluate
cardiac function (Porenta G. et al., J Nucl Med. 1995; 36(6):
1123-1129). The parameters from FDG PET are comparable with those
from MRI and CT studies. However, blood pool imaging probe such as
.sup.68Ga-DOTA-albumin has shown advantages over FDG PET because in
the infarcted heart, the cardiac wall is not intact due to
decreased or null uptake of FDG, leading to imprecise delineation
of the infarcted myocardium. In this study, the cardiac ventricles
and major vessels were successfully visualized by using
.sup.18F-AlF-NEB PET. After dividing each cardiac cycle into eight
equal time intervals, the volume of left ventricle in each interval
can be quantified to calculate the ejection fraction. Due to the
limitation of spatial resolution and partial volume effect, the
EFLV in MI mice based on PET quantification is higher than that
from ultrasound. Besides, the accuracy of ECG gated PET imaging is
also affected by the reconstruction algorithm, the software used
for outlining ROIs and ECG gating.
[0115] .sup.18F-AlF-NEB was applied to evaluate increased vascular
permeability in both turpentine induced acute inflammation and
xenografted tumor models. The inflamed muscle showed continuously
increased radioactivity accumulation, indicating the leakage of
serum albumin into surrounding interstitial tissues. The late time
point scans also provided better contrast between inflammatory
muscles and contralateral normal muscle.
[0116] Malignant tumors often show increased uptake and retention
of high molecular weight non-targeted drugs and prodrugs, which is
known as enhanced permeability and retention (EPR) effect.
Moreover, angiogenesis and vasculature vary in different tumor
types. Thus, the ability to non-invasively evaluate tumor
vasculature and permeability would be very helpful for patient
pre-selection and therapy response monitoring. With .sup.64Cu-NEB
PET, it was found that UM-22B tumors are very permeable. It was
noticed that TAC over the heart region showed a downslope and that
over tumor region showed a plateau. The difference between these
two slopes was thus used to quantify tumor vascular
permeability.
[0117] Evans blue has been used clinically to evaluate the blood
volume. The clinical practice has been discontinued due to the
toxicity of vital dye, especially potential pulmonary embolism
after intravenous injection. The mechanism of this toxicity is due
to an Evans blue dose related induction of platelet aggregation,
which begins with the threshold concentration of about 100 .mu.M.
For in vivo PET imaging, only trace amount of .sup.18F-AlF-NEB
(.about.3 .mu.g/mL or 3.6 .mu.M) was used, which would not elicit
toxicity. Even with multiple injections, the dose is expected to be
safe. A dosimetry study was also performed based on the PET imaging
data. For the sensitive organs such as red bone marrow, the
absorbed dose is 0.23 mSv for .sup.18F-AlF-NEB and 6.14 mSv for
.sup.64Cu-NEB if 185 MBq of radioactivity was injected into each
subject. This dose allows multiple PET scans without exceeding
recommended annual dose limit for diagnostic purpose.
[0118] The tracers of the invention could be used to visualize the
distribution and local accumulation of serum albumin non-invasively
by PET. ECG-gated .sup.18F-AlF-NEB PET could be used to evaluate
the loss of cardiac function in mice with myocardial infarction.
The vascular leakage induced by acute inflammation and increased
permeability in malignant tumors could also be visualized and
quantified with this strategy.
[0119] The invention further provides a lymphatic imaging agent by
mixing EB with the PET tracer .sup.18F-AlF-NEB. EB has been
extensively used as a visible dye. In fact, the quantum yield of EB
itself is rather low. However, like some other dye molecules, when
EB forms a complex with albumin, the fluorescence emission of the
complex increases dramatically (FIGS. 1A and 1B). It is widely
accepted that albumin binding sterically and electronically
stabilizes the fluorophore's ground state electronic distribution
and increases the quantum yield. In fact, the fluorescence signal
is more sensitive than the visible color. This phenomenon was taken
advantage of by performing fluorescence imaging after local
injection of .sup.18F-AlF-NEB/EB, which quickly forms a complex
with albumin within the interstitial fluid. The radioactive signal
reflects the behavior of endogenous albumin, avoiding the usage of
colloids, nanoparticles, and polymers. Thus, mixing
.sup.18F-AlF-NEB with the EB dye allows PET, visual, and
fluorescence tri-modality imaging. Local lymph nodes (LNs) and the
lymphatic vessels between LNs can be clearly visualized by the blue
color of the dye as well as optical imaging. Furthermore, the
sentinel lymph nodes can be detected by PET scans.
[0120] This imaging probe embodiment of the invention was first
applied to a turpentine oil-induced hind limb inflammation model.
With inflammatory stimulation, local lymph nodes undergo a series
of changes in order to clear debris and provide a site for
activated immune cells. This process is often coupled with an
increase in size and enhanced lymphatic drainage. Turpentine oil
induced tissue inflammatory responses peak at day 4. Therefore,
.sup.18F-AlF-NEB PET imaging was first performed in hind limb
inflammation model on day 5 after turpentine oil injection. The
popliteal and sciatic LNs on both sides can be clearly visualized
from 0.5 to 3 h after tracer injection with inflamed LNs
accumulating a higher amount of tracer (FIGS. 2A-2E). The imaging
results corroborate with the size and flow changes during local
inflammatory responses. The feasibility of imaging tumor draining
LNs in an orthotropic breast tumor model was next explored. After
intratumoral injection, SLNs were successfully detected by
.sup.18F-AlF-NEB PET with excellent image quality (FIG. 3).
[0121] The detection of SLNs is important in clinical cancer
classification and treatment. Currently, pre-surgical diagnosis of
SLNs is often based on the morphological changes observed by MR or
CT scans. However, it is very challenging for MRI or CT to
visualize SLNs when they are very small or have signal intensities
comparable with surrounding healthy soft tissues. Based on the
imaging results acquired in three different animal models, it is
believed that co-injection of .sup.18F-AlF-NEB and EB can be
applied clinically for SLN detection. After local administration,
PET imaging can be performed first to identify the distribution and
location of SLNs around the tumor. The surgeon can then rely on
visible blue color and fluorescence imaging during surgery for SLN
biopsy and removal. A hand-held detector can also be used for SLN
detection.
[0122] The trimodality imaging in accordance with the invention
provides an excellent, non-invasive pre-surgical visualization of
SLNs as well as intra-surgical guidance. The multimodal PET imaging
tracer that has been developed has a great potential for clinical
application due to its biosafety, excellent quality of imaging,
easy preparation, and cost-effectiveness.
[0123] Co-injection of .sup.18F-AlF-NEB and EB provides an easy
method of in vivo labeling of endogenous albumin in the
interstitial fluid thereby enabling PET, optical fluorescence and
visual trimodality imaging for highly sensitive detection of LNs
and lymphatic vessels. The excellent imaging quality, easy
preparation, multimodal applicability, and biosafety of this
approach warrant its clinical application to map SLNs and provide
intraoperative guidance.
[0124] The invention further provides a blood volume imaging agent
and its use in differentiating hemangioma from other focal hepatic
lesions. In a preferred embodiment, the imaging agent is
.sup.68Ga-NEB. This first-in-human study was based on a successful
in vivo albumin labeling strategy with a compound in accordance
with an embodiment of the invention, which forms a complex with
serum albumin after intravenous injection. The labeling is very
efficient without compromising the physiological behavior of the
protein, thus the emitted radioactive signal reflects accurately
the in vivo behavior of albumin. It also avoids unnecessary
cross-contamination from blood products as is the case with
labeling of RBCs for blood pool imaging. For imaging purpose, only
trace amount of the compound was administered so the possible
toxicity of the vital dye and potential pulmonary embolism after
intravenous injection is totally avoided.
[0125] All healthy volunteers and patients reported no discomfort
or adverse clinical events, no elicited toxicity, and no allergies.
Dosimetry study confirmed the safety with acceptable absorbed doses
by critical organs even with multiple injections for one patient.
With an injected dose of 3-4 mCi (121-148 MBq), a patient would be
exposed to a radiation dose of 2.65 mSv, which is much lower than
the dose limit as set by the by the Food and Drug Administration
(FDA).
[0126] After intravenous injection, majority of the radioactivity
was retained in blood circulation due to the stable complexation of
.sup.68Ga-NEB with serum albumin. Within a few minutes after tracer
injection, it reached an equilibrium reflected by the constant high
SUV value in the blood. A slow but steady clearance of the
radioactivity from the blood was observed. This was mainly caused
by the turnover of albumin from blood circulation to the
interstitial space and slight dissociation of .sup.68Ga-NEB from
albumin. The heart to liver ratios at different time points are
very close to that of in vitro labeled RBCs and is significantly
higher than that of in vivo labeled RBC, which confirmed the role
of .sup.68Ga-NEB as a blood pool PET imaging agent.
[0127] There are several major applications for a blood pool
imaging agent including evaluation of the cardiac function,
detection of vascular anomalies, and localization of neoplasms.
Hepatic hemangioma is a vascular anomaly, characterized with
multiple vascular channels with a single layer of benign
endothelial cells. Consequently, high level of local accumulation
of .sup.68Ga-NEB was observed, making tumors highly visible against
the surrounding normal hepatic tissues. Meanwhile, lesions of HCC,
hepatic cyst and hepatic metastases of neuroendocrine tumors showed
"negative" contrast because of the relatively low accumulation of
.sup.68Ga-NEB. The high specificity of .sup.68Ga-NEB PET would play
a very important role in differentiating hemangioma from other
focal hepatic lesions.
[0128] The accurate diagnosis of hemangioma with .sup.68Ga-NEB PET
can avoid unnecessary over-treatment and biopsy, which has the risk
of hemorrhage. In combination with .sup.18F-FDG PET, .sup.68Ga-NEB
PET/CT can be of great value for differential diagnosis of cysts,
hemangioma and other benign hepatic lesions from malignancy,
especially in patients with history of malignancy. It is also
predictable that .sup.68Ga-NEB PET will be helpful in diagnosing
hemangioma occurring in other organs.
[0129] The invention also provides an imaging composition
comprising a compound of formula (I) and a pharmaceutically
acceptable carrier.
[0130] The pharmaceutically acceptable carrier can be any of those
conventionally used and is limited only by chemico-physical
considerations, such as solubility and lack of reactivity with the
compound, and by the route of administration. It will be
appreciated by one of skill in the art that, in addition to the
following described imaging compositions; the compounds of the
present invention can be formulated as inclusion complexes, such as
cyclodextrin inclusion complexes, or liposomes.
[0131] The pharmaceutically acceptable carriers described herein,
for example, vehicles, adjuvants, excipients, or diluents, are well
known to those who are skilled in the art and are readily available
to the public. It is preferred that the pharmaceutically acceptable
carrier be one which is chemically inert to the compound and one
which has no detrimental side effects or toxicity under the
conditions of use.
[0132] The choice of carrier will be determined in part by the
compound, as well as by the particular method used to administer
the composition. Accordingly, there is a wide variety of suitable
formulations of the imaging composition of the present invention.
In a preferred embodiment, the imaging composition is administered
parenterally. The following formulations for parenteral
administration are merely exemplary and are in no way limiting.
[0133] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The compound can be
administered in a physiologically acceptable diluent in a
pharmaceutical carrier, such as a sterile liquid or mixture of
liquids, including water, saline, aqueous dextrose and related
sugar solutions, an alcohol, such as ethanol, isopropanol, or
hexadecyl alcohol, glycols, such as propylene glycol or
polyethylene glycol, glycerol ketals, such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as
poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester
or glyceride, or an acetylated fatty acid glyceride with or without
the addition of a pharmaceutically acceptable surfactant, such as a
soap or a detergent, suspending agent, such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
[0134] Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters. Suitable soaps for use in parenteral formulations
include fatty alkali metal, ammonium, and triethanolamine salts,
and suitable detergents include (a) cationic detergents such as,
for example, dimethyl dialkyl ammonium halides, and alkyl
pyridinium halides, (b) anionic detergents such as, for example,
alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and
monoglyceride sulfates, and sulfosuccinates, (c) nonionic
detergents such as, for example, fatty amine oxides, fatty acid
alkanolamides, and polyoxyethylene-polypropylene copolymers, (d)
amphoteric detergents such as, for example,
alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary
ammonium salts, and (3) mixtures thereof.
[0135] The parenteral formulations will typically contain from
about 0.5 to about 25% by weight of the compound in solution.
Suitable preservatives and buffers can be used in such
formulations. In order to minimize or eliminate irritation at the
site of injection, such compositions may contain one or more
nonionic surfactants having a hydrophile-lipophile balance (HLB) of
from about 12 to about 17. The quantity of surfactant in such
formulations ranges from about 5 to about 15% by weight. Suitable
surfactants include polyethylene sorbitan fatty acid esters, such
as sorbitan monooleate and the high molecular weight adducts of
ethylene oxide with a hydrophobic base, formed by the condensation
of propylene oxide with propylene glycol. The parenteral
formulations can be presented in unit-dose or multi-dose sealed
containers, such as ampoules and vials, and can be stored in a
freeze-dried (lyophilized) condition requiring only the addition of
the sterile liquid carrier, for example, water, for injections,
immediately prior to use. Extemporaneous injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
[0136] The compounds of the present invention may be made into
injectable formulations. The requirements for effective
pharmaceutical carriers for injectable compositions are well known
to those of ordinary skill in the art. See Pharmaceutics and
Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker
and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on
Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).
[0137] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
[0138] General Materials and Instrumentation
[0139] The monosodium salt of 1-amino-8-naphthol-2, 4-disulfonic
acid was purchased from TCI America (Portland, Oreg.) and all other
chemicals were from Sigma-Aldrich (St. Louis, Mo.). Mass spectra
(MS) were obtained on a Waters Acquity UPLC system coupled with
Waters QT of Premier MS (LC-MS). Semi-preparative reversed-phase
HPLC was performed on a Waters 600 gradient system with a Waters
996 Photodiode Array (PDA) detector using a Waters Nova-Pak HR
C.sub.18 column (6 .mu.m, 300.times.7.8 mm). Analytical
reversed-phase HPLC was performed on a Perkin-Elmer Series 200 LC
gradient system with a Waters 2784 Dual Absorbance UV detector plus
a Bioscan radioisotope detector using a Waters Symmetry column (5
.mu.m, 150.times.3.9 mm). The flow rate was 6 mL/min for the
semi-preparative column and 1 mL/min for the analytical column
running the same linear gradient starting from 5% A (0.1% TFA in
acetonitrile) and 95% B (0.1% TFA in water) for 5 min and
increasing A to 65% at 35 min. Varian BOND ELUT C.sub.18 column
(100 mg) was used for solid-phase extraction of the labeled
product. .sup.18F-fluoride and .sup.64CuCl.sub.2 were obtained from
the NIH cyclotron facility.
[0140] Animal Models
[0141] All animal studies were conducted in accordance with the
principles and procedures outlined in the Guide for the Care and
Use of Laboratory Animals and were approved by the Institutional
Animal Care and Use Committee of the Clinical Center, NIH. The
UM-22B human head and neck carcinoma cancer cell line was grown in
DMEM medium supplemented with 10% fetal bovine serum (FBS), 100
IU/mL penicillin, and 100 .mu.g/mL streptomycin (Invitrogen), and
in a humidified atmosphere containing 5% CO.sub.2 at 37.degree. C.
The tumor model was developed in 5 to 6 week old female athymic
nude mice (Harlan Laboratories) by injection of 5.times.10.sup.6
cells into their right shoulders. The mice underwent small-animal
PET studies when the tumor volume reached 100-300 mm.sup.3 (2-3
weeks after inoculation).
[0142] Myocardial infarction (MI) model was prepared in male Balb/c
mice aged from 8 to 10 weeks. MI was induced by ligation of the
left anterior descending coronary artery 1-3 mm from the tip of the
left auricle with a 7-0 polypropylene suture. The occlusion and
reperfusion were confirmed by ST-segment elevation on an
electrocardiogram (ECG) monitor (EC-60 model; Silogic) after
surgery. For mice in the control group, the surgery was performed
but without ligation of the left coronary artery.
[0143] The acute inflammation model was prepared by intramuscular
injection of turpentine. Up to 30 .mu.L turpentine was injected in
the caudal thigh muscles of left hind limb. The PET imaging was
performed 24 h after turpentine injection.
[0144] Small-Animal PET Imaging and Analysis
[0145] PET scans and image analysis were performed using an Inveon
small animal PET scanner (Siemens Medical Solutions). About 3.7 MBq
of .sup.18F-AlF-NEB or .sup.18F-FB-Albumin or 7.4 MBq of
.sup.64Cu-NEB was administered via tail vein injection under
isoflurane anesthesia. For normal mice, 60-min dynamic PET scans
were acquired. For tumor mice, 60-min dynamic PET scans were
acquired, followed by a series of late time point scans at 2, 4,
and 24 h (.sup.64Cu only) after tracer injection. With acute
inflammation model, 5-min static PET images were acquired at 30
min, 1 h and 2 h postinjection (n=3-5 per group). The images were
reconstructed using a two-dimensional ordered-subset expectation
maximum (2D OSEM) algorithm, and no correction was applied for
attenuation or scatter. For each scan, regions of interest (ROIs)
were drawn using vendor software (ASI Pro 5.2.4.0) on
decay-corrected whole-body coronal images. The radioactivity
concentrations (accumulation) within the heart, muscle, liver, and
kidneys were obtained from mean pixel values within the multiple
ROI volume and then converted to MBq per milliliter. These values
were then divided by the administered activity to obtain (assuming
a tissue density of 1 g/ml) an image-ROI-derived percent injected
dose per gram (% ID/g).
[0146] For ECG gated PET studies, mice were imaged in a prone
position within the PET scanner, and were kept at 37.degree. C.
using a heating pad with continuous rectal measurement of body
temperature. ECG electrodes were placed on the forepaws and the
left hindpaw. Respiration was measured using a small pressure
detector lying under the thorax of the mice. The cardiac excitation
and respiration were recorded with a Biovet system (Spin Systems
Pty Ltd.) throughout the scan. A list-mode PET scan of 30 min was
acquired at 15 min after intravenous injection of around 3.7 MBq of
.sup.18F-NEB (both control and MI models), respectively. The
cardiac cycle from the .sup.18F-NEB list-mode acquisitions was
separated into eight equal intervals using the Siemens Inveon
Acquisition Workplace and reconstructed using OSEM 3D with four
iterations and MAP 3D with 32 iterations. The measurements of LV
function from the PET data sets were calculated and quantified
using the Inveon Research Workplace (IRW, Siemens Preclinical
Solutions). A 55% intensity threshold was used for the quantitation
of LVEF.
[0147] Preclinical PET Imaging and Dosimetry Evaluation.
[0148] For dynamic PET scans, four BALB/C mice were injected
intravenously with 1.85 MBq (50 .mu.Ci) of .sup.68Ga-NEB under
isoflurane anesthesia. A 60 min list mode acquisition was performed
with an Inveon PET scanner (Siemens Preclinical Solutions, PA,
USA). Image reconstruction was done by the 2-dimensional ordered
subsets expectation maximum (OSEM) algorithm without attenuation or
scatter correction. Regions of interest (ROIs) over major organs
were drawn on decay-corrected whole-body coronal images for each
PET scan, using vendor software (ASI Pro 5.2.4.0). The
radioactivity concentration (accumulation) within a tumor or an
organ was obtained from mean pixel values within the multiple ROI
volume, after conversion of the values to MBq/mL/min by using a
conversion factor. The conversion to MBq/g/min assumed a tissue
density of 1 g/mL. Imaging ROI-derived % ID/g was calculated by
dividing the ROIs by the administered activity.
[0149] For ex vivo biodistribution and dosimetry evaluation, each
BALB/C mouse was injected with 1.85 MBq of .sup.68Ga-NEB. At
different time points after tracer injection (5 min, 30 min, 1 h, 3
h and 6 h, n=5/time point), the mice were sacrificed for tissue and
organ collection, including blood, muscle, bone, liver, kidneys,
spleen, pancreas, stomach, intestine, heart and lung. The samples
were wet weighed, and measured for radioactivity in a
.gamma.-counter (Wallac 1470-002, Perkin-Elmer). The results were
presented as percentage injected dose per gram of tissue (% ID/g).
For each mouse, the radioactivity of the tissue samples was
calibrated against a known aliquot of the injected tracer and
normalized to a mean body mass of each group. Values were expressed
as mean.+-.SD (n=5/group). Determination of organ doses for a
reference human male was made using the OLINDA/EXM program
(Vanderbilt University, Nashville, Tenn.).
[0150] Volunteers and Patients Recruiting.
[0151] This clinical study was approved by the Institute Review
Board of Peking Union Medical College Hospital, Chinese Academy of
Medical Sciences and Peking Union Medical College. Three healthy
volunteers (2 males and 1 female) were enrolled with written
informed consent to validate the safety of .sup.68Ga-NEB. No
fasting, hydration or other specific preparation was requested on
the day of imaging. Any unusual or adverse clinical symptoms were
recorded on the day of imaging and during the 2-week follow-up
period.
[0152] All patients were 18 years old or above, capable of
understanding the study and signed a written informed consent. A
total of 11 patients with hepatic space-occupying lesions were
recruited. The final diagnosis was based on pathological result of
surgical removal or biopsy. Among the recruited patients, 4
patients were diagnosed with hemangioma, 5 with primary hepatic
carcinoma, 1 with neuroendocrine tumor liver metastases and 1 with
hepatic cyst.
[0153] PET Imaging Procedures.
[0154] All three healthy volunteers underwent whole-body PET
acquisitions (Biograph mCT PET/CT system, Siemens) at multiple time
points after tracer injection. After the whole-body low-dose CT
scan (140 kV, 35 mA, pitch 1:1, layer 5 mm, layer spacing 3 mm,
matrix 512.times.512, FOV 70 cm), 111-148 MBq (3-4 mCi) of
.sup.68Ga-NEB was injected intravenously. The whole body (from the
top of skull to the middle of femur) of each volunteer was covered
by 7 bed positions. The acquisition duration was 40 sec/bed
position for the 5 min, 10 min and 15 min time points, 2 min/bed
position for the 30 min, 45 min, 60 min, 75 min and 90 min time
points.
[0155] All patients underwent whole-body PET/CT acquisitions at
30-45 min after intravenous injection of 111-148 MBq (3-4 mCi) of
.sup.68Ga-NEB with each bed position lasting for 2 min. The
acquisition field covered from the top of skull to the middle of
femur with 6 or 7 bed positions, depending on the height of the
patient. A standard .sup.18F-FDG PET/CT was acquired with the same
patients within one week.
[0156] Image and Data Analysis.
[0157] A Siemens MMWP workstation was used for post-processing.
Visual analysis was used to determine the general biodistribution
and the temporal and inter-subject stability. The volume of
interest (VOI) of 12 normal organs/tissues and concerned lesions
were drawn on the serial images. The radioactivity concentration
and standardized uptake value (SUV) in the VOIs were obtained
through the software. Organ dosimetry was calculated by using
organ-level internal dose assessment software. All quantitative
data were expressed as mean.+-.standard deviation.
[0158] Hematoxylin and Eosin Staining
[0159] Tissues were collected and fixed in Z-fix (buffered zinc
formalin fixatives, Anatech, Mich.) for at least 24 h, then
embedded in paraffin for sectioning. The slices (10 .mu.m) were
stained with hematoxylin and eosin by standard techniques. The
stained tissue sections were observed with a BX41 bright field
microscopy (Olympus).
[0160] Statistical Analysis
[0161] Quantitative data were expressed as mean.+-.SD. Means were
compared using Student's t test provided by Excel (Microsoft) or
GraphPad Prism (GraphPad Software, Inc.). P value of <0.05 was
considered statistically significant.
Example 1
[0162] This example demonstrates a synthesis of the compound:
##STR00019##
in accordance with an embodiment of the invention.
[0163] To a 4 mL glass vial containing 20.0 mg of o-tolidine (94
.mu.mol) and 20.0 mg of 1, 4,
7-triazacyclononane-N,N',N''-triacetic acid-3HCl ("NOTA") (48 mop
in 1 mL of DMSO was added 3.6 .mu.L of diethyl cyanophosphonate (24
.mu.mol) and 25 .mu.L of diisopropylethylamine (DIPEA). The mixture
was stirred at room temperature for 40 min and another 3.6 .mu.L of
diethyl cyanophosphonate was added and stirred at room temperature
overnight. The mixture was then purified with semi-preparative
HPLC. The peak containing the desired product was collected
(R.sub.t=10.0 min) and the solution was frozen over dry ice and
lyophilized overnight to give 12.2 mg pure product in 26.4% yield.
LC-MS (C.sub.26H.sub.35N.sub.5O.sub.5): [MH].sup.+=498.2467 (m/z),
calc: 497.2638.
Example 2
[0164] This example demonstrates a synthesis of the compound:
##STR00020##
in accordance with an embodiment of the invention.
[0165] To a 20 mL glass vial containing 2.5 mg of NOTA-tolidine
(5.0 .mu.mol) in 0.3 mL of water was added 18 .mu.mol of HCl in 0.1
mL of water. The mixture was cooled in ice bath and 0.5 mg of
sodium nitrite (7.2 .mu.mol) in 0.1 mL of water was added to the
vial. The mixture was stirred in ice bath for 20 min and the yellow
diazonium salt solution was added dropwise to another vial in ice
bath containing 4.0 mg of 1-amino-8-naphthol-2,4-disulfonic acid
(10.0 .mu.mol) and 2.4 mg of sodium bicarbonate (28.5 .mu.mol) in
0.2 mL of water. The mixture was stirred in ice bath for 2 h and
purified with semi-preparative HPLC. The product (denoted as NEB)
was collected (R.sub.t=19.0 min) and lyophilized overnight to give
1.4 mg pure product in 46.6% yield. LC-MS
(C.sub.36H.sub.41N.sub.7O.sub.12S.sub.2): [M-H].sup.-=826.2415
(m/z), calc: 827.2255.
Example 3
[0166] This example demonstrates a synthesis of the compound:
##STR00021##
in accordance with an embodiment of the invention.
[0167] To a 1 mL plastic tube containing 3 .mu.L of 2 mM aluminum
chloride in 0.5 M pH 4.0 sodium acetate buffer and 6 .mu.L of 3 mM
NEB in 0.5 M pH 4.0 sodium acetate buffer was added 0.13 mL
acetonitrile and 0.05 mL of aqueous .sup.18F-fluoride (0.3-0.9
GBq). The mixture was vortexed and heated in a 105.degree. C.
heating block for 10 min. The vial was cooled, and the solution was
diluted with 10 mL of water and trapped on a Varian Bond Elut
C.sub.18 column (100 mg). The radioactivity trapped on the C.sub.18
column was eluted with 0.3 mL of 80% ethanol/water containing 1 mM
HCl. The ethanol solution was evaporated with argon flow, and the
final product was dissolved in PBS and analyzed by HPLC.
Example 4
[0168] This example demonstrates a synthesis of the compound:
##STR00022##
in accordance with an embodiment of the invention.
[0169] To a 1 mL plastic tube containing 11.0 .mu.g of NEB in 100
.mu.L of 0.4 M pH 5.5 sodium acetate buffer was added 5 .mu.L, of
aqueous .sup.64Cu-CuCl.sub.2 solution (262.7 MBq). The mixture was
vortexed and heated on an 80.degree. C. heating block for 10 min.
The tube was cooled, and the radioactive solution transferred to a
10 mL syringe containing 10 mL of water. This solution was passed
through a Varian Bond Elut C.sub.18 cartridge (100 mg) and the
desired product was trapped on the cartridge. The radioactivity
trapped on the C.sub.18 column was eluted with 0.45 mL of 80%
ethanol/water with 1 mM HCl to give 185 MBq of the desired product
in 70% radiochemical yield. The ethanol solution was evaporated
with argon flow, and the final product was dissolved in PBS and
analyzed by HPLC. The radiochemical purity was >95%.
Example 5
[0170] This example demonstrates a synthesis of the compound:
##STR00023##
in accordance with an embodiment of the invention.
[0171] The NOTA conjugate of truncated form of Evans blue (NEB) was
synthesized according to a method described in our previous
publication (Niu, G. et al., J. Nucl. Med., 2014, 55(7):
1150-1156). .sup.68Ga was eluted from a .sup.68Ge/.sup.68Ga
generator (ITG, Berlin, Germany) using 0.05 M HCl and mixed with
1.25 M NaOAc buffer to adjust the pH value to 4.0. The mixture was
then directly transferred to a 1 mL plastic tube containing 30
.mu.s of NEB. After shaking, the mixture was incubated in a heating
block at 100.degree. C. for 10 min. The reaction mixture was then
cooled down, dissolved in sterile phosphate-buffered saline (PBS)
and passed through 0.22 .mu.m aseptic filtration membrane. The
quality control was performed with analytical HPLC and thin layer
chromatography (BIOSCAN, USA). CH.sub.3OH:NH.sub.4OAc (v/v 1:1) was
used as the developing solution for TLC. The radiochemical purity
was greater than 95%.
Example 6
[0172] This example demonstrates a preparation of
.sup.18F-fluorobenzyl albumin.
[0173] N-succinimidyl 4-.sup.18F-fluorobenzoate (.sup.18F-SFB) was
prepared with an Eckert & Ziegler synthesizer according to a
published procedure (Chen, X. et al., Eur. J Nucl. Med. Mol.
Imaging, 2004, 31: 1081-1089). The HPLC purified .sup.18F-SFB was
trapped on a Waters C-18 cartridge and eluted with 1 mL methylene
chloride into a 1 mL plastic tube. For a typical run, after
evaporation of solvent, the radioactivity (148 MBq) was
re-dissolved in 5 .mu.L of acetonitrile and 0.5 mg of mouse serum
albumin (MSA) in 100 .mu.L of pH 8.5 borate buffer was added to the
tube and reacted at 37.degree. C. for 10 min. The reaction mixture
was purified on a PD-10 size exclusion column to give 92.5 MBq of
product in 62.5% radiochemical (non-decay-corrected) yield.
Example 7
[0174] This example demonstrates in vivo PET imaging of normal
mice, in accordance with an embodiment of the invention.
[0175] The in vivo pharmacokinetics of .sup.18F-AlF-NEB was
evaluated with dynamic PET in healthy Balb/C mice. As expected,
most of the radioactivity was retained in the circulation system
during the first 60 min postinjection (p.i.). Ventricles of the
heart and major arteries were clearly visualized on PET images. The
locations of liver, kidneys, and spleen were also identified due to
the abundant blood supply of these organs (FIG. 4). Based on the
time-activity curves generated by PET images, whole blood
radioactivity was only decreased by 10% from 10 to 60 min p.i.
Organs with a large blood volume such as liver, spleen and kidneys
showed higher uptakes than the skeletal muscle but were
considerably less than the blood cavities within the heart and main
branches of the blood vessels. Counts in the heart VOI rapidly
peaked at 1 min p.i., then declined gradually but remained higher
than those in the liver and kidneys. The radioactivity in the
bladder increased continuously with time (FIG. 5A). HPLC analysis
of urine sample found that the radioactivity came from both intact
.sup.18F-AlF-NEB and its metabolites. In blood samples, the
majority of the radioactivity was from the intact
.sup.18F-AlF-NEB.
[0176] The in vivo labeling by .sup.18F-AlF-NEB was also compared
with in vitro labeled .sup.18F-FB-MSA. The PET results showed very
similar distribution pattern between the two probes. The blood and
liver time-activity curves from .sup.18F-FB-MSA showed slightly
less but non-significant (p>0.05) decline rate than those of
.sup.18F-AlF-NEB. The bladder showed almost no uptake of
.sup.18F-FB-MSA within the first 20 min but increased dramatically
afterwards. At 60 min p.i. of .sup.18F-FB-MSA, the radioactivity in
the bladder was much higher than that in the blood (FIGS. 5A and
5B).
[0177] After PET imaging, the animals were sacrificed and major
tissues and organs were collected. The radioactivity was measured
and the results were presented in Table 1. With both
.sup.18F-AlF-NEB and .sup.18F-FB-MSA, the blood had the highest
counts. The radioactivity levels in the liver, kidneys and spleen
for .sup.18F-FB-MSA (in vitro labeling) were significantly higher
than those for .sup.18F-AlF-NEB (in vivo labeling).
TABLE-US-00001 TABLE 1 Tracer Blood Muscle Liver Kidney Spleen
Pancreas Intestine .sup.18F-AlF- 26.35 .+-. 1.52 1.80 .+-. 0.48
5.05 .+-. 0.13 6.80 .+-. 1.16 3.75 .+-. 0.48 2.17 .+-. 1.38 4.42
.+-. 3.00 NEB .sup.18F-FB- 34.71 .+-. 3.29 0.80 .+-. 0.09 7.42 .+-.
053 12.01 .+-. 05.59 8.52 .+-. 1.71 2.09 .+-. 0.15 3.10 .+-. 0.28
MSA
Example 8
[0178] This example demonstrates ECG-gated PET imaging and its use
in measuring left ventricular volume over the cardiac cycle, in
accordance with an embodiment of the invention.
[0179] One of the major applications of blood pool imaging is to
evaluate cardiac function. The probe was first tested in normal
Sprague-Dawley rats. On the representative transaxial and coronal
images of .sup.18F-AlF-NEB, the fine septal borders between the
left and right ventricle were clearly identified. The major
arteries and vena cava were also visualized. After dividing each
cardiac cycle into eight fragments, the end-systole and
end-diastole can be easily distinguished.
[0180] A mouse myocardial infarction (MI) model was also developed
and performed ECG gated PET with the similar procedure. The cardiac
cycle with eight fragmentations is presented in FIG. 6. Although
limited by the much smaller size of mouse heart and spatial
resolution of PET, the ventricles can be clearly distinguished. The
end-systole volume of MI mice was significantly higher than that in
the control mice. Based on PET images, left ventricular (LV) volume
over the cardiac cycle was generated and shown in FIG. 7A. The MI
mice showed much lower LVEF than the control mice (79.54.+-.2.95%
vs. 60.24.+-.6.88%, P<0.01) (FIG. 7B). Ultrasound was also
performed with the same two groups of mice. The LVEF results
determined by US were consistent with those from PET imaging.
Example 9
[0181] This example demonstrates PET imaging of vascular leakage,
in accordance with an embodiment of the invention.
[0182] An acute skeletomuscular inflammation model was developed by
local intramuscular injection of turpentine and .sup.18F-AlF-NEB
PET was performed to evaluate the images for vascular leakage. The
inflammation induced by local intramuscular injection of turpentine
was indicated by neutrophil infiltration on HE staining (FIG. 8).
As shown in FIG. 9, high level of radioactivity accumulation was
observed within inflamed muscles at 1 and 2.5 h after
.sup.18F-AlF-NEB administration. Indicated by the time activity
curves (TACs) of 60-min dynamic imaging, the tracer uptake in the
inflammatory muscles increased gradually along with time while no
apparent changes were observed in TACs of the collateral muscles
(FIG. 10). Quantitative analysis of PET images indicated an uptake
of 5.94.+-.0.69% ID/g at 1 h after .sup.18F-AlF-NEB injection and
7.50.+-.0.69% ID/g at 2.5 h p.i. (FIG. 11).
Example 10
[0183] This example demonstrates PET imaging of tumor vasculature,
in accordance with an embodiment of the invention.
[0184] Malignant tumors are characterized by torturous blood
vessels and high vascular permeability. Besides, the
anti-angiogenesis and "normalization" of tumor vasculature have
been intensively investigated. Unlike radiolabeled RBCs, albumin
can be used to study vascular permeability in tumors. NEB was
labeled with .sup.64Cu, a positron emitter with longer half-life
(t.sub.1/2=12.6 h) than .sup.18F (t.sub.1/2=109.8 min). A UM-22B
xenograft model was developed and performed 60-min dynamic scan and
then static scans at late time points up to 24 h. The in vivo
distribution of .sup.64Cu-NEB was very similar to that of
.sup.18F-AlF-NEB with most of radioactivity retained in the
circulation system (FIG. 4A). Even at 4 h after tracer injection,
radioactivity within heart region and major vessels was still
dominant. The tumor uptake was 5.73.+-.1.11% ID/g at 1 h p.i. and
increased to 8.03.+-.0.77% ID/g at 2 h p.i. At 24 h after tracer
injection, the tumor uptake was still at a relatively high level
(8.07.+-.1.01% ID/g). The tracer uptake over the heart region was
16.09.+-.0.51% ID/g at 1 h p.i., which dropped to 8.58.+-.0.81%
ID/g at 24 h (FIG. 5B).
Example 11
[0185] This example demonstrates PET imaging of inflamed lymph
nodes, in accordance with an embodiment of the invention.
[0186] .sup.18F-AlF-NEB PET imaging was performed on day 5 after
turpentine injection. As shown in FIG. 2A, popliteal lymph nodes
(LNs) on both sides were clearly seen on PET images with a high
signal to background ratio at all the time points examined. Due to
the inflammatory stimulation, the left popliteal LNs had an
obviously higher tracer uptake than the contralateral normal LNs.
The left sciatic LNs also showed slightly higher signal intensity.
Corresponding T.sub.2-weighted MR images confirmed swelling of the
popliteal LNs (FIG. 2B) but not the sciatic LNs. Overlay of PET
images with x-ray confirmed the anatomic location of the popliteal
LNs (FIG. 2C). Quantification of the PET images showed uptake of
.sup.18F-AlF-NEB in the left popliteal LN was 0.195.+-.0.039% ID,
which was significantly higher than that in the right popliteal LN
(0.09.+-.0.035% ID, p<0.05) at 0.5 h p.i. The signal intensity
in the left popliteal LN dropped to 0.116.+-.0.052% ID at 3 h time
point (FIG. 2D). As shown in FIG. 2E, although the left sciatic LN
had somewhat higher tracer uptake than the right sciatic LN, no
significant difference was found (p>0.05).
Example 12
[0187] This example demonstrates PET imaging of tumor draining
lymph nodes, in accordance with an embodiment of the invention.
[0188] Thirty days after tumor inoculation, female nude mice
bearing orthotropic MDA-MB-435 breast cancer tumors were scanned
following intra-tumoral injection of .sup.18F-AlF-NEB. As shown in
FIG. 3A-3C, besides the tracer injection site, a satellite spot
with high signal intensity was identified on PET images from three
orientations (coronal, sagittal and transaxial) of the same mouse.
Using a reference map of the lymphatic system of rodent mammary fat
pad, the hot spot was identified as the accessory axillary LN. In
order to confirm this, one mouse was sacrificed after PET imaging
and the right accessory axillary LN was removed (FIG. 3D). Ex vivo
PET image showed that tumor draining-axillary LN had apparent
uptake of .sup.18F-AlF-NEB (FIG. 3E). Furthermore, another hot spot
was observed in the neck area, which, according to the anatomy of
murine LNs, might be a LN belonging to the cervical LN group (FIG.
3F). PET imaging of mice at day 60 after tumor inoculation was also
performed; both axillary LN and cervical LN could be detected by
.sup.18F-AlF-NEB PET (FIGS. 14A and 14B). However, no tumor
metastasis was observed with H&E staining of axillary LNs.
Example 13
[0189] This example demonstrates PET imaging of metastatic lymph
nodes, in accordance with an embodiment of the invention.
[0190] .sup.18F-AlF-NEB PET was also applied to image tumor
metastatic LNs. Four weeks after inoculation of Fluc.sup.+ 4T1
cells via hock injection, obvious bioluminescence signal could be
seen at the popliteal fossa by bioluminescence imaging (BLI) (FIG.
15A). T.sub.2-weighted MR image also showed enlarged tumor-side
popliteal LNs (FIG. 15B). Immunofluorescence staining with
anti-luciferase antibody confirmed the existence of tumor
metastasis in the left popliteal LN (FIG. 15C). The average
long-axis diameter of left LN measured by MRI was also
significantly larger than that of the right one (FIG. 16).
[0191] .sup.18F-AlF-NEB PET was performed one day after MRI. Both
popliteal LNs could be visualized in 4 out of 6 mice. As seen in
FIG. 17A, there were dramatically higher tracer uptake in tumor
draining popliteal LNs compared with the contralateral LNs at all
the time points measured. Additionally, the signal intensity of
left LNs remained high after 1 h and then decreased slowly over a 3
h period. The contralateral LNs showed a similar trend but with
much lower signal intensity. Autoradiography at 3 h after tracer
injection displayed heterogeneity of tracer distribution inside the
LN. The decreased radioactivity area observed on LN slice may be
due to local tumor metastasis (FIG. 17B). Quantitative results
demonstrated that the total tracer uptake of tumor metastatic LNs
dropped slightly with time from 0.5 to 3 h. The values were
significantly higher than those of LNs from right side (FIG. 17C).
In two of the six mice, no apparent tracer uptake in the tumor-side
popliteal LNs was detected. However, both the sciatic and inguinal
LNs from the tumor side could be clearly seen on PET images and had
much higher signal intensity than the LNs on the contralateral side
(FIGS. 18A and 18B). To confirm the quantitative PET results, an ex
vivo biodistribution study was carried out and the results are
presented in Fig. S3. Thirty min after tracer injection, majority
of radioactivity remained at the injection sites in both paws.
Consistent with PET, direct tissue sampling showed significantly
higher tracer accumulation in the left popliteal LNs than that in
the contralateral LNs (p<0.05).
[0192] Tumor metastasis in the draining LNs was confirmed by
H&E staining. As shown in FIG. 18C, healthy LNs consisted of
mainly immune cells with relatively large nuclei and small amount
of cytoplasm. Conversely, part of the tumor draining LNs,
especially subscapular sinus area, was occupied by cells with
irregular nuclei, which were tumor-metastatic foci (FIG. 18D). Foci
of micrometastasis were also found inside some of the tumor
draining LNs (FIGS. 18E and 18F).
Example 14
[0193] This example demonstrates multimodal imaging of lymph nodes,
in accordance with an embodiment of the invention.
[0194] Since NEB showed similar albumin binding compared with EB
dye, LN visual imaging was performed after co-injection of
.sup.18F-AlF-NEB and EB. Ninety min after local injection, both
popliteal LN sites could be distinguished clearly by the apparent
blue color, indicating the local accumulation of the dye molecules.
The left sciatic LNs could also be seen but with much lower uptake
of dye (FIGS. 19A and 19B). There was significant difference in
weight between the popliteal LNs on the tumor side and the
contralateral side but not between the sciatic LNs (popliteal LNs:
3.582.+-.0.762 vs. 1.995.+-.0.759 mg, p<0.05; sciatic LNs:
1.558.+-.0.731 vs. 1.403.+-.0.632 mg, p>0.05) (FIG. 20). The
total amount of EB dye in each group of LNs was measured and the
results are shown in FIG. 21. Left popliteal LNs contained
0.144.+-.0.034 .mu.g EB dye on average, which was significantly
higher than that of the right ones (0.091.+-.0.029 .mu.g,
p<0.05). However, there was no difference in the amount of EB
between two sciatic LNs (0.030.+-.0.008 .mu.g vs. 0.028.+-.0.015
.mu.g, p>0.05). These ex vivo results were consistent with in
vivo PET data.
[0195] After forming a complex with serum albumin, both NEB and EB
became fluorescent. Since only trace amount of NEB was mixed with
EB, the majority of the fluorescence came from EB. EB showed a
strong absorbance peak at 620 nm with or without albumin. However,
only with albumin, EB had a fluorescence emission peak at 680 nm
(Fig. S5). With optical imaging, the migration of the injected
EB/NEB in lymphatics could be clearly observed after local
injection. The fluorescence signal first reached the popliteal LN
then migrated to the sciatic LN (FIG. 22). Ninety min after tracer
injection, both LNs were clearly visualized by fluorescence optical
imaging. Under bright light, apparent blue dye accumulation could
also be seen by the naked eye (FIGS. 23A and 23B). PET and optical
imaging were also performed with the same animal after injection of
.sup.18F-AlF-NEB/EB. An overlay of the two images provided high
positional correlation of the LNs (FIGS. 24A and 24B).
Example 15
[0196] This example demonstrates in vivo dynamic PET imaging and
biodistribution in normal mice, in accordance with an embodiment of
the invention.
[0197] To investigate the pharmacokinetics of .sup.68Ga-NEB, 1 h
dynamic PET was performed in healthy BALB/C mice. After tail vein
injection, most of the radioactivity from .sup.68Ga-NEB was
retained in the blood circulation, including the ventricles of the
heart, major arteries and blood-enriched organs, during the entire
period of observation (FIGS. 25 and 26).
[0198] In order to estimate the safe dose for clinical use, an ex
vivo biodistribution study was performed in normal Balb/C mice.
Absorbed doses for major organs and whole body were then
extrapolated to adult human male of a body weight of 73.7 Kg using
OLINDA EXM software. The mean dose ranges from 5 mice at each time
point are listed in Table 2. The kidneys received the highest
absorbed doses (mean absorbed dose, 0.104.about.0.135 mSv/MBq),
resulting from abundant blood supply and tracer excretion through
the renal urinary tract. The mean effective dose of .sup.68Ga-NEB
was 0.0151 to 0.0159 mSv/MBq. With an injected dose of 185 MBq (5
mCi), the patient would be exposed to an effective radiation dose
of 2.94 mSv, which is much lower than the dose limit of 20 mSv for
the second risk category defined by the 2007 International
Commission on Radiological Protection.
TABLE-US-00002 TABLE 2 Target organ Absorbed Dose Adrenals
0.01370~0.01410 Brain 0.00868~0.01040 Breasts 0.00881~0.01000
Gallbladder Wall 0.01470~0.01490 LLI Wall 0.01040~0.01190 Small
Intestine 0.01140~0.01280 Stomach Wall 0.01170~0.01270 ULI Wall
0.01150~0.01280 Heart Wall 0.03290~0.04530 Kidneys 0.10400~0.13500
Liver 0.04440~0.05750 Lungs 0.01970~0.02360 Muscle 0.00946~0.01100
Ovaries 0.01080~0.01220 Pancreas 0.06250~0.08210 Red Marrow
0.00882~0.00973 Osteogenic Cells 0.01330~0.01530 Skin
0.00804~0.00925 Spleen 0.02630~0.03170 Testes 0.00896~0.01030
Thymus 0.01010~0.01120 Thyroid 0.00936~0.01070 Urinary Bladder Wall
0.01020~0.01170 Uterus 0.01080~0.01230 Total Body 0.01460~0.01460
Effective Dose Equivalent 0.02510~0.02970 Effective Dose
0.01510~0.01590
Example 16
[0199] This example demonstrates dosimetry of an imaging compound
in healthy volunteers, in accordance with an embodiment of the
invention.
[0200] With a mean injected dose of 3.77.+-.0.28 mCi, no adverse
symptoms were noticed and/or reported during the entire procedure
and 2 weeks follow-up, demonstrating the safety of the tracer. A
representative PET image acquired at 30 min after intravenous
administration of .sup.68Ga-NEB is presented in FIGS. 27A and 27B.
Cardiac ventricles, major arteries and veins showed the highest
signal density. Vessel branches in and out of major organs and
limbs can also be clearly identified. The liver, spleen and kidneys
are also visible with relatively lower activity whereas the bladder
showed high activity.
[0201] From 5 to 90 min, no dramatic distribution change was
observed, confirming the in vivo stability and long blood pool
retention of .sup.68Ga-NEB (FIG. 28). Increased bladder
accumulation was observed over time. The average standardized
uptake values in the major organs and tissues are listed in Table
3. Although the blood vessels in the brain showed high
radioactivity, the normal brain tissue had negligible accumulation
of .sup.68Ga-NEB, indicating that the tracer does not cross the
blood-brain barrier.
[0202] The mean absorbed radiation doses based on multiple time
point PET imaging of three volunteers were similar to those based
on mouse biodistribution data. The major organs that received
relatively high doses were kidneys, liver, spleen, and heart wall.
The bladder wall also received high exposure due to renal excretion
of the radioactivity (0.0683.+-.0.0090 mSv/MBq). The whole body
absorbed dose was 0.0151.+-.0.0001 mSv/MBq with an effective dose
of 0.0179.+-.0.0003 mSv/MBq.
TABLE-US-00003 TABLE 3 Time (min) Blood Lung Liver Spleen Kidneys
Stomach S. Intestine Pancreas Bone Muscle 5 7.97 .+-. 0.15 1.53
.+-. 0.15 3.80 .+-. 0.44 4.10 .+-. 0.36 4.40 .+-. 0.26 1.23 .+-.
0.15 1.73 .+-. 0.25 2.13 .+-. 0.40 2.10 .+-. 0.26 0.53 .+-. 0.21 10
7.53 .+-. 0.25 1.47 .+-. 0.29 3.70 .+-. 0.61 4.07 .+-. 0.47 4.23
.+-. 0.49 1.50 .+-. 0.20 1.57 .+-. 0.12 2.03 .+-. 0.15 1.93 .+-.
0.12 0.60 .+-. 0.10 15 7.17 .+-. 0.35 1.4 .+-. 0.17 3.73 .+-. 0.59
4.00 .+-. 0.52 4.03 .+-. 0.50 1.23 .+-. 0.12 1.73 .+-. 0.12 1.90
.+-. 0.26 1.83 .+-. 0.15 0.60 .+-. 0.17 30 6.93 .+-. 0.21 1.30 .+-.
0.17 3.57 .+-. 0.46 3.90 .+-. 0.61 3.77 .+-. 0.29 1.23 .+-. 0.06
1.63 .+-. 0.15 1.67 .+-. 0.15 1.67 .+-. 0.15 0.57 .+-. 0.12 45 6.70
.+-. 0.26 1.30 .+-. 0.20 3.50 .+-. 0.52 3.77 .+-. 0.57 3.87 .+-.
0.32 1.23 .+-. 0.06 1.63 .+-. 0.06 1.67 .+-. 0.15 1.50 .+-. 0.20
0.53 .+-. 0.06 60 6.20 .+-. 0.17 1.23 .+-. 0.06 3.47 .+-. 0.47 3.73
.+-. 0.42 3.60 .+-. 0.10 1.10 .+-. 0.00 1.47 .+-. 0.15 1.43 .+-.
0.15 1.37 .+-. 0.06 0.50 .+-. 0.00 75 6.03 .+-. 0.12 1.10 .+-. 0.17
3.43 .+-. 0.49 4.93 .+-. 2.10 3.43 .+-. 0.15 1.07 .+-. 0.12 1.33
.+-. 0.21 1.37 .+-. 0.21 1.27 .+-. 0.12 0.47 .+-. 0.06 90 5.97 .+-.
0.15 1.07 .+-. 0.12 3.33 .+-. 0.58 3.57 .+-. 0.55 3.30 .+-. 0.20
1.03 .+-. 0.15 1.17 .+-. 0.32 1.37 .+-. 0.31 1.23 .+-. 0.15 0.77
.+-. 0.64
Example 17
[0203] This example demonstrates the differential diagnosis of
focal hepatic lesions, in accordance with an embodiment of the
invention.
[0204] The widespread use of imaging studies has led to an increase
in detection of incidental focal hepatic lesions (FLLs).
Differential diagnosis of malignant and benign solid and cystic
liver lesions is very important for patient management. Among the
11 patients with focal hepatic lesion(s) diagnosed by enhanced CT
and/or MRI, 4 were with hemangioma. All hemangiomas showed much
higher .sup.68Ga-NEB signal intensity than the surrounding normal
hepatic tissues, while no apparent difference between lesions and
hepatic tissues was identified on FDG PET. The lesions were not
discernable on regular CT but showed signal enhancement with CT
contrast agent (FIG. 29A-29H). Hepatocellular carcinoma (HCC)
showed high tracer uptake on FDG PET but with big variance from
patient to patient. .sup.68Ga-NEB showed consistently lower HCC
uptake than normal hepatic tissue (FIG. 30 and FIGS. 31A-31C).
Similarly, hepatic cysts and neuroendocrine liver metastases also
showed low lesion/background ratio with .sup.68Ga-NEB PET (FIG.
30).
[0205] Due to the abundant blood supply, normal liver tissue showed
prominent .sup.68Ga-NEB accumulation with a standard uptake value
(SUV) of 3.73.+-.0.47 (Table 4). The SUV of .sup.68Ga-NEB in
hemangiomas (6.83.+-.1.38) was much higher than that in the
surrounding hepatic tissue (P<0.01). All other focal hepatic
lesions including HCC, hepatic cysts and neuroendocrine tumor liver
metastases showed negative contrast to hepatic tissues with SUVs of
2.12.+-.0.16, 2.13, and 2.69.+-.0.44, respectively.
TABLE-US-00004 TABLE 4 .sup.18Ga-NEB .sup.18F-FDG HCC (n = 7) 2.12
.+-. 0.16 5.96 .+-. 2.90 NET Met (n = 4) 2.69 .+-. 0.44 2.85 .+-.
0.70 Hemangioma (n = 5) 6.83 .+-. 1.38 1.19 .+-. 0.19 Cyst (n = 1)
2.13 1.21 Normal hepatic tissue (n = 11) 3.69 .+-. 0.53 1.80 .+-.
0.41
[0206] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0207] The use of the terms "a" and "an" and "the" and "at least
one" and similar referents in the context of describing the
invention (especially in the context of the following claims) are
to be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
use of the term "at least one" followed by a list of one or more
items (for example, "at least one of A and B") is to be construed
to mean one item selected from the listed items (A or B) or any
combination of two or more of the listed items (A and B), unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0208] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
* * * * *