U.S. patent application number 15/832371 was filed with the patent office on 2018-07-12 for positron emitting radionuclide labeled peptides for human upar pet imaging.
The applicant listed for this patent is CURASIGHT APS. Invention is credited to Andreas Kjaer, Jacob Madsen, Morten Persson.
Application Number | 20180193495 15/832371 |
Document ID | / |
Family ID | 50882823 |
Filed Date | 2018-07-12 |
United States Patent
Application |
20180193495 |
Kind Code |
A1 |
Kjaer; Andreas ; et
al. |
July 12, 2018 |
POSITRON EMITTING RADIONUCLIDE LABELED PEPTIDES FOR HUMAN UPAR PET
IMAGING
Abstract
There is provided a positron-emitting radionuclide labelled
peptide for non-invasive PET imaging of the Urokinase-type
Plasminogen Activator Receptor (uPAR) in humans. More specifically
the invention relates to human uPAR PET imaging of any solid cancer
disease for diagnosis, staging, treatment monitoring and especially
as an imaging biomarker for predicting prognosis, progression and
recurrence.
Inventors: |
Kjaer; Andreas;
(Frederiksberg, DK) ; Persson; Morten;
(Copenhagen, DK) ; Madsen; Jacob; (Copenhagen,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CURASIGHT APS |
Copenhagen |
|
DK |
|
|
Family ID: |
50882823 |
Appl. No.: |
15/832371 |
Filed: |
December 5, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14649113 |
Jun 2, 2015 |
9884131 |
|
|
PCT/DK2013/050402 |
Nov 29, 2013 |
|
|
|
15832371 |
|
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|
61732443 |
Dec 3, 2012 |
|
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 51/08 20130101;
A61K 51/088 20130101; A61K 38/08 20130101; A61K 51/083
20130101 |
International
Class: |
A61K 51/08 20060101
A61K051/08; A61K 38/08 20060101 A61K038/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 3, 2012 |
DK |
PA 2012 70751 |
Claims
1. A conjugate comprising a positron-emitting radionuclide labelled
peptide, wherein said conjugate comprises a uPAR binding peptide
coupled via NOTA, to a .sup.68Ga radionuclide.
2. The conjugate according to claim 1, wherein the peptide is
selected from the group consisting of:
(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(-
Trp)-(Ser),
(Ser)-(Leu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)--
(Trp)-(Ser),
(D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(-
Trp)-(Ser),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Tr-
p)-(Ser),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Le-
u)-(Trp)-(Ser),
(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp-
)-(Ser),
(D-Thr)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-
-(Leu)-(Trp)-(Ser),
(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(-
[beta]-2-naphthyl-L-alanine)-(Ser),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-
-(Ser),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(L-
eu)([beta]-1-naphthyl-L-alanine)-(Ser),
(D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Tr-
p)-(Ser),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)--
(Leu)-(Trp)-(D-His),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([beta]-cyclohe-
xyl-L-alanine)-(Leu)-(Trp)-(Ile),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([be-
ta]-1-naphthyl-L-alanine)-(D-His),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimetho-
xybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)g-
lycine),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-
-dimethoxybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[beta]thoxyethyl)-
glycine),
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,-
3-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-met-
hoxyethyl)glycine), and
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimetho-
xybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile),
wherein the C-terminal is either a carboxylic acid or an amide.
3. The conjugate according to claim 1, wherein the peptide is
(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(-
Trp)-(Ser).
4. The conjugate according to claim 1, having the formula:
##STR00005##
5. The conjugate of claim 1, further comprising at least one
pharmaceutical acceptable adjuvant, excipient or diluents.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/649,113, filed 2 Jun. 2015, which is a National Stage
Application of PCT/DK2013/050402, filed 29 Nov. 2013, which claims
benefit of Serial No. 2012 70751, filed 3 Dec. 2012 in Denmark, and
which claims benefit of Ser. No. 61/732,443, filed 3 Dec. 2012 in
the United States, and which application(s) are incorporated herein
by reference. To the extent appropriate, a claim of priority is
made to each of the above disclosed applications.
FIELD OF THE INVENTION
[0002] The present invention relates to a positron-emitting
radionuclide labelled peptide for noninvasive PET imaging of the
Urokinase-type Plasminogen Activator Receptor (uPAR) in humans.
More specifically the invention relates to human uPAR PET imaging
of any solid cancer disease for diagnosis, staging, treatment
monitoring and especially as an imaging biomarker for predicting
prognosis, progression and recurrence.
BACKGROUND OF THE INVENTION
[0003] Urokinase-type plasminogen activator receptor (uPAR) is
over-expressed in a variety of human cancers.sup.1, including
prostate cancer (PC), where uPAR expression in tumor biopsies and
shed forms of uPAR in plasma have been found to be associated with
advanced disease and poor prognosis.sup.2-9. Moreover, in patients
with localized PC, high preoperative plasma uPAR levels have been
shown to correlate with early progression.sup.10. Consistent with
uPARs important role in cancer pathogenesis, through extracellular
matrix degradation facilitating tumor invasion and metastasis, uPAR
is considered an attractive target for both therapy.sup.11-13 and
imaging 14 and the ability to non-invasively quantify uPAR density
in vivo is therefore crucial.
[0004] Radiolabeling and in vivo evaluation of a small peptide
radiolabeled with Cu-64.sup.15 and Ga-68.sup.16 have been described
for PET imaging of uPAR in various human xenograft cancer models.
Such tracers could specifically differentiate between tumors with
high and low uPAR expression and furthermore established a clear
correlation between tumor uptake of the uPAR PET probe and the
expression of uPAR.sup.15. However, .sup.18F (t.sub.1/2=109.7 min;
.beta.+, 99%) is considered the ideal short-lived PET isotope for
labeling of small molecules and peptides due to the high positron
abundance, optimal half-life and short positron range.
[0005] Recently, an elegant one step radiolabeling approach was
developed for radiofluorination of both small peptides and proteins
based on complex binding of (Al.sup.18F).sup.2+ using
1,4,7-triazacyclononane (NOTA) chelator.sup.17-20. In this method,
the traditional critical azeotropic drying step for 18F-fluoride is
not necessary, and the labeling can be performed in water. A number
of recently published studies have illustrated the potential of
this new .sup.18F-labeling method, where successful labeling of
ligands for PET imaging of angiogenesis.sup.21,22, Bombesin.sup.23,
EGFR.sup.24 and hypoxia.sup.25 have been demonstrated.
[0006] Various radio-labelled peptide compositions have been
developed or are under development for site-specific targeting of
various antigens, receptors and transporters for PET imaging. The
general principle involves attaching a selected positron emitting
radionuclide to a peptide and/or protein having a high specificity
for a particular antigen for visualize and quantify the expressing
and/or activity level using PET imaging. This field of research has
shown particular applicability for tumor diagnosis, staging and
treatment monitoring. A particularly desirable tumor antigen is
uPAR in many different solid tumors including but not limited to
non-small cell lung carcinomas, brain tumors, prostate tumors,
breast tumors, colorectal tumors, pancreatic tumors and ovarian
tumors.
[0007] DOTA (1,4,7, 10-tetrakis(carboxymethyl)-1,4,7, 10
tetraazacyclo dodecane) and its derivatives constitute an important
class of chelators for biomedical applications as they accommodate
very stably a variety of di- and trivalent metal ions. An emerging
area is the use of chelator conjugated bioactive peptides for
labelling with radiometals in different fields of diagnostic and
therapeutic nuclear oncology. NOTA and its derivatives constitute
another important class of chelators for biomedical
applications.
[0008] uPAR PET imaging has been exploited in several human cancer
xenograft models using a small linear DOTA-conjugated peptide,
DOTA-AE105 radiolabeled with .sup.64Cu (Li et al, 2008, Persson et
al, 2011) and .sup.68Ga (Persson et al, 2012) and using NODAGA
(NODAGA-AE105) radiolabeled with .sup.68Ga (Persson et al,
2012).
[0009] Malignant tumors are capable of degrading the surrounding
extracellular matrix, resulting in local invasion or metastasis.
Urokinase-type plasminogen activator (uPA) and its cell surface
receptor (uPAR) are central molecules for cell surface-associated
plasminogen activation both in vitro and in vivo. High expression
of uPA and uPAR in many types of human cancers correlate with
malignant tumor growth and associate with a poor prognosis,
possibly indicating a causal role for the uPA/uPAR system in cancer
progression and metastasis. Studies by immunohistochemistry and in
situ hybridization indicate that expression levels of the
components from the uPA system are generally very low in normal
tissues and benign lesions. It has also been reported that the
uPA/uPAR system is involved in regulating cell-extracellular matrix
interactions by acting as an adhesion receptor for vitronectin and
by modulating integrin function. Based on these properties, the
uPA/uPAR system is consequently considered an attractive target for
cancer therapy.
[0010] WO 01/25410 describes diagnostically or therapeutically
labelled uPAR-targeting proteins and peptides. The peptide or
protein comprises at least 38 amino acid residues, including
residues 13-30 of the uPAR binding site of uPA.
[0011] U.S. Pat. No. 6,277,818 describes uPAR-targeting cyclic
peptide compounds that may be conjugated with a diagnostic label.
The peptides are based on the amino acid residues 20-30 of uPA.
[0012] U.S. Pat. No. 6,514,710 is also directed to cyclic peptides
having affinity for uPAR. The peptides may carry a detectable
label. The peptide comprises 11 amino acids joined by a linking
unit.
[0013] Ploug et al. in Biochemistry 2001, 40, 12457-12168 describes
uPAR targeting peptides but not in the context of imaging,
including amino acid sequences as described in the present
document. Similar disclosure is provided in U.S. Pat. No.
7,026,282.
[0014] The efficient targeting of uPAR demands a selective
high-affinity vector that is chemically robust and stable.
SUMMARY OF THE INVENTION
[0015] The present inventors have surprisingly found that
[.sup.68Ga]-, [.sup.64Cu]- and [Al.sup.18F]-NOTA-AE105 have
superior in vivo characteristics as a uPAR PET ligand, with high
and specific tumor uptake, thus resulting in a high
tumor-to-background ratio and thereby superior contrast as a PET
ligand for uPAR expression tumors. The inventors have found that
both [.sup.68Ga]- and [.sup.64Cu]-NOTA-AE105 was able to
specifically detect uPAR expressing human-derived brain tumor
lesions in a orthotropic human cancer mouse model. Moreover, [Al
18F]-NOTA-AE105 was useful to detect uPAR positive human prostate
cancer lesions after subcutaneously inoculation in mice. Overall,
the radiolabeling of NOTA-AE105 with .sup.18F, .sup.68Ga and
.sup.64Cu, thus enable the visualization and quantification of uPAR
using PET Imaging. This is a major improvement in PET Imaging.
[0016] The present invention thus provides a positron-emitting
radionuclide labelled peptide conjugate for use in the
prediction/diagnosis of aggressiveness, prognosis, progression or
recurrence by PET imaging of uPAR expressing, and in particular
uPAR overexpressed tumors, said conjugate comprising a uPAR binding
peptide coupled via a chelating agent or covalently to a
radionuclide selected from 18F, 64Cu, 68Ga, 66Ga, 60Cu, 61Cu, 62Cu,
89Zr, 1241, 76Br, 86Y, and 94mTc, wherein the conjugate is
administered in a diagnostically effective amount, such as a dose
of 100-500 MBq followed by PET scan 1/2-24 h after the conjugate
has been administered, and quantification through SUVmax and/or
SUVmean.
[0017] In a preferred embodiment the peptide is selected from the
group consisting of: [0018]
(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(-
Trp)-(Ser), [0019]
(Ser)-(Leu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Gln)-(Tyr)(Leu)--
(Trp)-(Ser), [0020]
(D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Tyr)-(Tyr)-(Leu)-(-
Trp)-(Ser), [0021]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(Tr-
p)-(Ser), [0022]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp)--
(Ser), [0023]
(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(Ser)-(D-Arg)-(Tyr)-Leu)-(Trp-
)-(Ser), [0024]
(D-Thr)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(-
Trp)-(Ser), [0025]
(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)-(-
[beta]-2-naphthyl-L-alanine)-(Ser), [0026]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Arg)-(Tyr)-(Leu)-(Trp)-
-(Ser), [0027]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([be-
ta]-1-naphthyl-L-alanine)-(Ser), [0028]
(D-Glu)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(Tyr)-(Tyr)-(Leu)-(Tr-
p)-(Ser), [0029]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Leu)-(Leu)-(Tr-
p)-(D-His), [0030]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-([beta]-cyclohe-
xyl-L-alanine)-(Leu)-(Trp)-(Ile), [0031]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)([be-
ta]-1-naphthylL-alanine)-(D-His), [0032]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimetho-
xybenzyl)glycine)-(D-Phe)-(N-(3-indolylethyl)glycine)-(N-(2-methoxyethyl)g-
lycine), [0033]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimetho-
xybenzyl)glycine)-(D-Phe)-(N-benzylglycine)-(N-(2[beta]thoxyethyl)glycine)-
, [0034]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-
-dimethoxybenzyl)glycine)-(D-Phe)-(N-(methylnaphthalyl)glycine)-(N-(2-meth-
oxyethyl)glycine), and [0035]
(Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(N-(2,3-dimetho-
xybenzyl)glycine)-(D-Phe)-(N-(2,3-dimethoxybenzyl)glycine)-(Ile).
[0036] For all peptides mentioned, the C-terminal can be either
with a carboxylic acid or an amide.
[0037] Preferably the chelating agent is DOTA, NOTA, CB-TE2A or
NODAGA, and preferably the peptide is
(D-Asp)-([beta]-cyclohexyl-L-alanine)-(Phe)-(D-Ser)-(D-Arg)-(Tyr)-(Leu)(T-
rp)-(Ser).
[0038] Particularly preferred are the conjugates having the
formulas:
##STR00001## ##STR00002## ##STR00003##
[0039] The present inventors have surprisingly found that the
conjugates of the present invention are particularly useful in
predicting aggressiveness, prognosis, progression or recurrence by
PET imaging of uPAR expressing tumors, in particular prostate,
breast, pancreatic, lung, brain and colorectal cancer.
[0040] The present invention also provides a method for
predicting/diagnosing the aggressiveness, prognosis, progression or
recurrence of uPAR overexpressed tumors, wherein the method
comprises the steps of: [0041] administrating a conjugate of the
present invention in a diagnostically effective amount, such as a
dose of 100-500 MBq; [0042] PET scanning 1/2-24 h after the
conjugate has been administered. [0043] quantifying through SUVmax
and/or SUVmean the absorption/binding of the conjugate in the
tumor.
[0044] The steps carried out in accordance with the present
invention can be summarized with the flow diagram shown in FIG.
8.
[0045] The present conjugates for use in accordance with the
present invention can discriminate between uPAR expression levels
in the primary tumor and metastases. Also, the use of
quantification e.g. SUVmean and especially SUVmax can predict
prognosis, progression and recurrence. The positron-emitting
radionuclide labelled peptides of the present invention
specifically target uPAR-positive cancer cells and/or uPAR positive
stroma cells surrounding the cancer such as neutrophils and
macrophages, and in particular the most aggressive (metastatic)
cells. Moreover, the peptides of the present invention can be used
for non-invasive detection and quantification of the expression
level of uPAR using PET imaging. No current methods measuring uPAR
is capable of this non-invasively in humans.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1A shows In vitro competitive inhibition of the
uPA:uPAR binding for AE105 and AE152 using surface Plasmon
resonance.
[0047] FIG. 1B shows Radiolabeling method for
.sup.18F-AlF-NOTAAE105.
[0048] FIG. 2A shows representative HPLC UV chromatograms of
NOTA-AE105.
[0049] FIG. 2B shows cold standard AlF-NOTA-AE105.
[0050] FIG. 2C shows radio chromatograms for the final product
.sup.18F-AlFNOTA-AE105.
[0051] FIG. 2D shows radio chromatograms for the final product
.sup.18F-AlFNOTA-AE105 and after 30 min in PBS.
[0052] FIG. 3A shows Representative PET images after 0.5 h, 1.0 h
and 2.0 h p.i of .sup.18F-AlF-NOTA-AE105 (top) and
.sup.18F-AlF-NOTA-AE105 with a blocking dose of AE152. White arrows
indicate tumor.
[0053] FIG. 3B shows quantitative ROI analysis with tumor uptake
values (% ID/g). A significant higher tumor uptake was found at all
three time points. Results are shown as % ID/g.+-.SEM
(n=4mice/group). ** p<0.01, *** p<0.001 vs blocking group at
same time point.
[0054] FIG. 4 shows biodistribution results for
.sup.18F-AlF-NOTA-AE105 (normal) and
.sup.18F-AlFNOTA-AE105+blocking dose of AE152 (Blocking) in nude
mice bearing PC-3 tumors at 2.5 h p.i. Results are shown as %
ID/g.+-.SEM (n=4mice/group). *p<0.05 vs blocking group.
[0055] FIG. 5A shows uPAR expression level found using ELISA in
PC-3 cells.
[0056] FIG. 5B shows uPAR expression level found using ELISA in
PC-3 cells in resected PC-3 tumors.
[0057] FIG. 5C shows a significant correlation between uPAR
expression and tumor uptake was found in the four mice injected
with 18F-AlF-NOTA-AE105 (p<0.05, r=0.93, n=4 tumors).
[0058] FIG. 6 shows in vivo uPAR PET imaging with
[.sup.64Cu]NOTA-A.E105 in a orthotropic human glioblastoma mouse
model
[0059] FIG. 7 shows in vivo uPAR PET imaging with
[.sup.68Ga]NOTA-AE105 in a orthotropic 5 human glioblastoma mouse
model.
[0060] FIG. 8 shows a flow diagram summarizing the steps carried
out in accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0061] Surprisingly, a radiolabeled peptide of the present
invention is very useful in the prediction of cancer metastasis of
uPAR expressing tumors.
[0062] The peptides selected for use in the conjugates of the
present invention are typically radiolabeled by coupling a
chelating agent to the peptide. The chelating agent is capable of
binding a selected radionuclide thereto. The chelating agent and
radionuclide is coupled to the peptide in a manner that does not
interfere or adversely affect the binding properties or specificity
of the peptide. The use of various chelating agents for radio
labelling of peptides is well known in the art. The chelating agent
is coupled to the peptide by standard methodology known in the
field of the invention and may be added at any location on the
peptide provided that the biological activity of the peptide is not
adversely affected. Preferably, the chelating group is covalently
coupled to the amino terminal amino acid of the peptide. The
chelating group may advantageously be attached to the peptide
during solid phase peptide synthesis or added by solution phase
chemistry after the peptide has been obtained. Preferred chelating
groups include DOTA, NOTA, NODAGA or CB-TE2A.
[0063] Concerning the synthesis of the peptides used in the present
invention reference is made to U.S. Pat. No. 7,026,282.
[0064] The peptide/chelate conjugates of the invention are labeled
by reacting the conjugate with radionuclide, e.g. as a metal salt,
preferably water soluble. The reaction is carried out by known
methods in the art.
[0065] The conjugates of the present invention are prepared to
provide a radioactive dose of 35 between about 100-500 MBq (in
humans), preferably about 200-400 MBq, to the individual. As used
herein, "a diagnostically effective amount" means an amount of the
conjugate sufficient to permit its detection by PET. The conjugates
may be administered intravenously in any conventional medium for
intravenous injection. Imaging of the biological site may be
effected within about 30-60 minutes post-injection, but may also
take place several hours post-injection. Any conventional method of
imaging for diagnostic purposes may be utilized.
[0066] The following example focuses on the specific conjugate
denoted .sup.18F-AlF-NOTA-AE105. Other conjugates within the scope
of the claims herein will be apparent to one skilled in the art
from consideration of the specification or practice of the
invention as disclosed herein.
[0067] The following chemistry applies to the Examples: [0068]
AE105 (Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH) (1)
[0069] The peptide according to the above mentioned sequence was
synthesized by standard solid-phase peptide chemistry. [0070]
NOTA-AE105 (NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH)
##STR00004##
[0071] The product is purified by RP-HPLC and analysed by RP-HPLC
(retension time: 11.5 min, purity >98%) and electrospray-MS
(1510.8 m.u.).
Example 1
[0072] The aim of the present study was to synthesize a
NOTA-conjugated peptide and use the Al.sup.18F method for
development for the first .sup.18F-labeled PET ligand for uPAR PET
imaging and to perform a biological evaluation in human prostate
cancer xenograft tumors. To achieve this, the present inventors
synthesized high-affinity uPAR binding peptide denoted AE105 and
conjugated NOTA in the N-terminal. .sup.18F-labeling was done
according to a recently optimized protocol.sup.26. The final
product (.sup.18F-AlF-NOTA-AE105) was finally evaluated in vivo
using both microPET imaging in human prostate tumor bearing animals
and after collection of organs for biodistribution study.
Chemical Reagents
[0073] All chemicals obtained commercially were of analytical grade
and used without further purification. No-carrier-added
.sup.18F-fluoride was obtained from an in-house PETtrace cyclotron
(GE Healthcare). Reverse-phase extraction C18 Sep-Pak cartridges
were obtained from Waters (Milford, Mass., USA) and were pretreated
with ethanol and water before use. The syringe filter and
polyethersulfone membranes (pore size 0.22 .mu.m, diameter 13 mm)
were obtained from Nalge Nunc International (Rochester, N.Y., USA).
The reverse-phase HPLC using a Vydac protein and peptide column
(218TP510; 5 .mu.m, 250.times.10 mm) was performed as previously
described.sup.21.
[0074] MicroPET scans were performed on a microPET R4 rodent model
scanner (Siemens Medical Solutions USA, Inc., Knoxville, Tenn.,
USA). The scanner has a computer-controlled bed and 10.8-cm
transaxial and 8-cm axial fields of view (FOVs). It has no septa
and operates exclusively in the three-dimensional (3-D) list mode.
Animals were placed near the center of the FOV of the scanner.
Peptide Synthesis, Conjugation and Radiolabeling
[0075] NOTA-conjugated AE105
(NOTA-Asp-Cha-Phe-(D)Ser-(D)Arg-Tyr-Leu-Trp-Ser-COOH) was purchased
from ABX GmbH. The purity was characterized using HPLC analysis and
the mass was confirmed using matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) (Se Suppl. FIG. 1A). The radiolabeling of NOTAAE105
with .sup.18F-AlF is shown in FIG. 1 and was done according to a
recently published protocol with minor modifications.sup.26.
[0076] In brief, a QMA Sep-Pak Light cartridge (Waters, Milford,
Ma, USA) was fixed with approximately 3 GBq of 18F-fluoride and
then washed with 2.5 ml of metal free water. Na18F was then eluted
from the cartridge with 1 ml saline, from which 100 .mu.l fraction
was taken. Then amounts of 50 .mu.l 0.1 M Na-Acetate buffer (pH=4),
3 .mu.l 0.1 M AlCl3 and 100 .mu.l of Na.sup.18F in 0.9% saline (300
MBq) were first reacted in a 1 ml centrifuge tube (sealed) at
100.degree. c. for 15 min. The reaction mixture was cooled. 50
.mu.l ethanol and 30 nmol NOTA-AE105 in 3 .mu.l DMSO were added and
the reaction mixture were heated to 95.degree. C. for 5 min. The
crude mixture was purified with a semi-preparative HPLC. The
fractions containing .sup.18F-AlF-NOTA-AE105 were collected and
combined in a sterile vial. The product was diluted in
phosphate-buffered saline (PBS, pH=7.4) so any organic solvents
were below 5% (v/v) and used for in vivo studies.
Cell Line and Animal Model
[0077] Human prostate cancer cell line PC-3 was obtained from the
American Type Culture Collection (Manassas, Va., USA) and culture
media DMEM was obtained from Invitrogen Co. (Carlsbad, Calif.,
USA). The cell line was cultured in DMEM supplemented with 10%
(v/v) fetal bovine serum and 1% (v/v) penicillin/Streptomycin at
37.degree. C. and 5% CO.sub.2. Xenografts of human PC-3 prostate
cancer cells were established by injection of 200 .mu.l cells
(1.times.10.sup.8 cells/ml) suspended in 100 .mu.l Matrigel (BO
Biosciences, San Jose, Calif., USA), subcutaneously in the right
flank of male nude mice obtained from Charles River Laboratory
(Wilmington, Mass. USA), Tumors were allowed to grow to a size of
200-500 mg (3-4 weeks).
MicroPET Imaging
[0078] Three min static PET scans were acquired 0.5, 1.0 and 2.0 h
post injection (p.i) of .sup.18FAlF-NOTA-AE105 via tail-vein
injection of 2-3 MBq (n=4). Similar, the blocking study was
performed by injection of the ligand together with 100 .mu.g of
AE152 (uPAR antagonist) through the tail vein (n=4) and PET scanned
at the same time points. During each three minutes PET scan, mice
were anesthetized with isoflurane (5% induction and 2% 30
maintenance in 100% O.sub.2). Images were reconstructed using a
two-dimensional ordered subsets expectation maximization (OSEM-2D)
algorithm. No background correction was performed. All results were
analyzed using Inveon software (Siemens Medical Solutions) and PET
data was expressed as percent of injected dose per gram tissue (%
ID/g) based on manual region-of-interest drawing on PET images and
the use of a calibration constant. An assumption of a tissue
density of 1 g/ml was used. No attenuation correction was
performed.
Biodistribution Studies
[0079] After the last PET scan, all PC-3 bearing mice were
euthanized. Blood, tumor and major organs were collected
(wet-weight) and the radioactivity was measured using a y-counter
from Perkin Elmer, MA, USA (N=4 mice/group).
uPAR ELISA
[0080] uPAR ELISA on resected PC-3 tumors was done as described
previously in detail.sup.15. All results were performed as
duplicate measurements.
Statistical Analysis
[0081] All quantitative data are expressed as mean.+-.SEM (standard
error of the mean) and means were compared using Student t-test.
Correlation statistics was done using linear regression analysis. A
P-value of s 0.05 were considered statistically significant.
uPAR Binding Affinity
[0082] The uPAR binding affinity of AE105 and AE152.sup.27 (used
for blocking studies) was in this 20 study found to be 14.1 nM and
2.9 nM, respectively (FIG. 1A). A high uPAR binding affinity for
AE105 with different chelators conjugated in the N-terminal,
including the NOTA analogue NODAGA, has been confirmed in our
previously studies.sup.15, 16, thus confirming the ability to make
modifications in the N-terminal of AE105 without losing affinity
towards human uPAR.sup.14, 27, 28.
Radiochemistry
[0083] The .sup.18F-labeling of NOTA-AE105 was synthesized based on
a recently published procedure with some modification (FIG. 1B).
During our labeling optimization, we found that 33% ethanol (v/v)
was optimal using 30 nmol NOTA-AE105. We first formed the 18F-AlF
complex in buffer at 100.degree. C. for 15 min. Secondly was the
NOTA-conjugated peptide added and incubated together with Ethanol
at 95.degree. C. for 5 min. By adding ethanol we were able to
increase the overall yield to above 92.7% (FIG. 2C), whereas the
yield without ethanol was only 30.4%, with otherwise same
conditions. No further increase in the overall yield was observed
using longer incubation time and/or different ethanol
concentrations or using less than 30 nmol conjugated peptide. Two
isomers were observed for .sup.18F-AlF-NOTA-AE105.
[0084] In order to ensure the formation of the right product, a
cold standard of the final product was synthesis (AlF-NOTA-AE105).
The HPLC analysis of the precursor (NOTA-AE105, FIG. 2A) confirmed
the purity of the NOTA-conjugated precursor (>97%) and MALDI-MS
confirmed the mass (1511.7 Da) (See suppl. FIG. 1). The cold
standard (AlFNOTA-AE105, FIG. 2B), with the right mass confirmed by
MALDI-MS (1573.6 Da) (See suppl. FIG. 1B), corresponded well in
regards to retention time with the `hot` product (FIG. 2C), thus
confirming the formation of 18F-AlF-NOTA-AE105 (FIG. 2C). No
degradation of the final product was found after 30 min in PBS
(FIG. 2D). The radioactive peaks were collected and diluted in PBS
and used for in vivo studies. The specific activity in the final
product was above 25 GBq/.mu.mol.
In Vivo PET Imaging
[0085] .sup.18F-AlF-NOTA-AE105 was injected i.v. in four mice
bearing PC-3 tumors and PET scan were performed 0.5, 1.0 and 2.0 h
post injection (p.i). Tumor lesions were easily identified from the
reconstructed PET images (FIG. 3A) and ROI analysis revealed a high
tumor uptake, with 5.90.+-.0.35% ID/g after 0.5 h, declining to
4.22.+-.0.13% ID/g and 2.54.+-.0.24% ID/g after 1.0 and 2.0 h,
respectively (FIG. 3B).
[0086] In order to ensure that the found tumor uptake did indeed
reflect specific uPAR mediated uptake, four new PC-3 tumor bearing
mice were then injected with a mixed solution containing
.sup.18F-AlF-NOTA-AE105 and 100 .mu.g of the high-affinity uPAR
binding peptide denoted AE152, in order to see if the tumor uptake
could be inhibited. A significant lower amount of
.sup.18F-AlF-NOTA-AE105 tumor uptake was found at all three time
points investigate (FIG. 3B) and tumor lesions were not as easily
identified in the PET images (FIG. 3A). At 1.0 h p.i a tumor uptake
of 1.86.+-.0.14% ID/g was found in the blocking group compared with
4.22.+-.0.13% ID/g found in the group of mice receiving only
.sup.18F-AlFNOTA-AE105 (p<0.001, 2.3 fold reduction).
Biodistribution
[0087] After the last PET scan, each group of mice where euthanized
and selected organs and tissues were collected to investigate the
biodistribution profile 2.5 h p.i. (FIG. 4). A significant higher
tumor uptake in the group of mice receiving .sup.18F-AlF-NOTA-AE105
was found compared with blocking group (1.02.+-.0.37% ID/g vs.
0.30.+-.0.06% ID/g, p<0.05), thus confirming the specificity of
.sup.18F-AlF-NOTA-AE105 for human uPAR found in the PET study.
Highest activity was found in the kidneys for both groups of mice,
confirming the kidneys to be the primarily route of excretion.
Beside kidneys, the bone, well known to accumulate fluoride, also
had a relatively high uptake of 3.54.+-.0.32% ID/g and
2.34.+-.0.33% ID/g for normal and blocking group, respectively.
uPAR Expression
[0088] Both the PC-3 cells used for tumor inoculation and all PC-3
tumors at the end of the study (n=8) were finally analyzed for
confirming expression of human uPAR (FIG. 5). An expression in the
cells of 6.53.+-.1.6 ng/mg protein was found (FIG. 5A), whereas the
expression level in the resected tumors was 302.+-.129 .mu.g/mg
tumor tissue (FIG. 5B). A significant correlation between tumor
uptake of 18F-AlF-NOTA-AE105 and uPAR expression was found
(p<0.05, r=0.93) (FIG. 5C).
Data Interpretation
[0089] The above experiments provide evidence for the applicability
of an 18F-labeled ligand for 15 uPAR PET. The ligand was
characterized in a human prostate cancer xenograft mouse model.
Based on the obtained results, similar tumor uptake, specificity
and tumor-to-background contrast were found compared to our
previously published studies using 64Cu- and 68Ga-based ligands for
PET.sup.15, 16. Based on the superior physical characteristics of
18F and the high tumor-to-background contrast found already after 1
h p.i, our new 18F-based ligand must be considered the so far most
promising uPAR PET candidate for translation into clinical use in
order to non-invasively characterize invasive potential of e.g.
prostate cancer.
[0090] .sup.18F-labeling of peptides using the AlF-approach has
previously been described to be performed at 100.degree. c. for 15
min, at pH=4.sup.17-20. This protocol was modified, since
degradation of the NOTA-conjugated peptide was observed using these
conditions. The present inventors therefore first produced the
.sup.18F-AlF complex using the above mentioned conditions and next
added the NOTA-conjugated peptide and lowered the temperature to
95.degree. C., and within 5 min obtained a labeling yield of 92.7%
and with no degradation of the peptide. Two isomers of
.sup.18F-AlF-NOTA-AE105 were produced. Same observations have been
reported by others for .sup.18F-AlF-NOTA-Octreotide.sup.18 and all
NOTA-conjugated IMP peptide analogues described.sup.19. The ratio
of the two peaks were nearly constant for each labeling and both
radioactive peaks were collected and used for further in vivo
studies. This approach was recently also described by
others.sup.26.
[0091] Besides optimizing the temperature and time, the present
inventors found that the addition of ethanol, to a final
concentration of 33% (v/v), resulted in a significant higher
labeling yield, compared with radiolabeling without ethanol (30.4%
vs 92.7%), using the same amount of NOTA-conjugated peptide. Same
observations have recently been described by others.sup.26. Here
the effect of lowering the ionic strength was investigated using
both acetonitrile, ethanol, dimethylforamide (DMF) and
tetrahydrofuran (THF) at different concentrations. A labeling yield
of 97% was reported using ethanol at a concentration of 80% (v/v).
However, they used between 76-383 nmol NOTA-conjugated peptide,
whereas in this study only used 30 nmol was used. The amount needed
for optimal labeling yield therefore seems to be dependent on the
peptide and on the amount of peptide used for labeling.
[0092] The tumor uptake of .sup.18F-AlF-NOTA-AE105 was similarly
compared with previously published results pertaining to
.sup.64Cu-based ligands.sup.15. The tumor uptake 1 h p.i was
4.79.+-.0.7% ID/g, 3.48.+-.0.8% ID/g and 4.75.+-.0.9% ID/g for
.sup.64Cu-DOTA-AE105, .sup.64Cu-CB-TE2A-AE105,
.sup.64Cu-CB-TE2A-PA-AE105 compared to 4.22.+-.0.1% ID/g for
.sup.18F-AlF-NOTA-AE105. However, all .sup.64Cu-based ligands were
investigated using the human glioblastoma cell line U87MG, whereas
in this study, the prostate cancer cell line PC-3 was used.
Considering that the data show that the level of uPAR in the two
tumor types is not similar, with PC-3 having around 300 pg uPAR/mg
tumor tissue (FIG. 5B) and U87MG having approximately 1,700 pg/mg
tumor tissue (unpublished), the tumor uptake of
.sup.18F-AlF-NOTA-AE105 seems to be relatively higher per pg uPAR,
However, a direct comparison between the two independent studies is
difficult, considering the different cancer cell line used.
However, the present inventors have previously shown a significant
correlation between uPAR expression and tumor uptake across three
tumor types.sup.15, which is confirmed in the present study using
PC-3 xenografts (FIG. 5C), further validating the ability of
.sup.18F-AlF-NOTA-AE105 to quantify uPAR expression using PET
imaging. The uPAR specific binding of .sup.18F-AlF-NOTA-AE105 in
the present study was confirmed by a 2.3-fold reduction in tumor
uptake of .sup.18F-AlF-NOTA-AE105 1 h p.i. when co-administration
of an uPAR antagonist (AE152) was performed for blocking study.
[0093] The biodistribution study of 18F-AlF-NOTA-AE105 confirmed
the kidneys to be the primary route of excretion and the organ with
highest level of activity (FIG. 4). Same excretion profiles have
been found for .sup.68Ga-DOTA/NODAGA-AE105.sup.16,
.sup.177Lu-DOTAAE105.sup.30. Besides the kidneys and tumor, the
bone also had a relatively high accumulation of activity. Bone
uptake following injection of 18F-based ligands is a well-described
phenomenon and used clinically in NaF bone scans.sup.31. A bone
uptake of 3.54% ID/g 2.5 h p.i was found, which is similar to the
bone uptake following .sup.18F-FDG injection in mice, where 2.49%
ID/g have been reported 1.5 h p.i..sup.17.
[0094] The development of the first .sup.18F-based ligand for uPAR
PET provides of number of advantages compared to previously
published .sup.64Cu-based uPAR PET ligands. Considering the optimal
tumor-to-background contrast as early as 1 h p.i. as found in this
study and in previously studies using 64Cu, the relatively shorter
half-life of .sup.18F (T.sub.1/2=1.83 h) compared with .sup.64Cu
(T.sub.1/2=12.7 h) seems to be optimal consider the much lower
radiation burden to future patients using .sup.18F-AlF-NOTA-AE105.
Moreover, is the production of .sup.18F well established in a
number of institutions worldwide, whereas the production of
.sup.64Cu still is limited to relatively few places.
Example 2
[.sup.64Cu]NOTA-AE105
(NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH)
[0095] .sup.64CuCl2 dissolved in 50 ul metal-free water was added
to a solution containing 10 nmol NOTA-AE105 and 2.5 mg gentisic
acid dissolved in 500 ul 0.1 M NH4OAc buffer (pH 5.5) and left at
room temperature for 10 minutes resulting in 375 MBq
[64Cu]NOTA-AE105 20 with a radiochemical purity above 99%. The
radiochemical purity decreased to 94% after 48 hours storage.
Example 3
[0096] In Vivo uPAR PET Imaging with [.sup.64Cu]NOTA-AE105 in a
Orthotropic Human Glioblastoma Mouse Model
[0097] A mouse was inoculated with human derived glioblastoma cells
in the brain. 3 weeks later a small tumor was visible using microCT
scan A microPET images was recorded 1 hr post i.v. injection of
approximately 5 MBq [.sup.64Cu]NOTA-AE105. Uptake in the tumor and
background brain tissue was quantified. Moreover, was a control
mouse (with no tumor inoculated) also PET scanned using the same
procedure, to investigate the uptake in normal brain tissue with
intact blood brain barrier. See FIG. 6.
Example 4
[68Ga]NOTA-AE105 (NOTA-Asp-Cha-Phe-Ser-Arg-Tyr-Leu-Trp-Ser-OH)
[0098] A 1 ml fraction of the eluate form a .sup.68Ge/68Ga
generator for added to a solution containing 20 nmol NOTA-AE105
dissolved in 1000 ul 0.7M NaOAc buffer (pH 3.75) and heated to
60.degree. C. for 10 minutes. The corresponding mixture could be
purified on a C18 SepPak column resulting in 534MBq
[.sup.68Ga]NOTA-AE105 with a radiochemical purity above 98%.
Example 5
[0099] In Vivo uPAR PET Imaging with [.sup.68Ga]NOTA-AE105 in a
Orthotropic Human Glioblastoma Mouse Model
[0100] A mouse was inoculated with human derived glioblastoma cells
in the brain. 3 weeks 15 later a small tumor was visible using
microCT scan A microPET images was recorded 1 hr post i.v.
injection of approximately 5 MBq [68Ga]NOTA-AE105. Uptake in the
tumor and background brain tissue was quantified. Moreover, was a
control mouse (with no tumor inoculated) also PET scanned using the
same procedure, to investigate the uptake in normal brain tissue
with intact blood brain barrier. See FIG. 7.
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