U.S. patent application number 16/934414 was filed with the patent office on 2021-02-11 for 68ga-labeled nota-chelated psma-targeted imaging and therapeutic agents.
The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Ronnie C. Mease, Martin G. Pomper, Sangeeta Ray.
Application Number | 20210040126 16/934414 |
Document ID | / |
Family ID | 1000005164063 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210040126 |
Kind Code |
A1 |
Pomper; Martin G. ; et
al. |
February 11, 2021 |
68Ga-LABELED NOTA-CHELATED PSMA-TARGETED IMAGING AND THERAPEUTIC
AGENTS
Abstract
PSMA-targeted PET/SPECT agents for imaging PSMA-positive cancer
and or tumor neovasculature and PSMA-targeted radiotherapeutic
agent for the treatment of PSMA-positive cancer or tumor
neovasculature are disclosed. Methods of imaging PSMA expressing
tumors, or cells and kits also are disclosed.
Inventors: |
Pomper; Martin G.;
(Baltimore, MD) ; Mease; Ronnie C.; (Fairfax,
VA) ; Ray; Sangeeta; (Ellicott City, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
BALTIMORE |
MD |
US |
|
|
Family ID: |
1000005164063 |
Appl. No.: |
16/934414 |
Filed: |
July 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15557854 |
Sep 13, 2017 |
10717750 |
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PCT/US16/22309 |
Mar 14, 2016 |
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16934414 |
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14243535 |
Apr 2, 2014 |
9776977 |
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15557854 |
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13257499 |
Sep 19, 2011 |
9056841 |
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PCT/US2010/028020 |
Mar 19, 2010 |
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14243535 |
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62132955 |
Mar 13, 2015 |
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61248934 |
Oct 6, 2009 |
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61248067 |
Oct 2, 2009 |
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61161485 |
Mar 19, 2009 |
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61161484 |
Mar 19, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07F 5/003 20130101;
A61K 51/0497 20130101 |
International
Class: |
C07F 5/00 20060101
C07F005/00; A61K 51/04 20060101 A61K051/04 |
Goverment Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
CA148901, CA151838, CA134675, CA184228, CA183031, and CA092871
awarded by the National Institutes of Health. The government has
certain rights in the invention.
Claims
1. A compound of Formula (I): ##STR00008## wherein: Z is tetrazole
or CO.sub.2Q; Q is H or a protecting group; X and Y are each
independently O or S; a is an integer selected from the group
consisting of 1, 2, 3 and 4; b and c are each independently an
integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, and 10; W is selected from the group consisting of
--C(.dbd.O)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--,
--NR.sub.4--C(.dbd.O)--NR.sub.4--,
--NR.sub.4--C(.dbd.S)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--O--,
--O--C(.dbd.O)--NR.sub.4--, --O--C(.dbd.O)--, and --C(.dbd.O)--O;
each R.sub.1 is independently H or C.sub.1-C.sub.4 alkyl; each
R.sub.2 is independently H or --COOR.sub.3, wherein each R.sub.3 is
independently H or a C.sub.1-C.sub.6 alkyl; each R.sub.4 is
independently H or C.sub.1-C.sub.4 alkyl; each R is independently
selected from the group consisting of hydrogen, halogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, alkyaryl, arylakyl, and
alkylheteroaryl; n is an integer selected from the group consisting
of 0, 1, 2, 3 and 4; and M is a metal; and pharmaceutically
acceptable salts thereof.
2. The compound of claim 1, wherein the metal (M) is selected from
the group consisting of Tc-94m, Tc-99m, In-111, Ga-67, Ga-68, Y-86,
Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57, Sc-47,
Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, Gd-152, or
Dy-166.
3. The compound of claim 2, wherein the metal (M) is Ga-68.
4. The compound of claim 1, wherein the compound of Formula (I) is
.sup.68Ga-2.
5. A method for imaging one or more prostate-specific membrane
antigen (PSMA) tumors, or cells the method comprising contacting
the one or more tumors, or cells, with an effective amount of a
compound of Formula (I) and making an image, the compound of
Formula (I) comprising: ##STR00009## wherein: Z is tetrazole or
CO.sub.2Q; Q is H or a protecting group; X and Y are each
independently O or S; a is an integer selected from the group
consisting of 1, 2, 3 and 4; b and c are each independently an
integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, and 10; W is selected from the group consisting of
--C(.dbd.O)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--,
--NR.sub.4--C(.dbd.O)--NR.sub.4--,
--NR.sub.4--C(.dbd.S)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--O--,
--O--C(.dbd.O)--NR.sub.4--, --O--C(.dbd.O)--, and --C(.dbd.O)--O--;
each R.sub.1 is independently H or C.sub.1-C.sub.4 alkyl; each
R.sub.2 is independently H or --COOR.sub.3, wherein each R.sub.3 is
independently H or a C.sub.1-C.sub.6 alkyl; each R.sub.4 is
independently H or C.sub.1-C.sub.4 alkyl; each R is independently
selected from the group consisting of hydrogen, halogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, alkyaryl, arylakyl, and
alkylheteroaryl; n is an integer selected from the group consisting
of 0, 1, 2, 3 and 4; and M is a radioactive metal suitable for
imaging; and pharmaceutically acceptable salts thereof.
6. The method of claim 5, wherein the radioactive metal suitable
for imaging (M) is selected from the group consisting of Tc-94m,
Tc-99m, In-111, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186, Re-188,
Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212,
Sm-153, Ho-166, Gd-152, or Dy-166.
7. The method of claim 5, wherein the radioactive metal suitable
for imaging (M) is Ga-68.
8. The method of claim 5, wherein the compound of Formula (I) is
.sup.68Ga-2.
9. The method of claim 5, wherein the imaging comprises positron
emission tomography (PET).
10. The method of claim 5, wherein the imaging comprises
single-photon emission computed tomography (SPECT).
11. The method of claim 5, wherein the one or more PSMA-expressing
tumors or cells is selected from the group consisting of: a
prostate tumor or cell, a metastasized prostate tumor or cell, a
lung tumor or cell, a renal tumor or cell, a glioblastoma, a
pancreatic tumor or cell, a bladder tumor or cell, a sarcoma, a
melanoma, a breast tumor or cell, a colon tumor or cell, a germ
cell, a pheochromocytoma, an esophageal tumor or cell, a stomach
tumor or cell, and combinations thereof.
12. The method of claim 5, wherein the one or more PSMA-expressing
tumors or cells is a prostate tumor or cell.
13. The method of claim 5, wherein the one or more PSMA-expressing
tumors or cells is in vitro, in vivo or ex-vivo.
14. The method of claim 5, wherein the one or more PSMA-expressing
tumors or cells is present in a subject.
15. The method of claim 14, wherein the compound of formula (I)
comprising the radioactive metal suitable for imaging is cleared
from the tumor or cell in the subject.
16. The method of claim 14, wherein the compound of formula (I)
comprising the radioactive metal suitable for imaging is cleared
more rapidly from a subject's kidneys than from a tumor of the
subject.
17. The method of claim 5, wherein the compound of formula (I)
comprising the radioactive metal suitable for imaging substantially
localizes to the tumor or cell within about 60 minutes of
administration.
18. A kit comprising a compound according to claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/557,854, filed Sep. 13, 2017, now U.S. Pat.
No. 10,717,750, which is a 35 U.S.C. .sctn. 371 National Stage
Entry of International Application No. PCT/US16/022309 having an
international filing date of Mar. 14, 2016, which claims the
benefit of U.S. Provisional Application No. 62/132,955, filed Mar.
13, 2015, the contents of which are incorporated herein by
reference in their entirety.
BACKGROUND
[0003] According to the National Cancer Institute approximately
220,800 cases of prostate cancer will be diagnosed in 2015, with
over 27,540 cases proving to be lethal (about 12.5%) (Institute, N.
C. Cancer Statistics (2015)). Existing imaging techniques for
detection and therapeutic monitoring of prostate cancer are
inadequate for effective management of the disease. The
transmembrane glycoprotein prostate-specific membrane antigen
(PSMA) is increasingly recognized as an important target for both
imaging and therapy of prostate cancer (Afshar-Oromieh et al.
(2014) Eur J Nucl Med Mol Imaging; Afshar-Oromieh et al., (2013)
Eur J Nucl Med Mol Imaging 40, 486-95). PSMA is found in benign, as
well as in malignant prostate tissue (Murphy et al., (1995)
Prostate 26, 164-8; Murphy et al., (1998) Urology 51, 89-97; Murphy
et al., (1998) J Urol 160, 2396-401). However, expression of PSMA
is greatest in prostate adenocarcinoma, particularly in
castration-resistant disease (Sweat et al. (1998) Urology 52,
637-40; Silver et al. (1997) Clin Cancer Res 3, 81-5). PSMA is also
present in the neovasculature of solid tumors including kidney,
lung (Wang et al. (2015) PLoS ONE 10.), stomach, colon, and breast
(Haffner et al. (2009) Hum Pathol 40, 1754-61; Haffner et al (2012)
Mod Pathol 25, 1079-85; Baccala et al. (2007) Urology 70, 385-90).
Expression of PSMA is associated with the neovascular endothelium
in non-prostate tumors (Chang et al. (1999) Mol Urol 3, 313-320;
Chang et al. (1999) Clin Cancer Res 5, 2674-81).
[0004] PSMA-targeted agents to image patients with prostate cancer
using positron emission tomography (PET) have been reported by
several groups (Cho et al. (2012) J Nucl Med 53, 1883-91;
Afshar-Oromieh et al. (2012) Eur J Nucl Med Mol Imaging 39, 1085-6;
Afshar-Oromieh et al. (2013) Eur J Nucl Med Mol Imaging 40,
1629-30; Afshar-Oromieh et al. (2014) Eur J Nucl Med Mol Imaging
41, 887-97; Afshar-Oromieh et al. (2015) Eur. J Nucl. Med. Mol.
Imaging 42, 197-209; Eiber et al. (2015) J Nucl Med 56, 668-74;
Eiber et al. (2014) Abdom Imaging; Rowe et al. (2015) J Nucl Med
56, 1003-10). Although there are debatable advantages and
disadvantages with respect to which isotope to use for detection
with PET, namely .sup.18F vs. .sup.68Ga, the radiometal .sup.68Ga
can be produced on-site with a generator, followed by simple
synthesis of the radiotracer (Fani et al. (2008) Contrast media
& molecular imaging 3, 67-77). .sup.68Ga-1, a radiotracer that
employed the DOTA-mono-amide chelator with conjugation to
H.sub.2N-Lys-(CH.sub.2).sub.3-Lys-urea-Glu for targeting to PSMA
(FIG. 1) has been previously reported (Banerjee et al. (2010) J Med
Chem 53, 5333-41). That chelator has been chosen to make it
possible to complex imaging radiometals, such as .sup.68Ga,
.sup.86Y, .sup.203Pb, as well as therapeutic radiometal nuclides,
such as .sup.177Lu, .sup.90Y, .sup.212Pb or .sup.225Ac, within the
same scaffold.
[0005] Two .sup.68Ga-based agents have demonstrated excellent
clinical results for detection of prostate cancer, namely,
.sup.68Ga-DKFZ-PSMA-11 (Glu-urea-Lys-(Ahx)-HBED-CC) and
EuK-Sub-kff-.sup.68Ga-DOTAGA (.sup.68Ga-PSMA I&T) (Herrmann et
al. (2015) Journal of Nuclear Medicine; Eder et al. (2012)
Bioconjug Chem 23, 688-97; Weineisen et al. (2015) J Nucl Med;
Weineisen et al. (2014) EJNMMI Res 4, 63). Those compounds both
employ the Glu-Lys-urea-based PSMA-targeted moiety, while
.sup.68Ga-DOTA-DUPA-Pep, also recently tested clinically, uses
DOTA-monoamide as the chelating agent and Glu-Glu-urea as the
PSMA-targeting moiety (Reske et al. (2013) Mol Imaging 40, 969-70).
A recent preclinical study also evaluated
.sup.68Ga-(CHX-A''-DTPA)-Pep using CHX-A''-DTPA as the chelating
agent (Baur et al. (2014) Pharmaceuticals (Basel) 7, 517-29). Among
the agents, .sup.68Ga-DKFZ-PSMA-11 has been most widely studied
clinically (Afshar-Oromieh et al. (2013) Eur J Nucl Med Mol Imaging
40, 486-95; Afshar-Oromieh et al. (2012) Eur J Nucl Med Mol Imaging
39, 1085-6; Afshar-Oromieh et al. (2013) Eur J Nucl Med Mol Imaging
40, 1629-30; Afshar-Oromieh et al. (2014) Eur J Nucl Med Mol
Imaging 41, 887-97; Afshar-Oromieh et al. (2015) Eur. J. Nucl. Med.
Mol. Imaging 42, 197-209; Afshar-Oromieh et al. (2013) Eur J Nucl
Med Mol Imaging 40, 971-2; Mottaghy et al. (2015) European Journal
of Nuclear Medicine and Molecular Imaging 1-3. The growing number
of clinical trials employing .sup.68Ga-based, PSMA-targeted PET
provides rationale to investigate structural elements that could
promote the least off-target uptake of this class of
radiotracers.
SUMMARY
[0006] In some aspects, the presently disclosed subject matter
provides a compound of Formula (I):
##STR00001##
wherein: Z is tetrazole or CO.sub.2Q; Q is H or a protecting group;
X and Y are each independently O or S; a is an integer selected
from the group consisting of 1, 2, 3 and 4; b and c are each
independently an integer selected from the group consisting of 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; W is selected from the group
consisting of --C(.dbd.O)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--,
--NR.sub.4--C(.dbd.O)--NR.sub.4--,
--NR.sub.4--C(.dbd.S)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--O--,
--O--C(.dbd.O)--NR.sub.4--, --O--C(.dbd.O)--, and --C(.dbd.O)--O--;
each R.sub.1 is independently H or C.sub.1-C.sub.4 alkyl; each
R.sub.2 is independently H or --COOR.sub.3, wherein each R.sub.3 is
independently H or a C.sub.1-C.sub.6 alkyl; each R.sub.4 is
independently H or C.sub.1-C.sub.4 alkyl; each R is independently
selected from the group consisting of hydrogen, halogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, alkyaryl, arylakyl, and
alkylheteroaryl; n is an integer selected from the group consisting
of 0, 1, 2, 3 and 4; and M is a metal; and pharmaceutically
acceptable salts thereof.
[0007] In particular aspects of the compound of the Formula (I),
the metal (M) is selected from the group consisting of Tc-94m,
Tc-99m, In-111, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186, Re-188,
Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212,
Sm-153, Ho-166, Gd-152, or Dy-166.
[0008] In yet more particular aspects, the metal (M) is Ga-68.
[0009] In still more particular aspects, the compound of Formula
(I) is .sup.68Ga-SRV168.
[0010] In other aspects, the presently disclosed subject matter
provides a method for imaging one or more prostate-specific
membrane antigen (PSMA) tumors or cells, the method comprising
contacting the one or more tumor or cells, with an effective amount
of a compound of Formula (I) and making an image, the compound of
Formula (I) comprising:
##STR00002##
wherein: Z is tetrazole or CO.sub.2Q; Q is H or a protecting group;
X and Y are each independently O or S; a is an integer selected
from the group consisting of 1, 2, 3 and 4; b and c are each
independently an integer selected from the group consisting of 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; W is selected from the group
consisting of --C(.dbd.O)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--,
--NR.sub.4--C(.dbd.O)--NR.sub.4--,
--NR.sub.4--C(.dbd.S)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--O--,
--O--C(.dbd.O)--NR.sub.4--, --O--C(.dbd.O)--, and --C(.dbd.O)--O--;
each R.sub.1 is independently H or C.sub.1-C.sub.4 alkyl; each
R.sub.2 is independently H or --COOR.sub.3, wherein each R.sub.3 is
independently H or a C.sub.1-C.sub.6 alkyl; each R.sub.4 is
independently H or C.sub.1-C.sub.4 alkyl; each R is independently
selected from the group consisting of hydrogen, halogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, alkyaryl, arylakyl, and
alkylheteroaryl; n is an integer selected from the group consisting
of 0, 1, 2, 3 and 4; and M is a radioactive metal suitable for
radiotherapy; and pharmaceutically acceptable salts thereof.
[0011] In some other aspects, the presently disclosed subject
matter provides a method for treating or preventing a disease or
condition associated with one or more PSMA expressing tumors or
cells, the method comprising administering to a subject in need of
treatment thereof, at least one compound of Formula (I), in an
amount effective to treat or prevent the disease or condition. In
yet other aspects, the presently disclosed subject matter provides
a kit comprising a compound of Formula (I).
[0012] Certain aspects of the presently disclosed subject matter
having been stated hereinabove, which are addressed in whole or in
part by the presently disclosed subject matter, other aspects will
become evident as the description proceeds when taken in connection
with the accompanying Examples and Figures as best described herein
below.
BRIEF DESCRIPTION OF THE FIGURES
[0013] Having thus described the presently disclosed subject matter
in general terms, reference will now be made to the accompanying
Figures, which are not necessarily drawn to scale, and wherein:
[0014] FIG. 1 shows chemical structures of representative
radiotracers used for the presently disclosed studies;
[0015] FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D show the comparison
of selected tissue uptake of .sup.68Ga-1, .sup.68Ga-2 and
.sup.68Ga-DKFZ-PSMA-11 in male SCID-NOD mice (n=4) bearing both
PSMA+ PC3 PIP and PSMA- PC3 flu tumors: (A) PSMA+ PC3 PIP tumor;
(B) kidney; (C) salivary gland; and (D) spleen; (*, P<0.05; **,
P<0.001; ***, P<0.0001;
[0016] FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D show the comparison
of PSMA+ PC3 PIP tumor-to-PSMA- PC3 flu tumor (A); PSMA+ PC3
PIP-to-kidney (B); PSMA+ PC3 PIP-to-salivary gland (C); and, PSMA+
PC3 PIP-to-blood (D) of .sup.68Ga-1, .sup.68Ga-2 and
.sup.68Ga-DKFZ-PSMA-11; (*, P<0.05; **, P<0.001; ***,
P<0.0001);
[0017] FIG. 4 shows the .sup.1H NMR spectrum of Ga-2 in DMSO at
room temperature;
[0018] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, and FIG. 5E show
preparative HPLC chromatograms of .sup.68Ga-1 (A), .sup.68Ga-2 (B,
C) and .sup.68Ga-DKFZ-PSMA-11 (D, E) for radio-HPLC (A,B,D) and UV
(C, E) peaks; and
[0019] FIG. 6 shows whole-body PET-CT imaging at 1 h post injection
for the radiotracers, .sup.68Ga-1, .sup.68Ga-2 and
.sup.68Ga-DKFZ-PSMA-11 using NOD-SCID male mice bearing both PSMA+
PC3 PIP (right) and PSMA- flu (left) tumor xenografts within the
upper flanks; PSMA+ PC3 tumor uptake for .sup.68Ga-2 was further
blocked by injecting ZJ43 (50 mg/kg), 30 min prior to injection of
the radiotracer.
DETAILED DESCRIPTION
[0020] The presently disclosed subject matter now will be described
more fully hereinafter with reference to the accompanying Figures,
in which some, but not all embodiments of the inventions are shown.
Like numbers refer to like elements throughout. The presently
disclosed subject matter may be embodied in many different forms
and should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will satisfy applicable legal requirements. Indeed, many
modifications and other embodiments of the presently disclosed
subject matter set forth herein will come to mind to one skilled in
the art to which the presently disclosed subject matter pertains
having the benefit of the teachings presented in the foregoing
descriptions and the associated Figures. Therefore, it is to be
understood that the presently disclosed subject matter is not to be
limited to the particular embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims.
[0021] I. .sup.68Ga-Labeled NOTA-Chelated PSMA-Targeted Imaging and
Therapeutic Agents
[0022] Prostate-specific membrane antigen (PSMA) is an increasingly
important target for imaging and therapy of prostate cancer. A
variety of high affinity radiohalogenated, urea-based PSMA
inhibitors that selectively image prostate tumors in experimental
models has been previously synthesized. Chelated
radiometal-linker-urea conjugates also have been synthesized. These
compounds also selectively image prostate tumors in experimental
models. .sup.68Ga-Labeled, low-molecular-weight imaging agents that
target the prostate-specific membrane antigen (PSMA) are
increasingly used clinically to detect prostate and other cancers
with positron emission tomography (PET). The DOTA ligand was
selected because it can chelate both imaging and therapeutic
nuclides. The growing number of clinical trials employing
.sup.68Ga-based, PSMA-targeted PET has encouraged the investigation
of structural elements that could promote the least off-target
uptake of this class of radiotracers.
[0023] Accordingly, the presently disclosed subject matter
provides, in some embodiments, a head-to-head, preclinical
comparison of radiometal-chelate-linker-urea based PSMA binding
imaging agents, wherein the chelating agents are DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and NOTA
(1,4,7-triazacyclononane-1,4,7-trisacetic acid), with a known
imaging agent, DKFZ-PSMA-11, when radiolabeled with .sup.68Ga.
[0024] More particularly, the presently disclosed subject matter,
in some embodiments, directly compares the tumor uptake,
selectivity and pharmacokinetics of a known radiotracer,
.sup.68Ga-1, the presently disclosed, .sup.68Ga-2, which in
contrast to .sup.68Ga-1, employs the NOTA chelator, and
.sup.68Ga-DKFZ-PSMA-11, which is currently in imaging clinical
trials for prostate cancer agent. Specific attention was given to
decrease activity within renal and salivary gland tissue, commonly
seen with these agents. It is believed that a preclinical study
such as this retains value as it is carefully controlled and all of
the aforementioned agents were evaluated for pharmacokinetics in
preclinical studies (Banerjee et al. (2010) J Med Chem 53, 5333-41;
Eder et al. (2012) Bioconjug Chem 23, 688-97)--with similar
comparisons performed--before their successful move to the clinic
(Eder et al. (2012) Bioconjug Chem 23, 688-97; Weineisen et al.
(2014) EJNMMI Res 4, 63).
[0025] The preparation and use of PSMA binding ureas conjugated to
chelated radiometals via various linking groups for imaging and
possible radiotherapy of PSMA expressing tumors has been described.
Institute, N. C. Cancer Statistics (2015); Afshar-Oromieh et al.,
(2014) Eur J Nucl Med Mol Imaging; Afshar-Oromieh et al., (2013)
Eur J Nucl Med Mol Imaging, 40, 486-95; Murphy et al., (1995)
Prostate, 26, 164-8; Murphy et al., (1998) Urology, 51, 89-97; and
Murphy et al., (1998) J Urol, 160, 2396-401. See also,
international PCT patent application publication nos. WO
2009/002529 A2 and WO2010/108125A2, each of which is incorporated
herein by reference in their entirety.
[0026] In the presently disclosed subject matter, .sup.68Ga-1,
which is disclosed in WO 2010108125 A2 20100923, and the new
radiotracer .sup.68Ga-2 were compared with .sup.68Ga-DKFZ-PSMA-11,
which is currently in clinical trial in Europe. Structures of the
representative agents are shown in FIG. 1.
[0027] The precursors for the agents .sup.68Ga-1 and .sup.68Ga-2
were reported earlier, Banerjee, et al., J. Medicinal Chem. (2010);
Banerjee, et al., J. Med. Chem. (2014), although no .sup.68Ga agent
has been reported. In vivo biological properties of the presently
disclosed agents are disclosed herein, demonstrating superior
biodistribution properties in comparison to .sup.68Ga-DKFZ-PSMA-11.
See Eder et al., Bioconjugate chemistry (2012).
[0028] A. Compounds of Formula (I)
[0029] Accordingly, in some embodiments, the presently disclosed
subject matter provides a compound of Formula (I):
##STR00003##
wherein: Z is tetrazole or CO.sub.2Q; Q is H or a protecting group;
X and Y are each independently O or S; a is an integer selected
from the group consisting of 1, 2, 3 and 4; b and c are each
independently an integer selected from the group consisting of 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; W is selected from the group
consisting of --C(.dbd.O)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--,
--NR.sub.4--C(.dbd.O)--NR.sub.4--,
--NR.sub.4--C(.dbd.S)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--O--,
--O--C(.dbd.O)--NR.sub.4--, --O--C(.dbd.O)--, and --C(.dbd.O)--O--;
each R.sub.1 is independently H or C.sub.1-C.sub.4 alkyl; each
R.sub.2 is independently H or --COOR.sub.3, wherein each R.sub.3 is
independently H or a C.sub.1-C.sub.6 alkyl; each R.sub.4 is
independently H or C.sub.1-C.sub.4 alkyl; each R is independently
selected from the group consisting of hydrogen, halogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, alkyaryl, arylakyl, and
alkylheteroaryl; n is an integer selected from the group consisting
of 0, 1, 2, 3 and 4; and M is a metal; and pharmaceutically
acceptable salts thereof.
[0030] Formula (I) does not include compounds disclosed in WO
2009/002529 and WO 2010/108125.
[0031] In particular embodiments of the compound of Formula (I),
the metal (M) is selected from the group consisting of Tc-94m,
Tc-99m, In-111, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186, Re-188,
Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212,
Sm-153, Ho-166, Gd-152, or Dy-166.
[0032] In yet more particular embodiments, the metal (M) is
Ga-68.
[0033] In still more particular embodiments, the compound of
Formula (I) is .sup.68Ga-SRV168.
[0034] B. Methods of Using Compounds of Formula (I) for Imaging One
or More PSMA-Expressing Tumors or Cells
[0035] In other embodiments, the presently disclosed subject matter
provides a method for imaging one or more prostate-specific
membrane antigen (PSMA) tumors or cells, the method comprising
contacting to the one or more tumors or cells, with an effective
amount of a compound of Formula (I) and making an image, the
compound of Formula (I) comprising:
##STR00004##
wherein: Z is tetrazole or CO.sub.2Q; Q is H or a protecting group;
X and Y are each independently O or S; a is an integer selected
from the group consisting of 1, 2, 3 and 4; b and c are each
independently an integer selected from the group consisting of 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; W is selected from the group
consisting of --C(.dbd.O)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--,
--NR.sub.4--C(.dbd.O)--NR.sub.4--,
--NR.sub.4--C(.dbd.S)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--O--,
--O--C(.dbd.O)--NR.sub.4--, --O--C(.dbd.O)--, and --C(.dbd.O)--O--;
each R.sub.1 is independently H or C.sub.1-C.sub.4 alkyl; each
R.sub.2 is independently H or --COOR.sub.3, wherein each R.sub.3 is
independently H or a C.sub.1-C.sub.6 alkyl; each R.sub.4 is
independently H or C.sub.1-C.sub.4 alkyl; each R is independently
selected from the group consisting of hydrogen, halogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, alkyaryl, arylakyl, and
alkylheteroaryl; n is an integer selected from the group consisting
of 0, 1, 2, 3 and 4; and M is a radioactive metal suitable for
imaging; and pharmaceutically acceptable salts thereof.
[0036] Formula (I) does not include compounds disclosed in WO
2009/002529 and WO 2010/108125.
[0037] "Contacting" means any action which results in at least one
compound comprising the imaging agent of the presently disclosed
subject matter physically contacting at least one PSMA-expressing
tumor or cell. Contacting can include exposing the cell(s) or
tumor(s) to the compound in an amount sufficient to result in
contact of at least one compound with at least one cell or tumor.
The method can be practiced in vitro or ex vivo by introducing, and
preferably mixing, the compound and cell(s) or tumor(s) in a
controlled environment, such as a culture dish or tube. The method
can be practiced in vivo, in which case contacting means exposing
at least one cell or tumor in a subject to at least one compound of
the presently disclosed subject matter, such as administering the
compound to a subject via any suitable route.
[0038] According to the presently disclosed subject matter,
contacting may comprise introducing, exposing, and the like, the
compound at a site distant to the cells to be contacted, and
allowing the bodily functions of the subject, or natural (e.g.,
diffusion) or man-induced (e.g., swirling) movements of fluids to
result in contact of the compound and cell(s) or tumor(s).
[0039] By "making an image", it is meant using positron emission
tomography (PET) or single-photon emission computed tomography
(SPECT) imaging to form an image of a cell, tissue, tumor, part of
body, and the like. The presently disclosed methods include
radioactive metal capable of emitting radiation suitable for
detection with PET or SPECT.
[0040] In some embodiments, the radioactive metal suitable for
imaging (M) is selected from the group consisting of Tc-94m,
Tc-99m, In-111, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186, Re-188,
Cu-64, Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212,
Sm-153, Ho-166, Gd-152, or Dy-166. In particular embodiments, the
radioactive metal suitable for imaging (M) is Ga-68. In more
particular embodiments, the compound of Formula (I) is
.sup.68Ga-2.
[0041] In some embodiments, the imaging comprises positron emission
tomography (PET). In other embodiments, the imaging comprises
single-photon emission computed tomography (SPECT).
[0042] In some embodiments, the one or more PSMA-expressing tumors
or cells is selected from the group consisting of: a prostate tumor
or cell, a metastasized prostate tumor or cell, a lung tumor or
cell, a renal tumor or cell, a glioblastoma, a pancreatic tumor or
cell, a bladder tumor or cell, a sarcoma, a melanoma, a breast
tumor or cell, a colon tumor or cell, a germ cell, a
pheochromocytoma, an esophageal tumor or cell, a stomach tumor or
cell, and combinations thereof. In particular embodiments, the one
or more PSMA-expressing tumors or cells is a prostate tumor or
cell.
[0043] In other embodiments, the one or more PSMA-expressing tumors
or cells is in vitro, in vivo or ex-vivo. In yet other embodiments,
the one or more PSMA-expressing tumors, cells organs, or tissues is
present in a subject.
[0044] The subject treated by the presently disclosed methods in
their many embodiments is desirably a human subject, although it is
to be understood that the methods described herein are effective
with respect to all vertebrate species, which are intended to be
included in the term "subject." Accordingly, a "subject" can
include a human subject for medical purposes, such as for the
treatment of an existing condition or disease or the prophylactic
treatment for preventing the onset of a condition or disease, or an
animal (non-human) subject for medical, veterinary purposes, or
developmental purposes. Suitable animal subjects include mammals
including, but not limited to, primates, e.g., humans, monkeys,
apes, and the like; bovines, e.g., cattle, oxen, and the like;
ovines, e.g., sheep and the like; caprines, e.g., goats and the
like; porcines, e.g., pigs, hogs, and the like; equines, e.g.,
horses, donkeys, zebras, and the like; felines, including wild and
domestic cats; canines, including dogs; lagomorphs, including
rabbits, hares, and the like; and rodents, including mice, rats,
and the like. An animal may be a transgenic animal. In some
embodiments, the subject is a human including, but not limited to,
fetal, neonatal, infant, juvenile, and adult subjects. Further, a
"subject" can include a patient afflicted with or suspected of
being afflicted with a condition or disease. Thus, the terms
"subject" and "patient" are used interchangeably herein.
[0045] In some embodiments, a detectably effective amount of the
imaging agent of the presently disclosed methods is administered to
a subject. In accordance with the presently disclosed subject
matter, "a detectably effective amount" of the imaging agent is
defined as an amount sufficient to yield an acceptable image using
equipment which is available for clinical use. A detectably
effective amount of the imaging agent may be administered in more
than one injection. The detectably effective amount of the imaging
agent can vary according to factors such as the degree of
susceptibility of the individual, the age, sex, and weight of the
individual, idiosyncratic responses of the individual, the
dosimetry, and instrument and film-related factors. Optimization of
such factors is well within the level of skill in the art. In some
embodiments, the compound of formula (I) comprising the radioactive
metal suitable for imaging substantially localizes to the tumor or
cell within about 60 minutes of administration.
[0046] It is preferable that the compounds of the presently
disclosed subject matter are excreted from tissues of the body
quickly. In some embodiments, the presently disclosed methods
comprise clearance of the compound comprising the imaging agent
from the tumor or cell in the subject. In some other embodiment,
the imaging agent is cleared more rapidly from a subject's kidneys
than from a tumor in the subject.
[0047] C. Methods of Using Compounds of Formula (I) for Treating a
Disease or Condition Associated with One or More One or More
PSMA-Expressing Tumors or Cells In other embodiments, the presently
disclosed subject matter provides a method for treating or
preventing a disease or condition associated with one or more PSMA
expressing tumors or cells, the method comprising administering to
a subject in need of treatment thereof, at least one compound of
Formula (I), in an amount effective to treat or prevent the disease
or condition, the compound of formula (I) comprising:
##STR00005##
wherein: Z is tetrazole or CO.sub.2Q; Q is H or a protecting group;
X and Y are each independently 0 or S; a is an integer selected
from the group consisting of 1, 2, 3 and 4; b and c are each
independently an integer selected from the group consisting of 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; W is selected from the group
consisting of --C(.dbd.O)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--,
--NR.sub.4--C(.dbd.O)--NR.sub.4--,
--NR.sub.4--C(.dbd.S)--NR.sub.4--, --NR.sub.4--C(.dbd.O)--O--,
--O--C(.dbd.O)--NR.sub.4--, --O--C(.dbd.O)--, and --C(.dbd.O)--O--;
each R.sub.1 is independently H or C.sub.1-C.sub.4 alkyl; each
R.sub.2 is independently H or --COOR.sub.3, wherein each R.sub.3 is
independently H or a C.sub.1-C.sub.6 alkyl; each R.sub.4 is
independently H or C.sub.1-C.sub.4 alkyl; each R is independently
selected from the group consisting of hydrogen, halogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, alkyaryl, arylakyl, and
alkylheteroaryl; n is an integer selected from the group consisting
of 0, 1, 2, 3 and 4; and M is a radioactive metal suitable for
radiotherapy; and pharmaceutically acceptable salts thereof.
[0048] Formula (I) does not include compounds disclosed in WO
2009/002529 and WO 2010/108125.
[0049] As used herein, the term "treating" can include reversing,
alleviating, inhibiting the progression of, preventing or reducing
the likelihood of the disease, disorder, or condition to which such
term applies, or one or more symptoms or manifestations of such
disease, disorder or condition. Preventing refers to causing a
disease, disorder, condition, or symptom or manifestation of such,
or worsening of the severity of such, not to occur. Accordingly,
the presently disclosed compounds can be administered
prophylactically to prevent or reduce the incidence or recurrence
of the disease, disorder, or condition.
[0050] In general, the "effective amount" of an active agent refers
to the amount necessary to elicit the desired biological response.
As will be appreciated by those of ordinary skill in this art, the
effective amount of an agent or device may vary depending on such
factors as the desired biological endpoint, the agent to be
delivered, the makeup of the pharmaceutical composition, the target
tissue, and the like.
[0051] The term "combination" is used in its broadest sense and
means that a subject is administered at least two agents, more
particularly a compound of Formula (I) and at least one other
active agent. More particularly, the term "in combination" refers
to the concomitant administration of two (or more) active agents
for the treatment of a, e.g., single disease state. As used herein,
the active agents may be combined and administered in a single
dosage form, may be administered as separate dosage forms at the
same time, or may be administered as separate dosage forms that are
administered alternately or sequentially on the same or separate
days. In one embodiment of the presently disclosed subject matter,
the active agents are combined and administered in a single dosage
form. In another embodiment, the active agents are administered in
separate dosage forms (e.g., wherein it is desirable to vary the
amount of one but not the other). The single dosage form may
include additional active agents for the treatment of the disease
state.
[0052] In particular embodiments, the disease or condition is a
prostate cancer, renal cancer, head cancer, neck cancer, head and
neck cancer, lung cancer, breast cancer, prostate cancer,
colorectal cancer, esophageal cancer, stomach cancer,
leukemia/lymphoma, uterine cancer, skin cancer, endocrine cancer,
urinary cancer, pancreatic cancer, gastrointestinal cancer, ovarian
cancer, cervical cancer, adenomas, and tumor neovasculature. In
more particular embodiments, the disease or condition is prostate
cancer. Accordingly, the presently disclosed compounds can be
administered prophylactically to prevent or reduce the incidence or
recurrence of the cancer or the tumor neovasculature.
[0053] A "cancer" in a subject refers to the presence of cells
possessing characteristics typical of cancer-causing cells, for
example, uncontrolled proliferation, loss of specialized functions,
immortality, significant metastatic potential, significant increase
in anti-apoptotic activity, rapid growth and proliferation rate,
and certain characteristic morphology and cellular markers. In some
circumstances, cancer cells will be in the form of a tumor; such
cells may exist locally within a subject, or circulate in the blood
stream as independent cells, for example, leukemic cells.
[0054] A cancer can include, but is not limited to, renal cancer,
head cancer, neck cancer, head and neck cancer, lung cancer, breast
cancer, prostate cancer, colorectal cancer, esophageal cancer,
stomach cancer, leukemia/lymphoma, uterine cancer, skin cancer,
endocrine cancer, urinary cancer, pancreatic cancer,
gastrointestinal cancer, ovarian cancer, cervical cancer, and
adenomas. In more particular embodiments, the disease or condition
is prostate cancer. In some embodiments, a detectably effective
amount of the therapeutic agent of the presently disclosed methods
is administered to a subject.
[0055] D. Kits
[0056] In yet other embodiments, the presently disclosed subject
matter provides a kit comprising a compound of Formula (I). Formula
(I) does not include compounds disclosed in WO 2009/002529 and WO
2010/108125.
[0057] In certain embodiments, the kit provides packaged
pharmaceutical compositions comprising a pharmaceutically
acceptable carrier and a compound of the invention. In certain
embodiments the packaged pharmaceutical composition will comprise
the reaction precursors necessary to generate the compound of the
invention upon combination with a radio labeled precursor. Other
packaged pharmaceutical compositions provided by the present
invention further comprise indicia comprising at least one of:
instructions for preparing compounds according to the invention
from supplied precursors, instructions for using the composition to
image cells or tissues expressing PSMA, or instructions for using
the composition to image glutamatergic neurotransmission in a
patient suffering from a stress-related disorder, or instructions
for using the composition to image prostate cancer.
[0058] E. Pharmaceutical Compositions and Administration
[0059] In another aspect, the present disclosure provides a
pharmaceutical composition including one compounds of Formula (I)
alone or in combination with one or more additional therapeutic
agents in admixture with a pharmaceutically acceptable excipient.
One of skill in the art will recognize that the pharmaceutical
compositions include the pharmaceutically acceptable salts of the
compounds described above. Pharmaceutically acceptable salts are
generally well known to those of ordinary skill in the art, and
include salts of active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituent moieties found on the compounds described herein. When
compounds of the present disclosure contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent or by ion
exchange, whereby one basic counterion (base) in an ionic complex
is substituted for another. Examples of pharmaceutically acceptable
base addition salts include sodium, potassium, calcium, ammonium,
organic amino, or magnesium salt, or a similar salt.
[0060] When compounds of the present disclosure contain relatively
basic functionalities, acid addition salts can be obtained by
contacting the neutral form of such compounds with a sufficient
amount of the desired acid, either neat or in a suitable inert
solvent or by ion exchange, whereby one acidic counterion (acid) in
an ionic complex is substituted for another. Examples of
pharmaceutically acceptable acid addition salts include those
derived from inorganic acids like hydrochloric, hydrobromic,
nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-toluenesulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, for
example, Berge et al, "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds
of the present disclosure contain both basic and acidic
functionalities that allow the compounds to be converted into
either base or acid addition salts.
[0061] Accordingly, pharmaceutically acceptable salts suitable for
use with the presently disclosed subject matter include, by way of
example but not limitation, acetate, benzenesulfonate, benzoate,
bicarbonate, bitartrate, bromide, calcium edetate, carnsylate,
carbonate, citrate, edetate, edisylate, estolate, esylate,
fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,
hydroxynaphthoate, iodide, isethionate, lactate, lactobionate,
malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate,
pamoate (embonate), pantothenate, phosphate/diphosphate,
polygalacturonate, salicylate, stearate, subacetate, succinate,
sulfate, tannate, tartrate, or teoclate. Other pharmaceutically
acceptable salts may be found in, for example, Remington: The
Science and Practice of Pharmacy (20.sup.th ed.) Lippincott,
Williams & Wilkins (2000).
[0062] In therapeutic and/or diagnostic applications, the compounds
of the disclosure can be formulated for a variety of modes of
administration, including systemic and topical or localized
administration. Techniques and formulations generally may be found
in Remington: The Science and Practice of Pharmacy (20.sup.th ed.)
Lippincott, Williams & Wilkins (2000).
[0063] Depending on the specific conditions being treated, such
agents may be formulated into liquid or solid dosage forms and
administered systemically or locally. The agents may be delivered,
for example, in a timed- or sustained-slow release form as is known
to those skilled in the art. Techniques for formulation and
administration may be found in Remington: The Science and Practice
of Pharmacy (20.sup.th ed.) Lippincott, Williams & Wilkins
(2000). Suitable routes may include oral, buccal, by inhalation
spray, sublingual, rectal, transdermal, vaginal, transmucosal,
nasal or intestinal administration; parenteral delivery, including
intramuscular, subcutaneous, intramedullary injections, as well as
intrathecal, direct intraventricular, intravenous, intra-articular,
intra-sternal, intra-synovial, intra-hepatic, intralesional,
intracranial, intraperitoneal, intranasal, or intraocular
injections or other modes of delivery.
[0064] For injection, the agents of the disclosure may be
formulated and diluted in aqueous solutions, such as in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological saline buffer. For such
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0065] Use of pharmaceutically acceptable inert carriers to
formulate the compounds herein disclosed for the practice of the
disclosure into dosages suitable for systemic administration is
within the scope of the disclosure. With proper choice of carrier
and suitable manufacturing practice, the compositions of the
present disclosure, in particular, those formulated as solutions,
may be administered parenterally, such as by intravenous injection.
The compounds can be formulated readily using pharmaceutically
acceptable carriers well known in the art into dosages suitable for
oral administration. Such carriers enable the compounds of the
disclosure to be formulated as tablets, pills, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral
ingestion by a subject (e.g., patient) to be treated.
[0066] For nasal or inhalation delivery, the agents of the
disclosure also may be formulated by methods known to those of
skill in the art, and may include, for example, but not limited to,
examples of solubilizing, diluting, or dispersing substances, such
as saline; preservatives, such as benzyl alcohol; absorption
promoters; and fluorocarbons.
[0067] Pharmaceutical compositions suitable for use in the present
disclosure include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein. Generally, the compounds
according to the disclosure are effective over a wide dosage range.
For example, in the treatment of adult humans, dosages from 0.01 to
1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to
40 mg per day are examples of dosages that may be used. A
non-limiting dosage is 10 to 30 mg per day. The exact dosage will
depend upon the route of administration, the form in which the
compound is administered, the subject to be treated, the body
weight of the subject to be treated, the bioavailability of the
compound(s), the adsorption, distribution, metabolism, and
excretion (ADME) toxicity of the compound(s), and the preference
and experience of the attending physician.
[0068] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions.
[0069] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipients, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethyl-cellulose (CMC), and/or polyvinylpyrrolidone (PVP:
povidone). If desired, disintegrating agents may be added, such as
the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a
salt thereof such as sodium alginate.
[0070] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol
gel, polyethylene glycol (PEG), and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dye-stuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0071] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin, and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols (PEGs). In
addition, stabilizers may be added.
[0072] II. Definitions
[0073] Although specific terms are employed herein, they are used
in a generic and descriptive sense only and not for purposes of
limitation. Unless otherwise defined, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this presently described
subject matter belongs.
[0074] While the following terms in relation to compounds of
Formula (I) are believed to be well understood by one of ordinary
skill in the art, the following definitions are set forth to
facilitate explanation of the presently disclosed subject matter.
These definitions are intended to supplement and illustrate, not
preclude, the definitions that would be apparent to one of ordinary
skill in the art upon review of the present disclosure.
[0075] The terms substituted, whether preceded by the term
"optionally" or not, and substituent, as used herein, refer to the
ability, as appreciated by one skilled in this art, to change one
functional group for another functional group on a molecule,
provided that the valency of all atoms is maintained. When more
than one position in any given structure may be substituted with
more than one substituent selected from a specified group, the
substituent may be either the same or different at every position.
The substituents also may be further substituted (e.g., an aryl
group substituent may have another substituent off it, such as
another aryl group, which is further substituted at one or more
positions).
[0076] Where substituent groups or linking groups are specified by
their conventional chemical formulae, written from left to right,
they equally encompass the chemically identical substituents that
would result from writing the structure from right to left, e.g.,
--CH.sub.2O-- is equivalent to --OCH.sub.2--; --C(.dbd.O)O-- is
equivalent to --OC(.dbd.O)--; --OC(.dbd.O)NR-- is equivalent to
--NRC(.dbd.O)O--, and the like.
[0077] When the term "independently selected" is used, the
substituents being referred to (e.g., R groups, such as groups
R.sub.1, R.sub.2, and the like, or variables, such as "m" and "n"),
can be identical or different. For example, both R.sub.1 and
R.sub.2 can be substituted alkyls, or R.sub.1 can be hydrogen and
R.sub.2 can be a substituted alkyl, and the like.
[0078] The terms "a," "an," or "a(n)," when used in reference to a
group of substituents herein, mean at least one. For example, where
a compound is substituted with "an" alkyl or aryl, the compound is
optionally substituted with at least one alkyl and/or at least one
aryl. Moreover, where a moiety is substituted with an R
substituent, the group may be referred to as "R-substituted." Where
a moiety is R-substituted, the moiety is substituted with at least
one R substituent and each R substituent is optionally
different.
[0079] A named "R" or group will generally have the structure that
is recognized in the art as corresponding to a group having that
name, unless specified otherwise herein. For the purposes of
illustration, certain representative "R" groups as set forth above
are defined below.
[0080] Description of compounds of the present disclosure are
limited by principles of chemical bonding known to those skilled in
the art. Accordingly, where a group may be substituted by one or
more of a number of substituents, such substitutions are selected
so as to comply with principles of chemical bonding and to give
compounds which are not inherently unstable and/or would be known
to one of ordinary skill in the art as likely to be unstable under
ambient conditions, such as aqueous, neutral, and several known
physiological conditions. For example, a heterocycloalkyl or
heteroaryl is attached to the remainder of the molecule via a ring
heteroatom in compliance with principles of chemical bonding known
to those skilled in the art thereby avoiding inherently unstable
compounds.
[0081] Unless otherwise explicitly defined, a "substituent group,"
as used herein, includes a functional group selected from one or
more of the following moieties, which are defined herein:
[0082] The term hydrocarbon, as used herein, refers to any chemical
group comprising hydrogen and carbon. The hydrocarbon may be
substituted or unsubstituted. As would be known to one skilled in
this art, all valencies must be satisfied in making any
substitutions. The hydrocarbon may be unsaturated, saturated,
branched, unbranched, cyclic, polycyclic, or heterocyclic.
Illustrative hydrocarbons are further defined herein below and
include, for example, methyl, ethyl, n-propyl, isopropyl,
cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl,
cyclohexyl, and the like.
[0083] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight (i.e.,
unbranched) or branched chain, acyclic or cyclic hydrocarbon group,
or combination thereof, which may be fully saturated, mono- or
polyunsaturated and can include di- and multivalent groups, having
the number of carbon atoms designated (i.e., C.sub.1-C.sub.10 means
one to ten carbons, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10
carbons). In particular embodiments, the term "alkyl" refers to
C.sub.1-20 inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons, linear (i.e.,
"straight-chain"), branched, or cyclic, saturated or at least
partially and in some cases fully unsaturated (i.e., alkenyl and
alkynyl) hydrocarbon radicals derived from a hydrocarbon moiety
containing between one and twenty carbon atoms by removal of a
single hydrogen atom.
[0084] Representative saturated hydrocarbon groups include, but are
not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl,
neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl,
n-undecyl, dodecyl, cyclohexyl, (cyclohexyl)methyl,
cyclopropylmethyl, and homologs and isomers thereof.
[0085] "Branched" refers to an alkyl group in which a lower alkyl
group, such as methyl, ethyl or propyl, is attached to a linear
alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to
about 8 carbon atoms (i.e., a C.sub.1-8 alkyl), e.g., 1, 2, 3, 4,
5, 6, 7, or 8 carbon atoms. "Higher alkyl" refers to an alkyl group
having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments,
"alkyl" refers, in particular, to C.sub.1-8 straight-chain alkyls.
In other embodiments, "alkyl" refers, in particular, to C.sub.1-8
branched-chain alkyls.
[0086] Alkyl groups can optionally be substituted (a "substituted
alkyl") with one or more alkyl group substituents, which can be the
same or different. The term "alkyl group substituent" includes but
is not limited to alkyl, substituted alkyl, halo, arylamino, acyl,
hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl,
aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There
can be optionally inserted along the alkyl chain one or more
oxygen, sulfur or substituted or unsubstituted nitrogen atoms,
wherein the nitrogen substituent is hydrogen, lower alkyl (also
referred to herein as "alkylaminoalkyl"), or aryl.
[0087] Thus, as used herein, the term "substituted alkyl" includes
alkyl groups, as defined herein, in which one or more atoms or
functional groups of the alkyl group are replaced with another atom
or functional group, including for example, alkyl, substituted
alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro,
amino, alkylamino, dialkylamino, sulfate, and mercapto.
[0088] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon group, or combinations
thereof, consisting of at least one carbon atoms and at least one
heteroatom selected from the group consisting of O, N, P, Si and S,
and wherein the nitrogen, phosphorus, and sulfur atoms may
optionally be oxidized and the nitrogen heteroatom may optionally
be quaternized. The heteroatom(s) O, N, P and S and Si may be
placed at any interior position of the heteroalkyl group or at the
position at which alkyl group is attached to the remainder of the
molecule. Examples include, but are not limited to,
--CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3,
--CH.sub.2--CH.sub.25--S(O)--CH.sub.3,
--CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3,
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3, O--CH.sub.3,
--O--CH.sub.2--CH.sub.3, and --CN. Up to two or three heteroatoms
may be consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3
and --CH.sub.2--O--Si(CH.sub.3).sub.3.
[0089] As described above, heteroalkyl groups, as used herein,
include those groups that are attached to the remainder of the
molecule through a heteroatom, such as --C(O)NR', --NR'R'', --OR',
--SR, --S(O)R, and/or --S(O.sub.2)R'. Where "heteroalkyl" is
recited, followed by recitations of specific heteroalkyl groups,
such as --NR'R or the like, it will be understood that the terms
heteroalkyl and --NR'R'' are not redundant or mutually exclusive.
Rather, the specific heteroalkyl groups are recited to add clarity.
Thus, the term "heteroalkyl" should not be interpreted herein as
excluding specific heteroalkyl groups, such as --NR'R'' or the
like.
[0090] "Cyclic" and "cycloalkyl" refer to a non-aromatic mono- or
multicyclic ring system of about 3 to about 10 carbon atoms, e.g.,
3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can
be optionally partially unsaturated. The cycloalkyl group also can
be optionally substituted with an alkyl group substituent as
defined herein, oxo, and/or alkylene. There can be optionally
inserted along the cyclic alkyl chain one or more oxygen, sulfur or
substituted or unsubstituted nitrogen atoms, wherein the nitrogen
substituent is hydrogen, unsubstituted alkyl, substituted alkyl,
aryl, or substituted aryl, thus providing a heterocyclic group.
Representative monocyclic cycloalkyl rings include cyclopentyl,
cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include
adamantyl, octahydronaphthyl, decalin, camphor, camphane, and
noradamantyl, and fused ring systems, such as dihydro- and
tetrahydronaphthalene, and the like.
[0091] The term "cycloalkylalkyl," as used herein, refers to a
cycloalkyl group as defined hereinabove, which is attached to the
parent molecular moiety through an alkyl group, also as defined
above. Examples of cycloalkylalkyl groups include cyclopropylmethyl
and cyclopentylethyl.
[0092] The terms "cycloheteroalkyl" or "heterocycloalkyl" refer to
a non-aromatic ring system, unsaturated or partially unsaturated
ring system, such as a 3- to 10-member substituted or unsubstituted
cycloalkyl ring system, including one or more heteroatoms, which
can be the same or different, and are selected from the group
consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P),
and silicon (Si), and optionally can include one or more double
bonds.
[0093] The cycloheteroalkyl ring can be optionally fused to or
otherwise attached to other cycloheteroalkyl rings and/or
non-aromatic hydrocarbon rings. Heterocyclic rings include those
having from one to three heteroatoms independently selected from
oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur
heteroatoms may optionally be oxidized and the nitrogen heteroatom
may optionally be quaternized. In certain embodiments, the term
heterocyclic refers to a non-aromatic 5-, 6-, or 7-membered ring or
a polycyclic group wherein at least one ring atom is a heteroatom
selected from O, S, and N (wherein the nitrogen and sulfur
heteroatoms may be optionally oxidized), including, but not limited
to, a bi- or tri-cyclic group, comprising fused six-membered rings
having between one and three heteroatoms independently selected
from the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered
ring has 0 to 2 double bonds, each 6-membered ring has 0 to 2
double bonds, and each 7-membered ring has 0 to 3 double bonds,
(ii) the nitrogen and sulfur heteroatoms may be optionally
oxidized, (iii) the nitrogen heteroatom may optionally be
quaternized, and (iv) any of the above heterocyclic rings may be
fused to an aryl or heteroaryl ring. Representative
cycloheteroalkyl ring systems include, but are not limited to
pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl,
pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl,
quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl,
tetrahydrofuranyl, and the like.
[0094] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like. The terms "cycloalkylene" and
"heterocycloalkylene" refer to the divalent derivatives of
cycloalkyl and heterocycloalkyl, respectively.
[0095] An unsaturated alkyl group is one having one or more double
bonds or triple bonds. Examples of unsaturated alkyl groups
include, but are not limited to, vinyl, 2-propenyl, crotyl,
2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,
3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the
higher homologs and isomers. Alkyl groups which are limited to
hydrocarbon groups are termed "homoalkyl."
[0096] More particularly, the term "alkenyl" as used herein refers
to a monovalent group derived from a C.sub.1-20 inclusive straight
or branched hydrocarbon moiety having at least one carbon-carbon
double bond by the removal of a single hydrogen molecule. Alkenyl
groups include, for example, ethenyl (i.e., vinyl), propenyl,
butenyl, 1-methyl-2-buten-1-yl, pentenyl, hexenyl, octenyl,
allenyl, and butadienyl.
[0097] The term "cycloalkenyl" as used herein refers to a cyclic
hydrocarbon containing at least one carbon-carbon double bond.
Examples of cycloalkenyl groups include cyclopropenyl,
cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl,
1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and
cyclooctenyl.
[0098] The term "alkynyl" as used herein refers to a monovalent
group derived from a straight or branched C.sub.1-20 hydrocarbon of
a designed number of carbon atoms containing at least one
carbon-carbon triple bond. Examples of "alkynyl" include ethynyl,
2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, and heptynyl
groups, and the like.
[0099] The term "alkylene" by itself or a part of another
substituent refers to a straight or branched bivalent aliphatic
hydrocarbon group derived from an alkyl group having from 1 to
about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group
can be straight, branched or cyclic. The alkylene group also can be
optionally unsaturated and/or substituted with one or more "alkyl
group substituents." There can be optionally inserted along the
alkylene group one or more oxygen, sulfur or substituted or
unsubstituted nitrogen atoms (also referred to herein as
"alkylaminoalkyl"), wherein the nitrogen substituent is alkyl as
previously described. Exemplary alkylene groups include methylene
(--CH.sub.2--); ethylene (--CH.sub.2--CH.sub.2--); propylene
(--(CH.sub.2).sub.3--); cyclohexylene (--C.sub.6H.sub.10--);
--CH.dbd.CH--CH.dbd.CH--; --CH.dbd.CH--CH.sub.2--;
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH.dbd.CHCH.sub.2--, --CH.sub.2CsCCH.sub.2--,
--CH.sub.2CH.sub.2CH(CH.sub.2CH.sub.2CH.sub.3)CH.sub.2--,
--(CH.sub.2).sub.q--N(R)--(CH.sub.2).sub.r--, wherein each of q and
r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20,
and R is hydrogen or lower alkyl; methylenedioxyl
(--O--CH.sub.2--O--); and ethylenedioxyl
(--O--(CH.sub.2).sub.2--O--). An alkylene group can have about 2 to
about 3 carbon atoms and can further have 6-20 carbons. Typically,
an alkyl (or alkylene) group will have from 1 to 24 carbon atoms,
with those groups having 10 or fewer carbon atoms being some
embodiments of the present disclosure. A "lower alkyl" or "lower
alkylene" is a shorter chain alkyl or alkylene group, generally
having eight or fewer carbon atoms.
[0100] The term "heteroalkylene" by itself or as part of another
substituent means a divalent group derived from heteroalkyl, as
exemplified, but not limited by,
--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms also can occupy either or both
of the chain termini (e.g., alkyleneoxo, alkylenedioxo,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula --C(O)OR'--
represents both --C(O)OR'-- and --R'OC(O)--.
[0101] The term "aryl" means, unless otherwise stated, an aromatic
hydrocarbon substituent that can be a single ring or multiple rings
(such as from 1 to 3 rings), which are fused together or linked
covalently. The term "heteroaryl" refers to aryl groups (or rings)
that contain from one to four heteroatoms (in each separate ring in
the case of multiple rings) selected from N, O, and S, wherein the
nitrogen and sulfur atoms are optionally oxidized, and the nitrogen
atom(s) are optionally quaternized. A heteroaryl group can be
attached to the remainder of the molecule through a carbon or
heteroatom. Non-limiting examples of aryl and heteroaryl groups
include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,
2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,
pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,
3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,
5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,
3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,
purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below. The terms "arylene" and "heteroarylene" refer to
the divalent forms of aryl and heteroaryl, respectively.
[0102] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the terms
"arylalkyl" and "heteroarylalkyl" are meant to include those groups
in which an aryl or heteroaryl group is attached to an alkyl group
(e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like)
including those alkyl groups in which a carbon atom (e.g., a
methylene group) has been replaced by, for example, an oxygen atom
(e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,
and the like). However, the term "haloaryl," as used herein is
meant to cover only aryls substituted with one or more
halogens.
[0103] Where a heteroalkyl, heterocycloalkyl, or heteroaryl
includes a specific number of members (e.g. "3 to 7 membered"), the
term "member" refers to a carbon or heteroatom.
[0104] Further, a structure represented generally by the
formula:
##STR00006##
as used herein refers to a ring structure, for example, but not
limited to a 3-carbon, a 4-carbon, a 5-carbon, a 6-carbon, a
7-carbon, and the like, aliphatic and/or aromatic cyclic compound,
including a saturated ring structure, a partially saturated ring
structure, and an unsaturated ring structure, comprising a
substituent R group, wherein the R group can be present or absent,
and when present, one or more R groups can each be substituted on
one or more available carbon atoms of the ring structure. The
presence or absence of the R group and number of R groups is
determined by the value of the variable "n," which is an integer
generally having a value ranging from 0 to the number of carbon
atoms on the ring available for substitution. Each R group, if more
than one, is substituted on an available carbon of the ring
structure rather than on another R group. For example, the
structure above where n is 0 to 2 would comprise compound groups
including, but not limited to:
##STR00007##
and the like.
[0105] A dashed line representing a bond in a cyclic ring structure
indicates that the bond can be either present or absent in the
ring. That is, a dashed line representing a bond in a cyclic ring
structure indicates that the ring structure is selected from the
group consisting of a saturated ring structure, a partially
saturated ring structure, and an unsaturated ring structure.
[0106] The symbol () denotes the point of attachment of a moiety to
the remainder of the molecule.
[0107] When a named atom of an aromatic ring or a heterocyclic
aromatic ring is defined as being "absent," the named atom is
replaced by a direct bond.
[0108] Each of above terms (e.g., "alkyl," "heteroalkyl,"
"cycloalkyl, and "heterocycloalkyl", "aryl," "heteroaryl,"
"phosphonate," and "sulfonate" as well as their divalent
derivatives) are meant to include both substituted and
unsubstituted forms of the indicated group. Optional substituents
for each type of group are provided below.
[0109] Substituents for alkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl monovalent and divalent derivative groups
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one
or more of a variety of groups selected from, but not limited to:
--OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --C(O)NR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''', --NR''C(O)OR',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such groups. R', R'', R''' and R'''' each may
independently refer to hydrogen, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted
aryl (e.g., aryl substituted with 1-3 halogens), substituted or
unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl
groups. As used herein, an "alkoxy" group is an alkyl attached to
the remainder of the molecule through a divalent oxygen. When a
compound of the disclosure includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R'' and R'''' groups when more than one of these groups is
present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 4-, 5-, 6-,
or 7-membered ring. For example, --NR'R'' is meant to include, but
not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0110] Similar to the substituents described for alkyl groups
above, exemplary substituents for aryl and heteroaryl groups (as
well as their divalent derivatives) are varied and are selected
from, for example: halogen, --OR', --NR'R'', --SR', --SiR'R''R''',
--OC(O)R', --C(O)R', --CO.sub.2R', --C(O)NR'R'', --OC(O)NR'R'',
--NR''C(O)R', --NR'--C(O)NR''R''', --NR''C(O)OR',
--NR--C(NR'R''R''').dbd.NR'''', --NR--C(NR'R'').dbd.NR'''--S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and
--NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxo, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
aromatic ring system; and where R', R'', R''' and R'''' may be
independently selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl and substituted
or unsubstituted heteroaryl. When a compound of the disclosure
includes more than one R group, for example, each of the R groups
is independently selected as are each R', R'', R''' and R''''
groups when more than one of these groups is present.
[0111] Two of the substituents on adjacent atoms of aryl or
heteroaryl ring may optionally form a ring of the formula
--T--C(O)--(CRR').sub.q--U--, wherein T and U are independently
--NR--, --O--, --CRR'-- or a single bond, and q is an integer of
from 0 to 3. Alternatively, two of the substituents on adjacent
atoms of aryl or heteroaryl ring may optionally be replaced with a
substituent of the formula --A--(CH.sub.2).sub.r--B--, wherein A
and B are independently --CRR'--, --O--, --NR--, --S--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2NR'-- or a single bond, and r is an
integer of from 1 to 4.
[0112] One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula
--(CRR').sub.s--X'--(C''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X' is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'' and R''' may be independently selected from
hydrogen, substituted or unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, and
substituted or unsubstituted heteroaryl.
[0113] As used herein, the term "acyl" refers to an organic acid
group wherein the --OH of the carboxyl group has been replaced with
another substituent and has the general formula RC(.dbd.O)--,
wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocyclic,
heterocyclic, or aromatic heterocyclic group as defined herein). As
such, the term "acyl" specifically includes arylacyl groups, such
as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetyl group. Specific
examples of acyl groups include acetyl and benzoyl. Acyl groups
also are intended to include amides, --RC(.dbd.O)NR', esters,
--RC(.dbd.O)OR', ketones, --RC(.dbd.O)R', and aldehydes,
--RC(.dbd.O)H.
[0114] The terms "alkoxyl" or "alkoxy" are used interchangeably
herein and refer to a saturated (i.e., alkyl-O--) or unsaturated
(i.e., alkenyl-O-- and alkynyl-O--) group attached to the parent
molecular moiety through an oxygen atom, wherein the terms "alkyl,"
"alkenyl," and "alkynyl" are as previously described and can
include C.sub.1-20 inclusive, linear, branched, or cyclic,
saturated or unsaturated oxo-hydrocarbon chains, including, for
example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl,
sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl,
and the like.
[0115] The term "alkoxyalkyl" as used herein refers to an
alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl
group.
[0116] "Aryloxyl" refers to an aryl-O-- group wherein the aryl
group is as previously described, including a substituted aryl. The
term "aryloxyl" as used herein can refer to phenyloxyl or
hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl
substituted phenyloxyl or hexyloxyl.
[0117] "Aralkyl" refers to an aryl-alkyl-group wherein aryl and
alkyl are as previously described, and included substituted aryl
and substituted alkyl. Exemplary aralkyl groups include benzyl,
phenylethyl, and naphthylmethyl.
[0118] "Aralkyloxyl" refers to an aralkyl-O-- group wherein the
aralkyl group is as previously described. An exemplary aralkyloxyl
group is benzyloxyl, i.e., C.sub.6H.sub.5--CH.sub.2--O--. An
aralkyloxyl group can optionally be substituted.
[0119] "Alkoxycarbonyl" refers to an alkyl-O--C(.dbd.O)-- group.
Exemplary alkoxycarbonyl groups include methoxycarbonyl,
ethoxycarbonyl, butyloxycarbonyl, and tert-butyloxycarbonyl.
[0120] "Aryloxycarbonyl" refers to an aryl-O--C(.dbd.O)-- group.
Exemplary aryloxycarbonyl groups include phenoxy- and
naphthoxy-carbonyl.
[0121] "Aralkoxycarbonyl" refers to an aralkyl-O--C(.dbd.O)--
group. An exemplary aralkoxycarbonyl group is
benzyloxycarbonyl.
[0122] "Carbamoyl" refers to an amide group of the formula
--C(.dbd.O)NH.sub.2. "Alkylcarbamoyl" refers to a R'RN--C(.dbd.O)--
group wherein one of R and R' is hydrogen and the other of R and R'
is alkyl and/or substituted alkyl as previously described.
"Dialkylcarbamoyl" refers to a R'RN--C(.dbd.O)-- group wherein each
of R and R' is independently alkyl and/or substituted alkyl as
previously described.
[0123] The term carbonyldioxyl, as used herein, refers to a
carbonate group of the formula --O--C(.dbd.O)--OR.
[0124] "Acyloxyl" refers to an acyl-O-- group wherein acyl is as
previously described.
[0125] The term "amino" refers to the --NH.sub.2 group and also
refers to a nitrogen containing group as is known in the art
derived from ammonia by the replacement of one or more hydrogen
radicals by organic radicals. For example, the terms "acylamino"
and "alkylamino" refer to specific N-substituted organic radicals
with acyl and alkyl substituent groups respectively.
[0126] An "aminoalkyl" as used herein refers to an amino group
covalently bound to an alkylene linker. More particularly, the
terms alkylamino, dialkylamino, and trialkylamino as used herein
refer to one, two, or three, respectively, alkyl groups, as
previously defined, attached to the parent molecular moiety through
a nitrogen atom. The term alkylamino refers to a group having the
structure --NHR' wherein R' is an alkyl group, as previously
defined; whereas the term dialkylamino refers to a group having the
structure --NR'R'', wherein R' and R'' are each independently
selected from the group consisting of alkyl groups. The term
trialkylamino refers to a group having the structure --NR'R''R''',
wherein R', R'', and R'' are each independently selected from the
group consisting of alkyl groups. Additionally, R', R'', and/or
R''' taken together may optionally be --(CH.sub.2).sub.k-- where k
is an integer from 2 to 6. Examples include, but are not limited
to, methylamino, dimethylamino, ethylamino, diethylamino,
diethylaminocarbonyl, methylethylamino, isopropylamino, piperidino,
trimethylamino, and propylamino.
[0127] The amino group is --NR'R'', wherein R' and R'' are
typically selected from hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted aryl, or substituted
or unsubstituted heteroaryl.
[0128] The terms alkylthioether and thioalkoxyl refer to a
saturated (i.e., alkyl-S--) or unsaturated (i.e., alkenyl-S-- and
alkynyl-S--) group attached to the parent molecular moiety through
a sulfur atom. Examples of thioalkoxyl moieties include, but are
not limited to, methylthio, ethylthio, propylthio, isopropylthio,
n-butylthio, and the like.
[0129] "Acylamino" refers to an acyl-NH-- group wherein acyl is as
previously described. "Aroylamino" refers to an aroyl-NH-- group
wherein aroyl is as previously described.
[0130] The term "carbonyl" refers to the --C(.dbd.O)-- group, and
can include an aldehyde group represented by the general formula
R--C(.dbd.O)H.
[0131] The term "carboxyl" refers to the --COOH group. Such groups
also are referred to herein as a "carboxylic acid" moiety.
[0132] The terms "halo," "halide," or "halogen" as used herein
refer to fluoro, chloro, bromo, and iodo groups. Additionally,
terms such as "haloalkyl," are meant to include monohaloalkyl and
polyhaloalkyl. For example, the term "halo(C.sub.1-C.sub.4)alkyl"
is mean to include, but not be limited to, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0133] The term "hydroxyl" refers to the --OH group.
[0134] The term "hydroxyalkyl" refers to an alkyl group substituted
with an --OH group.
[0135] The term "mercapto" refers to the --SH group.
[0136] The term "oxo" as used herein means an oxygen atom that is
double bonded to a carbon atom or to another element.
[0137] The term "nitro" refers to the --NO.sub.2 group.
[0138] The term "thio" refers to a compound described previously
herein wherein a carbon or oxygen atom is replaced by a sulfur
atom.
[0139] The term "sulfate" refers to the --SO.sub.4 group.
[0140] The term thiohydroxyl or thiol, as used herein, refers to a
group of the formula --SH.
[0141] More particularly, the term "sulfide" refers to compound
having a group of the formula --SR.
[0142] The term "sulfone" refers to compound having a sulfonyl
group --S(O.sub.2)R.
[0143] The term "sulfoxide" refers to a compound having a sulfinyl
group --S(O)R
[0144] The term ureido refers to a urea group of the formula
--NH--CO--NH.sub.2.
[0145] Throughout the specification and claims, a given chemical
formula or name shall encompass all tautomers, congeners, and
optical- and stereoisomers, as well as racemic mixtures where such
isomers and mixtures exist.
[0146] Certain compounds of the present disclosure may possess
asymmetric carbon atoms (optical or chiral centers) or double
bonds; the enantiomers, racemates, diastereomers, tautomers,
geometric isomers, stereoisometric forms that may be defined, in
terms of absolute stereochemistry, as (R)- or (S)- or, as D- or L-
for amino acids, and individual isomers are encompassed within the
scope of the present disclosure. The compounds of the present
disclosure do not include those which are known in art to be too
unstable to synthesize and/or isolate. The present disclosure is
meant to include compounds in racemic, scalemic, and optically pure
forms. Optically active (R)- and (S)-, or D- and L-isomers may be
prepared using chiral synthons or chiral reagents, or resolved
using conventional techniques. When the compounds described herein
contain olefenic bonds or other centers of geometric asymmetry, and
unless specified otherwise, it is intended that the compounds
include both E and Z geometric isomers.
[0147] Unless otherwise stated, structures depicted herein are also
meant to include all stereochemical forms of the structure; i.e.,
the R and S configurations for each asymmetric center. Therefore,
single stereochemical isomers as well as enantiomeric and
diastereomeric mixtures of the present compounds are within the
scope of the disclosure.
[0148] It will be apparent to one skilled in the art that certain
compounds of this disclosure may exist in tautomeric forms, all
such tautomeric forms of the compounds being within the scope of
the disclosure. The term "tautomer," as used herein, refers to one
of two or more structural isomers which exist in equilibrium and
which are readily converted from one isomeric form to another.
[0149] Unless otherwise stated, structures depicted herein are also
meant to include compounds which differ only in the presence of one
or more isotopically enriched atoms. For example, compounds having
the present structures with the replacement of a hydrogen by a
deuterium or tritium, or the replacement of a carbon by .sup.13C-
or .sup.14C-enriched carbon are within the scope of this
disclosure.
[0150] The compounds of the present disclosure may also contain
unnatural proportions of atomic isotopes at one or more of atoms
that constitute such compounds. For example, the compounds may be
radiolabeled with radioactive isotopes, such as for example tritium
(.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C). All
isotopic variations of the compounds of the present disclosure,
whether radioactive or not, are encompassed within the scope of the
present disclosure.
[0151] The compounds of the present disclosure may exist as salts.
The present disclosure includes such salts. Examples of applicable
salt forms include hydrochlorides, hydrobromides, sulfates,
methanesulfonates, nitrates, maleates, acetates, citrates,
fumarates, tartrates (e.g. (+)-tartrates, (-)-tartrates or mixtures
thereof including racemic mixtures, succinates, benzoates and salts
with amino acids such as glutamic acid. These salts may be prepared
by methods known to those skilled in art. Also included are base
addition salts such as sodium, potassium, calcium, ammonium,
organic amino, or magnesium salt, or a similar salt. When compounds
of the present disclosure contain relatively basic functionalities,
acid addition salts can be obtained by contacting the neutral form
of such compounds with a sufficient amount of the desired acid,
either neat or in a suitable inert solvent or by ion exchange.
Examples of acceptable acid addition salts include those derived
from inorganic acids like hydrochloric, hydrobromic, nitric,
carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,
dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or
phosphorous acids and the like, as well as the salts derived
organic acids like acetic, propionic, isobutyric, maleic, malonic,
benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like. Certain
specific compounds of the present disclosure contain both basic and
acidic functionalities that allow the compounds to be converted
into either base or acid addition salts.
[0152] The neutral forms of the compounds may be regenerated by
contacting the salt with a base or acid and isolating the parent
compound in the conventional manner. The parent form of the
compound differs from the various salt forms in certain physical
properties, such as solubility in polar solvents.
[0153] Certain compounds of the present disclosure can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are encompassed within the scope of the present
disclosure. Certain compounds of the present disclosure may exist
in multiple crystalline or amorphous forms. In general, all
physical forms are equivalent for the uses contemplated by the
present disclosure and are intended to be within the scope of the
present disclosure.
[0154] In addition to salt forms, the present disclosure provides
compounds, which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present disclosure. Additionally, prodrugs can be converted to
the compounds of the present disclosure by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present disclosure when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0155] Following long-standing patent law convention, the terms
"a," "an," and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a subject" includes a plurality of subjects, unless the context
clearly is to the contrary (e.g., a plurality of subjects), and so
forth.
[0156] Throughout this specification and the claims, the terms
"comprise," "comprises," and "comprising" are used in a
non-exclusive sense, except where the context requires otherwise.
Likewise, the term "include" and its grammatical variants are
intended to be non-limiting, such that recitation of items in a
list is not to the exclusion of other like items that can be
substituted or added to the listed items.
[0157] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing amounts, sizes,
dimensions, proportions, shapes, formulations, parameters,
percentages, quantities, characteristics, and other numerical
values used in the specification and claims, are to be understood
as being modified in all instances by the term "about" even though
the term "about" may not expressly appear with the value, amount or
range. Accordingly, unless indicated to the contrary, the numerical
parameters set forth in the following specification and attached
claims are not and need not be exact, but may be approximate and/or
larger or smaller as desired, reflecting tolerances, conversion
factors, rounding off, measurement error and the like, and other
factors known to those of skill in the art depending on the desired
properties sought to be obtained by the presently disclosed subject
matter. For example, the term "about," when referring to a value
can be meant to encompass variations of, in some embodiments,
.+-.100% in some embodiments .+-.50%, in some embodiments .+-.20%,
in some embodiments .+-.10%, in some embodiments .+-.5%, in some
embodiments .+-.1%, in some embodiments .+-.0.5%, and in some
embodiments .+-.0.1% from the specified amount, as such variations
are appropriate to perform the disclosed methods or employ the
disclosed compositions.
[0158] Further, the term "about" when used in connection with one
or more numbers or numerical ranges, should be understood to refer
to all such numbers, including all numbers in a range and modifies
that range by extending the boundaries above and below the
numerical values set forth. The recitation of numerical ranges by
endpoints includes all numbers, e.g., whole integers, including
fractions thereof, subsumed within that range (for example, the
recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as
fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and
any range within that range.
EXAMPLES
[0159] The following Examples have been included to provide
guidance to one of ordinary skill in the art for practicing
representative embodiments of the presently disclosed subject
matter. In light of the present disclosure and the general level of
skill in the art, those of skill can appreciate that the following
Examples are intended to be exemplary only and that numerous
changes, modifications, and alterations can be employed without
departing from the scope of the presently disclosed subject matter.
The synthetic descriptions and specific examples that follow are
only intended for the purposes of illustration, and are not to be
construed as limiting in any manner to make compounds of the
disclosure by other methods.
Example 1
Overview
[0160] .sup.68Ga-Labeled, low-molecular-weight imaging agents that
target the prostate-specific membrane antigen (PSMA) are
increasingly used clinically to detect prostate and other cancers
with positron emission tomography (PET). The presently disclosed
subject matter compares the pharmacokinetics of three PSMA-targeted
radiotracers: .sup.68Ga-1, using DOTA-monoamide as the chelating
agent; .sup.68Ga-2, containing the macrocyclic chelating agent
p-SCN-Bn-NOTA, and .sup.68Ga-DKFZ-PSMA-11, currently in clinical
trials, which uses the acyclic chelating agent, HBED-CC. The
PSMA-targeting scaffold for all three agents utilizes a similar
Glu-Lys-urea-linker construct. Each radiotracer enabled
visualization of PSMA+ PC3 PIP tumor, kidney, and urinary bladder
as early as 15 min post-injection using small animal PET/computed
tomography (PET/CT). .sup.68Ga-2 demonstrated the highest PSMA+ PC3
PIP tumor uptake, at 42.2.+-.6.7 percentage injected dose per gram
(% ID/g) of tissue at 1 h post-injection, and the fastest rate of
clearance from all tissues. .sup.68Ga-1 and .sup.68Ga-DKFZ-PSMA-11
displayed similar uptake and retention patterns in PSMA+ PC3 PIP
tumors up to 3 h post-injection. .sup.68Ga-DKFZ-PSMA-11
demonstrated the highest uptake and retention in normal tissues,
including kidney, blood, spleen, salivary glands and PSMA-negative
PC3 flu tumors up to 3 h post-injection. In this preclinical
evaluation .sup.68Ga-2 had the most advantageous characteristics
for PSMA-targeted PET imaging. The biodistribution profile of
.sup.68Ga-1 indicates promise for future therapeutic radionuclides
that could employ a similar combination of chelator and targeting
scaffold.
Example 2
Material and Methods
[0161] Solvents and chemicals purchased from commercial sources
were of analytical grade or better and used without further
purification. [.sup.68Ga]GaCl.sub.3 was obtained from the
University of Wisconsin. DOTA-tris(t-butyl ester)-monoacid and
p-SCN-Bn-NOTA were received from Macrocyclics, Inc. (Dallas, Tex.).
Compounds 1 and 2 were synthesized following our previous report
(Banerjee et al. (2010) J Med Chem 53, 5333-41). DKFZ-PSMA-11 and
the corresponding stable Ga-DKFZ-PSMA-11 were purchased from ABX
(Radeberg, Germany). Triethylsilane (Et.sub.3SiH),
diisopropylethylamine (DIEA) and triethylamine (TEA) were purchased
from Sigma-Aldrich (St. Louis, Mo.). All other chemicals were
purchased from Thermo Fisher Scientific (Pittsburgh, Pa.) unless
otherwise specified.
[0162] Analytical thin-layer chromatography (TLC) was performed
using Aldrich aluminum-backed 0.2 mm silica gel Z19, 329-1 plates
and was visualized by ultraviolet light (254 nm), I.sub.2 and 1%
ninhydrin in EtOH. Flash chromatography was performed using silica
gel (MP SiliTech 32-63 D 60 .ANG.) purchased from Bodman (Aston,
Pa.). All in vitro PSMA binding studies and determination of
partition coefficients were performed in triplicate to ensure
reproducibility, as previously reported (Banerjee et al. (2011)
Angewandte Chemie 50, 9167-70). .sup.1H NMR spectra were recorded
on a Bruker Ultrashield.TM. 400 MHz spectrometer. Chemical shifts
(.delta.) are reported in ppm downfield in reference to proton
resonances resulting from incomplete deuteration of the NMR
solvent. Quantitative .sup.1H NMR was used to prove that all
synthesized compounds were at >95% chemical purity.
[0163] Low resolution ESI mass spectra were obtained on a Bruker
Daltonics Esquire 3000 Plus spectrometer. High resolution mass
spectra were obtained by the University of Notre Dame Mass
Spectrometry & Proteomics Facility, Notre Dame, Ind., using
electrospray ionization (ESI) mass spectrometry either by direct
infusion on a Bruker microTOF-II or by LC elution via an ultra-high
pressure Dionex RSLC C.sub.18 column coupled to a Bruker microTOF-Q
II. The purity of tested compounds was also determined by
analytical high performance liquid chromatography (HPLC) with
absorbance at 220 nm and were all again determined to be
>95%.
[0164] HPLC purification of stable compounds was performed using a
Phenomenex C.sub.18 Luna 10.times.250 mm.sup.2 column and elution
with water (0.1% TFA) (A) and CH.sub.3CN (0.1% TFA) (B) on a Waters
600E Delta LC system with a Waters 486 variable wavelength UV/Vis
detector, both controlled by Empower software (Waters Corporation,
Milford, Mass.). HPLC purifications of .sup.68Ga-1, .sup.68Ga-2 and
.sup.68Ga-DKFZ-PSMA-11 were performed on a Varian Prostar System
(Palo Alto, Calif.), equipped with a Varian ProStar 325 UV-Vis
variable wavelength detector and a Bioscan Flow-count in-line
Radioactivity detector (Washington DC), all controlled by Galaxie
software (Varian Inc., Walnut Creek, Calif.).
[0165] All radiotracers were purified using a Varian microsob-MV
100-5 C.sub.8 25.times.4.6 mm column with a flow rate 1 mL/min with
water (0.1% TFA) (A) and CH.sub.3CN (0.1% TFA) (B) as the eluting
solvents. In order to ensure uniform purity of the compounds
undergoing comparison, various HPLC methods were applied to
separate excess ligand from the radiolabeled compound. For
.sup.68Ga-1, an isocratic solution of 80% A and 20% B was used. For
.sup.68Ga-2 and .sup.68Ga-DKFZ-PSMA-11, an isocratic solution of
85% water and 15% B was employed.
[0166] Retention times of the radiolabeled compound and unlabeled
free ligands are listed in Table 1. The radiochemical yield and
purity of the radiotracers were further checked by withdrawing 1
.mu.L aliquots of the radiolabeled solution and were analyzed by
radio-TLC on RP-18 thin layer plates using 5/1 saline/methanol as
the mobile phase. The specific radioactivity was calculated as the
radioactivity eluting at the retention time of product during the
preparative HPLC purification divided by the mass corresponding to
the area under the curve of the UV absorption.
[0167] Radiolabeling Methods. .sup.68Ga-Labeling of target ligands
was performed according to a method previously reported (Banerjee
et al. (2010) J Med Chem 53, 5333-41) and following other
literature procedures (Eder et al. (2012) Bioconjug Chem 23,
688-97; Zhernosekov et al. (2007) J Nucl Med 48, 1741-8. Briefly,
488 MBq (13 mCi) of .sup.68GaCl.sub.3 in 7 mL of 0.1 N HCl were
obtained from an 18-month-old 1,850 MBq (50 mCi)
.sup.68Ge/.sup.68Ga generator, Eckert-Ziegler (Berlin, DE).
Pre-concentration was performed on a cation-exchange cartridge. The
purified .sup.68Ga(III)Cl.sub.3 was obtained in a total volume of
400 .mu.L, eluted in 2.4/97.6 0.05 N HCl/acetone. The
.sup.68Ga(III) in HCl/acetone was used immediately for the
radiolabeling of 1, 2 or DKFZ-PSMA-11.
[0168] Two radiolabeling techniques were investigated. The first
was undertaken in water (without any added buffer solution), as
reported earlier (Banerjee et al. (2010) J Med Chem 53, 5333-41),
and the second used 2.1 M HEPES buffer at pH about 4, as reported
by Eder et al. (Eder et al. (2012) Bioconjug Chem 23, 688-97).
Using HEPES buffer, each ligand (12.5 .mu.g), was radiolabeled in
>95% yield in a total volume of about 120 .mu.L, however this
yield was dependent on the total volume of the radiolabeling
solution. In water the pre-concentrated .sup.68Ga(III)Cl.sub.3
solution could be directly used for radiolabeling at pH about 3-4
(Banerjee et al. (2010) J Med Chem 53, 5333-41). The total volume
of the radiolabeling solution was about 300-350 .mu.L to produce
>93% yield using about 4-6 .mu.g of each precursor ligand.
[0169] In a typical reaction 50 .mu.L of the concentrated
radioactivity was added to 250 .mu.L of deionized H.sub.2O in a 1.5
mL polypropylene vial, followed by addition of 3-5 .mu.L of a
solution of precursor ligand (2 .mu.g/.mu.L in water, pH about
3.5-4). The reaction vial was heated at 95.degree. C. for 10 min
for ligand 1, about 3 min for ligand 2 and the complex was allowed
to form at room temperature for 10 min for both 2 and DKFZ-PSMA-11.
Complex formation was monitored by iTLC as above, using 5/1
saline/methanol.
[0170] For the comparison studies, all three radiotracers were
purified by HPLC to remove excess precursor ligand so that all
three radioligands could be obtained in >98% purity. The acidic
eluate was neutralized with 50 .mu.L 1 M Na.sub.2CO.sub.3 and the
volume of the eluate was reduced under vacuum to dryness. The solid
residue was diluted with saline to the desired radioactivity
concentration for biodistribution and imaging studies.
[0171] PSMA Inhibition Assay. The PSMA inhibitory activity of 1, 2
and DKFZ-PSMA-11 and the corresponding natural Ga-labeled analogs
Ga-1 and Ga-2 were determined using a fluorescence-based assay
according to a previously reported procedure (Banerjee et al.
(2011) Angewandte Chemie 50, 9167-70) (Table 1). Briefly, lysates
of LNCaP cell extracts (25 .mu.L) were incubated with the inhibitor
(12.5 .mu.L) in the presence of 4 .mu.M N-acetylaspartylglutamate
(NAAG) (12.5 .mu.L) for 2 h. The amount of the glutamate released
by NAAG hydrolysis was measured by incubating with a working
solution (50 .mu.L) of the Amplex Red Glutamic Acid Kit (Life
Technologies, Grand Island, N.Y.) for 1 h.
[0172] Fluorescence was measured with a VICTOR3V multilabel plate
reader (Perkin Elmer Inc., Waltham, Mass.) with excitation at 490
nm and emission at 642 nm. Inhibition curves were determined using
semi-log plots and IC50 values were determined at the concentration
at which enzyme activity was inhibited by 50%. Enzyme inhibitory
constants (K.sub.i values) were generated using the Cheng-Prusoff
conversion (Cheng et al. (1973) Biochemical pharmacology 22,
3099-108). Assays were performed in triplicate. Data analysis was
performed using GraphPad Prism version 4.00 for Windows (GraphPad
Software, San Diego, Calif.).
[0173] Cell Lines. Sublines of the androgen-independent PC3 human
prostate cancer cell line, originally derived from an advanced
androgen independent bone metastasis, were used. Those sublines
have been modified to express high levels of PSMA [PSMA-positive
(+) PC3 PIP] or are devoid of target [PSMA-negative (-) PC3 flu].
They were generously provided by Dr. Warren Heston (Cleveland
Clinic). Cells were grown in RPMI 1640 medium (Corning Cellgro,
Manassas, Va.) containing 10% fetal bovine serum (FBS)
(Sigma-Aldrich, St. Louis, Mo.) and 1% penicillin-streptomycin
(Coming Cellgro, Manassas, Va.). PSMA+ PC3 PIP cells were grown in
the presence of 20 .mu.g/mL of puromycin to maintain PSMA
expression. All cell cultures were maintained in an atmosphere
containing 5% carbon dioxide (CO.sub.2), at 37.0.degree. C. in a
humidified incubator.
[0174] Tumor Models. Animal studies were undertaken in compliance
with the regulations of the Johns Hopkins University Animal Care
and Use Committee. Six-to eight-week-old male, Nonobese Diabetic
(NOD)/Severe Combined immunodeficient (SCID) mice (Johns Hopkins
Immune Compromised Animal Core) were implanted subcutaneously (sc)
with PSMA+ PC3 PIP and PSMA- PC3 flu cells (1.times.10.sup.6 in 100
.mu.L of HBSS (Corning Cellgro, Manassas, Va.) at the forward right
and left flanks, respectively. Mice were imaged or used in
biodistribution assays when the xenografts reached 5 to 7 mm in
diameter.
[0175] Small-animal PET Imaging and Analysis. Whole-body PET and CT
images were acquired on a SuperArgus PET-CT preclinical imaging
system (SEDECAL SA4R PET-CT, Madrid, Spain). For imaging studies,
mice were anesthetized with 3% and maintained under 1.5% isoflurane
(v/v). PET-CT Imaging studies were performed on NOD/SCID mice
bearing PSMA+ PC3 PIP and PSMA- PC3 flu tumors. After intravenous
injection of .sup.68Ga-1, .sup.68Ga-2 or .sup.68Ga-DKFZ-PSMA-11,
whole-body PET emission images (two bed positions, 15 min per
position) were acquired at the indicated (30 min, 1 h, 2 h and 3 h)
time points after injection of radiotracer.
[0176] For binding specificity studies, a mouse was subcutaneously
administered a blocking dose of the known PSMA inhibitor ZJ43
(Olszewski et al. (2004) Journal of neurochemistry 89, 876-85) (50
mg/kg) at 30 min before the injection of .sup.68Ga-2, and another
mouse was injected with .sup.68Ga-2 alone. A CT scan was acquired
after each PET scan in 512 projections using a 50 keV beam for
anatomic co-registration. PET emission data were corrected for
decay and dead time and were reconstructed using the 3-dimensional
ordered-subsets expectation maximization algorithm. Data were
displayed and analyzed using AMIDE software
(http://sourceforge.net/amide).
[0177] Biodistribution. Mice bearing PSMA+ PC3 PIP and PSMA- PC3
flu xenografts were injected via the tail vein with 740 kBq (20
.mu.Ci) of .sup.68Ga-1, .sup.68Ga-2 and .sup.68Ga-DKFZ-PSMA-11 in
150 .mu.L of saline (n=4). At 1 h, 2 h, and 3 h post-injection,
mice were sacrificed by cervical dislocation and the blood was
immediately collected by cardiac puncture. The heart, lungs, liver,
stomach, pancreas, spleen, fat, kidney, muscle, small and large
intestines, urinary bladder, PSMA+ PC3 PIP and PSMA- PC3 flu tumors
were collected. Each organ was weighed, and the tissue
radioactivity was measured with an automated gamma counter (1282
Compugamma CS, Pharmacia/LKBNuclear, Inc., Mt. Waverly, Vic.
Australia). The percentage of injected dose per gram of tissue (%
ID/g) was calculated by comparison with samples of a standard
dilution of the initial dose. All measurements were corrected for
decay.
[0178] Data Analysis. Data are expressed as mean.+-.standard
deviation (SD). Prism software (GraphPAD, San Diego, Calif.) was
used to determine statistical significance. Statistical
significance was calculated using a two-tailed Student's t test. A
P-value.ltoreq.0.05 was considered significant.
Example 3
Results
[0179] Chemical and Radiochemical Syntheses and Characterization.
Structures of the radioligands used for the study are shown in FIG.
1. Lys-Glu urea was used as the PSMA-targeting moiety in all cases.
Selected physical properties of 1, 2 and the corresponding natural
Ga-complexes are summarized in Table 1. Since NOTA is a hexadentate
N.sub.3O.sub.3 macrocyclic chelator, [.sup.68Ga(III)]2 was expected
produce a neutral compound (Broan et al. (1991) J. Chem. Soc.
Perkin Trans. 2, 87-99.). DKFZ-PSMA-11 chelated with HBED-CC is
reported to provide a uni-negative, hexadentate chelation
(N.sub.2O.sub.4) to Ga(III), with two carboxylates and two
phenolates (L'Eplattenier et al. (1967) Journal of the American
Chemical Society 89; Zoller et al. (1992) Journal of Nuclear
Medicine 33, 1366-1372; Eder et al. (2008) European Journal of
Nuclear Medicine and Molecular Imaging 35, 1878-1886). All three
radiotracers were synthesized in high radiochemical yield (about
95-99%) and purity (>98%), with specific radioactivity>168
GBq/.mu.mol (4.05 mCi/.mu.mol).
[0180] Two radiolabeling methods have been investigated, one in the
presence of HEPES buffer as reported by Eder et al. (Eder et al.
(2012) Bioconjug Chem 23, 688-97) and the other by a method
reported by Banerjee et al. (Banerjee et al. (2010) J Med Chem 53,
5333-41) following the literature (Zhernosekov et al. (2007) J Nucl
Med 48, 1741-8). For the latter method, pre-concentrated
.sup.68Ga(III)Cl.sub.3 could be directly used for radiolabeling,
without adjusting pH, and radiolabeling could be done in a total
volume of 300-350 .mu.L using as low as 4 .mu.g of any of the three
ligands. Based on the HPLC retention time (Table 1), the
non-radiolabeled precursor DKFZ-PSMA-11 was the least hydrophilic,
although, after radiolabeling, .sup.68Ga-DKFZ-PSMA-11 became the
most hydrophilic compound in the series.
TABLE-US-00001 TABLE 1 Selected physical properties of the
presently disclosed agents. HPLC (RP C.sub.8) Molar Mass K.sub.i
95% CI of K.sub.i retention time (g/mol) (nM) (nM) (min) 1 1284.4
0.70 0.42-1.16 19.2-19.8.sup.a Ga-1 1352.5 0.33 0.17-0.66
21.8-23.8.sup.a 2 1054.2 0.81 0.35-1.89 23.9-24.9.sup.b Ga-2 1120.9
0.38 2.26-6.28 20.8-22.5.sup.b DKFZ-PSMA-11 947.0 0.03 0.016-0.06
34.5-40.0.sup.b Ga-DKFZ-PSMA-11 1013.7 N.A. N.A. 14.5-20.0.sup.b
ZJ43 304.3 0.31 0.20-0.48 N.A. Compounds 1 and 2 are the unlabeled
agents containing DOTA-monoamide and NOTA-Bn-SCN chelating agents,
respectively. .sup.aIsocratic solution of 80% A and 20% B.
.sup.bIsocratic solution of 85% water and 15% B. Flow rate was 1
mL/min for both methods.
[0181] Precursor ligands and the corresponding stable metal-labeled
compounds demonstrated high binding affinity to PSMA, with K.sub.i
values ranging from 0.03 to 0.81 nM (Table 1). The known,
high-affinity PSMA inhibitor ZJ43 (Olszewski et al. (2004) Journal
of neurochemistry 89, 876-85) was used as a reference ligand and
exhibited a K.sub.i of 0.31 nM (Table 1). DKFZ-PSMA-11 displayed
the highest PSMA-binding affinity from the compounds tested in this
comparative study, ten-fold higher than either of Ga-1 and Ga-2. In
addition, based on HPLC data, it was observed that DKFZ-PSMA-11 is
the most lipophilic in the series, although after complexation with
gallium (III), the agent Ga-DKFZ-PSMA-11, is the most hydrophilic
in the series. The order of hydrophilicity is
Ga-DKFZ-PSMA-11>Ga-2>Ga-SR2.
[0182] Biodistribution. Tables 2, 3 and 4 show the pharmacokinetics
in selected organs for .sup.68Ga-1, .sup.68Ga-2 and
.sup.68Ga-DKFZ-PSMA-11, respectively. All compounds exhibited clear
PSMA-dependent binding in PSMA+ PC3 PIP tumor xenografts. The tumor
uptake for .sup.68Ga-1 was 19.46.+-.1.81% ID/g at 1 h, highest at 2
h (24.75.+-.1.05% ID/g) and remained high at 3 h post-injection
(19.46.+-.5.12% ID/g) (Table 2). PSMA+ PC3 PIP-to-PSMA- PC3 flu
tumor uptake ratios were 83.60.+-.3.59 at 1 h and 148.75.+-.16.43
at 2 h. The distribution within normal organs and tissues was also
favorable, with low blood and normal tissue uptake and rapid
clearance. The highest non-specific accumulation of radioactivity
was observed in the kidneys, where uptake was expectedly high and
peaked at 26.45.+-.6.85% ID/g at 1 h and decreased to
11.88.+-.0.99% ID/g by 2 h and remained roughly the same at 3 h
post-injection.
TABLE-US-00002 TABLE 2 Tissue biodistribution of .sup.68Ga-1 in
mice bearing PSMA + PC3 PIP and PSMA - PC3 flu tumors (n = 4).
Values are expressed as mean .+-. SD. Tissue 1 h 2 h 3 h blood 0.45
.+-. 0.05 0.25 .+-. 0.01 0.16 .+-. 0.03 heart 0.16 .+-. 0.03 0.10
.+-. 0.01 0.07 .+-. 0.02 lung 0.43 .+-. 0.03 0.21 .+-. 0.01 0.14
.+-. 0.04 liver 0.19 .+-. 0.03 0.17 .+-. 0.02 0.13 .+-. 0.02 spleen
1.03 .+-. 0.28 0.39 .+-. 0.04 0.40 .+-. 0.19 kidney 26.45 .+-. 6.85
11.88 .+-. 0.99 12.09 .+-. 5.56 muscle 0.14 .+-. 0.10 0.04 .+-.
0.00 0.03 .+-. 0.01 small intestine 0.17 .+-. 0.03 0.10 .+-. 0.00
0.08 .+-. 0.01 salivary gland 0.25 .+-. 0.03 0.16 .+-. 0.00 0.11
.+-. 0.02 PSMA + PC3 PIP 19.46 .+-. 1.81 24.75 .+-. 1.05 19.46 .+-.
5.12 PSMA - PC3 flu 0.23 .+-. 0.03 0.17 .+-. 0.01 0.16 .+-. 0.03
PIP:flu 83.60 .+-. 3.59 148.75 .+-. 16.43 122.32 .+-. 31.28
PIP:kidney 0.82 .+-. 0.25 2.09 .+-. 0.09 1.77 .+-. 0.60 PIP:blood
59.17 .+-. 24.48 100.91 .+-. 10.12 236.06 .+-. 235.66 PIP:salivary
gland 76.91 .+-. 2.73 157.24 .+-. 2.55 172.83 .+-. 27.29
[0183] Table 3 shows the organ-related % ID/g of uptake for
.sup.68Ga-2. .sup.68Ga-2 showed the highest PSMA-dependent tumor
uptake with 42.18.+-.6.66% ID/g at 1 h post-injection. Tumor uptake
remained high, with faster clearance from 1 h to 2 h. The PSMA+ PC3
PIP-to-PSMA- PC3 flu tumor ratios were 109.82.+-.21.61 at 1 h,
232.14.+-.25.99 at 2 h and 182.27.+-.14.59 at 3 h. Renal uptake for
.sup.68Ga-2 was highest at 1 h, 106.37.+-.23.29% ID/g, much higher
than that seen for .sup.68Ga-1 and showed faster renal clearance,
which decreased to 34.73.+-.5.74% ID/g by 2 h post-injection. In
addition, non-target organs, such as blood, heart, liver, spleen,
stomach, pancreas, showed lower uptake (.ltoreq.1% ID/g at 1 h,
except for spleen) and faster clearance than for .sup.68Ga-1.
TABLE-US-00003 TABLE 3 Tissue biodistribution of .sup.68Ga-2 in
mice bearing PSMA + PC3 PIP and PSMA - PC3 flu tumors (n = 4).
Values are expressed as mean .+-. SD. Tissue 1 h 2 h 3 h Blood 0.38
.+-. 0.18 0.07 .+-. 0.02 0.09 .+-. 0.02 heart 0.23 .+-. 0.08 0.04
.+-. 0.01 0.04 .+-. 0.01 lung 1.00 .+-. 0.20 0.25 .+-. 0.02 0.23
.+-. 0.07 liver 0.52 .+-. 0.15 0.16 .+-. 0.01 0.16 .+-. 0.03 spleen
4.88 .+-. 0.68 0.79 .+-. 0.23 0.69 .+-. 0.11 kidney 106.37 .+-.
23.29 34.73 .+-. 5.74 12.68 .+-. 4.92 muscle 0.12 .+-. 0.02 0.03
.+-. 0.01 0.03 .+-. 0.02 small intestine 0.21 .+-. 0.08 0.04 .+-.
0.01 0.05 .+-. 0.02 salivary gland 0.23 .+-. 0.08 0.16 .+-. 0.11
0.08 .+-. 0.01 PSMA + PC3 PIP 42.18 .+-. 6.66 21.66 .+-. 3.68 17.39
.+-. 5.55 PSMA - PC3 flu 0.40 .+-. 0.14 0.10 .+-. 0.01 0.09 .+-.
0.03 PIP:flu 109.82 .+-.2 1.61 232.14 .+-. 25.99 182.27 .+-. 14.59
PIP:kidney 0.40 .+-. 0.06 0.67 .+-. 0.14 1.63 .+-. 0.05 PIP:blood
119.62 .+-. 28.54 320.90 .+-. 78.52 206.66 .+-. 32.95 PIP:salivary
gland 188.14 .+-. 35.20 211.89 .+-. 2.83 222.32 .+-. 35.18
[0184] Table 4 lists the organ-related % ID/g of uptake for
.sup.68Ga-DKFZ-PSMA-11. Unlike .sup.68Ga-1, .sup.68Ga-DKFZ-PSMA-11
showed the highest PSMA-dependent tumor uptake with 26.86.+-.5.59%
ID/g at 3 h post-injection. Tumor uptake was nearly comparable from
1 to 3 h post-injection. The PSMA+ PC3 PIP-to-PSMA- PC3 flu ratios
were 46.62.+-.7.57% ID/g at 1 h, 57.68.+-.27.10% ID/g at 2 h and
110.57.+-.21.27% ID/g at 3 h post-injection.
TABLE-US-00004 TABLE 4 Tissue biodistribution of
.sup.68Ga-DKFZ-PSMA-11 in mice bearing PSMA + PC3 PIP and PSMA -
PC3 flu tumors (n = 4). Values are expressed as mean .+-. SD.
Tissue 1 h 2 h 3 h blood 0.75 .+-. 0.20 0.41 .+-. 0.06 0.34 .+-.
0.06 heart 0.42 .+-. 0.16 0.28 .+-. 0.06 0.20 .+-. 0.04 lung 2.21
.+-. 0.48 2.07 .+-. 0.68 1.26 .+-. 0.20 liver 0.75 .+-. 0.19 0.33
.+-. 0.05 0.38 .+-. 0.15 spleen 12.35 .+-. 3.75 8.87 .+-. 1.50 7.18
.+-. 2.25 kidney 133.24 .+-. 21.08 88.56 .+-. 20.09 119.54 .+-.
15.49 muscle 0.32 .+-. 0.12 0.19 .+-. 0.09 0.14 .+-. 0.02 small
intestine 0.39 .+-. 0.09 0.19 .+-. 0.07 0.17 .+-. 0.06 salivary
gland 1.42 .+-. 0.33 1.02 .+-. 0.22 0.86 .+-. 0.12 PSMA + PC3 PIP
25.96 .+-. 9.69 21.08 .+-. 1.17 26.86 .+-. 5.59 PSMA - PC3 flu 0.57
.+-. 0.14 0.41 .+-. 0.17 0.25 .+-. 0.07 PIP:flu 46.62 .+-. 17.57
57.68 .+-. 27.10 110.57 .+-. 21.27 PIP:kidney 0.20 .+-. 0.09 0.24
.+-. 0.04 0.22 .+-. 0.04 PIP:blood 35.45 .+-. 13.43 51.88 .+-.
10.82 79.55 .+-. 9.83 PIP:salivary gland 18.57 .+-. 8.78 20.99 .+-.
3.40 31.40 .+-. 4.85
[0185] FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D summarize several
comparative tissue uptake properties of the three agents. PSMA+ PC3
PIP tumor uptake of .sup.68Ga-2 was significantly higher than
.sup.68Ga-1 at 1 h post-injection (P<0.004) (FIG. 2A). There was
no significant difference in PSMA+ PIP tumor uptake between
.sup.68Ga-1 and .sup.68Ga-DKFZ-PSMA-11 or between .sup.68Ga-2 and
.sup.68Ga-DKFZ-PSMA-11 (P<0.09). In addition, there were no
significant differences in tumor uptake at 2 h and 3 h
post-injection between the compounds.
[0186] As shown in FIG. 2B, renal uptake of .sup.68Ga-1 was
significantly lower than .sup.68Ga-2 (P<0.006) and
.sup.68Ga-DKFZ-PSMA-11 (P<0.001) at 1 h, although there was no
significant difference between .sup.68Ga-2 and
.sup.68Ga-DKFZ-PSMA-11. At 2 h post-injection renal uptake of both
.sup.68Ga-1 and .sup.68Ga-2 were significantly lower than for
.sup.68Ga-DKFZ-PSMA-11 (P<0.003) and renal uptake of .sup.68Ga-1
was still significantly lower than .sup.68Ga-2 (P<0.005). At 3 h
post-injection renal uptake of both .sup.68Ga-1 and .sup.68Ga-2
were significantly lower than for .sup.68Ga-DKFZ-PSMA-11.
[0187] FIG. 2C reveals that .sup.68Ga-DKFZ-PSMA-11 demonstrated
significantly higher salivary gland uptake up to 3 h after
injection compared to .sup.68Ga-1 and .sup.68Ga-2 (P<0.001).
[0188] FIG. 2D shows higher spleen uptake for
.sup.68Ga-DKFZ-PSMA-11 compared to either 68.sub.Ga-1 or 68Ga-2
(P<0.04) at all time-points. Between .sup.68Ga-1 and 68Ga-2, the
former showed significantly lower spleen (P<0.03) uptake at 1 h
and 2 h post-injection compared to the latter. Selected PSMA+ PC3
PIP tumor-to-background for the three agents at 1-3 h
post-injection are shown in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D.
As anticipated from the biodistribution data, PSMA+ PC3 PIP
tumor-to-salivary gland (.ltoreq.0.002) and PSMA+ PC3 PIP
tumor-to-kidney ratios proved significantly higher for .sup.68Ga-1
and .sup.68Ga-2 than for .sup.68Ga-DKFZ-PSMA-11
(P.ltoreq.0.04).
[0189] PSMA+ PC3 PIP tumor-to-PSMA- PC3 flu tumor ratios were also
significantly higher for .sup.68Ga-1 and .sup.68Ga-2 compared to
.sup.68Ga-DKFZ-PSMA-11 at 1 h post-injection (P.ltoreq.0.02). The
data show that PSMA+ PC3 PIP tumor-to-blood ratios were highest for
.sup.68Ga-2 at all three time points.
[0190] Small Animal PET-CT Imaging. Whole body PET-CT images were
studied for .sup.68Ga-1, .sup.68Ga-2 and .sup.68Ga-DKFZ-PSMA-11 in
intact male NOD/SCID mice (FIG. 6) bearing both PSMA+ PC3 PIP and
PSMA- PC3 flu xenografts in opposite, upper flanks. Irrespective of
charge and lipophilicity, all radiotracers enabled visualization of
PSMA+ PC3 PIP tumor and kidneys (FIG. 6). As anticipated from the
biodistribution results, for all three agents PSMA+ PC3 PIP tumor
was visible as early as 15 min post-injection. Renal uptake of the
radiotracers is partially due to the route of excretion of these
agents as well as to specific uptake from the expression of PSMA in
mouse proximal renal tubules (Silver et al. (1997) Clin Cancer Res
3, 81-5.). All three agents showed significant bladder activity,
indicating rapid renal clearance. A reduction of the tumor and
kidney uptake to background levels was observed with the blocking
agent ZJ43 for .sup.68Ga-2, indicating the receptor-mediated
accumulation of the agents.
Example 4
Discussion
[0191] Structural optimization of low-molecular-weight imaging and
therapeutic agents targeting PSMA is under active investigation
(Banerjee et al. (2010) J Med Chem 53, 5333-41; Kularatne et al.
(2009) Molecular pharmaceutics 6, 780-789; Kularatne et al. (2009)
Mol Pharm 6, 790-800; Nedrow-Byers et al. (2012) Prostate 72,
904-12; Nedrow-Byers et al. (2013) Prostate 73, 355-62; Nguyen
& Tsien (2013) Nat Rev Cancer 13, 653-62; Zhang et al. (2010) J
Am Chem Soc 132, 12711-6; Banerjee et al. (2014) J Med Chem 57,
2657-69; Ray Banerjee et al. (2013) J Med Chem 56, 6108-21;
Anderson et al. (2007) Bioorg Med Chem 15, 6678-86; Benesova et al.
(2015) J Nucl Med 56, 914-20). Such optimization is geared toward
high tumor uptake with minimal off-target, namely, renal and
salivary gland, uptake at times convenient for imaging and
endoradiotherapy. High salivary gland uptake in particular has
proved to be a concern. .sup.68Ga-1 (Banerjee et al. (2010) J Med
Chem 53, 5333-41) was originally synthesized as its scaffold with
the DOTA chelator to enable imaging or therapy, depending on the
radionuclide employed. It has been previously showed that a linker
to DOTA containing a p-isothiocyanatobenzyl function provided the
most suitable pharmacokinetics in a small series of compounds
generated for imaging PSMA with .sup.86Y- and .sup.64Cu-PET
(Banerjee et al. (2014) J Med Chem 57, 2657-69; Banerjee et al.
(2015) Journal of nuclear medicine: official publication, Society
of Nuclear Medicine 56, 628-34). NOTA has been shown to be an
effective chelating agent for .sup.68Ga, (stability constant,
K.sub.ML=31.1), compared to for DOTA (K.sub.ML=21.3) (Studer &
Meares (1992) Bioconjugate Chemistry 3, 337-341; Roesch & Riss
(2010) Curr Top Med Chem 10, 1633-68). The commercially available
p-isothiocyanatobenzyl derivative of NOTA has been used in
.sup.68Ga-2 for its mild radiolabeling conditions in the hope of
creating a .sup.68Ga-based agent with improved pharmacokinetics
that could be generated simply, as in a kit-like preparation. The
in vivo performance characteristics of .sup.68Ga-1, .sup.68Ga-2 and
.sup.68Ga-DKFZ-PSMA-11 (Eder et al. (2012) Bioconjug Chem 23,
688-97) were compared, the latter of which has been used throughout
Europe in clinical trials.
[0192] To improve precision with respect to the comparison, all
three radiotracers were purified by HPLC to remove unlabeled
ligand. The results obtained from biodistribution and imaging
experiments indicated that there were no differences in absolute
uptake between the three agents in PSMA-expressing tumors (FIG. 2A,
FIG. 2B, FIG. 2C, and FIG. 2D, and FIG. 3A, FIG. 3B, FIG. 3C, and
FIG. 3D), except for highest uptake for .sup.68Ga-2 at 1 h
post-injection (P<0.007 between .sup.68Ga-2 and .sup.68Ga-1).
Higher non-target uptake for .sup.68Ga-DKFZ-PSMA-11 than for
.sup.68Ga-1 or .sup.68Ga-2 were found, contrary to Eder et al.
(Eder et al. (2012) Bioconjug Chem 23, 688-97), in which
.sup.68Ga-1 was compared to .sup.68Ga-DKFZ-PSMA-11 (FIG. 2A, FIG.
2B, FIG. 2C, and FIG. 2D, and FIG. 3A, FIG. 3B, FIG. 3C, and FIG.
3D). Without wishing to be bound to any one particular theory, it
is believed that discrepancy derived from the lack of HPLC
purification for .sup.68Ga-1 in Eder et al. (Eder et al. (2012)
Bioconjug Chem 23, 688-97), which could negatively impact its
effective specific activity. Another possibility could be the
discrepant tumor models used, with LNCaP used in the earlier report
(Eder et al. (2012) Bioconjug Chem 23, 688-97), and PSMA+ PC3 PIP
for the PSMA-expressing positive control. However, it has been
previously reported that the levels of expression of PSMA in PSMA+
PC3 PIP tumors were very similar to that in LNCaP (Banerjee et al.
(2011) Angewandte Chemie 50, 9167-70.
[0193] As previously shown by several groups in the field of PSMA
imaging with low-molecular-weight agents (Eder et al. (2012)
Bioconjug Chem 23, 688-97; Reske et al. (2013) Mol Imaging 40,
969-70; Banerjee, et al. (2014) J Med Chem 57, 2657-69; Weineisen
et al. (2014) EJNMMI Res. 4, 1-15; Banerjee et al. (2015) J Nucl
Med accepted; Banerjee et al. (2011) Oncotarget 2, 1244-53; 57;
Banerjee et al. International Symposium on Radiopharmaceutical
Sciences, Amsterdam, The Netherlands, 2011; Vol. 2011, p S65;
Nedrow et al. (2015) Mol Imaging Biol), the key parameter of
non-specific tissue uptake depends on the overall physicochemical
properties of the radiolabeled agent, including the metabolic
stability of the metal-chelate complex, charge and lipophilicity.
Both the chelating agent and the linker employed to attach the
radionuclide to the targeting agent are important in establishing
those physicochemical features--particularly for compounds<1,500
Da. For example, it has been shown that certain .sup.99mTc-oxo
cores with different combinations of NxSy-based chelating agents
demonstrated high retention in kidney and spleen for more than 6 h
(Ray Banerjee et al. (2013) J Med Chem 56, 6108-21). Such agents
displayed high PSMA+ tumor retention. On the other hand,
.sup.99mTc(CO).sub.3-based agents showed much faster clearance from
most normal tissues including kidneys, although, these agents
showed slightly higher gastrointestinal uptake at initial
time-points (<2 h) (Banerjee et al. (2013) J. Med. Chem.
(submitted). High kidney uptake and retention for NOTA-chelated
.sup.64Cu-labeled PSMA-inhibitor were observed, compared to the
CB-TE2A-conjugated .sup.64Cu-labeled agent (Banerjee et al. (2014)
J Med Chem 57, 2657-69) although both chelating agents are known to
form a copper complex with comparable stability (Dumont et al.
(2011) Journal of nuclear medicine: official publication, Society
of Nuclear Medicine 52, 1276-84; Fani et al. (2011) Journal of
nuclear medicine: official publication, Society of Nuclear Medicine
52, 1110-8). Modifying linker and chelating agent indeed revealed
significant changes in biodistribution pattern as reported by Eder
et al. (Benesova et al. (2015) J Nucl Med 56, 914-20). A direct
comparison of DOTA-mono amide chelated PSMA-targeting agent,
.sup.68Ga-DKFZ-PSMA-617 vs HBED-CC-conjugated
.sup.68Ga-DKFZ-PSMA-11 in preclinical studies demonstrated higher
tumor uptake at later time points, lower spleen accumulation, and
fast activity clearance from the kidneys.
[0194] In summary, a preclinical comparative study to evaluate the
in vivo pharmacokinetics of three .sup.68Ga-labeled PSMA-targeting
PET radiopharmaceuticals has been reported. The macrocyclic NOTA
chelated agent .sup.68Ga-2 demonstrated the highest PSMA+ tumor
accumulation at clinically convenient times post-injection, and
showed rapid clearance from most normal tissues, including kidney
and salivary gland. .sup.68Ga-2 is a clinically viable imaging
agent for detecting PSMA+ lesions.
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[0195] All publications, patent applications, patents, and other
references mentioned in the specification are indicative of the
level of those skilled in the art to which the presently disclosed
subject matter pertains. All publications, patent applications,
patents, and other references are herein incorporated by reference
to the same extent as if each individual publication, patent
application, patent, and other reference was specifically and
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art-accepted meanings of terms are used herein unless indicated
otherwise. Standard abbreviations for various terms are used
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[0260] Although the foregoing subject matter has been described in
some detail by way of illustration and example for purposes of
clarity of understanding, it will be understood by those skilled in
the art that certain changes and modifications can be practiced
within the scope of the appended claims.
* * * * *
References