U.S. patent application number 17/079013 was filed with the patent office on 2021-02-25 for 18f - tagged inhibitors of prostate specific membrane antigen (psma) and their use as imaging agents for prostate cancer.
The applicant listed for this patent is DEUTSCHES KREBSFORSCHUNGSZENTRUM, RUPRECHT-KARLS-UNIVERSITAET HEIDELBERG. Invention is credited to Ulrike BAUDER-WUEST, Martina BENESOVA, Jens CARDINALE, Matthias EDER, Michael EISENHUT, Frederik L. GIESEL, Uwe HABERKORN, Klaus KOPKA, Martin SCHAEFER.
Application Number | 20210053922 17/079013 |
Document ID | / |
Family ID | 1000005197030 |
Filed Date | 2021-02-25 |
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United States Patent
Application |
20210053922 |
Kind Code |
A1 |
CARDINALE; Jens ; et
al. |
February 25, 2021 |
18F - TAGGED INHIBITORS OF PROSTATE SPECIFIC MEMBRANE ANTIGEN
(PSMA) AND THEIR USE AS IMAGING AGENTS FOR PROSTATE CANCER
Abstract
The present invention generally relates to the field of
radiopharmaceuticals and their use in nuclear medicine as tracers
and imaging agents for various disease states of prostate
cancer.
Inventors: |
CARDINALE; Jens; (Wien,
AT) ; SCHAEFER; Martin; (Neckarsteinach, DE) ;
KOPKA; Klaus; (Dossenheim, DE) ; EDER; Matthias;
(Freiburg, DE) ; BAUDER-WUEST; Ulrike;
(Schriesheim, DE) ; EISENHUT; Michael;
(Heidelberg, DE) ; BENESOVA; Martina;
(Neckarsteinach, DE) ; HABERKORN; Uwe;
(Schwetzingen, DE) ; GIESEL; Frederik L.;
(Heidelberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DEUTSCHES KREBSFORSCHUNGSZENTRUM
RUPRECHT-KARLS-UNIVERSITAET HEIDELBERG |
Heidelberg
Heidelberg |
|
DE
DE |
|
|
Family ID: |
1000005197030 |
Appl. No.: |
17/079013 |
Filed: |
October 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15915978 |
Mar 8, 2018 |
10815200 |
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17079013 |
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PCT/EP2016/001573 |
Sep 19, 2016 |
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15915978 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 213/61 20130101;
A61K 51/04 20130101; C07K 7/02 20130101; A61P 35/00 20180101; C07B
2200/05 20130101; A61K 38/00 20130101; G01N 33/57434 20130101; A61K
51/0455 20130101; C07B 59/002 20130101 |
International
Class: |
C07D 213/61 20060101
C07D213/61; A61K 51/04 20060101 A61K051/04; C07B 59/00 20060101
C07B059/00; G01N 33/574 20060101 G01N033/574; C07K 7/02 20060101
C07K007/02; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 30, 2015 |
EP |
15002800.9 |
Apr 6, 2016 |
EP |
16164090.9 |
Aug 4, 2016 |
EP |
16182764.7 |
Claims
1. A precursor or a solvate of the compound of Formula I:
##STR00056## with: TABLE-US-00005 i, j 0, 1 m 1-5 n 0-3 R H,
CH.sub.3 AS Natural or non-natural amino acid, Z: --CO.sub.2H,
--SO.sub.2H, --SO.sub.3H, --SO.sub.4H, --PO.sub.2H, --PO.sub.3H,
--PO.sub.4H.sub.2 X: Naphthyl, Phenyl, Biphenyl, Indolyl,
Benzothiazolyl, Quinoyl Y: Aryl, Alkylaryl, Cyclopentyl,
Cyclohexyl, Cycloheptyl, N-Piperidyl and N-methylated Piperidyl
salt .sup.18F-Tag: ##STR00057## ##STR00058## ##STR00059##
##STR00060## ##STR00061## ##STR00062## ##STR00063## ##STR00064##
##STR00065## ##STR00066## ##STR00067## ##STR00068## With: x = 1-5
Carbohydrate: ##STR00069## ##STR00070## ##STR00071## ##STR00072##
##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077##
##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082##
##STR00083## ##STR00084## ##STR00085## ##STR00086## R.sub.1: Any
alkyl, aryl or arylalkyl linker R.sub.2: Any alkyl or aryl group
##STR00087## R.sub.1: Any alkyl, aryl or arylalkyl linker R.sub.2:
Any alkyl or aryl group ##STR00088## R.sub.1: Any alkyl, aryl or
arylalkyl linker R.sub.2: Any alkyl or aryl group ##STR00089##
R.sub.1: Any alkyl, aryl or arylalkyl linker R.sub.2: Any alkyl or
aryl group ##STR00090## R.sub.1: Any alkyl, aryl or arylalkyl
linker R.sub.2: Any alkyl or aryl group ##STR00091## R.sub.1: Any
alkyl, aryl or arylalkyl linker R.sub.2: Any alkyl or aryl group
##STR00092## R.sub.1: Any alkyl, aryl or arylalkyl linker R.sub.2:
Any alkyl, or aryl group.
2. The precursor or the solvate of claim 1, wherein the compound
has the structure: ##STR00093## wherein the linker A is selected
from the group consisting of: ##STR00094## wherein
R.sup.1=(Glu)-(Urea)-(Lys) and R.sup.2=(Linker B).sub.m-.sup.18F
Tag, m=1-5, and linker B is selected from the group consisting of:
##STR00095## ##STR00096##
3. The precursor or the solvate of claim 1, wherein the compound is
selected from the following ##STR00097## ##STR00098## ##STR00099##
##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104##
##STR00105## ##STR00106##
4. The precursor or the solvate of claim 1, wherein the non-natural
amino acid has the formula ##STR00107## and R'=H, CO.sub.2H,
CH.sub.2CO.sub.2H, C.sub.2H.sub.4CO.sub.2H, CH(CO.sub.2H).sub.2,
CH(CH.sub.2CO.sub.2H).sub.2, CH(CO.sub.2H)(CH.sub.2CO.sub.2H),
CH.sub.2CH(CO.sub.2H).sub.2, SO.sub.3H; o=1-3; R=H, CH.sub.3.
5. The precursor or the solvate of claim 1, wherein R.sub.1 is
selected from the group consisting of: methyl, 2-ethyl, 3-propyl,
2-,3-,4-phenyl, 2-,3-,4-phenylmethyl, and 2,3-,4-phenylpropyl.
6. The precursor or the solvate of claim 1, wherein R.sub.2 is
selected from the group consisting of methyl, isopropyl,
tert-butyl, phenyl, and 1-naphtyl.
7. A pharmaceutical composition comprising the precursor or the
solvate of claim 1, and a pharmaceutically acceptable carrier.
8. A method of imaging a prostate region in a patient comprising
the steps of: (i) administering to a patient a diagnostically
effective amount of the precursor or the solvate of claim 1, (ii)
exposing the prostate region of the patient to a scanning device to
detect the 18F-tag; and (iii) obtaining an image of the prostate
region of the patient by the scanning device.
Description
[0001] This application is continuation of U.S. application Ser.
No. 15/915,978, filed Mar. 8, 2018; which is a continuation of
PCT/EP2016/001573, filed Sep. 19, 2016, which claims priority of EP
15002800.9, filed Sep. 30, 2015, EP 16164090.9, filed Apr. 6, 2016,
and EP16182764.7, filed Aug. 4, 2016. The contents of the
above-identified applications are incorporated herein by reference
in their entirety.
[0002] The present invention generally relates to the field of
radiopharmaceuticals and their use in nuclear medicine as tracers
and imaging agents and for the various disease states of prostate
cancer.
BACKGROUND OF THE INVENTION
[0003] Prostate cancer (PCa) is the leading cancer in the US and
European population. At least 1-2 million men in the western
hemisphere suffer from prostate cancer and it is estimated that the
disease will strike one in six men between the ages of 55 and 85.
There are more than 300.000 new cases of prostate cancer diagnosed
each year in USA. The mortality from the disease is second only to
lung cancer. Currently anatomic methods, such as computed
tomography (CT), magnetic resonance (MR) imaging and ultrasound,
predominate for clinical imaging of prostate cancer. An estimated
$2 billion is currently spent worldwide on surgical, radiation,
drug therapy and minimally invasive treatments. However, there is
presently no effective therapy for relapsing, metastatic,
androgen-independent prostate cancer.
[0004] A variety of experimental low molecular weight PCa imaging
agents are currently being pursued clinically, including
radiolabeled choline analogs ([.sup.11C]Choline, [.sup.18F]FECh,
[.sup.18F] FMC, [.sup.18F]fluorodihydrotestosterone
([.sup.18F]FDHT),
anti-1-amino-3-[.sup.18F]fluorocyclobutyl-1-carboxylic acid
(anti[.sup.18F]F-FACBC, [.sup.11C]acetate and
1-(2-deoxy-2-[.sup.18F]flouro-L-arabinofuranosyl)-5-methyluracil
(-[.sup.18F]FMAU) (Scher, B.; et al. Eur J Nucl Med Mol Imaging
2007, 34, 45-53; Rinnab, L.; et al. BJU Int 2007, 100, 786,793;
Reske, S. N.; et al. J Nucl Med 2006, 47, 1249-1254; Zophel, K.;
Kotzerke, J. Eur J Nucl Med Mol Imaging 2004, 31, 756-759; Vees,
H.; et al. BJU Int 2007, 99, 1415-1420; Larson, S. M.; et al. J
Nucl Med 2004, 45, 366-373; Schuster, D. M.; et al. J Nucl Med
2007, 48, 56-63; Tehrani, O. S.; et al. J Nucl Med 2007, 48,
1436-1441). Each operates by a different mechanism and has certain
advantages, e.g., low urinary excretion for [.sup.11C]choline, and
disadvantages, such as the short physical half-life of
positron-emitting radionuclides.
[0005] It is well known that tumors may express unique proteins
associated with their malignant phenotype or may over-express
normal constituent proteins in greater number than normal cells.
The expression of distinct proteins on the surface of tumor cells
offers the opportunity to diagnose and characterize disease by
probing the phenotypic identity and biochemical composition and
activity of the tumor. Radioactive molecules that selectively bind
to specific tumor cell surface proteins provide an attractive route
for imaging and treating tumors under non-invasive conditions. A
promising new series of low molecular weight imaging agents targets
the prostate-specific membrane antigen (PSMA) (Mease R. C. et al.
Clin Cancer Res. 2008, 14, 3036-3043; Foss, C. A.; et al. Clin
Cancer Res 2005, 11, 4022-4028; Pomper, M. G.; et al. Mol Imaging
2002, 1, 96-101; Zhou, J.; etr al. Nat Rev Drug Discov 2005, 4,
1015-1026; WO 2013/022797).
[0006] PSMA is a trans-membrane, 750 amino acid type II
glycoprotein that has abundant and restricted expression on the
surface of PCa, particularly in androgen-independent, advanced and
metastatic disease (Schulke, N.; et al. Proc Natl Acad Sci USA
2003, 100, 12590-12595). The latter is important since almost all
PCa become androgen independent over the time. PSMA possesses the
criteria of a promising target for therapy, i.e., abundant and
restricted (to prostate) expression at all stages of the disease,
presentation at the cell surface but not shed into the circulation
and association with enzymatic or signaling activity (Schulke, N.;
et al. Proc. Natl. Acad. Sci. USA 2003, 100, 12590-12595). The PSMA
gene is located on the short arm of chromosome 11 and functions
both as a folate hydrolase and neuropeptidase. It has
neuropeptidase function that is equivalent to glutamate
carboxypeptidase II (GCPII), which is referred to as the "brain
PSMA", and may modulate glutamatergic transmission by cleaving
N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and
glutamate (Nan, F.; et al. J Med Chem 2000, 43, 772-774). There are
up to 10.sup.6 PSMA molecules per cancer cell, further suggesting
it as an ideal target for imaging and therapy with
radionuclide-based techniques (Tasch, J.; et al. Crit Rev Immunol
2001, 21, 249-261).
[0007] The radio-immunoconjugate of the anti-PSMA monoclonal
antibody (mAb) 7E11, known as the PROSTASCINT.RTM. scan, is
currently being used to diagnose prostate cancer metastasis and
recurrence. However, this agent tends to produce images that are
challenging to interpret (Lange, P. H. PROSTASCINT scan for staging
prostate cancer. Urology 2001, 57, 402-406; Haseman, M. K.; et al.
Cancer Biother Radiopharm 2000, 15, 131-140; Rosenthal, S. A.; et
al. Tech Urol 2001, 7, 27-37). More recently, monoclonal antibodies
have been developed that bind to the extracellular domain of PSMA
and have been radiolabeled and shown to accumulate in PSMA-positive
prostate tumor models in animals. However, diagnosis and tumor
detection using monoclonal antibodies has been limited by the low
permeability of the monoclonal antibody in solid tumors.
[0008] The selective targeting of cancer cells with
radiopharmaceuticals for imaging or therapeutic purposes is
challenging. A variety of radionuclides are known to be useful for
radio-imaging or cancer radiotherapy, including .sup.11C, .sup.18F,
.sup.111In, .sup.90Y, .sup.68Ga, .sup.177Lu, .sup.99mTC, .sup.123I
and .sup.131I. Recently it has been shown that some compounds
containing a glutamate-urea-glutamate (GUG) or a
glutamate-urea-lysine (GUL) recognition element linked to a
radionuclide-ligand conjugate exhibit high affinity for PSMA.
[0009] Several .sup.18F-tagged compounds which show PSMA
interaction and are suitable for the detection of prostate cancer
are shown in EP 14 003 570.0. These compounds, however, show high
lipophilic properties which make them, to a certain extent,
difficult to handle and to administer.
[0010] New agents that will enable rapid visualization of prostate
cancer are needed. Thus, the object of the present invention is to
develop ligands that interact with PSMA and carry appropriate
radionuclides which provide a promising and novel targeting option
for the detection, treatment and management of prostate cancer.
SUMMARY OF THE INVENTION
[0011] The solution of said object is achieved by providing the
embodiments characterized in the claims.
[0012] The inventors found new compounds which are useful
radiopharmaceuticals and their use in nuclear medicine as tracers
and imaging agents and for various disease states of prostate
cancer.
[0013] The novel imaging agents with structural modifications in
the linker region have improved tumor targeting properties and
pharmacokinetics. The pharmacophore presents three carboxylic
groups able to interact with the respective side chains of PSMA and
an oxygen as part of zinc complexation in the active center.
Besides these obligatory interactions, the inventors were able to
optimize the lipophilic interactions in the linker region in
comparison to the compounds described in EP 14003570.0. Moreover,
the inventors added some hydrophilic building blocks to the linkler
for an enhancement of the pharmakokinetics.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1: .sup.18F-Labelling of macromolecules
[0015] FIG. 2: .sup.18F-Fluorination via prosthetic groups
[0016] FIG. 3: .sup.18F-prosthetic groups for peptides
[0017] FIG. 4: .sup.18F-Labelled Prosthetic Groups using "Click
Chemistry"
[0018] FIG. 5: Formation of [.sup.18F]aryltrifluoroboronates and
fluoride sensors
[0019] FIG. 6: Complexes of .sup.18F-Fluorides
[0020] FIG. 7: .sup.18F-Labelling of RGD peptides
[0021] FIG. 8: Organ distribution of [.sup.18F]PSMA-1007 in
PSMA-positive LNCaP mice (normal and blocked) and in PSMA negative
PC3 mice vs. [.sup.18F]PSMA-1009 ([.sup.18F]DCFPyL) in PSMA
positive LNCaP mice
[0022] FIG. 9: Organ distribution of [.sup.18F]PSMA-1007 in
PSMA-positive LNCaP mice (non-blocked and blocked) vs.
[.sup.18F]PSMA1003 in PSMA positive LNCaP mice (non-blocked)
[0023] FIG. 10: MIP of [.sup.18F]PSMA-1007 in a PSMA-positive LNCaP
mouse 120-140 min p.i.
[0024] FIG. 11: Time-activity curve of [.sup.18F]PSMA-1007 in a
PSMA-positive LNCaP mouse, including SUV values 120-140 min
p.i.
[0025] FIG. 12: Structure of PSMA-1003
[0026] FIG. 13: Structure of PSMA-1009
[0027] FIG. 14: MIPs of [.sup.18F]PSMA-1007 in a healthy
volunteer
[0028] FIG. 15: Blood (black) and serum (gray) time-activity-curves
expressed as percent injected dose in a healthy volunteer
[0029] FIG. 16: Time-activity-curves of normal organs with
PET-delineable volume-of-interest in a healthy volunteer
[0030] FIG. 17: Organ distribution [.sup.18F]PSMA-1007 in ten
patients suffering from prostate cancer expressed as
SUV.sub.max
[0031] FIG. 18: Tumor-to-Background ratios of [.sup.18F]PSMA-1007
in ten patients suffering from prostate cancer calculated from the
respective SUV.sub.max values
[0032] FIG. 19: MIP of a 77-year old prostate cancer patient (PSA
40 ng/ml) scanned with [.sup.18F]PSMA-1007 1 and 3 h p.i. showing a
large tumor mass on the mid and apex area of prostate and several
lymph node metastases. Outside of the pelvic region there was no
metastasis found
[0033] FIG. 20: MIP of a 72-year old patient (PSA 15 ng/ml)
diagnosed with Gleason 9 (5+4) prostate cancer scanned with
[.sup.18F]PSMA-1007 1 and 3 h p.i. Patient presents a large tumor
mass in the whole prostate gland with infiltration in the left
seminal vesicles and several lymph nodes in the pelvic region. Two
metastatic lymph nodes are located outside the pelvic region, both
paraaortic at level L3/4 and L5.
[0034] FIG. 21: Transaxial PET/CT-scan of a patient (a,b) and
corresponding histopathology of the subsequent prostatectomy
specimen; H&E staining (c); PSMA-immunostaining with outlined
tumor contours circumscribed by the broken line (d).
[0035] FIG. 22: Structure of preferred compounds of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention relates to radiopharmaceuticals and
their use in nuclear medicine as tracers and imaging agents for the
various disease states of prostate cancer.
[0037] Thus, the present invention concerns compounds that are
represented by the general Formula I
##STR00001##
[0038] with:
TABLE-US-00001 i, j 0, 1 m 1-5 n 0-3 R H, CH.sub.3 AS Natural or
non-natural amino acid Z: --CO.sub.2H, --SO.sub.2H, --SO.sub.3H,
--SO.sub.4H, --PO.sub.2H, --PO.sub.3H, --PO.sub.4H.sub.2 X:
Naphthyl, Phenyl, Biphenyl, Indolyl (=2,3-benzopyrrolyl),
Benzothiazolyl, Quinoyl Y: Aryl, Alkylaryl, Cyclopentyl,
Cyclohexyl, Cycloheptyl, N-Piperidyl and N-methylated Piperidyl
salt .sup.18F-Tag: ##STR00002## ##STR00003## ##STR00004##
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012## With: x = 1-5 Carboxylate:
##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029## ##STR00030## R.sub.1: Any alkyl, aryl or
arylalkyl linker especially methyl, 2-ethyl, 3-propyl,
2-,3-,4-phenyl, 2-,3-,4-phenylmethyl, 2-,3-,4-phenylpropyl R.sub.2:
Any alkyl or aryl group especially methyl isopropyl, tert-butyl,
phenyl or 1-naphthyl ##STR00031## R.sub.1: Any alkyl, aryl or
arylalkyl linker especially methyl, 2-ethyl, 3-propyl,
2-,3-,4-phenyl, 2-,3-,4-phenylmethyl, 2-,3-,4-phenylpropyl R.sub.2:
Any alkyl or aryl group especially methyl isopropyl, tert-butyl,
phenyl or 1-naphtyl ##STR00032## R.sub.1: Any alkyl, aryl or
arylalkyl linker especially methyl, 2-ethyl, 3-propyl,
2-,3-,4-phenyl, 2-,3-,4-phenylmethyl, 2-,3-,4-phenylpropyl R.sub.2:
Any alkyl or aryl group especially methyl isopropyl, tert-butyl,
phenyl or 1-naphtyl ##STR00033## R.sub.1: Any alkyl, aryl or
arylalkyl linker especially methyl, 2-ethyl, 3-propyl,
2-,3-,4-phenyl, 2-,3-,4-phenylmethyl, 2-,3-,4-phenylpropyl R.sub.2:
Any alkyl or aryl group especially methyl isopropyl, tert-butyl,
phenyl or 1-naphtyl ##STR00034## R.sub.1: Any alkyl, aryl or
arylalkyl linker especially methyl, 2-ethyl, 3-propyl,
2-,3-,4-phenyl, 2-,3-,4-phenylmethyl, 2-,3-,4-phenylpropyl R.sub.2:
Any alkyl or aryl group especially methyl isopropyl, tert-butyl,
phenyl or 1-naphtyl ##STR00035## R.sub.1: Any alkyl, aryl or
arylalkyl linker especially methyl, 2-ethyl, 3-propyl,
2-,3-,4-phenyl, 2-,3-,4-phenylmethyl, 2-,3-,4-phenylpropyl R.sub.2:
Any alkyl or aryl group especially methyl isopropyl, tert-butyl,
phenyl or 1-naphtyl ##STR00036## R.sub.1: Any alkyl, aryl or
arylalkyl linker especially methyl, 2-ethyl, 3-propyl,
2-,3-,4-phenyl, 2-,3-,4-phenylmethyl, 2-,3-,4-phenylpropyl R.sub.2:
Any alkyl or aryl group especially methyl isopropyl, tert-butyl,
phenyl or 1-naphtyl
[0039] If not stated otherwise, in the present invention the term
"alkyl" by itself or as part of another molecule, means a straight
or branched chain, or cyclic hydrocarbon radical, or combination
thereof, which may be fully saturated, mono- or polyunsaturated and
can include di- and multivalent radicals, The "alkyl" residue is
preferably C.sub.1 to C.sub.10 and may be unsubstituted or
substituted (e.g with halogen). Preferred alkyl residues are
methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, n-pentyl,
n-hexyl, n-hepyl or n-octyl or the like. The same also applies to
the corresponding cycloalkyl compounds having preferably 3 to 10
carbon atoms, e.g. cycloproyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, etc. 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. The term "alkyl," unless otherwise
noted, is also meant to include those derivatives of alkyl, such as
"heteroalkyl", "haloalkyl" and "homoalkyl".
[0040] The term "aryl", as used herein, refers to a closed ring
structure which has at least one ring having a conjugated pi
electron system and includes both carbocyclic aryl and heterocyclic
aryl (or "heteroaryl" or "heteroaromatic") groups. The carbocyclic
or heterocyclic aromatic group may contain from 5 to 20 ring atoms.
The term includes monocyclic rings linked covalently or fused-ring
polycyclic (i.e., rings which share adjacent pairs of carbon atoms)
groups. An aromatic group can be unsubstituted or substituted.
Non-limiting examples of "aromatic" or "aryl", groups include
phenyl, 1-naphthyl, 2-naphthyl, 2-biphenyl, 3-biphenyl, 4-biphenyl,
anthracenyl, and phenanthracenyl. Substituents for each of the
above noted aryl and heteroaryl ring systems are selected from the
group of acceptable substituents (e.g. alkyl, carbonyl, carboxyl or
halogen) described herein. The term "aryl" when used in combination
with other terms (including but not limited to, aryloxy,
arylthioxy, aralkyl) includes both aryl and heteroaryl rings. Thus,
the term "aralkyl" or "alkaryl" is meant to include those radicals
in which an aryl group is attached to an alkyl group (including but
not limited to, benzyl, phenethyl, pyridylmethyl and the like)
including those alkyl groups in which a carbon atom (including but
not limited to, a methylene group) has been replaced by a
heteroatom, by way of example only, by an oxygen atom. Examples of
such aryl groups include, but are not limited to, phenoxymethyl,
2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like.
[0041] "Heteroaryl" refers to aryl groups which contain at least
one heteroatom selected from N, O, and S; wherein the nitrogen and
sulfur atoms may be optionally oxidized, and the nitrogen atom(s)
may be optionally quaternized. Heteroaryl groups may be substituted
or unsubstituted. A heteroaryl group may be attached to the
remainder of the molecule through a heteroatom. Non-limiting
examples of suitable groups include 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, 4-benzothiazolyl,
5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl, purinyl,
2-benzimidazolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl,
1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,
2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl,
7-quinolyl or 8-quinolyl.
[0042] The term "amino acid" refers to naturally occurring and
non-natural amino acids, as well as amino acid analogs and amino
acid mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally amino acids are the 20 common
amino acids in their D- or L-form (alanine, arginine, asparagine,
aspartic acid, cysteine, glutamine, glutamic acid, glycine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, and valine) and
pyrolysine and selenocysteine. Amino acid analogs refers to
compounds that have the same basic chemical structure as a
naturally occurring amino acid, by way of example only, an
ex-carbon that is bound to a hydrogen, a carboxyl group, an amino
group, and an R group. Such analogs may have modified R groups (by
way of example, norleucine) or may have modified peptide backbones,
while still retaining the same basic chemical structure as a
naturally occurring amino acid. Non-limiting examples of amino acid
analogs include homoserine, norleucine, methionine sulfoxide,
methionine methyl sulfonium. Amino acids may be referred to herein
by either their name, their commonly known three letter symbols or
by the one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission. A "non-natural amino acid" refers to an
amino acid that is not one of the 20 common amino acids or
pyrolysine or selenocysteine. Other terms that may be used
synonymously with the term "non-natural amino acid" is
"non-naturally encoded amino acid," "unnatural amino acid,"
"non-naturally-occurring amino acid" or "artificial amino acid. The
term "non-natural amino acid" includes, but is not limited to,
amino acids which occur by modification of a naturally encoded
amino acid in their backbone or side chains. In some embodiments
the non-natural amino acid comprises a carbonyl group, an acetyl
group, an aminooxy group, a hydrazine group, a hydrazide group, a
semicarbazide group, an azide group or an alkyne group. In a
preferred embodiment the non-natural amino acid has the formula
##STR00037##
[0043] and R'=H, CO.sub.2H, CH.sub.2CO.sub.2H,
C.sub.2H.sub.4CO.sub.2H, CH(CO.sub.2H).sub.2,
CH(CH.sub.2CO.sub.2H).sub.2, CH(CO.sub.2H)(CH.sub.2CO.sub.2H),
CH.sub.2CH(CO.sub.2H).sub.2, SO.sub.3H; o=.sub.1-3; R=H,
CH.sub.3
[0044] Preferred are those amino acids which bring a hydrophilic
element into the compound of Formula I.
[0045] Some of the residues herein (including, but not limited to
non-natural amino acids), may exist in several tautomeric forms.
All such tautomeric forms are considered as part of the compounds
described herein. Also, for example all enol-keto forms of any
compounds herein are considered as part of the compositions
described herein.
[0046] The linker B, i.e. the natural amino acids and/or
non-naturally occurring amino acids, may be bound within the
molecule via a peptide or amide linkage. In case of acidic amino
acids (e.g. glutamic acid, aspartic acid) however, the binding may
be alternatively via the .alpha.-, .beta. or .gamma.-position.
[0047] Although it is preferred that the Z-Group is --CO.sub.2H it
may be easily replaced with biosteric replacements such as
--SO.sub.2H, --SO.sub.3H, --SO.sub.4H, --PO.sub.2H, --PO.sub.3H,
--PO.sub.4H.sub.2, see e.g. "The Practice of Medicinal Chemistry"
(Academic Press New York, 1996), page 203.
[0048] Within the meaning of the invention, all residues are
considered combinable unless stated otherwise in the definition of
the residues. All conceivable subgroupings thereof are considered
to be disclosed.
[0049] The .sup.18F-Tags of the above Table comprising triazoles
exist in two isomeric forms which belong both to the invention and
are illustrated by the given formulas.
[0050] Thus, preferred molecules of the present invention consist
of three principle components (as shown in FIG. 22): the
hydrophilic PSMA binding motif (Glu-Urea-Lys=Glu-NH--CO--NH-Lys),
two variable linkers (Linker A and Linker B) and the
.sup.18F-Tag.
[0051] Some preferred lipophilic linkers (linker A) are shown
below, wherein R.sup.1=Glu-urea-Lys (PSMA binding motif) and
R.sup.2=(Linker B).sub.m-[.sup.18F-Tag] with m=1-5,
##STR00038##
[0052] The different preferred building blocks for hydrophilic
linkers (linker B) are shown below with their preferred
connectivity exemplified on the basis of the respective single
amino acids (m=1 in the generic structure)
##STR00039## ##STR00040##
[0053] The preferred hydrophilic linkers may also be formed from
two or more building blocks (m=2-5 in the generic formula),
preferably selected from the acidic building blocks listed above.
Preferred are 1-3 building blocks.
[0054] A number of preferred structures are listed in the table
below:
TABLE-US-00002 Lipophilic Hydrophilic Num- Linker (= Linker (=
Linker B) ber Linker A) Pos. 1 Pos.2 Pos. 3 .sup.18F-Tag 1
(2-Nal)-(Bn) (Glu) (Glu) -- [.sup.18F]FN 2 (2-Nal) (Glu) (Glu) --
[.sup.18F]FN 3 (2-Nal) (.gamma.Glu) (Glu) -- [.sup.18F]FN 4
(2-Nal)-(Bn) (Glu) -- -- [.sup.18F]FN 5 (2-Nal)-(AcAMP)
(.gamma.Glu) (.gamma.Glu) -- [.sup.18F]FN 6 (2-Nal)-(Bn) (Glu)
(.gamma.Glu) -- [.sup.18F]FN 7 (2-Nal)-(Bn) (.gamma.Glu) (Glu) --
[.sup.18F]FN 8 (2-Nal)-(Bn) (.gamma.Glu) (.gamma.Glu) --
[.sup.18F]FN 9 (2-Nal)-(Bn) (Asp) (Glu) -- [.sup.18F]FN 10
(2-Nal)-(Bn) (.beta.Asp) (Glu) -- [.sup.18F]FN 11 (2-Nal)-(Bn)
(Asp) (.gamma.Glu) -- [.sup.18F]FN 12 (2-Nal)-(Bn) (.beta.Asp)
(.gamma.Glu) -- [.sup.18F]FN 13 (2-Nal)-(Bn) (Mal) (Glu) --
[.sup.18F]FN 14 (2-Nal)-(Bn) (Mal) (.gamma.Glu) -- [.sup.18F]FN 15
(2-Nal)-(Bn) (Gla) (Glu) -- [.sup.18F]FN 16 (2-Nal)-(Bn)
(.gamma.Gla) (Glu) -- [.sup.18F]FN 17 (2-Nal)-(Bn) (Gla)
(.gamma.Glu) -- [.sup.18F]FN 18 (2-Nal)-(Bn) (.gamma.Gla)
(.gamma.Glu) -- [.sup.18F]FN
[0055] The structures of the preferred compounds 1-18 as
exemplified in the table above are shown below:
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##
##STR00051## ##STR00052## ##STR00053##
[0056] As may be understood by a person skilled in the art, the
above mentioned preferred compounds 1-18 and 1a-18a are not limited
to the .sup.18F-Tags as shown but the .sup.18F-Tags are easily
interchangeable by standard techniques and any of the .sup.18F-Tags
exemplified in connection with Formula I may be used instead.
[0057] The invention also relates to precursors or pharmaceutically
acceptable salts of the compounds of general formula I. The
invention also relates to solvates of the compounds, including the
salts as well as the active metabolites thereof and, where
appropriate, the tautomers thereof according to general formula I
include prodrug formulations.
[0058] A "pharmaceutically acceptable salt" is a pharmaceutically
acceptable, organic or inorganic acid or base salt of a compound of
the invention. Representative pharmaceutically acceptable salts
include, e.g., alkali metal salts, alkali earth salts, ammonium
salts, water-soluble and water-insoluble salts, such as the
acetate, carbonate, chloride, gluconate, glutamate, lactate,
laurate, malate or tartrate.
[0059] The term "prodrug" refers to a precursor of a drug that is a
compound which upon administration to a patient, must undergo
conversion by metabolic processes before becoming an active
pharmacological agent. Prodrugs are generally drug precursors that,
following administration to a subject and subsequent absorption,
are converted to an active, or a more active species via some
process, such as conversion by a metabolic pathway. Some prodrugs
have a chemical group present on the prodrug that renders it less
active and/or confers solubility or some other property to the
drug. Once the chemical group has been cleaved and/or modified from
the prodrug the active drug is generated. Prodrugs are converted
into active drug within the body through enzymatic or non-enzymatic
reactions. Prodrugs may provide improved physiochemical properties
such as better solubility, enhanced delivery characteristics, such
as specifically targeting a particular cell, tissue, organ or
ligand, and improved therapeutic value of the drug. Illustrative
prodrugs of compounds in accordance with general formula I are
esters and amides, preferably alkyl esters of fatty acid esters.
Prodrug formulations here comprise all substances which are formed
by simple transformation including hydrolysis, oxidation or
reduction either enzymatically, metabolically or in any other way.
A suitable prodrug contains e.g. a substance of general formula I
bound via an enzymatically cleavable linker (e.g. carbamate,
phosphate, N-glycoside or a disulfide group) to a
dissolution-improving substance (e.g. tetraethylene glycol,
saccharides, formic acids or glucuronic acid, etc.). Such a prodrug
of a compound according to the invention can be applied to a
patient, and this prodrug can be transformed into a substance of
general formula I so as to obtain the desired pharmacological
effect.
[0060] Some compounds of general formula I are encompassed in form
of the racemates, their enantiomers and optionally in form of their
diastereomers and all possible mixtures thereof.
[0061] According to the invention all chiral C-atoms shall have D-
and/or L-configuration; also combinations within one compound shall
be possible, i.e. some of the chiral C-atoms may be D- and others
may be L-configuration.
[0062] The obtained compounds can be optionally separated by known
methods (e.g. A/linger, N. L. and Elliel E. L. in "Topics in
Stereochemistry" Vol. 6, Wiley Interscience, 1971) in their
enantiomers and/or diastereomers. One possible method of
enantiomeric separation is the use of chromatography.
[0063] The invention encompasses also precursors of the compounds
of general formula I. The term "precursor" refers to any compound
which can be used to produce the compounds of Formula I. An
exemplary precursor may be a compound having no .sup.18F-tag which
is added at a later stage to provide the complete compound.
[0064] The invention also relates to pharmaceutical preparations
which contain a diagnostically or therapeutically effective amount
of the active ingredients (compound according to the invention of
formula I) together with organic or inorganic solid or liquid,
pharmaceutically acceptable carriers which are suited for the
intended administration and which interact with the active
ingredients without drawbacks.
[0065] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, material, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of a patient without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0066] A "patient" includes an animal, such as a human, monkey,
cow, horse, cat or dog. The animal can be a mammal such as a
non-primate and a primate (e.g., monkey and human). In one
embodiment, a patient is a human being.
[0067] In general, the formula I compound or pharmaceutical
compositions thereof, may be administered orally or via a
parenteral route, usually injection or infusion.
[0068] A "parenteral administration route" means modes of
administration other than enteral and topical administration,
usually by injection, and includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticluare, subcapsular, subarachnoid, intraspinal and
intrasternal injection and infusion.
[0069] The dosage of the compounds according to the invention is
determined by the physician on the basis of the patient-specific
parameters, such as age, weight, sex, severity of the disease, etc.
Corresponding to the kind of administration, the medicament is
suitably formulated, e.g. in the form of solutions or suspensions,
simple tablets or dragees, hard or soft gelatine capsules,
suppositories, ovules, preparations for injection, which are
prepared according to common galenic methods.
[0070] The compounds according to the invention can be formulated,
where appropriate, together with further active substances and with
excipients and carriers common in pharmaceutical compositions,
e.g.--depending on the preparation to be produced--talcum, gum
arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous
and non-aqueous carriers, fatty bodies of animal or vegetable
origin, paraffin derivatives, glycols (in particular polyethylene
glycol), various plasticizers, dispersants or emulsifiers,
pharmaceutically compatible gases (e.g. air, oxygen, carbon
dioxide, etc.), preservatives.
[0071] In order to produce liquid preparations, additives, such as
sodium chloride solution, ethanol, sorbitol, glycerine, olive oil,
almond oil, propylene glycol or ethylene glycol, can be used.
[0072] When solutions for infusion or injection are used, they are
preferably aqueous solutions or suspensions, it being possible to
produce them prior to use, e.g. from lyophilized preparations which
contain the active substance as such or together with a carrier,
such as mannitol, lactose, glucose, albumin and the like. The ready
made solutions are sterilized and, where appropriate, mixed with
excipients, e.g. with preservatives, stabilizers, emulsifiers,
solubilizers, buffers and/or salts for regulating the osmotic
pressure. The sterilization can be obtained by sterile filtration
using filters having a small pore size according to which the
composition can be lyophilized, where appropriate. Small amounts of
antibiotics can also be added to ensure the maintenance of
sterility.
[0073] The phrases "effective amount" or "therapeutically-effective
amount" as used herein means that amount of a compound, material,
or composition comprising a compound of the invention, or other
active ingredient which is effective for producing some desired
therapeutic effect in at least a sub-population of cells in an
animal at a reasonable benefit/risk ratio applicable to any medical
treatment. A therapeutically effective amount with respect to a
compound of the invention means that amount of therapeutic agent
alone, or in combination with other therapies, that provides a
therapeutic benefit in the treatment of prevention of a disease.
Used in connection with a compound of the invention, the term can
encompass an amount that improves overall therapy, reduces or
avoids symptoms or causes of disease, or enhances the therapeutic
efficacy of or synergies with another therapeutic agent.
[0074] As used herein, the terms "treating" or "treatment" is
intended to encompass also diagnosis, prophylaxis, prevention,
therapy and cure.
[0075] The terms "prevent", "preventing," and "prevention" refer to
the prevention of the onset, recurrence, or spread of the disease
in a patient resulting from the administration of a prophylactic or
therapeutic agent.
[0076] As noted above, compounds according general formula I are
suitable for use as radio-imaging agents or as therapeutics for the
treatment of rapidly proliferating cells, for example, PSMA
expressing prostate cancer cells. According to the present
invention they are called "radiopharmaceuticals".
[0077] Preferred imaging methods are positron emission tomography
(PET) or single photon emission computed tomography (SPECT).
[0078] Accordingly, in one embodiment, a pharmaceutical composition
is provided including a compound of formula I, a salt, solvate,
stereoisomer, or tautomer thereof, and a pharmaceutically
acceptable carrier. Accordingly, a pharmaceutical composition is
provided, which is suitable for in vivo imaging and radiotherapy.
Suitable pharmaceutical compositions may contain the compound of
formula I in an amount sufficient for imaging, together with a
pharmaceutically acceptable radiological vehicle. The radiological
vehicle should be suitable for injection or aspiration, such as
human serum albumin; aqueous buffer solutions, e.g.,
tris(hydromethyl) aminomethane (and its salts), phosphate, citrate,
bicarbonate, etc; sterile water physiological saline; and balanced
ionic solutions containing chloride and or dicarbonate salts or
normal blood plasma cautions such as calcium potassium, sodium and
magnesium.
[0079] The concentration of the imaging agent or the therapeutic
agent in the radiological vehicle should be sufficient to provide
satisfactory imaging. For example, when using an aqueous solution,
the dosage is about 1.0 to 100 millicuries. The actual dose
administered to a patient for imaging or therapeutic purposes,
however, is determined by the physician administering treatment.
The imaging agent or therapeutic agent should be administered so as
to remain in the patient for about 1 hour to 10 days, although both
longer and shorter time periods are acceptable. Therefore,
convenient ampoules/vials containing 1 to 10 mL of aqueous solution
may be prepared.
[0080] Imaging may be carried out in the normal manner, for example
by injecting a sufficient amount of the imaging composition to
provide adequate imaging and then scanning with a suitable imaging
or scanning machine, such as a tomograph or gamma camera. In
certain embodiments, a method of imaging a region in a patient
includes the steps of: (i) administering to a patient a
diagnostically effective amount of a compound labeled with a
radionuclide; exposing a region of the patient to the scanning
device; and (ii) obtaining an image of the region of the
patient.
[0081] The amount of the compound of the present invention, or its
salt, solvate, stereoisomer, or tautomer that is administered to a
patient depends on several physiological factors that are routinely
used by the physician, including the nature of imaging to be
carried out, tissue to be targeted for imaging or therapy and the
body weight and medical history of the patient to be imaged or
treated using a radiopharmaceutical.
[0082] Specifically, the cell proliferative disease or disorder to
be treated or imaged using a compound, pharmaceutical composition
or radiopharmaceutical in accordance with this invention is a
cancer, for example, prostate cancer and/or prostate cancer
metastasis in e.g. lung, liver, kidney, bones, brain, spinal cord,
bladder, etc.
[0083] The synthesis of the compounds of the present invention is
carried out according to methods well known in the prior art (e.g.
Hugenberg et al., J. Med. Chem. 2013, 56, pp. 6858-6870). General
methods for .sup.18F-labelling of various macromolecules are shown
in FIG. 1-7. In addition, reference is made to Schubiger et al.,
PET Chemistry: The Driving Force in Molecular Imaging, Ernst
Schering Research Foundation, Workshop 62, Springer Verlag, ISSN
0947-6075; Ross et al., Current Radiopharmaceuticals, 2010, 3,
202-223; Kuhnast et al., Current Radiopharm 3, 2010, 174;
Bernard-Gauthier et al., BioMed Research International, 2014, 1;
Maschauer and Prante, BioMed Research International, 2014, 1;
Olberg et al., J. Med. Chem., 2010, 53, 1732; Rostovtsev et al.,
Angew. Chem., 2002, 114, 2708; Smith and Greaney, Org. Lett., 2013,
15, 4826. Thus, a person skilled in the art would be able to choose
the right .sup.18F-labelling depending on the starting molecule.
The synthesis of the specific linker molecules is shown in EP
13004991 to which reference is made.
[0084] The synthesized compounds are chemically characterized by
RP-HPLC, MS, and/or NMR.
[0085] The novel .sup.18F-tagged imaging agents with structural
modifications in the linker region have improved tumor targeting
properties and pharmacokinetics. The pharmacophore presents three
carboxylic group able to interact with the respective side chains
of PSMA and an oxygen as part of zinc complexation in the active
center. Besides these obligatory interactions, the inventors were
able to optimize the lipophilic interactions in the linker region
compared to the compounds as described in EP 14003570.0. Further
hydrophilic building blocks have been added to the linker of the
compounds of the present invention (linker B), leading to another
enhancement of the pharmacokinetics. Those compounds were evaluated
in in vitro assays (affinity, internalization) and in vivo
experiments (.mu.PET screening and organ distribution).
[0086] Four preferred compounds with particularly promising results
are .sup.18F-PSMA1007, .sup.18F-PSMA1011, .sup.18F-PSMA 1012 and
.sup.18F-PSMA 1015:
##STR00054## ##STR00055##
[0087] All compounds were labelled with fluorine-18 via
2-[.sup.18F]fluoronicotinic acid TFP ester in good radiochemical
yields. Table A shows that the binding affinity of the PSMA
inhibitors prepared so far are essentially the same and in the
typical range. Further, all compounds were specifically
internalized at 37.degree. C. with rather high cell uptake and
internalization values (Table B). Thus, the compounds investigated
exhibit optimal in vitro characteristics for a high contrast PET
imaging.
[0088] The compounds of the present invention were investigated in
organ distribution studies in mice carrying a LNCaP tumor (PSMA
positive) with and without PMPA block. The results which have been
obtained exemplary with PSMA1007 (this does not mean that the
invention is limited in any way to this specific compound only) are
summarised in the FIGS. 8 and 9. The tumor uptake was about
8.0.+-.2.4% ID/g. Since a quantitative blocking of the binding was
not observed in the blocking experiment, the organ distribution
experiment was repeated with mice carrying a PC3-tumor (PSMA
negative; Results shown FIG. 8). Here, practically no tumor uptake
was observed. Thus, the tumor uptake is considered specifically.
Additionally, compared to the organ distribution observed with the
control compound PSMA-1003 which has been described in EP 14 003
570.0 and is shown in FIG. 12, the novel compound PSMA-1007 showed
a significantly reduced uptake in non-target tissue such as the
liver and the small intestine. Thus, PSMA-1007 was further
evaluated in microPET experiment. The results are shown in FIGS. 10
and 11. The LNCaP tumor was clearly visualised in this experiment
(SUV.sub.max=3.1 at 120-140 min p.i.). The undesired uptake in the
gallbladder may be an indicator for metabolites. Overall the novel
class of fluorine-18 labelled PSMA inhibitors showed a great
potential as possible tracer for the detection of prostate cancer
and its metastases.
[0089] By the application of [.sup.18F]PSMA-1007 to a healthy
volunteer several important insights were gained. First, the
effective dose of a PET/CT scan with 200-250 MBq is with 4.3-5.4
mSv (2.15 mSv/MBq; FIG. 14). More than 95% of the blood pool
activity is found in the serum (no or negligible infiltration of
red blood cells) and more than 90% clearance from the blood pool
within the first 3 h after injection. So far, those results are
comparable to other important PSMA-tracers. However, in comparison
to the other known PSMA-tracers the compounds of the present
invention, particularly [.sup.18F]PSMA-1007, provide a very unique
hepatobiliary clearance with very small clearance via the renal
pathway. This outstanding low urinary clearance enables an
excellent assessment of the prostatic bed. Thus, the tracers
according to the present invention are perfectly suited for the
primary diagnosis of prostate cancer and local recurrence.
[0090] This was further demonstrated by the results of the
first-in-man study. Here, excellent tumor-to-background ratios of
up to 10 were observed in the primary tumor without any
interference from tracer accumulation in the bladder. Further,
lymph node metastases with a diameter of down to 1 mm could be
detected, which is in the range of the resolution of PET-Scans with
F-18. Correlation with the samples gained by pelvic lymphadenectomy
analysis revealed a specificity of 95%. Moreover, the samples
gained from the prostatectomy were analyzed by histopathology
revealing a nearly perfect correlation with the images acquired by
PET. Those results clearly demonstrate the feasibility of prostate
cancer imaging with the compounds of the present invention,
particularly with [.sup.18F]PSMA-1007.
[0091] The below example explains the invention in more detail but
are not construed to limit the invention in any way to the
exemplified embodiments only.
EXAMPLES
Example 1: Synthesis of Precursors and Cold Reference Compounds of
the .sup.18F-Conjugated Inhibitors
[0092] The isocyanate of the glutamyl moiety was generated in situ
by adding a mixture of 3 mmol of bis(tert-butyl) L-glutamate
hydrochloride and 1.5 mL of N-ethyldiisopropylamine (DIPEA) in 200
mL of dry CH.sub.2Cl.sub.2 to a solution of 1 mmol triphosgene in
10 mL of dry CH.sub.2Cl.sub.2 at 0.degree. C. over 4 h. After
agitation of the reaction mixture for 1 h at 25.degree. C., 0.5
mmol of the resin-immobilized (2-chloro-tritylresin)
.epsilon.-allyloxycarbonyl protected lysine in 4 mL DCM was added
and reacted for 16 h with gentle agitation. The resin was filtered
off and the allyloxy-protecting group was removed using 30 mg
tetrakis(triphenyl)palladium(0) and 400 .mu.L morpholine in 4 mL
CH.sub.2Cl.sub.2 for 3 hours.
[0093] The following coupling of the "linker amino acids" (as
defined in Formula I and Scheme 1 as shown above) was performed
stepwise using 2 mmol of the Fmoc-protected acid, 1.96 mmol of HBTU
and 2 mmol of N-ethyldiisopropylamine in a final volume of 4 mL
DMF.
[0094] The product was cleaved from the resin in a 2 mL mixture
consisting of trifluoroacetic acid, triisopropylsilane, and water
(95:2.5:2.5). Purification was performed using RP-HPLC and the
purified product was analysed by analytical RP-HPLC and
MALDI-MS.
[0095] For the preparation of the non-radioactive reference
compounds 50 mg of HBTU/DIPEA (0.98 and 1 Eq.) activated
6-Fluoronicotinic-3-acid was coupled in a final volume of 4 ml DMF
and agitated for 1 h at room temperature and the product was the
cleaved of the resin as described above.
[0096] In some preparations a .sup.18F-tag reactive moieties (e.g.
pent-4-ynoic acid or (Boc-aminooxy)acetic acid) were attached to
the terminal amino-group for subsequent .sup.18F labeling.
Example 2: Production and Activation of the [.sup.18F]Fluoride
[0097] Preparation and activation of the [.sup.18F]fluoride
Fluorine-18 was produced by the irradiation of .sup.18O-enriched
water with 16.5 MeV protons using the .sup.18O (p,n).sup.18F
nuclear reaction. Irradiations were performed with the Scanditronix
MC32NI cyclotron at the department of Radiopharmaceutical Chemistry
(E030) at the German Cancer Research Center Heidelberg.
[0098] After transfer of the irradiated water to an automated
system (Trasis All In One 32) the [.sup.18F]F.sup.- was separated
from the [.sup.18O]H.sub.2O by passing through an previously
conditioned (5 ml 1 M K.sub.2CO.sub.3 and 10 ml water) anion
exchange cartridge (Waters Accel.TM. Plus QMA Cartridge light) and
subsequently eluted with a mixture of 800 .mu.l acetonitrile and
150 .mu.l tetrabutylammonium bicarbonate solution (320 mM in
water). The mixture was evaporated to dryness an a temperature of
100.degree. C. under a stream of nitrogen. This distillation was
subsequently repeated two times by the adding 1.8 ml of
acetonitrile for each step. After applying maximum achievable
vacuum to the residue for 5 minutes at 100.degree. C. and
subsequent cooling to 50.degree. C. the dry residue was dissolved
in 2 ml of tert-butanol/acetonitrile (8:2) and used for the
labelling reactions.
[0099] Alternatively, the system was also used with the classical
Kryptofix.RTM. 2.2.2/K.sub.2CO.sub.3 activation system (20 mg
2.2.2+28 .mu.l 1 M K.sub.2CO.sub.3) in acetonitrile. Further, the
solvent for dissolving the activated [.sup.18F]F.sup.- was changed
to dry DMF, DMSO or acetonitrile for some experiments.
Example 3: Radiosynthesis of 6-['.sup.8F]Fluoronicotinic Acid
Tetrafluorophenyl Ester ([.sup.18F]FN-TFP)
[0100] To 10 mg
N,N,N-Trimethyl-5-((2,3,5,6-tetrafluorophenoxy)-carbonyl)pyridin-2-aminiu-
m trifluoromethanesulfonate (prepared as described by Olberg et al.
J. Med. Chem., 2010, 53, 1732) 1 ml of tert-butanol/Acetonitrile
(8:2) containing the dried [.sup.18F]KF-Kryptofix 2.2.2 komplex
(0.1-10 GBq .sup.18F) was added and the mixture was heated at
40.degree. C. After 10 minutes the mixture was diluted with 3 ml of
water and the product loaded on a preconditioned Oasis MCX Plus
Sep-Pak (Waters). The cartridge was rinsed with 10 mL of water and
the purified 6-[.sup.18F]Fluoronicotinic acid tetrafluorophenyl
ester was eluted back to the reaction vessel using 2 mL of
water/acetonitrile (7:13). For achieving a higher activity
concentration a fractionized elution was done in some cases.
Therefore the loaded cartridge was rinsed with 500 .mu.l of
solvent, which were discarded, and then eluted with 400-800 .mu.l
of solvent for further reactions. Usually more than 50% of the
initial activity were eluted with the second fraction.
Example 4: PSMA-1007
[0101] The synthesis of the precursor and the cold reference was
performed as described under example 1.
[0102] Radiosynthesis of [.sup.18F]PSMA-1007
[0103] 200 .mu.l of ([.sup.18F]FN-TFP) were added to 50 .mu.l of a
2 mg/ml solution of PSMA-1007-VL in DMSO. Then 50 .mu.l of buffer
(0.2 M phosphate buffer, pH 8.0) were added and the mixture heated
at 60.degree. C. for 20 minutes. The products were separated by
semipreparative radio-HPLC and identified by analytical radio-HPLC
and comparison of the retention times with the respective
non-radioactive reference compounds.
[0104] PSMA1007-VL (Precursor):
[0105] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Glu)-(Glu);
(C.sub.43H.sub.53N.sub.7O.sub.15; 907.93 g/mol) particularly
(Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(L-Glu)-(L-Glu)
[0106] MS (MALDI): m/z=908.7 [M+H].sup.+
[0107] PSMA-1007
[0108] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Glu)-(Glu)-FN;
C.sub.49H.sub.55FN.sub.8O.sub.16; (1031 g/mol) particularly
(Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(L-Glu)-(L-Glu)-FN;
[0109] MS (MALDI): m/z=1032.1 [M+H].sup.+
[0110] [.sup.18F]PSMA-1007
[0111] RCA: ca. 6%
[0112] HPLC (Gradient: 5% A/95% B-95% A/5% B in 12.5 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 4.56 min (t.sub.dead: 0.56 min)
Example 5: PSMA-1011
[0113] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1011 was performed as described under example 4.
[0114] PSMA-1011-VL (precursor):
[0115] (Glu)-(Urea)-(Lys)-(2-Naphthylalanine)-(Glu)-(Glu);
C.sub.35H.sub.46N.sub.8O.sub.14 (774.78 g/mol)
[0116] MS (MALDI): m/z=775.3 [M+H].sup.+
[0117] PSMA-1011:
[0118] (Glu)-(Urea)-(Lys)-(2-Naphthylalanine)-(Glu)-(Glu)-FN;
C.sub.41H.sub.48F N.sub.7O.sub.15 (897.86 g/mol)
[0119] MS (MALDI): m/z=898.3 [M+H].sup.+
[0120] [.sup.18F]PSMA-1011:
[0121] RCA: ca. 7%
[0122] HPLC (Gradient: 5% A/95% B-50% A/50% B in 10.0 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 5.72 min (t.sub.dead: 0.56 min)
Example 6: PSMA-1012
[0123] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1012 was performed as described under example 4.
[0124] PSMA-1012-VL (precursor):
[0125] (Glu)-(Urea)-(Lys)-(2-Naphthylalanine)-(.gamma.Glu)-(Glu);
C.sub.35H.sub.46N.sub.8O.sub.14 (774.78 g/mol)
[0126] MS (MALDI): m/z=775.0 [M+H].sup.+
[0127] PSMA-1012:
[0128]
(Glu)-(Urea)-(Lys)-(2-Naphthylalanine)-(.gamma.Glu)-(Glu)-FN;
C.sub.41H.sub.48FN.sub.7O.sub.15 (897.86 g/mol)
[0129] MS (MALDI): m/z=897.9 [M+H].sup.+
[0130] [.sup.18F]PSMA-1012:
[0131] RCA: ca. 4%
[0132] HPLC (Gradient: 5% A/95% B-50% A/50% B in 10.0 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 5.61 min (t.sub.dead: 0.56 min)
Example 7: PSMA-1015
[0133] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1015 was performed as described under example 4.
[0134] PSMA-1015-VL (precursor):
[0135] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Glu);
C.sub.38H.sub.46N.sub.6O.sub.12 (778.80 g/mol)
[0136] MS (MALDI): m/z=779.4 [M+H].sup.+
[0137] PSMA-1015:
[0138] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Glu)-FN;
C.sub.44H.sub.48FN.sub.7O.sub.13 (901.89 g/mol)
[0139] MS (MALDI): m/z=902.5 [M+H].sup.+
[0140] [.sup.18F]PSMA-1015:
[0141] RCA: ca. 7%
[0142] HPLC (Gradient: 5% A/95% B-50% A/50% B in 10.0 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 6.48 min (t.sub.dead: 0.56 min)
Example 8: Synthesis of Dry [.sup.18F]FN-TFP
[0143] The radiosynthesis was performed as described under example
3 until elution of the [.sup.18F]FN-TFP from the cartridge. After
the washing the cartridge with 10 ml water (example 3) the
cartridge was blown dry with 20-40 ml air. Then the MCX cartridge
was connected to a SepPak SodSulf drying cartridge and the product
was eluted with 2 ml of dry acetonitrile. For achieving a higher
activity concentration a fractionized elution was done in some
cases. Therefore the loaded MCX cartridge was rinsed with 500 .mu.l
of solvent (after blowing the cartridge dry), which were discarded,
and then the drying cartridge was connected to the MCX cartridge
and the product was eluted with 0.8-1.2 ml of acetonitrile for
further reactions. Usually more than 50% of the initial activity
were eluted with the second fraction.
Example 9: PSMA-1018
[0144] The synthesis of the precursor and the cold reference was
performed as described under example 1.
[0145] Radiosynthesis of [.sup.18F]PSMA-1018
[0146] 200 .mu.l of dry [.sup.18F]FN-TFP (example 8) were added to
50 .mu.l of a 4 mg/ml solution of PSMA-1018-VL in DMSO. Then 10
.mu.l of DIPEA were added and the mixture heated at 60.degree. C.
for 20 minutes. Then the reaction mixture was acidified by the
addition of 10 .mu.l TFA, the products separated by semipreparative
radio-HPLC and identified by analytical radio-HPLC and comparison
of the retention times with the respective non-radioactive
reference compounds.
[0147] PSMA-1018-VL (precursor):
[0148] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Glu)-(Glu)-(Glu);
C.sub.48H.sub.60N.sub.8O.sub.18 (1037.03 g/mol)
[0149] MS (MALDI): m/z=1037.6 [M+H].sup.+
[0150] PSMA-1018:
[0151] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Glu)-(Glu)-(Glu)-FN;
C.sub.54H.sub.62FN.sub.9O.sub.19 (1160.12 g/mol)
[0152] MS (MALDI): m/z=1160.8 [M+H].sup.+
[0153] [.sup.18F]PSMA-1018:
[0154] RCA: ca. 20%
[0155] HPLC (Gradient: 5% A/95% B-95% A/5% B in 10.0 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 3.87 min (t.sub.dead: 0.56 min)
Example 10: PSMA-1019
[0156] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1019 was performed as described under example 9.
[0157] PSMA-1019-VL (precursor):
[0158] (Glu)-(Urea)-(Lys)-(2-Nal)-(Chx)-(Glu)-(Glu);
C.sub.43H.sub.59N.sub.7O.sub.15 (913.97 g/mol)
[0159] MS (MALDI): m/z=914.4 [M+H].sup.+
[0160] PSMA-1019:
[0161] (Glu)-(Urea)-(Lys)-(2-Nal)-(Chx)-(Glu)-(Glu)-FN;
C.sub.49H.sub.61FN.sub.8O.sub.16 (1037.05 g/mol)
[0162] MS (MALDI): m/z=1037.7 [M+H].sup.+
[0163] [.sup.18F]PSMA-1019:
[0164] RCA: ca. 47%
[0165] HPLC (Gradient: 5% A/95% B-95% A/5% B in 12.5 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 4.43 min (t.sub.dead: 0.56 min)
Example 11: PSMA-1020
[0166] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1020 was performed as described under example 9.
[0167] PSMA-1020-VL (precursor):
[0168] (Glu)-(Urea)-(Lys)-(2-Nal)-(.gamma.Glu)-(Glu)-(Glu);
C.sub.40H.sub.53N.sub.7O.sub.17 (903.89 g/mol)
[0169] MS (MALDI): m/z=904.9 [M+H].sup.+
[0170] PSMA-1020:
[0171] (Glu)-(Urea)-(Lys)-(2-Nal)-(.gamma.Glu)-(Glu)-(Glu)-FN;
C.sub.46H.sub.55FN.sub.8O.sub.18 (1026.97 g/mol)
[0172] MS (MALDI): m/z=1027.9 [M+H].sup.+
[0173] [.sup.18F]PSMA-1020:
[0174] RCA: ca. 60%
[0175] HPLC (Gradient: 5% A/95% B-95% A/5% B in 12.5 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 6.48 min (t.sub.dead: 0.56 min)
Example 12: PSMA-1022
[0176] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1022 was performed as described under example 9.
[0177] PSMA-1022-VL (precursor):
[0178] (Glu)-(Urea)-(Lys)-(2-Nal)-(.gamma.Glu)-(.gamma.Glu);
C.sub.35H.sub.46N.sub.8O.sub.14 (774.78 g/mol)
[0179] MS (MALDI): m/z=775.4 [M+H].sup.+
[0180] PSMA-1022:
[0181] (Glu)-(Urea)-(Lys)-(2-Nal)-(.gamma.Glu)-(.gamma.Glu)-FN;
C.sub.41H.sub.48FN.sub.7O.sub.15 (897.86 g/mol)
[0182] MS (MALDI): m/z=898.8 [M+H].sup.+
[0183] [.sup.18F]PSMA-1022:
[0184] RCA: ca. 29%
[0185] HPLC (Gradient: 5% A/95% B-95% A/5% B in 10.0 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 3.92 min (t.sub.dead: 0.56 min)
Example 13: PSMA-1023
[0186] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1023 was performed as described under example 9
[0187] PSMA-1023-VL (precursor):
[0188] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(D-Glu)-(D-Glu);
C.sub.43H.sub.53N.sub.7O.sub.15 (907.93 g/mol)
[0189] MS (MALDI): m/z=907.8 [M+H].sup.+
[0190] PSMA-1023:
[0191] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(D-Glu)-(D-Glu)-FN;
C.sub.49H.sub.55FN.sub.8O.sub.16 (1031.00 g/mol)
[0192] MS (MALDI): m/z=1031.8 [M+H].sup.+
[0193] [.sup.18F]PSMA-1023:
[0194] RCA: ca. 24%
[0195] HPLC (Gradient: 5% A/95% B-50% A/50% B in 10.0 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 3.87 min (t.sub.dead: 0.56 min)
Example 14: PSMA-1024
[0196] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1024 was performed as described under example 9.
[0197] PSMA-1024-VL (precursor):
[0198] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(D-Glu)-(Glu);
C.sub.43H.sub.53N.sub.7O.sub.15 (907.93 g/mol)
[0199] MS (MALDI): m/z=908.6 [M+H].sup.+
[0200] PSMA-1024:
[0201] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(D-Glu)-(Glu)-FN;
C.sub.49H.sub.55FN.sub.8O.sub.16 (1031.00 g/mol)
[0202] MS (MALDI): m/z=1031.5 [M+H].sup.+
[0203] [.sup.18F]PSMA-1024:
[0204] RCA: ca. 27%
[0205] HPLC (Gradient: 5% A/95% B-95% A/5% B in 10.0 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 3.85 min (t.sub.dead: 0.56 min)
Example 15: PSMA-1025
[0206] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1025 was performed as described under example 9.
[0207] PSMA-1025-VL (precursor):
[0208] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Gla);
C.sub.39H.sub.46N.sub.6O.sub.14 (822.21 g/mol)
[0209] MS (MALDI): m/z=822.8 [M+H].sup.+
[0210] PSMA-1025:
[0211] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Gla)-FN;
C.sub.49H.sub.55FN.sub.8O.sub.16 (945.32 g/mol)
[0212] MS (MALDI): m/z=946.0 [M+H].sup.+
[0213] [.sup.18F]PSMA-1025:
[0214] RCA: ca. 24%
[0215] HPLC (Gradient: 5% A/95% B-95% A/5% B in 12.5 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 4.51 min (t.sub.dead: 0.56 min)
Example 16: PSMA-1026
[0216] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1026 was performed as described under example 9.
[0217] PSMA-1026-VL (precursor):
[0218] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Sala);
C.sub.36H.sub.44N.sub.6O.sub.13S (800.83 g/mol)
[0219] MS (MALDI): m/z=801.8 [M+H].sup.+
[0220] PSMA-1026:
[0221] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Sala)-FN;
C.sub.42H.sub.46FN.sub.7O.sub.14S (923.92 g/mol)
[0222] MS (MALDI): m/z=924.7 [M+H].sup.+
[0223] [.sup.18F]PSMA-1026:
[0224] RCA: ca. 57%
[0225] HPLC (Gradient: 5% A/95% B-95% A/5% B in 12.5 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 4.03 min (t.sub.dead: 0.56 min)
Example 17: PSMA-1027
[0226] The synthesis of the precursor and the cold reference was
performed as described under example 1. The synthesis
[.sup.18F]PSMA-1027 was performed as described under example 9.
[0227] PSMA-1027-VL (precursor):
[0228] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Sala)-(Sala);
C.sub.39H.sub.49N.sub.7O.sub.17S.sub.2 (951.97 g/mol)
[0229] MS (MALDI): m/z=952.7 [M+H].sup.+
[0230] PSMA-1027:
[0231] (Glu)-(Urea)-(Lys)-(2-Nal)-(Bn)-(Sala)-(Sala)-FN;
C.sub.45H.sub.51FN.sub.8O.sub.18S.sub.2 (1075.06 g/mol)
[0232] MS (MALDI): m/z=1075.8 [M+H].sup.+
[0233] [.sup.18F]PSMA-1027:
[0234] RCA: ca. 62%
[0235] HPLC (Gradient: 5% A/95% B-95% A/5% B in 10.0 min; Flow: 3
ml/min; Column: Merck Chromolith.RTM. Performance RP-18e 100-4.6
mm; Solvent A: Acetonitrile, Solvent B: 0.1% aqueous TFA):
t.sub.ret: 3.10 min (t.sub.dead: 0.56 min)
Example 18: Cell Culture
[0236] For binding studies and in vivo experiments LNCaP cells
(metastatic lesion of human prostatic adenocarcinoma, ATCC
CRL-1740) were cultured in RPMI medium supplemented with 10% fetal
calf serum and Glutamax (PAA, Austria). During cell culture, cells
were grown at 37.degree. C. in an incubator with humidified air,
equilibrated with 5% CO2. The cells were harvested using
trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA; 0.25%
trypsin, 0.02% EDTA, all from PAA, Austria) and washed with
PBS.
Example 19: Cell Binding and Internalization
[0237] The competitive cell binding assay and internalization
experiments were performed as described previously (Eder et al.
Bioconjugate Chem. 2012, 23 (4), 688-697). Briefly, the respective
cells (10.sup.5 per well) were incubated with the radioligand
(.sup.68Ga-labeled [Glu-urea-Lys(Ahx)].sub.2-HBED-CC (Schafer et
al. EJNMMI Research 2012, 2:23 doi:10.1186/2191-219X-2-23))) in the
presence of 12 different concentrations of analyte (0-5000 nM, 100
.mu.L/well). After incubation, washing was carried out using a
multiscreen vacuum manifold (Millipore, Billerica, Mass.).
Cell-bound radioactivity was measured using a gamma counter
(Packard Cobra II, GMI, Minnesota, USA). The 50% inhibitory
concentration (IC.sub.50) was calculated by fitting the data using
a nonlinear regression algorithm (GraphPad Software). Experiments
were performed three times. Reference is made to Table A below.
[0238] To determine the specific cell uptake and internalization,
10.sup.5 cells were seeded in poly-L-lysine coated 24-well cell
culture plates 24 h before incubation. After washing, the cells
were incubated with 30 nM of the radiolabeled compounds for 45 min
at 37.degree. C. Specific cellular uptake was determined by
competitive blocking with 2-(phosphonomethyl)pentanedioic acid (500
.mu.M final concentration, PMPA, Axxora, Loerrach, Germany).
Cellular uptake was terminated by washing 3 times with 1 mL of
ice-cold PBS. Cells were subsequently incubated twice with 0.5 mL
glycine-HCl in PBS (50 mM, pH=2.8) for 5 min to remove the
surface-bound fraction. The cells were washed with 1 mL of ice-cold
PBS and lysed using 0.3 N NaOH (0.5 mL). The surface-bound and the
internalized fractions were measured in a gamma counter. The cell
uptake was calculated as percent of the initially added
radioactivity bound to 10.sup.5 cells [% ID/10.sup.5 cells]. The
main results are given in Table B.
TABLE-US-00003 TABLE A Binding Affinity Assay Compound IC.sub.50
[nM] PSMA-1007 5 PSMA-1011 7 PSMA-1012 7 PSMA-1015 4 PSMA-1018 14
PSMA-1019 12 PSMA-1020 9 PSMA-1022 8 PSMA-1023 8 PSMA-1024 8
PSMA-1025 9 PSMA-1026 14 PSMA-1027 7
TABLE-US-00004 TABLE B Specific Internalization Cell surface
Internalised Internalized Compound [% ID/10.sup.5 cells] [%
ID/10.sup.5 cells] fraction [%]* PSMA-1007 2.7 6.5 71 PSMA-1011 4.1
0.7 15 PSMA-1012 6.9 2.7 28 PSMA-1015 14.3 6.0 30 PSMA-1018 1.1 0.9
45 PSMA-1019 5.5 1.9 26 PSMA-1020 6.8 1.5 18 PSMA-1022 3.5 1.0 22
PSMA-1023 6.5 3.2 33 PSMA-1024 5.3 2.1 28 PSMA-1025 4.3 3.1 42
PSMA-1026 1.3 1.3 50 PSMA-1027 0.9 1.4 60 *(Internalized
activity/total activity)*100
Example 20: MicroPET
[0239] For the microPET studies, 10-25 MBq of the radiolabeled
compounds in a volume of 0.10 ml (60 pmol) were injected via a
lateral tail vein into mice bearing LNCaP tumor xenografts. The
anesthetized animals (2% sevoflurane, Abbott, Wiesbaden, Germany)
were placed in prone position into the Inveon small animal PET
scanner (Siemens, Knoxville, Tenn., USA) to perform dynamic
microPET scans. The results are shown in FIGS. 10 and 11.
Example 21: Plasma Binding and Stability
[0240] For the determination of the plasma binding 3 .mu.l of 6
.mu.molar c.a. [.sup.18F]PSMA solution was added to 300 .mu.l human
serum AB and incubated at 37.degree. C. for 1 h. Subsequently the
product mixture was analyzed by size-exclusion chromatography.
[0241] No plasma binding was observed with any of the
compounds.
[0242] For the determination of the plasma stability 50 .mu.l of 6
.mu.molar c.a. [.sup.18F]PSMA solution was added to 450 .mu.l human
serum AB and incubated at 37.degree. C. At 1, 2 and 4 h samples
were prepared. Therefore 100 .mu.l of the tracer/plasma mixture
were added to 100 .mu.l of acetonitrile. Subsequently the mixture
was centrifuged at 13000 rpm for 3 minutes. 100 .mu.l of the
supernatant were added to 100 .mu.l of acetonitrile, centrifuged at
13000 rpm for 5 minutes, the liquid separated from any residual
solids and analyzed by HPLC.
[0243] All of the compounds were stable in human plasma at
37.degree. C. for at least 4 hours.
Example 22: In Vivo Experiments
[0244] For in vivo experiments, 8 week old BALB/c nu/nu mice were
subcutaneously inoculated into the right trunk with
5.times.10.sup.6 LNCaP- or PC3-cells in 50% Matrigel. When the size
of tumor was approximately 1 cm.sup.3, the radiolabeled compound
was injected via the tail vein (ca. 30 MBq, 60 pmol for .mu.PET
imaging; ca. 1 MBq, 60 pmol for organ distribution).
[0245] Organ Distribution
[0246] The F-18 labeled compounds were injected via tail vein (1-2
MBq per mouse; 60 pmol). At 1 h after injection, the animals were
sacrificed. Organs of interest were dissected, blotted dry, and
weighed. The radioactivity was measured with a gamma counter
(Packard Cobra II, GMI, Minnesota, USA) and calculated as % ID/g.
The main results are given in FIGS. 8 and 9.
[0247] The compounds PSMA 1003 and PSMA 1009 are for comparison and
are shown in FIGS. 12 and 13.
Example 23: [.sup.18F]PSMA-1007 for Human Application
[0248] [.sup.18F]PSMA-1007 was produced by conjugation of dry
[.sup.18F]FN-TFP (Example 8) to PSMA1007-VL (Example 4) under dry
conditions (analogously Example 9) on an automated synthesis module
(Trasis AllInOne) and purified by semi-preparative HPLC. Radio-HPLC
was performed to determine the chemical identity and the chemical
and radiochemical purity of [.sup.18F]PSMA-1007. Residual solvents
were determined by gas chromatography. The radionuclide purity was
controlled by half-life measurement. The product solution was
tested for sterility, bacterial endotoxins (LAL-test), pH,
colorlessness and particles. The integrity of the sterile filter
after filtration was examined using the bubble-point test.
Example 24: Application of [.sup.18F]PSMA-1007 to a Healthy
Volunteer
[0249] The healthy subject was injected a total activity of 300 MBq
and subsequently PET scans were performed on a Biograph mCT Flow
scanner (Siemens, Erlangen, Germany) in three blocks. Block 1
contained PET-1 (start 5 min p.i.) to PET-7 (ending 140 min p.i.),
block 2 contained PET-8 (start 180 min p.i.) and PET-9 (240-270 min
p.i.), block 3 contained PET-10 (440-480 min p.i.). A non-enhanced
low-dose CT (estimate 1.43 mSv, respectively) for attenuation
correction was performed at the beginning of each block, followed
by serial emission scans without moving the volunteer in
between.
[0250] Kidneys, liver, spleen, whole heart, upper and lower large
intestine, parotid glands, submandibular glands and urinary bladder
were segmented into volumes of interest (VOIs) using a percentage
of maximum threshold between 20% and 30% using the corresponding CT
as guidance and then time activity curves (TACs) were calculated
for all organs. The TAC for red marrow was derived from the venous
blood samples and then the dose for the red marrow calculated (S.
Shen et al., JNM 2002, 43, 1245-1253; G. Sgouros et al., JNM 1993,
34, 689-694). The TAC for the urinary bladder content was a
combination of estimated activity in the urinary bladder VOI in PET
and activity measured in the voided urine. Curve fitting was
applied to all TACs. Kidneys, salivary glands, upper and lower
large intestine and heart were fitted with a bi-exponential
function. For liver, spleen and urinary bladder content a
mono-exponential fit to the last three time points was
performed.
[0251] Absorbed and effective dose calculations were performed
using the ICRP endorsed IDAC 1.0 package which is integrated in
QDOSE. Additionally, residence times of all included source organs
and remainder body were exported as an OLINDA case file for dose
calculation (OLINDA 1.1). The absorbed doses to the salivary glands
(parotid and submandibular glands) were determined using the
spherical model. The organ masses for the salivary glands were
estimated with 25 g for a parotid and 12.5 g for a submandibular
gland (ICRP publication 89). The results are summarized in FIGS.
14-16.
Example 25: Application of [.sup.18F]PSMA-1007 in a Patients
Suffering from Prostate Cancer
[0252] Ten patients (age 60-80 years) suffering from a newly
diagnosed high-risk prostate cancer with a gleason score of 7-9 and
initial PSA levels of 5-90 ng/ml were injected 100-360 MBq
[.sup.18F]-PSMA-1007 and subsequently PET scans were performed on a
Biograph mCT Flow scanner (Siemens, Erlangen, Germany) at 1 and 3 h
p.i. The results are depicted in FIGS. 17 and 18 as mean values of
the SUV.sub.mean and SUV.sub.max, respectively. The pictures gained
with the PET scans are exemplified in FIGS. 19 and 20 as maximum
intensity projections.
[0253] Eight of the patients underwent radical prostatectomy with
extended pelvic lymphadenectomy. Analyses of prostatectomy specimen
were performed blinded to PET-data under the supervision of
dedicated uropathologists, according to International Society of
Urological Pathology standards (T. H. van der Kwast et al. Mod.
Phathol. 2011, 24, 16-25). Representative sections were stained by
immunohistochemistry. The sections were deparaffinized in xylene
and rehydrated in a graded ethanol series. Antigen retrieval was
performed with a steam cooker using retrieval buffer (Target
Retrieval Solution, Dako). A mouse monoclonal antibody against PSMA
(clone 3E6, Dako) was used at a 1:100 dilution and incubated
overnight at 4.degree. C. ad subsequently immunodetection was
performed using the Histostain-Plus detection kit (Invitrogen).
Stained sections were scanned using a Nanozoomer 2.0-HT Scansystem
(Hamamatsu Photonics) to generate digital whole slide images. The
staining revealed a nearly perfect correlation with the PET scan,
as exemplified in FIG. 21.
* * * * *