U.S. patent application number 14/104476 was filed with the patent office on 2014-04-10 for cancer imaging and treatment.
This patent application is currently assigned to Mallinckrodt LLC. The applicant listed for this patent is Mallinckrodt LLC. Invention is credited to Martina Anton, Oliver Demmer, Horst Kassler, Norman Koglin, Burkhardt Laufer, Markus Schwaiger, Hans Jurgen Wester.
Application Number | 20140100172 14/104476 |
Document ID | / |
Family ID | 37963490 |
Filed Date | 2014-04-10 |
United States Patent
Application |
20140100172 |
Kind Code |
A1 |
Wester; Hans Jurgen ; et
al. |
April 10, 2014 |
Cancer Imaging and Treatment
Abstract
Provided herein are compounds useful for diagnostic imaging
and/or therapeutic purposes. Each compound comprises a ligand for
the chemokine receptor CXCR4, which has a binding affinity for the
CXCR4 receptor, measured as IC50 in the presence of
.sup.125I-CPCR4, of 250 nM or lower. The ligand is a cyclic
oligopeptide moiety having a motif B-Arg or B-(Me)Arg within the
cyclic moiety, wherein B is a basic amino acid, a derivative
thereof, or phenylalanine, provided that the motif is B-Arg when B
is a N.sup..alpha.-methyl derivative of a basic amino acid, and
provided that the cyclic oligopeptide moiety has a sequence other
than cyclo[D-Tyr-Arg-Arg-NaI-Gly] or
cyclo[D-Tyr-Orn-Arg-NaI-Gly].
Inventors: |
Wester; Hans Jurgen;
(Ilmmunster, DE) ; Koglin; Norman; (Berlin,
DE) ; Schwaiger; Markus; (Munchen, DE) ;
Kassler; Horst; (Garching, DE) ; Laufer;
Burkhardt; (Munchen, DE) ; Demmer; Oliver;
(Munchen, DE) ; Anton; Martina; (Vaterstetten,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mallinckrodt LLC |
Hazelwood |
MO |
US |
|
|
Assignee: |
Mallinckrodt LLC
Hazelwood
MO
|
Family ID: |
37963490 |
Appl. No.: |
14/104476 |
Filed: |
December 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12280829 |
Oct 14, 2010 |
8628750 |
|
|
PCT/GB2007/000684 |
Feb 27, 2007 |
|
|
|
14104476 |
|
|
|
|
Current U.S.
Class: |
514/19.9 ;
530/321 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/04 20180101; A61P 35/00 20180101; C07K 7/64 20130101; A61K
51/08 20130101; A61K 51/088 20130101 |
Class at
Publication: |
514/19.9 ;
530/321 |
International
Class: |
C07K 7/64 20060101
C07K007/64; A61K 51/08 20060101 A61K051/08; A61K 47/48 20060101
A61K047/48 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 2006 |
GB |
0603901.0 |
Jun 2, 2006 |
GB |
0610962.3 |
Claims
1. A compound, or a pharmaceutically acceptable salt or ester
thereof, the compound having a binding affinity for a CXCR4
receptor, measured as IC50 in the presence of .sup.125I-CPCR4, of
250 nM or lower, wherein the compound is a cyclic oligopeptide
moiety having a motif B-Arg or B-(Me)Arg within the cyclic moiety,
and wherein B is a basic amino acid, a derivative thereof, or
phenylalanine, provided that the motif is B-Arg when B is a
N.sup..alpha.-methyl derivative of a basic amino acid, and provided
that the cyclic oligopeptide moiety has a sequence other than
cyclo[D-Tyr-Arg-Arg-NaI-Gly] or cyclo[D-Tyr-Orn-Arg-NaI-Gly], NaI
being L-3-(2-naphthyl)alanine.
2. The compound of claim 1, wherein the cyclic oligopeptide moiety
has the sequence:
cyclo[D-Tyr/(Me)D-Tyr-B-Arg/(Me)Arg-Z-(Ala).sub.n-X] wherein: B is
as defined in claim 1; Z is an amino acid containing an aromatic
group in its side chain; X selected from Ala, Gly, Dap
(diaminopropionic acid), Dab (diaminobutyric acid), or
N-substituted derivatives thereof; and n is 1 or 0.
3. The compound of claim 2, wherein B is selected from Arg, Orn,
D-Orn, Cit and His, or N-substituted derivatives thereof.
4. The compound of claim 2, wherein B is N.sup..alpha.-substituted
with a Me group.
5. The compound of claim 2, wherein B is Orn or D-Orn, the
ornithine residue being substituted at N.sup..delta. with one or
two groups selected from fluorobenzoyl (FB), fluoropropionyl (FP),
acetyl (Ac), amido (Am), Me, 1-naphthylmethyl (N1),
2-naphthylmethyl (N2), benzyl (Bz) and acyl spacer moieties,
wherein the acyl spacer moiety is an acyl group containing a chain
of 1-14 carbons, optionally interrupted by heteroatoms, and having
a nucleophilic functional group at its end distal to the ornithine
N.sup..delta., or the ornithine residue being substituted at
N.sup..alpha. with a Me group.
6. The compound of claim 6, wherein the acyl spacer moiety is
selected from aminohexanoyl (Ahx), triethyleneglycolamino acyl
(TGAS), (Ahx).sub.2, (Ahx).sub.3, (TGAS).sub.2 and
(TGAS).sub.3.
7. The compound of claim 6, wherein Orn is substituted at
N.sup..delta. with FB, FP, Ac, Am, N1, N2, Me and N1, Me and N2,
Bz, Bz and FB, Bz and FP, Me and FB, Me and FP, or Me.
8. The compound of claim 6, wherein D-Orn is substituted at
N.sup..delta. with FB, FP, Me and FB, or Me and FP, and optionally
substituted at N.sup..alpha. with Me.
9. The compound of claim 2, wherein Z is selected from NaI,
(Me)NaI, Dap(FB) or AMS (FB) (an oxime of aminooxy serine and
4-fluorobenzaldehyde).
10. The compound of claim 2, wherein X is selected from Gly,
(Me)Gly, Ala, Dap Dap(FP), Dab, Dab(FP), Dab(FB), or Dap(FB).
11. The compound of claim 2, wherein the cyclic oligopeptide moiety
has the sequence: cyclo[D-Tyr B-Arg-Z-X], provided that not more
than one of the residues in the sequence is
N.sup..alpha.-methylated.
12. The compound of claim 2, wherein the cyclic oligopeptide moiety
has the sequence: cyclo[D-Tyr/(Me)D-Tyr-B-Arg/(Me)Arg-Z-X], wherein
B is selected from Arg, (Me)Arg, Orn, Cit, Orn(FB), Orn(FP),
Orn(Ac), Orn(Am), Orn(N1), Orn(N2), Orn(Me, N1), Orn(Me, N2),
Orn(Me), Orn(Bz), Orn(Bz,FB), Orn(Ahx), Orn(Ahx.sub.2),
Orn(Ahx.sub.3), Orn(TGAS), Orn(TGAS.sub.2), Orn(TGAS.sub.3),
Orn(Me,FB), D-Orn(FB), (Me)D-Orn(FB), (Me)D-Orn(Me,FB), His and
Phe, provided that not more than one of the residues in the said
sequence may be N.sup..alpha.-methylated.
13. The compound of claim 2, wherein the first residue is D-Tyr,
the third residue is Arg, Z is NaI, and X is Gly.
14. The compound of claim 2, wherein the cyclic oligopeptide moiety
has a sequence selected from: TABLE-US-00012
cyclo[D-Tyr-(Me)Arg-Arg-Nal-Gly] cyclo[D-Tyr-Arg-(Me)Arg-Nal-Gly]
cyclo[D-Tyr-Arg-Arg-Nal-(Me)Gly]; cyclo[D-Tyr-Cit-Arg-Nal-Gly]
cyclo[D-Tyr-Arg-Arg-Nal-Ala-Gly] cyclo[D-Tyr-Arg-Arg-Nal-Ala-Ala]
cyclo[D-Tyr-(Me)Arg-Arg-Nal-(Me)Gly]
cyclo[D-Tyr-(Me)Arg-Arg-(Me)Nal-Gly]
cyclo[(Me)D-Tyr-Arg-Arg-Nal-Ala-Gly]
cyclo[(Me)D-Tyr-Arg-Arg-Nal-Gly] cyclo[D-Tyr-Orn(FB)-Arg-Nal-Gly]
cyclo[D-Tyr-Orn(FP)-Arg-Nal-Gly] cyclo[D-Tyr-Orn(Ac)-Arg-Nal-Gly]
cyclo[D-Tyr-Orn(Am)-Arg-Nal-Gly] cyclo[D-Tyr-Arg-Arg-Nal-Dap(FP)]
cyclo[D-Tyr-Orn(N1)-Arg-Nal-Gly] cyclo[D-Tyr-Orn(N2)-Arg-Nal-Gly]
cyclo[D-Tyr-Orn(Me, N1)-Arg-Nal-Gly] cyclo[D-Tyr-Orn(Me,
N2)-Arg-Nal-Gly] cyclo[D-Tyr-Orn(Me)-Arg-Nal-Gly]
cyclo[D-Tyr-Orn(Bz)-Arg-Nal-Gly] cyclo[D-Tyr-Orn(Bz,
FB)-Arg-Nal-Gly] cyclo[D-Tyr-Orn(Ahx)-Arg-Nal-Gly]
cyclo[D-Tyr-Orn(Ahx3)-Arg-Nal-Gly]
cyclo[D-Tyr-Orn(TGAS)-Arg-Nal-Gly]
cyclo[D-Tyr-Orn(TGAS2)-Arg-Nal-Gly]
cyclo[D-Tyr-Orn(TGAS3)-Arg-Nal-Gly] cyclo[D-Tyr-D-Orn(Me,
FB)-Arg-Nal-Gly] cyclo[D-Tyr-D-Orn(FB)-Arg-Nal-Gly]
cyclo[D-Tyr-(Me)D-Orn(FB)-Arg-Nal-Gly] cyclo[D-Tyr-(Me)D-Orn(Me,
FB)-Arg-Nal-Gly] cyclo[D-Tyr-His-Arg-Nal-Gly] or
cyclo[D-Tyr-Phe-Arg-Nal-Gly].
15. The compound of claim 1 which has been modified by the
attachment of one or more hydrophilic moieties.
16. A pharmaceutical composition comprising the compound of claim 1
together with one or more pharmaceutically acceptable
excipients.
17. A method for treating a neoplastic condition, the method
comprising administering to a subject having a neoplasia the
compound of claim 1.
18. The compound of claim 1 further comprising a cytotoxic
moiety.
19. The compound of claim 18, wherein the cyclic oligopeptide
moiety has the sequence:
cyclo[D-Tyr/(Me)D-Tyr-B-Arg/(Me)Arg-Z-(Ala).sub.n-X] wherein: B is
selected from Arg, Orn, D-Orn, Cit and His, or N-substituted
derivatives thereof; Z is an amino acid containing an aromatic
group in its side chain; X selected from Ala, Gly, Dap, Dab, or
N-substituted derivatives thereof; and n is 1 or 0.
20. The compound of claim 19, wherein B is selected from Arg,
(Me)Arg, Orn, Cit, Orn(FB), Orn(FP), Orn(Ac), Orn(Am), Orn(N1),
Orn(N2), Orn(Me, N1), Orn(Me, N2), Orn(Me), Orn(Bz), Orn(Bz,FB),
Orn(Ahx), Orn(Ahx.sub.2), Orn(Ahx.sub.3), Orn(TGAS),
Orn(TGAS.sub.2), Orn(TGAS.sub.3), Orn(Me,FB), D-Orn(FB),
(Me)D-Orn(FB), (Me)D-Orn(Me,FB), His and Phe, provided that not
more than one of the residues in the said sequence may be
N.sup..alpha.-methylated.
21. The compound of claim 19, wherein Z is selected from NaI,
(Me)NaI, Dap(FB) or AMS (FB).
22. The compound of claim 19, wherein X is selected from Gly,
(Me)Gly, Ala, Dap Dap(FP), Dab, Dab(FP), Dab(FB), or Dap(FB).
23. The compound of claim 18, wherein the cytotoxic moiety is bound
directly to the cyclic oligopeptide moiety or the cytotoxic moiety
is attached to the cyclic oligopeptide moiety by a spacer.
24. The compound of claim 18, wherein the cytotoxic moiety is a
chemotherapeutic compound.
25. A method for treating a neoplastic condition, the method
comprising administering to a subject having a neoplasia the
compound of claim 18.
26. The method of claim 25, wherein the neoplasia has, or is
suspected of having, metastatic potential.
27. A pharmaceutical composition comprising the compound of claim
18 together with one or more pharmaceutically acceptable
excipients.
28. The compound of claim 1 further comprising a detectable
label.
29. The compound of claim 28, wherein the cyclic oligopeptide
moiety has the sequence:
cyclo[D-Tyr/(Me)D-Tyr-B-Arg/(Me)Arg-Z-(Ala).sub.n-X] wherein: B is
selected from Arg, Orn, D-Orn, Cit and His, or N-substituted
derivatives thereof; Z is an amino acid containing an aromatic
group in its side chain; X selected from Ala, Gly, Dap, Dab, or
N-substituted derivatives thereof; and n is 1 or 0.
30. The compound of claim 28, wherein the detectable label is a
fluorescent moiety, a magnetic or paramagnetic moiety, or a
radionuclide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 12/280,829, filed Aug. 27, 2008, which claims
the priority of PCT application PCT/GB2007/000684, filed Feb. 27,
2007, which claims the priority of GB Application No. 0603901.0
filed Feb. 27, 2006 and GB Application No. 0610962.3 filed Jun. 2,
2006, all of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the imaging and treatment
of cancer. In particular, though not exclusively, it relates to
compositions suitable for the targeting of radionuclides to cells
expressing the chemokine receptor CXCR4 for the purposes of imaging
and treatment thereof.
BACKGROUND OF THE INVENTION
[0003] A method for the early assessment of the metastatic
potential and metastatic spread of tumors would be a valuable tool
for therapy prediction and control. Recently a key role in
metastasis was attributed to the chemokine receptor CXCR4 (Muller
et al., Nature 410 (2001) 50). In a variety of tumors such as
breast and prostate cancer, CXCR4 has been found to play a
dominating role during tumor cell homing and was shown to be
expressed, both in primaries and metastases.
[0004] Stromal cell-derived factor 1.alpha. (SDF-1.alpha.) is the
endogenous ligand for CXCR4 (Nagasawa T. et al. PNAS. 91 (1994)
2305). Peptide-based antagonists for CXCR4 have been described,
including CPCR4 (also known as FC131, and having the sequence
cyclo[D-Tyr-Arg-Arg-NaI-Gly]) (SEQ ID NO:1) (see Fujii N. et al.,
Angew. Chem. Int. Ed 42 (2003) 3251). CXCR4 is a co-receptor for
HIV-1 and HIV-2, enabling entry of the viruses into cells. EP
1541585 describes radiolabeled SDF-1.alpha. for histology studies.
This document also discloses a number of relatively bulky synthetic
peptide antagonists of CXCR4. WO 2004/087608 discloses a CXCR4
antagonist labeled with biotin. Detection of such a compound
requires the addition of a second, streptavidin-bearing reporter
compound. The antagonists exemplified in WO 2004/087608 are
peptides of 14 amino acids cyclised by means of a disulfide bond
between Cys residues at positions 4 and 13. An Arg-Arg motif is
present at positions 1 and 2, i.e. outside the cyclic moiety.
[0005] Until now, investigations with antagonists for CXCR4 (both
peptide and non-peptide) have essentially been restricted to their
potential use as inhibitors of the metastatic process or HIV
infection.
SUMMARY OF THE INVENTION
[0006] Accordingly, a first aspect of the present invention
provides a compound, or a pharmaceutically acceptable salt or ester
thereof, comprising a ligand for the chemokine receptor CXCR4 and a
detectable label, the ligand having a binding affinity for the
CXCR4 receptor, measured as IC50 in the presence of
.sup.125I-CPCR4, of 250 nM or lower, wherein the ligand comprises a
cyclic oligopeptide moiety having the motif B-Arg or B-(Me)Arg
within the cyclic moiety, and wherein B is a basic amino acid, a
derivative thereof, or phenylalanine, provided that the motif is
B-Arg when B is a N.sup..alpha.-methyl derivative of a basic amino
acid.
[0007] In certain embodiments, the ligand for CXCR4 is preferably
synthetic. It is currently preferred that the ligand binds to CXCR4
with an affinity (IC50) of 200 nM or less, more preferably 100 nM
or less, and most preferably 50 nM or less. The term `IC50` refers
to the concentration of test compound required to reduce binding of
the radiolabeled reference peptide .sup.125I-CPCR4 to
CXCR4-expressing cells to 50% of maximum binding. The person of
ordinary skill in the art would readily be able to determine the
IC50 of a given compound, and a method for doing so is described
below. Compounds of the invention may bind to the CXCR4 receptor
without activating the receptor (i.e. antagonist properties).
Alternatively, compounds of the invention may compete with the
endogenous ligand for the receptor, but activate the receptor to a
lesser degree (i.e. partial agonist properties). As a further
alternative, compounds of the invention may bind to the CXCR4
receptor and reduce subsequent signal transduction below the
baseline, non-activated level (i.e. negative efficacy, or inverse
agonist properties). In certain preferred embodiments, the
compounds of the invention bind to the CXCR4 receptor without
activating the receptor. In other preferred embodiments, the
compounds of the invention do not comprise ligands with full
agonist properties at CXCR4.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 shows the fluorescence-activated cell sorting (FACS)
results from transfection of cells in vitro with a vector coding
for CXCR4 (A) and a GFP reporter (B).
[0009] FIG. 2 illustrates the .sup.125I-CPCR4 binding parameters at
CXCR4 on Jurkat cells (A, C) and a comparison thereof with
.sup.125I-SDF-1.alpha. (B, D)
[0010] FIG. 3 illustrates the biodistribution of .sup.125I-CPCR4
following intravenous injection thereof in nude mice (A, B).
[0011] FIG. 4 shows PET/SPECT images of radiolabelled CPCR4
distribution in mice bearing CXCR4 positive and negative
tumors.
DETAILED DESCRIPTION OF THE INVENTION
[0012] As used herein, the expression `(Me)Xaa` means an
N.sup..alpha.-methyl derivative of an amino acid. The expression
`Xaa(substituent)` means that the side chain of the amino acid is
derivatized with the indicated substituent. The expression
`Xaa/(Me)Xaa` means that the stated amino acid may be unmethylated
or may bear an N.sup..alpha.-methyl group. The amino acid
abbreviations used herein refer to the L-enantiomer of the
respective amino acid, unless the expression `D-Xaa` is used, in
which case the D enantiomer is denoted. The term `basic amino acid`
as used herein denotes a naturally occurring or synthetic
(preferably naturally occurring) amino acid having a side chain
capable of receiving a proton, and becoming positively charged,
under normal physiological conditions. Accordingly, basic amino
acids include lysine, arginine, citrulline (Cit), ornithine,
histidine, Dap (2,3-diaminopropionic acid) and Dab
(2,4-diaminobutyric acid). Preferred basic amino acids are lysine,
arginine, citrulline, ornithine and histidine, more preferably
arginine and ornithine.
[0013] Compounds of the invention may provide an efficient probe
for the in vivo targeting of the CXCR4 chemokine receptor. The
compounds bind with high affinity and specificity to their binding
site and allow ready imaging (by a variety of methods) and hence a
clear delineation of CXCR4 positive tumors (and any associated
metastases) in vivo. This new class of probes/tracers may provide
highly valuable tools for the investigation of the metastatic
potential of tumors and early imaging, and potentially radionuclide
therapy, of metastatic processes.
[0014] The detectable label is preferably selected from fluorescent
moieties, magnetic or paramagnetic moieties, or radionuclides. For
many applications, radionuclides are preferred. The label is
detectable without the addition of further reagents, by means of an
output of detectable electromagnetic radiation or other nuclear
radiation from the label itself, or as a result of its magnetic or
paramagnetic properties. The ligand and the detectable label may be
covalently bound to each other.
[0015] The cyclic oligopeptide moiety preferably comprises 20 amino
acid residues or less, more preferably 9 residues or less. In
preferred embodiments, the cyclic oligopeptide is a pentapeptide.
The cyclic oligopeptide is preferably cyclised via a peptide bond,
which may be between its N and C termini, or may be cyclised via a
disulfide bond between two cysteine residues when present. The
compound may include other moieties in addition to the cyclic
oligopeptide moiety and the detectable label. Accordingly,
additional peptide sequences may be attached, or groups capable of
altering the pharmacokinetic and/or physicochemical properties of
the compound (e.g. hydrophilic groups such as sugars or
polyethylene glycol chains). The ligand may include additional
components to the cyclic oligopeptide moiety. Alternatively, the
ligand may consist of the cyclic oligopeptide moiety.
[0016] In certain preferred embodiments, the cyclic oligopeptide
moiety has the sequence:
[0017] cyclo[D-Tyr/(Me)D-Tyr-B-Arg/(Me)Arg-Z-(Ala).sub.n-X]
[0018] wherein:
[0019] B is as defined above;
[0020] Z is an amino acid containing an aromatic group in its side
chain;
[0021] n is 1 or 0, provided that n is 1 only when the preceding
four amino acids in the cyclic moiety sequence are
D-Tyr/(Me)D-Tyr-Arg-Arg-NaI (SEQ ID NO:36), NaI being
L-3-(2-naphthyl)alanine; and
[0022] X is selected from Gly, (Me)Gly, Ala, Dap, Dap(FP)
((N-fluoropropionyl)-diaminopropionic acid), Dab, Dab(FP)
((N-fluoropropionyl)-diaminobutyric acid), Dab(FB)
((N-fluorobenzoyl)-diaminobutyric acid) and Dap(FB)
((N-fluorobenzoyl)-diaminopropionic acid).
[0023] Z may be selected from NaI, Dap(FB), AMS (FB) (an oxime of
aminooxy serine (O-amino serine) and 4-fluorobenzaldehyde), and,
when B is (Me)Arg, (Me)NaI. Z is preferably NaI.
[0024] X is preferably selected from Gly, (Me)Gly, Ala, Dap
(diaminopropionic acid) and Dap(FP)
((N-fluoropropionyl)-diaminopropionic acid). X is preferably Gly or
Dap(FP).
[0025] B is preferably a basic amino acid. The basic amino acid is
preferably selected from Arg, Orn, D-Orn, Cit and His, or
N-substituted derivatives thereof. Most preferably, B is Arg or
Orn. Ornithine residues confer the advantage of an amino-containing
side chain which is relatively straightforward to derivatize. In
certain embodiments, B may be N.sup..alpha.-substituted with a Me
group. Preferably, no more than one residue in the cyclic
oligopeptide moiety is N.sup..alpha.-substituted with a Me
group.
[0026] When B is Orn or D-Orn, the ornithine residue may be
substituted at N.sup..delta. with one or two groups which may be
selected from fluorobenzoyl (FB), fluoropropionyl (FP), acetyl
(Ac), amido (Am) (i.e. so as to form a urea-type moiety), methyl
(Me), 1-naphthylmethyl (N1), 2-naphthylmethyl (N2), benzyl (Bz) and
acyl spacer moieties. Preferably, the acyl spacer moiety is an acyl
group containing a chain of 1-14 carbons, optionally interrupted by
heteroatoms, and preferably having a nucleophilic functional group
at its end distal to the ornithine N.sup..delta.. The nucleophilic
functional group may be, for example, an amino or hydroxyl group.
This group enables further moieties to be added to the end of the
spacer, the purpose of the spacer being to minimize the effects of
any additional groups on the CXCR4 binding capability of the cyclic
oligopeptide. The acyl spacer moiety may be selected from
aminohexanoyl (Ahx), triethyleneglycolamino acyl (TGAS,
i.e.--COCH.sub.2(OCH.sub.2CH.sub.2).sub.2NH.sub.2), (Ahx).sub.2,
(Ahx).sub.3, (TGAS).sub.2 and (TGAS).sub.3. When multimers of these
spacers are present, the repeating units are joined together by
amide bonds. Currently preferred spacer groups are Ahx, TGAS,
(Ahx).sub.3, (TGAS).sub.2 and (TGAS).sub.3. The substituents
described for ornithine, including the acyl spacer moieties, may
also be employed when B is Lys, Dap or Dab. In such cases, the
spacer moiety preferably has a nucleophilic functional group at its
end distal to its point of attachment to the oligopeptide (i.e.,
the N.sup..epsilon. when B is Lys).
[0027] In certain embodiments, B is Orn or D-Orn, preferably D-Orn,
substituted at N.sup..alpha. with a Me group. When B is Orn, it may
be substituted at N.sup..delta. with FB, FP, Ac, Am, N1, N2, Me and
N1, Me and N2, Bz, Bz and FB, Bz and FP, Me and FB, Me and FP, or
Me.
[0028] In yet other embodiments, B is Orn or D-Orn, preferably
D-Orn, substituted at N.sup..delta. with FB, FP, Me and FB, or Me
and FP, and optionally substituted at N.sup..alpha. with a Me
group. Preferred substituents in this instance are FB, and Me and
FB, optionally in conjunction with substitution of N.sup..alpha.
with a Me group.
[0029] The cyclic oligopeptide moiety may have the sequence:
cyclo[D-Tyr-B-Arg-Z-X], wherein B, Z and X are selected from the
options listed above, provided that not more than one of the
residues in the said sequence may be N.sup..alpha.-methylated.
Preferably in such embodiments, B is Arg. Alternatively, the cyclic
oligopeptide moiety may have the sequence:
cyclo[D-Tyr/(Me)D-Tyr-B-Arg/(Me)Arg-Z-X], wherein Z and X are
selected from the options listed above and wherein B is selected
from Arg, (Me)Arg, Orn, Cit, Orn(FB), Orn(FP), Orn(Ac), Orn(Am),
Orn(N1), Orn(N2), Orn(Me, N1), Orn(Me, N2), Orn(Me), Orn(Bz),
Orn(Bz,FB), Orn(Ahx), Orn(Ahx.sub.2), Orn(Ahx.sub.3), Orn(TGAS),
Orn(TGAS.sub.2), Orn(TGAS.sub.3), Orn(Me,FB), D-Orn(FB),
(Me)D-Orn(FB), (Me)D-Orn(Me,FB), His and Phe, provided that not
more than one of the residues in the said sequence may be
N.sup..alpha.-methylated. In such embodiments, the first residue is
preferably D-Tyr. Also in such embodiments, Z is preferably NaI.
Also in such embodiments, X is preferably Gly. Also in such
embodiments, the third residue is preferably Arg.
[0030] In specific preferred embodiments, the cyclic oligopeptide
moiety has a sequence selected from:
TABLE-US-00001 (SEQ ID NO: 1) cyclo[D-Tyr-Arg-Arg-Nal-Gly] (SEQ ID
NO: 2) cyclo[D-Tyr-(Me)Arg-Arg-Nal-Gly] (SEQ ID NO: 3)
cyclo[D-Tyr-Arg-(Me)Arg-Nal-Gly] (SEQ ID NO: 4)
cyclo[D-Tyr-Arg-Arg-Nal-(Me)Gly] (SEQ ID NO: 5)
cyclo[D-Tyr-Orn-Arg-Nal-Gly] (SEQ ID NO: 6)
cyclo[D-Tyr-Cit-Arg-Nal-Gly] (SEQ ID NO: 7)
cyclo[D-Tyr-Arg-Arg-Nal-Ala-Gly] (SEQ ID NO: 8)
cyclo[D-Tyr-Arg-Arg-Nal-Ala-Ala] (SEQ ID NO: 9)
cyclo[D-Tyr-(Me)Arg-Arg-Nal-(Me)Gly] (SEQ ID NO: 10)
cyclo[D-Tyr-(Me)Arg-Arg-(Me)Nal-Gly] (SEQ ID NO: 11)
cyclo[(Me)D-Tyr-Arg-Arg-Nal-Ala-Gly] (SEQ ID NO: 12)
cyclo[(Me)D-Tyr-Arg-Arg-Nal-Gly] (SEQ ID NO: 13)
cyclo[D-Tyr-Orn(FB)-Arg-Nal-Gly] (SEQ ID NO: 14)
cyclo[D-Tyr-Orn(FP)-Arg-Nal-Gly] (SEQ ID NO: 15)
cyclo[D-Tyr-Orn(Ac)-Arg-Nal-Gly] (SEQ ID NO: 16)
cyclo[D-Tyr-Orn(Am)-Arg-Nal-Gly] (SEQ ID NO: 17)
cyclo[D-Tyr-Arg-Arg-Nal-Dap(FP)] (SEQ ID NO: 18)
cyclo[D-Tyr-Orn(N1)-Arg-Nal-Gly] (SEQ ID NO: 19)
cyclo[D-Tyr-Orn(N2)-Arg-Nal-Gly] (SEQ ID NO: 20)
cyclo[D-Tyr-Orn(Me, N1)-Arg-Nal-Gly] (SEQ ID NO: 21)
cyclo[D-Tyr-Orn(Me, N2)-Arg-Nal-Gly] (SEQ ID NO: 22)
cyclo[D-Tyr-Orn(Me)-Arg-Nal-Gly] (SEQ ID NO: 23)
cyclo[D-Tyr-Orn(Bz)-Arg-Nal-Gly] (SEQ ID NO: 24)
cyclo[D-Tyr-Orn(Bz, FB)-Arg-Nal-Gly] (SEQ ID NO: 25)
cyclo[D-Tyr-Orn(Ahx)-Arg-Nal-Gly] (SEQ ID NO: 26)
cyclo[D-Tyr-Orn(Ahx.sub.3)-Arg-Nal-Gly] (SEQ ID NO: 27)
cyclo[D-Tyr-Orn(TGAS)-Arg-Nal-Gly] (SEQ ID NO: 28)
cyclo[D-Tyr-Orn(TGAS.sub.2)-Arg-Nal-Gly] (SEQ ID NO: 29)
cyclo[D-Tyr-Orn(TGAS.sub.3)-Arg-Nal-Gly] (SEQ ID NO: 30)
cyclo[D-Tyr-Orn(Me, FB)-Arg-Nal-Gly] (SEQ ID NO: 31)
cyclo[D-Tyr-D-Orn(FB)-Arg-Nal-Gly] (SEQ ID NO: 32)
cyclo[D-Tyr-(Me)D-Orn(FB)-Arg-Nal-Gly] (SEQ ID NO: 33)
cyclo[D-Tyr-(Me)D-Orn(Me, FB)-Arg-Nal-Gly] (SEQ ID NO: 34)
cyclo[D-Tyr-His-Arg-Nal-Gly] (SEQ ID NO: 35)
cyclo[D-Tyr-Phe-Arg-Nal-Gly]
[0031] More preferred oligopeptide moieties include those having
the following sequences
TABLE-US-00002 (SEQ ID NO: 1) cyclo[D-Tyr-Arg-Arg-Nal-Gly] (SEQ ID
NO: 2) cyclo[D-Tyr-(Me)Arg-Arg-Nal-Gly] (SEQ ID NO: 3)
cyclo[D-Tyr-Arg-(Me)Arg-Nal-Gly] (SEQ ID NO: 5)
cyclo[D-Tyr-Orn-Arg-Nal-Gly] (SEQ ID NO: 6)
cyclo[D-Tyr-Cit-Arg-Nal-Gly] (SEQ ID NO: 13)
cyclo[D-Tyr-Orn(FB)-Arg-Nal-Gly] (SEQ ID NO: 14)
cyclo[D-Tyr-Orn(FP)-Arg-Nal-Gly] (SEQ ID NO: 15)
cyclo[D-Tyr-Orn(Ac)-Arg-Nal-Gly] (SEQ 1D NO: 16)
cyclo[D-Tyr-Orn(Am)-Arg-Nal-Gly] (SEQ ID NO: 18)
cyclo[D-Tyr-Orn(N1)-Arg-Nal-Gly] (SEQ ID NO: 19)
cyclo[D-Tyr-Orn(N2)-Arg-Nal-Gly] (SEQ ID NO: 20)
cyclo[D-Tyr-Orn(Me, N1)-Arg-Nal-Gly] (SEQ ID NO: 21)
cyclo[D-Tyr-Orn(Me, N2)-Arg-Nal-Gly] (SEQ ID NO: 32)
cyclo[D-Tyr-(Me)D-Orn(FB)-Arg-Nal-Gly] (SEQ ID NO: 34)
cyclo[D-Tyr-His-Arg-Nal-Gly]
[0032] Particularly preferred oligopeptide moieties include those
having a sequence selected from
TABLE-US-00003 (SEQ ID NO: 5) cyclo[D-Tyr-Orn-Arg-Nal-Gly] (SEQ ID
NO: 13) cyclo[D-Tyr-OrnArg-Nal-Gly] (SEQ ID NO: 32)
cyclo[D-Tyr-(Me)D-Orn(FB)-Arg-Nal-Gly]
[0033] In preferred embodiments, the label is a radiolabel. The
label may be covalently attached directly to the ligand, or may be
attached (e.g., in the case of a metal radiolabel) by means of a
complexation agent which is covalently attached to the ligand. When
a spacer group is used, as described above, the complexation agent
may be attached via the nucleophilic group at the distal end of the
spacer. Other intermediate groups to facilitate indirect attachment
between the ligand and the label would be apparent to the person of
ordinary skill in the art.
[0034] The cyclic pentapeptide cyclo(D-Tyr-Arg-Arg-NaI-Gly) (also
known as CPCR4 or FC131) (SEQ ID NO:1) binds to CXCR4 with high
affinity. It is also relatively easy to radiolabel, e.g. by using
iodine radionuclides attached to the tyrosine residue. In
preliminary animal studies, radiolabeled CPCR4 showed around 10
times increased accumulation in CXCR4/ tumors compared to control
tumors. The pharmacokinetic and other properties of CPCR4 may be
altered by modification of the amino acid residues. In particular,
N-methylation of an Arg residue, the substitution of Arg.sup.1 for
another cationic amino acid (e.g. ornithine), the insertion of Ala
between NaI and Gly and the N-methylation of Tyr in the resulting
hexapeptides all lead to modified CXCR4 antagonists maintaining
useful affinity for the receptor.
[0035] Preferably, the compound does not include an antibody or
fragment thereof as part of its structure.
[0036] In certain compounds of the invention, the radiolabel may be
selected from .sup.18F, .sup.123I, .sup.124I and .sup.125I.
.sup.123I is particularly useful when the compound is to be used
for in vivo single photon emission computed tomography (SPECT)
studies. .sup.125I may be preferred for in vitro or ex vivo uses of
the compound. .sup.18F and .sup.124I are particularly useful for in
vivo studies using positron emission tomography (PET) imaging.
[0037] When the compound of the invention contains one or more
Dap(FB), Dap(FP), Dab(FB), Dab(FP), FB or FP groups, the fluorine
substituent may be .sup.18F. This presents a convenient means for
radiolabelling such compounds. In preferred compounds of this type,
the .sup.18F is present on an FB or FP substituent at
N.sup..quadrature. of Orn or D-Orn.
[0038] Alternatively, the radiolabel may be selected from
.sup.211At, .sup.225Ac, .sup.211Bi and .sup.212Bi. These
radionuclides are all relatively low-range .alpha.-emitters which
allow the compounds of the invention to be used for targeted
radiotherapy. The low-range emission provides a safer
radiotherapeutic approach for metastases. For radiotherapy of
primary tumors using compounds of the present invention, it may be
preferred to use a radionuclide with longer-range emission and
hence, in this case, the radiolabel may be selected from
beta-emitters with low and higher range, e.g. .sup.177Lu or
.sup.90Y, .sup.188Re and .sup.131I, respectively.
[0039] In general, useful diagnostic isotopes (for PET and
SPECT-based detection and imaging) for use in accordance with the
present invention include: .sup.18F, .sup.47Sc, .sup.51Cr,
.sup.52Fe, .sup.52mMn, .sup.56Ni, .sup.57Ni, .sup.62Cu, .sup.64Cu,
.sup.67Ga, .sup.68Ga, .sup.72As, .sup.75Br, .sup.76Br, .sup.77Br,
.sup.82Br, .sup.89Zr, .sup.94mTc, .sup.97Ru, .sup.99mTc,
.sup.111In, .sup.123I, .sup.124I, .sup.131I, .sup.191Pt,
.sup.197Hg, .sup.201Tl, .sup.203Pb, .sup.110mIn, .sup.120I.
[0040] In general, useful therapeutic isotopes for use in
accordance with the present invention include: .sup.32P.sub.,
.sup.67Cu, .sup.77As, .sup.90Y, .sup.99Mo, .sup.103Ru, .sup.105Rh,
109Pd, .sup.111Ag, .sup.114mIn, .sup.117mSn, .sup.121Sn,
.sup.127Te, .sup.131I, .sup.140La, .sup.140Nd, .sup.142Pr,
.sup.143Pr, .sup.149Tb, .sup.149Pm, .sup.151Pm, .sup.153Sm,
.sup.159Gd, .sup.161Tb, .sup.166Ho, .sup.166Dy, .sup.169Er,
.sup.169Yb, .sup.172Tm, .sup.175Yb, .sup.177Lu, .sup.186Re,
.sup.188Re, 198Au, 199Au, .sup.211At, .sup.211Bi, .sup.212Bi,
.sup.213Bi, .sup.225Ac.
[0041] In certain compounds of the invention, the radiolabel is
bound to the ligand by means of a complex between an organic
complexation agent and a radionuclide, the complex being bound to
the ligand in such a way as not to destroy its binding properties
at the CXCR4 receptor. In such embodiments, the complexation agent
is preferably covalently bound to the ligand, whilst the radiolabel
may be covalently or non-covalently bound to the complexation
agent.
[0042] The use of complexation agents broadens the range of
radionuclides which may be bound to the compounds of the invention.
Preferred complexation agents include DOTA
(1,4,7,10-tetraazacyclododecane-N,N',N',N''-tetraacetic acid) and
derivatives thereof, TETA
(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), DTPA
(diethylene triamine pentaacetic acid) and HYNIC
(hydrazinonicotinamide). The complexation agents may be bound to
appropriate side chains of the amino acids of the cyclic
oligopeptides of the invention, or to linker groups bound to
appropriate side chains of the amino acids of the cyclic
oligopeptides of the invention, i.e. so as to minimise disruption
of the CXCR4-binding properties of the compound. Alternatively,
intervening spacer groups can be employed, as described above.
[0043] It is also possible to modify the compounds of the invention
by the addition of one or more hydrophilic moieties (e.g.
carbohydrates or polyethylene glycol chains). Such modifications
can be used to improve the pharmacokinetics of the compounds in
vivo. For example, a carbohydrate-modified peptide-based compound
of the invention is expected to exhibit reduced hepatic uptake and
thus, compared with a lipophilic peptide, should show somewhat
delayed blood clearance and predominantly renal excretion following
administration. This leads to the generation of an image which is
obtainable soon after administration and which is expected to be
higher in contrast between CXCR4 positive and CXCR4 negative
tissues.
[0044] In accordance with a second aspect of the present invention,
there is provided a compound, or a pharmaceutically acceptable salt
or ester thereof, comprising a cytotoxic moiety and a ligand for
the chemokine receptor CXCR4, the ligand having a binding affinity
for the CXCR4 receptor, measured as IC50 in the presence of
.sup.125I-CPCR4, of 250 nM or lower, wherein the ligand comprises a
cyclic oligopeptide moiety having the motif B-Arg or B-(Me)Arg
within the cyclic moiety, and wherein B is a basic amino acid, a
derivative thereof, or phenylalanine, provided that the motif is
B-Arg when B is a N.sup..alpha.-methyl derivative of a basic amino
acid.
[0045] The optional and preferred features of the compounds of the
first aspect of the invention are also to be understood to be
preferred, as appropriate, in compounds of this second aspect. In
particular, the cytotoxic moiety may be bound directly to the
ligand or may be attached via a spacer group. Compounds of this
aspect of the invention may be used for the targeted chemotherapy
of tumours having metastatic potential, and their associated
metastases, as a result of the relatively high expression of CXCR4
by such tissues. Preferred cytotoxic moieties may be selected from
any of those cytotoxic compounds generally used for chemotherapy of
the tumour concerned.
[0046] In accordance with a third aspect of the invention, there is
provided a compound, or a pharmaceutically acceptable salt or ester
thereof, comprising a ligand for the chemokine receptor CXCR4, the
ligand having a binding affinity for the CXCR4 receptor, measured
as IC50 in the presence of .sup.125I-CPCR4, of 250 nM or lower,
wherein the ligand comprises a cyclic oligopeptide moiety having
the motif B-Arg or B-(Me)Arg within the cyclic moiety, and wherein
B is a basic amino acid, a derivative thereof, or phenylalanine,
provided that the motif is B-Arg when B is a N.sup..alpha.-methyl
derivative of a basic amino acid, and provided that the cyclic
oligopeptide moiety does not have the sequence
cyclo[D-Tyr-Arg-Arg-NaI-Gly] (SEQ ID NO:1), nor the sequence
cyclo[D-Tyr-Orn-Arg-NaI-Gly] (SEQ ID NO:5).
[0047] The compounds of the third aspect of the invention are
useful for labeling for use in diagnostics and imaging. They may
also be coupled to cytotoxic moieties for targeted chemotherapy of
CXCR4-positive tumours. Furthermore, they may also be used for
chemotherapy on their own since they are capable of antagonistic
properties at the CXCR4 receptor. The optional and preferred
features of the compounds of the first aspect of the invention are
also to be understood to be preferred, as appropriate, in compounds
of this third aspect.
[0048] In accordance with a fourth aspect of the invention, there
is provided a compound, or a pharmaceutically acceptable salt or
ester thereof, comprising a ligand for the chemokine receptor
CXCR4, wherein the ligand comprises a cyclic oligopeptide moiety
having the sequence:
[0049] cyclo[D-Tyr/(Me)D-Tyr-B-Arg/(Me)Arg-Z-(Ala).sub.n-X]
[0050] wherein:
[0051] B is as defined above;
[0052] Z is an amino acid containing an aromatic group in its side
chain;
[0053] n is 1 or 0; and
[0054] X is selected from Gly, (Me)Gly, Ala, Dap, Dap(FP), Dab,
Dab(FP), Dab(FB) and Dap(FB), the compound optionally comprising a
detectable label, provided that, when the compound does not
comprise a detectable label, the cyclic oligopeptide moiety does
not have the sequence cyclo[D-Tyr-Arg-Arg-NaI-Gly] (SEQ ID NO:1),
nor the sequence cyclo[D-Tyr-Orn-Arg-NaI-Gly] (SEQ ID NO:5).
[0055] In this fourth aspect, Z may be selected from NaI, Dap(FB),
AMS (FB), and (Me)NaI. Z is preferably NaI. Preferably, n is 1 only
when the preceding four amino acids in the cyclic moiety sequence
are D-Tyr/(Me)D-Tyr-Arg-Arg-NaI (SEQ ID NO:36). Preferably, Z is
Me(NaI) only when B is Me(Arg). The other preferred and optional
features of the first, second and third aspects of the invention
are also applicable to this fourth aspect, as appropriate. In
particular, When B is Orn or D-Orn, the ornithine residue may be
substituted at N.sup..delta. with one or two groups which may be
selected from fluorobenzoyl (FB), fluoropropionyl (FP), acetyl
(Ac), palmitoyl (Palm; e.g. so as to form the peptide
cyclo[D-Tyr-Orn(Palm)-Arg-NaI-Gly]) (SEQ ID NO:37), amido (Am)
(i.e. so as to form a urea-type moiety), methyl (Me),
1-naphthylmethyl (N1), 2-naphthylmethyl (N2), benzyl (Bz) and acyl
spacer moieties. Preferably, the acyl spacer moiety is an acyl
group containing a chain of 1-16 carbons, more preferably 1-14
carbons, optionally interrupted by heteroatoms, and preferably
having a nucleophilic functional group at its end distal to the
ornithine N.sup..delta.. The nucleophilic functional group may be,
for example, an amino or hydroxyl group. This group enables further
moieties to be added to the end of the spacer, the purpose of the
spacer being to minimize the effects of any additional groups on
the CXCR4 binding capability of the cyclic oligopeptide. The acyl
spacer moiety may be selected from aminohexanoyl (Ahx),
triethyleneglycolamino acyl (TGAS, i.e.
--COCH.sub.2(OCH.sub.2CH.sub.2).sub.2NH.sub.2), (Ahx).sub.2,
(Ahx).sub.3, (TGAS).sub.2 and (TGAS).sub.3. When multimers of these
spacers are present, the repeating units are joined together by
amide bonds. Currently preferred spacer groups are Ahx, TGAS,
(Ahx).sub.3, (TGAS).sub.2 and (TGAS).sub.3. The substituents
described for ornithine, including the acyl spacer moieties, may
also be employed when B is Lys, Dap or Dab. In such cases, the
spacer moiety preferably has a nucleophilic functional group at its
end distal to its point of attachment to the oligopeptide (i.e.,
the N.sup..epsilon. when B is Lys).
[0056] In accordance with a fifth aspect of the invention, there is
provided a pharmaceutical composition comprising a compound of the
invention as described in the first, second, third or fourth
aspects above, together with one or more pharmaceutically
acceptable excipients. Preferably, the composition is suitable for
injection.
[0057] Pharmaceutical compositions of this invention comprise any
of the compounds of the present invention, or pharmaceutically
acceptable salts or esters thereof, with any pharmaceutically
acceptable carrier, adjuvant or vehicle. Pharmaceutically
acceptable carriers, adjuvants and vehicles that may be used in the
pharmaceutical compositions of this invention, depending on the
intended formulation and route of administration, include, but are
not limited to, ion exchangers, alumina, aluminium stearate,
lecithin, serum proteins, such as human serum albumin, buffer
substances such as phosphates, glycerine, sorbic acid, potassium
sorbate, partial glyceride mixtures of saturated vegetable fatty
acids, water, salts or electrolytes, such as protamine sulfate,
disodium hydrogen phosphate, potassium hydrogen phosphate, sodium
chloride, zinc salts, colloidal silica, magnesium trisilicate,
polyvinyl pyrrolidone, cellulose-based substances, polyethylene
glycol, sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat.
[0058] The pharmaceutical compositions of this invention may be
administered orally, parenterally, by inhalation spray, rectally,
nasally, buccally, vaginally or via an implanted reservoir. As
mentioned above, parenteral administration is preferred. The
pharmaceutical compositions of this invention may contain any
conventional non-toxic pharmaceutically-acceptable carriers,
adjuvants or vehicles. The term parenteral as used herein includes
subcutaneous, intracutaneous, intravenous, intramuscular,
intra-articular, intrasynovial, intrasternal, intrathecal,
intralesional and intracranial injection or infusion techniques.
For most applications, intravenous or intralesional (e.g.
intratumoral) injection is envisaged.
[0059] The pharmaceutical compositions may be in the form of a
sterile injectable preparation, for example, as a sterile
injectable aqueous or oleaginous suspension. This suspension may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents (such as, for example, Tween 80) and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are mannitol solution, water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose, any bland fixed oil
may be employed including synthetic mono- or diglycerides. Fatty
acids, such as oleic acid and its glyceride derivatives are useful
in the preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant such as Ph. Helv or a similar alcohol.
[0060] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, and aqueous suspensions and
solutions. In the case of tablets for oral use, carriers which are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions are administered
orally, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening and/or flavouring
and/or colouring agents may be added.
[0061] The pharmaceutical compositions of this invention may also
be administered in the form of suppositories for rectal
administration. These compositions can be prepared by mixing a
compound of this invention with a suitable non-irritating excipient
which is solid at room temperature but liquid at the rectal
temperature and therefore will melt in the rectum to release the
active components. Such materials include, but are not limited to,
cocoa butter, beeswax and polyethylene glycols.
[0062] The pharmaceutical compositions of this invention may be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other solubilizing or dispersing agents known in the
art.
[0063] In a further aspect, the present invention provides a method
of synthesis of a compound according to the first aspect of the
invention as described above, the method comprising treating the
ligand with a source of the detectable label under conditions such
that the detectable label, or a complex between an organic
complexation agent and the label, becomes bound to the ligand.
[0064] In yet another aspect, the present invention provides a
method of synthesis of a compound according to the second aspect as
described above, the method comprising treating the ligand with a
source of the cytotoxic moiety under conditions such that the
cytotoxic moiety becomes bound, directly or indirectly, to the
ligand.
[0065] The present invention also provides a compound according to
the invention as described above, for use in therapy or
diagnosis.
[0066] In a related aspect, the invention also provides the use of
a compound according to the invention as described above in the
preparation of a medicament for the treatment of a neoplastic
condition.
[0067] By targeting appropriate radionuclides or cytotoxic
components to CXCR4 receptor-bearing tissues, it should be possible
to provide a relatively selective chemotherapy of neoplasias having
metastatic potential. Any metastases or circulating tumor cells
resulting from such tumors should also be targeted by the targeted
radionuclide or cytotoxic component.
[0068] In a further aspect, the present invention provides the use
of a compound as described above in relation to the first aspect of
the invention in the preparation of a medicament for the diagnostic
imaging of a neoplastic condition. In preferred embodiments of this
aspect of the invention, the neoplasia has, or is suspected of
having, metastatic potential. In certain embodiments, the
neoplastic condition may be breast or prostate cancer.
[0069] As mentioned above, the compounds of the first aspect of the
invention provide a highly useful tool for the selective detection
and imaging of cells bearing CXCR4 receptors and hence having
metastatic potential. The compounds may be administered by routine
methods (e.g. i.v. injection) and images of the patient may be
taken after a short time, by which stage any tissues having a
relatively high expression of CXCR4 will show a relative
concentration of the detectable compound of the invention.
[0070] In a related aspect, the invention also provides a method of
imaging neoplastic tissue, the method comprising the
administration, to a subject having (or suspected of having) a
neoplasia, of a compound according to the first aspect of the
invention, and the detection of the compound following distribution
thereof in vivo.
[0071] The said method of imaging preferably includes the further
step, following the detection step, of generating an image of the
detected distributed compound. The detection step may in particular
be performed using PET or single photon emission computed
tomography (SPECT) when the label is a radionuclide. When magnetic
or paramagnetic labels are employed, magnetic resonance imaging is
preferred.
[0072] In accordance with yet another aspect, the present invention
provides a method of determining the metastatic potential of cells
of a neoplasia, the method comprising exposing the cells to a
compound according to the first aspect of the present invention, or
a composition as described above, so as to allow the compound to
bind to CXCR4 receptors on the surface of the cells, removing
unbound compound from the vicinity of the cells, and determining
the presence and/or amount of compound bound to the cells.
[0073] The said method of determining the metastatic potential of
cells may be carried out in vivo or in vitro (i.e. using a sample
of cells or tissue removed from a patient).
[0074] When the method of determining the metastatic potential of
cells is carried out using a compound according to the first aspect
of the invention, the imaging, or the determination of the presence
and/or amount of bound compound, may in particular be performed
using PET or single photon emission computed tomography (SPECT)
when the label is a radionuclide. When magnetic or paramagnetic
labels are employed, magnetic resonance imaging is preferred.
[0075] Other detectable labels for use in the compounds of the
present invention include fluorescent components (e.g. green
fluorescent protein (GFP), rhodamine).
[0076] The invention additionally provides, in yet another aspect,
a method of treatment of a neoplastic condition in a subject, the
neoplasia having, or being suspected of having, metastatic
potential, the method comprising the administration to the subject
of a compound according to the invention, or a composition as
described above. In certain embodiments, the neoplastic condition
may be breast or prostate cancer.
[0077] The invention will now be described in more detail by way of
example only and with reference to the appended drawings.
EXAMPLES
Example 1
Radiolabeled CPCR4 SPECT/PET Imaging
1.1 Summary
1.1.1 Materials and Methods:
[0078] A method for early assessment of the metastatic potential of
tumors would be a valuable tool for therapy prediction and control.
Recently a key role in metastasis was attributed to the chemokine
receptor CXCR4. In a variety of tumors such as breast and prostate
cancer, CXCR4 has been found to play a dominating role during tumor
cell homing and was shown to be expressed, both in primaries and
metastases. The aim of this study was to develop a novel
radiolabeled probe for the in vivo imaging of CXCR4 expression on
tumors and metastases by SPECT and PET imaging.
[0079] CPCR4, a cyclic peptide (cyclo(D-Tyr-Arg-Arg-NaI-Gly) (SEQ
ID NO:1), was radiolabeled and evaluated in binding assays on
CXCR4-expressing Jurkat cells. The tumorigenic fibrosarcoma cell
line CMS5 was retrovirally transduced for stable CXCR4/GFP
expression and characterized in fluorescence-activated cell sorting
(FAGS) and radioligand binding assays. Biodistribution studies and
SPECT/PET imaging were carried out in CMS5/CXCR4.sup.+ mice. Tumors
were further analyzed by autoradiography, IHC and GFP
fluorescence.
1.1.2 Results and Conclusions:
[0080] Radiolabeled CPCR4 binds with high affinity (K.sub.D:
0.4.+-.0.1 nM) and specificity (>90%) in an antagonistic manner
to endogenously CXCR4-expressing Jurkat cells and to transduced
CXCR4/GFP-expressing CMS5 cells. CMS5/CXCR4.sup.+-fibrosarcomas
were found to be a reliable CXCR4 tumor model in mice, as confirmed
by autoradiography, immunohistochemistry (IHC) and GFP
fluorescence. Biodistribution studies of i.v. injected radiolabeled
CPCR4 showed 1 h post-injection 5.5.+-.1.5% ID/g (injected dose/g)
in the CMS5/CXCR4.sup.+ tumor and 0.6.+-.0.2% ID/g in the
CMS5/CXCR4.sup.- control. Besides a rapid blood clearance and a low
background accumulation (<1.0% ID/g) a higher tracer uptake was
found in the liver 19.5.+-.2.8% ID/g, intestine 17.2.+-.2.9% ID/g
and kidneys 12.2.+-.2.3% ID/g. Using CPCR4-SPECT and animal PET
imaging of mice, a clear delineation of CXCR4.sup.+ tumors was
possible, whereas no activity accumulation was visible for
CXCR4.sup.- controls in the same animals.
[0081] In this study we succeeded in the development of the first
radiolabeled probe for in vivo targeting of the CXCR4 chemokine
receptor. The tracer binds with high affinity and specificity in an
antagonistic manner to its binding site and allowed a clear
delineation of CXCR4.sup.+ tumors in vivo. We hypothesize that this
new class of tracers will be very promising probes for the
investigation of the metastatic potential of tumors and early
imaging and radionuclide therapy of metastatic processes.
1.2 Detailed Description of Example 1
1.2.1 Materials and Methods
1.2.1.1 Peptide Synthesis and Radiolabeling
[0082] Peptides were synthesized by using standard solid-phase
peptide synthesis protocols according to the Fmoc strategy. The
Fmoc amino acids Fmoc-Arg(Pbf), Fmoc-D-Tyr(tBu) and Fmoc-Gly were
purchased from Novabiochem (Bad Soden, Germany),
Fmoc-2-naphthylalanine was obtained from Bachem (Bubendorf,
Switzerland). Peptide synthesis was performed manually on a TCP
(trityl chloride polystyrene) resin.
O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU) and diphenyl phosphoryl azide (DPPA) were
purchased from Alexis and Aldrich (Steinheim, Germany),
respectively. IodoGen
(1,3,4,6-tetrachloro-3R,6R-diphenylglycoluril) was obtained from
Pierce (Rockford, Ill., USA), sodium iodide-125 was purchased from
Hartmann-Analytic GmbH (Braunschweig, Germany) and sodium
iodide-123 was obtained from Amersham Health (Eindhoven, The
Netherlands). Sodium iodide-124 was kindly provided by Prof. W.
Brandau (Essen, Germany). All other reagents were purchased from
Merck (Darmstadt, Germany) or Sigma-Aldrich (Taufkirchen, Germany).
Unless specified otherwise, solvents were used without further
purification.
[0083] The synthesis of the cyclic pentapeptide CPCR4 and
derivatives thereof was performed as described recently with small
modifications.[1, 2] In brief, after attachment of Fmoc-Gly-OH to
the TCP-resin the remaining amino acids were coupled after
activation with TBTU and subsequent deprotection of the Fmoc group
by using 20% piperidine in DMF, respectively. After peptide chain
assembly, the resin-bound peptides were treated with of a mixture
of acetic acid, 2,2,2-trifluoroethanol and dichloromethane (2:2:6)
for 2 h at room temperature. Afterwards the resin was filtered and
washed twice with the cleavage mixture. The combined filtrates were
evaporated in the presence of petrol ether in vacuum.
[0084] For cyclization the side chain protected peptides were
dissolved in DMF at a concentration of 2.5 mM. At -40.degree. C., 5
equiv. NaHCO.sub.3 and 3 equiv. DPPA were added and the solution
was stirred overnight with warming to room temperature. After
filtration of the solid NaHCO.sub.3, DMF was evaporated in vacuum.
The residue was triturated with water, filtered and washed with
water and diethyl ether. The fully protected cyclized peptides were
treated with of a solution of 95% TFA and 5% water for 2 hours at
room temperature. The deprotected peptide was precipitated from ice
cold diethyl ether and centrifuged at 5.degree. C. For the
synthesis of the non-radioactive iodinated reference peptide the
amino acid building block Fmoc-D-3-iodo-Tyr-OH was synthesized as
described previously.[2] For the incorporation of this amino acid
and subsequent peptide cyclization, PyBOP
(Benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate)/collidine activation was used. Afterwards the
crude cyclic peptides were lyophilized and purified by preparative
RP-HPLC. Finally, the peptides were characterized by analytical
HPLC and HPLC-ESI/MS on a LCQ LC-MS system from Finnigan (Bremen,
Germany) using the Hewlett-Packard series 1100 HPLC system.
[0085] Additional details of the peptide syntheses are as
follows:
Materials and Methods
General
[0086] All commercially available chemical reagents were used
without further purification. Technical solvents were distilled
before use.
[0087] Trityl resins were purchased from PepChem and amino acid
derivatives from Iris Biotech GmbH, NovaBiochem, Merck, Bachem,
Neosystem, Aldrich, while all other chemicals were bought from
Aldrich, Fluke or Merck if not stated otherwise.
[0088] NMP (N-methylpyrrolidone) was obtained from BASF and used
without further distillation. Dry solvents were purchased from
Aldrich, Fluke or Merck. Dry dichloromethane was distilled from
calcium hydride under argon and kept over a 4 .ANG. molecular
sieve. Water for RP-HPLC was filtered through a 0.22 .mu.m filter
(Millipore, Millipak40).
[0089] RP-HPLC analyses were performed using an Omnicrom YMC column
(4.6 mm.times.250 mm, 5 .mu.m C.sub.18, 1 mL/min). The eluent was a
linear gradient from water (0.1% TFA) to acetonitrile (0.1% TFA)
over 30 minutes (10% to 100%, 10% to 60%, and 20% to 50%) and
detection at 220 nm and 254 nm. The retention time (Rt) of the
analytical RP-HPLC is given in minutes with the gradient in
percentage of acetonitrile. Semi-preparative RP-HPLC was done on a
Beckman System Gold equipped with high pressure module 125,
UV-detector 166, and using an Omnicrom ODS-A C18 (120 .ANG., 5
.mu.m, 250 mm.times.20 mm) column in combination with the same
solvents as stated above.
[0090] NMR spectra were recorded on a Bruker Avance 250 or Bruker
DMX 500 at 298K. The chemical shifts are reported in ppm on the
.delta. scale relative to the solvent signal used.
.sup.13C-NMR-spectra were recorded using .sup.1H-broad band
decoupling. Pulse programs were taken from the Bruker library or
developed by the inventors. Samples were prepared in tubes with a
diameter of 5 mm using 0.5 ml of deuterated solvent. The resulting
spectra were processed on a PC workstation using Bruker TOPSPIN 1.3
software.
[0091] ESI mass spectra were recorded on a Finnigan LCQ in
combination with an Agilent/HP 1100 RP-HPLC system using an
Omnicrom YMC ODS-A C18 column (120 .ANG., 3 .mu.m, 125 mm.times.2
mm) with a flow rate of 0.2 mL/min. The eluent was a linear
gradient (10% to 100% acetonitrile) from water to acetonitrile with
0.1% formic acid over 20 min with detection at 220 nm.
Loading of TCP-Resin (General Procedure)
[0092] Peptide synthesis was carried out using TCP-resin (1 mmol/g)
following standard Fmoc-strategy [13]. Fmoc-Xaa-OH (1.2 eq.) were
attached to the TCP resin with DIEA (diisopropylethylamine) (2.5
eq.) in anhydrous DCM (2 mL) at room temperature for 1 h. The
remaining trityl chloride groups were capped by addition of a
solution of MeOH, DIEA (5:1; v:v) for 15 min. The resin was
filtered and washed thoroughly with DCM (5.times.) and MeOH
(3.times.). The loading capacity was determined by weight after
drying the resin under vacuum and ranged from 0.4-0.9 mmol/g.
Fmoc Deprotection (General Procedure)
[0093] The resin-bound Fmoc peptide was treated with 20% piperidine
in NMP (v/v) for 10 minutes and a second time for 5 minutes. The
resin was washed with NMP (5.times.).
TBTU/HOBt Coupling (General Procedure)
[0094] A solution of Fmoc-Xaa-OH (2 eq.), TBTU (2 eq.), HOBt
(hydroxybenzotriazole) (2 eq.), DIEA (5.2 eq.) in NMP was added to
the resin-bound free amine peptide and shaken for 60 min at room
temperature and washed with NMP (5.times.).
o-Nitrobenzenesulfonyl (o-Ns) Protection
[0095] N-alkylation was carried out using an optimized protocol
[14]. A solution of o-Nitrobenzenelsulfonyl chloride (o-Ns-Cl) (5
eq.) and collidine (10 eq.) in NMP was added to the resin-bound
free amine peptide and shaken for 15 min at room temperature. The
resin was washed with NMP (3.times.) and dry THF (3.times.).
N-Alkylation Under Mitsunobu Conditions
[0096] A solution of triphenylphosphine (5 eq.), DIAD (diisopropyl
azodicarboxylate) (5 eq.) and alcohol (10 eq.) in dry THF was added
to the resin-bound o-Ns-protected peptides and shaken for 10 min at
room temperature. The resin was filtered off, and washed with dry
THF (3.times.) and NMP (3.times.).
o-Ns Deprotection
[0097] For o-Ns deprotection, the resin-bound
N-alkyl-N-o-Ns-peptides were treated with a solution of
mercaptoethanol (10 eq.) and DBU (5 eq.) in NMP for 5 minutes. The
deprotection procedure was repeated one more time and the resin was
washed with NMP (5.times.).
HATU/HOAt Coupling (General Procedure)
[0098] A solution of Fmoc-Xaa-OH (2 eq.), HATU (2 eq.), HOAt
(hydroxyazobenzotriazole) (2 eq.), DIEA (4 eq.) in NMP was added to
the resin-bound Ar-methylamine free peptides and shaken for 3 hours
at room temperature and washed with NMP (5.times.).
Alloc Deprotection
[0099] Pd(PPh.sub.3).sub.4 (0.125 eq.) in dry DCM (0.5 ml/g resin)
was added to the resin-bound Alloc (allyloxycarbonyl) peptide
followed by an addition of phenylsilan in dry DCM (0.5 ml/g resin)
and shaken for 1 hour. The resin was washed 5 times with DCM.
Peptide Cleavage
[0100] For complete cleavage from the resin the peptides were
treated three times with a solution of DCM and HFIP (4:1; v:v) at
room temperature for half an hour and the solvent evaporated under
reduced pressure.
Cyclization
[0101] To a 1 mM solution of peptide and NaHCO.sub.3 (5 eq.) DPPA
(diphenylphosphorylazide) (3 eq.) was added at room temperature and
stirred over night or until no linear peptide could be observed by
ESI-MS. The solvent was evaporated to a small volume under reduced
pressure and the peptides precipitated in saturated NaCl solution
and washed two times in HPLC grade water.
Removal of Acid Labile Side Chain Protecting Groups
[0102] Cyclized peptides were stirred in a solution of TFA, water
and TIPS (triisopropylsilane) (95:2.5:2.5) at room temperature for
one hour or until no more protected peptide could be observed by
ESI-MS and precipitated in diethylether and washed two more
times.
Acylation in Solution
[0103] For ornithine side chain acylation cyclized and fully
deprotected peptides were stirred with TBTU (1 eq) and the
corresponding acid (1 eq) in DMF for 15 minutes. The solution was
directly injected into the HPLC for purification.
Amino Acid Synthesis
N.sup..alpha.-Alloc-N.sup..epsilon.-Boc-L-Ornithine
[0104] N.sup..epsilon.-Boc-L-ornithine (1.00 g, 4.3 mmol) was
dissolved in a solution of Na.sub.2CO.sub.3 (1.14 g, 10.75 mmol) in
water and THF (50 ml, 1:1, v/v). After addition of allyl
chloroformate (0.46 ml, 4.3 mmol) the solution was stirred for 1.5
h. The THF was evaporated under reduced pressure and the aqueous
phase washed with diethylether (1.times.50 mL), acidified with
conc. HCl to pH 1 and the product extracted with EtOAc (3.times.50
mL). The combined organic layers were dried (Na.sub.2SO.sub.4),
filtered, concentrated and dried in vacuo to give a colourless,
sticky oil as sufficiently pure product (1.20 g, 90%). .sup.1H NMR
(250 MHz, DMSO-d.sub.6): .delta. 12.52 (s, 1H, OH), 7.49 (d, 7.72
Hz, 1H, NH.sup..alpha.), 6.78 (t, 5.05 Hz, 1H, N.sup..epsilon.),
5.91 (br m, 1H, CH.sup.Alloc), 5.30 (dd, 17.15 Hz, 1.69 Hz,
H.sup.AllocTerm1), 5.19 (dd, 10.17 Hz, 1.68 Hz, H.sup.AllocTerm2),
4.48 (m, 2H, CH.sub.2.sup.Alloc), 3.91 (br m, 1H, H.sup..alpha.),
2.91 (m, 2H, H.sup..beta.), 1.81-1.40 (br m, 4H, H.sup..gamma.,
H.sup..delta.), 1.38 (s, 9H, H.sup.Boc). .sup.13C NMR (250 MHz,
DMSO-d.sub.6): 174.4, 156.5, 156.1, 134.1, 117.4, 77.9, 65.1, 60.2,
54.1, 28.8, 26.7, 14.6. RP-HPLC: 16.7 min.
N.sup..alpha.-Alloc-N.sup..epsilon.-Fmoc-L-Ornithine
[0105] N.sup..alpha.-Alloc-N.sup..epsilon.-Boc-L-ornithine (1.20 g,
3.87 mmol) was dissolved in DCM (10 mL) and TFA (5 mL) was added
slowly. After stirring for 45 min the liquid was evaporated.
[0106] The crude product was dissolved in a solution of
Na.sub.2CO.sub.3 (1.02 g, 9.68 mmol) in water and THF (40 ml, 1:1,
v/v). After addition of Fmoc-N-Oxysuccinimid (1.31 g, 3.87 mmol)
the solution was stirred for 1.5 h. The THF was evaporated under
reduced pressure and the aqueous phase washed with diethylether
(1.times.50 mL), acidified with conc. HCl to pH 1 and the product
extracted with EtOAc (3.times.50 mL). The combined organic layers
were dried (Na.sub.2SO.sub.4), filtered, concentrated and dried in
vacuo to give a colourless solid as sufficiently pure product (1.65
g, 97%). .sup.1H NMR (250 MHz, DMSO-d.sub.6): .delta. 12.52 (s, 1H,
OH), 7.49 (d, 7.72 Hz, 1H, NH.sup..alpha.), 6.78 (t, 5.05 Hz, 1H,
NH.sup..epsilon.), 5.91 (br m, 1H, CH.sup.Alloc), 5.30 (dd, 17.15
Hz, 1.69 Hz, H.sup.AllocTerm1), 5.19 (dd, 10.17 Hz, 1.68 Hz,
H.sup.AllocTerm2), 4.48 (m, 2H, CH.sub.2.sup.Alloc), 3.91 (br m,
1H, H.sup..alpha.), 2.91 (m, 2H, H.sup..beta.), 1.81-1.40 (br m,
4H, H.sup..gamma., H.sup..delta.), 1.38 (s, 9H, H.sup.Boc).
.sup.13C NMR (250 MHz, DMSO-d.sub.6): 174.4, 156.5, 156.1, 134.1,
117.4, 77.9, 65.1, 60.2, 54.1, 28.8, 26.7, 14.6. RP-HPLC: 21.9
min.
[0107] The reaction scheme is shown below:
##STR00001##
1.2.1.2 Peptide Radioiodination
[0108] CPCR4 was labeled with .sup.123I-, .sup.124I- or
.sup.125I-iodide using the Iodogen method.[2] 0.2 mg of the peptide
was dissolved in 250 .mu.l phosphate buffered saline (PBS, pH 7.4).
This solution was added to Eppendorf cups coated with 150 .mu.g
Iodogen and was combined with the radioiodide solution. After 15
min at room temperature, the solution was removed from the solid
oxidizing reagent. Purification was performed using gradient
RP-HPLC. Radiochemical purity was generally >95%. For animal
experiments the fraction containing the radiolabeled peptide was
diluted with water and bound to a Sep-Pak C18 column. Afterwards
the column was washed with water and the radiolabeled peptide was
eluted with methanol. After removal of the methanol in vacuum the
residue was dissolved and diluted in PBS (pH 7.4). For storage at
4.degree. C. the solution was acidified with 0.1% trifluoroacetic
acid in H.sub.2O containing 20% ethanol.
1.2.1.3 Lipophilicity
[0109] For the determination of the lipophilicity 0.4-2.7 .mu.Ci of
.sup.125I-CPCR4 in 500 .mu.l PBS (pH 7.4) was mixed with 500 .mu.l
octanol and was vigorously vortexed. After centrifugation for
quantitative phase separation, 100 .mu.l from each phase were
withdrawn and radioactivity was determined in a gamma counter. The
experiment was performed in triplicates and repeated two times
independently.
1.2.1.4 Cell Lines and Tissue Culture
[0110] The murine fibrosarcoma cell line CMS5[3] and the human 293T
cell line[4] (kindly provided by R. Willemsen, Department of
Clinical and Tumour Immunology, Daniel den Hoed Cancer Center,
Rotterdam, The Netherlands) were both cultured in Dulbeccos's
modified Eagle's medium, supplemented with 10% (v/v) fetal calf
serum (PAA, Linz, Austria) and 1% (v/v) L-glutamine. The
T-lymphocyte Jurkat cell line (ATCC) was maintained in RPMI 1640
medium supplemented with 10% (v/v) fetal calf serum (FCS) and 1%
(v/v) L-glutamine. Media and supplements were obtained from
Biochrom (Berlin, Germany), unless otherwise mentioned.
1.2.1.5 Construction of the Retroviral Vector and Target Cell
Transduction
[0111] The cDNA coding for enhanced fluorescence protein was
excised from pEGFP (BD Biosciences Clontech, Germany) by NcoI StuI
digest, blunt ended using Klenow enzyme and inserted into the
unique SmaI site of pIRESneo3 (BD Biosciences Clontech, Germany) to
obtain pIRESeGFPneo3. In the next step the Nail fragment carrying
IRES-eGFP was cloned into the NotI site of pBullet (Schaff et al.
2003) to obtain pBulletlRESeGFP. The 1292 bp HindIII XbaI fragment
of pcDNA3CXCR4[5] carrying the human chemokine receptor type 4
(CXCR4) cDNA (kindly provided by B. Moser, Bern) was isolated and
cloned into the BamHI site of the retroviral vector pBulletIRESeGFP
after blunt ending all sites with Klenow enzyme. The resulting
vector was designated pBulletCXCR4-IRES-eGFP. Retrovirus production
by transient transfection of 293T cells and transduction of CMS5
cells have been described elsewhere.[6]
1.2.1.6 FACS Sorting and Analyses
[0112] EGFP and CXCR4 expression of trypsinized cells was analyzed
with a fluorescence activated cell sorter (Becton Dickinson FACS
Vantage, Heidelberg, Germany) using Argon Laser beam
(Spectra-Physics) of excitation energy 40 mW at 488 nm and the
CellQuest Software. EGFP expression was measured directly, using
FL1 (530130 nm) filter. Dead cells were determined by addition of
propidium iodide to the cells and fluorescence was determined using
a FL2 585/42 nm filter. The percentage of dead cells was always
.ltoreq.0.2%. The population of CXCR4 expressing CMS5 cells was
enriched by sorting CXCR4-EGFP-co-expressing cells for FL1 with a
minimum fluorescence of 20.
[0113] CXCR4 expression on the cell surface of trypsinized cells
was determined using a phycoerythrine (PE)-labeled monoclonal rat
antibody with specificity for human CXCR4 (109, BD Biosciences
Pharmingen, Heidelberg, Germany). Trypsinized cells were washed
with FACS buffer (PBS, 0.5% FCS) and 1.times.10.sup.6 cells were
stained with 0.5 .mu.g antibody for 30 min. in the dark at
4.degree. C. Cells were extensively washed with ice-cold FACS
buffer and analyzed by flow cytometry. Nonspecific staining was
assessed by PE-conjugated rat IgG.sub.2b,.kappa. (BD Biosciences
Pharmingen, Heidelberg, Germany). Detection of CXCR4 on the cell
surface was in the same samples as EGFP and was detected using a
575/26 nm filter (FL2). CXCR-4 staining was plotted against EGFP
fluorescence (FL1).
[0114] Where indicated cells were resuspended in medium
supplemented with 0.5% bovine serum albumin (BSA) (Sigma,
Taufkirchen, Germany), incubated with recombinant human 100 nM
SDF-1.alpha. (R&D Systems, Wiesbaden, Germany) for 1 hr at
37.degree. C. (adapted from protocols published previously)[7, 8];
controls were incubated with diluent (PBS/0.1% BSA). Samples were
immediately transferred to ice to avoid further internalization,
centrifuged, washed with PBS/0.5% BSA and FACS staining for CXCR4
was performed as indicated above.
1.2.1.7 Receptor Binding Assays
[0115] For receptor binding assays cells were resuspended in
PBS/0.2% BSA. A total of 200 .mu.l of the suspension containing
400,000 cells (Jurkat, CMS5) or 200,000 cells (CMS5/CXCR4) were
incubated with 25 .mu.l of the tracer solution (containing 3.1 kBq,
approx. 0.1 nM) and 25 .mu.l of the diluent or the competitor at
different concentrations. For determination of IC.sub.50 values,
.sup.125I-CPCR4 was used as a tracer. SDF-1.alpha. was obtained
from R&D Systems (Wiesbaden, Germany) and
.sup.125I-SDF-1.alpha. was purchased from Perkin-Elmer (Boston,
Mass., USA). For saturation curves the tracer concentration was
varied from 5 to 500 .mu.M whereas nonspecific binding was
determined in the presence of 1 .mu.M cold CPCR4. After shaking for
2 h at room temperature, the incubation was terminated by
centrifugation at 700.times.g and 4.degree. C. for 4 min. Cell
pellets were washed once with cold PBS followed by a second
centrifugation step or for internalization studies, two times with
an acidic wash buffer (20 mM NaOAc, pH5.0). Cell bound
radioactivity was determined by using a gamma counter. Experiments
were repeated 2-3 times in duplicates or triplicates. IC.sub.50
values of the binding curves were calculated by nonlinear
regression on a one-site or two-site competition based model using
Prism 3.0 (Graph Pad Software, Inc, San Diego). K.sub.D and Bmax
values were determined by nonlinear regression with the Prism 3.0
according to the manufacturer's protocol.
1.2.1.8 In Vivo Studies
[0116] For animal experiments parental CMS5 cells and transduced
CMS5/CXCR4 cells were injected subcutaneously in female Swiss nu/nu
mice (Charles River, France). Therefore for each mouse
1.5.times.10.sup.6 CMS5 cells and 2.times.10.sup.6 CMS5/CXCR4 cells
were resuspended in 75 .mu.l PBS, respectively and mixed with the
same volume Matrigel-Matrix HC (BD Biosciences, Heidelberg,
Germany) according to the manufacturer's protocol. Subsequently
cell suspension was inoculated at each shoulder, respectively.
After 14-16 days of tumour growth mice were used for imaging and
biodistribution purposes. All animal experiments were approved by
the local authorities and are in compliance with the institutions
guidelines.
1.2.1.9 Biodistribution Studies
[0117] 370 kBq (10 .mu.Ci) of .sup.125I-labeled CPCR4 were injected
intravenously into the tail vain of tumour bearing mice. The
animals were sacrificed and dissected 30, 60 and 120 min after
tracer injection. Organs of interest were removed and the
radioactivity was measured in weighted tissue samples using the
1480 Wizard3 gamma counter from Wallac (Turku, Finland). Results
are expressed as percent injected dose per gram tissue weight (%
ID/g). Each value represents the mean of four to six animals.
1.2.2 Results
1.2.2.1 CPCR4-Synthesis and Radiolabeling
[0118] The synthesis of CPCR4, the cyclic pentapeptide
cyclo(D-Tyr-Arg-Arg-NaI-Gly) (SEQ ID NO:1) that shows high affinity
and selectivity for the CXCR4 receptor, was carried out by using
standard Fmoc solid phase peptide synthesis protocols on an acid
labile tritylchloride resin as described previously.[1, 2]
Additional modifications by N-alkylation were done using a modified
protocol designed for N-methylation via a Fukuyama-Mitsunobu
reaction. [14] After peptide chain assembly the side chain
protected peptide was cleaved from the resin and was cyclized using
the DPPA method.[2] After removal of all protecting groups the
crude cyclic pentapeptide was further purified by preparative HPLC.
Analytical HPLC and HPLC/ESI-MS analyses proved homogeneity and
identity of the peptides.
[0119] The radiolabeling at the Tyr side chain of CPCR4 was
performed either with .sup.123I- or .sup.125I-iodide using the
Iodogen method and subsequent separation of the unlabeled precursor
by HPLC. The HPLC conditions applied allowed very efficient
separation of the radioiodinated peptide from the unlabeled
precursor and side products thus resulting in high radiochemical
purity (>99%) and specific activity. The specific activity of
the labeled peptides was assumed to be that of the radioiodide used
for labeling (>2000 Ci/mmol for .sup.125I, >5000 Ci/mmol for
.sup.123I). Whereas the radioiodide incorporation was usually
>95%, the overall radiochemical yield of the .sup.123I- and
.sup.125I-labeled peptides after HPLC purification and
biocompatible formulation was in the range of 50%. After
biocompatible formulation in PBS the lipophilicity of
.sup.125I-CPCR4 was determined as octanol/water(PBS) partition
coefficient. A log P value of -0.04 (.+-.0.01) was obtained.
1.2.2.2 CXCR4-Vector Construction and Viral Infection
[0120] The mouse fibrosarcoma cell line CMS5 was retrovirally
transduced with CXCR4-IRES-eGFP. In the cell pool 70-80% of the
retrovirally CXCR4-transduced CMS5 cells were positive for
eGFP-expression as determined by FACS analysis with a mean
fluorescence intensity of 130. Growth curves and survival assay
(XTT) demonstrated that both cell lines had similar growth kinetics
in vitro (data not shown). When CMS5 cells and CMS5/CXCR4 cells
were stained for human CXCR4, CMS5 showed a background staining of
2.2% whereas 61.6% of CMS5/CXCR4 cells stained positive for human
CXCR4, exhibiting a mean fluorescence intensity of 66 and 57.9% of
the cells were positive for both CXCR4 and eGFP. (FIG. 1A,B) The
cell line was stable over time as indicated by repeated FACS
analyses (data not shown).
1.2.2.3 Receptor Binding Studies
[0121] The suitability of .sup.125I-CPCR4 as a novel
CXCR4-radioligand was tested first at Jurkat cells that
endogenously express the CXCR4 receptor [9, 10] and subsequently at
CMS5/CXCR4 cells that were retrovirally transduced for CXCR4
expression. For both cell lines reproducible high specific binding
was found by using .sup.125I-SDF-1.alpha. (50-70%) and
.sup.125I-CPCR4 (>90%). At parental CMS5 cells both tracers
showed negligible binding in the range of the non-specific binding
of Jurkat and transduced CMS5/CXCR4 cells. From saturation binding
curves nearly identical K.sub.D values in the sub-nanomolar range
(0.3 to 0.4 nM) were obtained for both cell lines indicating high
affinity of .sup.125I-CPCR4 for the CXCR4 receptor. (FIG. 2A,C)
Furthermore a high number of .sup.125I-CPCR4 binding sites (Bmax)
was determined. Whereas for Jurkat cells the Bmax value was more
dependent on origin and varies stronger with culture conditions,
the number of binding sites (Bmax) on CMS5/CXCR4 cells was constant
and better reproducible (23.+-.6 fmol receptor protein).
[0122] With .sup.125I-CPCR4 as novel radioligand the affinity
profile of distinct CXCR4 selective ligands was ascertained in
competitive radioligand binding assays. (FIG. 2B,D) For
SDF-1.alpha., CPCR4 and its non-radioactive iodinated reference
compound Iodo-CPCR4 high affinities with nanomolar IC.sub.50 values
were found either with .sup.125I-CPCR4 or .sup.125I-SDF-1.alpha. at
the CXCR4 receptor. In comparison with SDF-1.alpha. and the cyclic
pentapeptides the CXCR4 selective bicyclam AMD3100 showed reduced
affinity with both tracers. Depending on tracer and competitor two
CXCR4 binding sites were monitored as reported previously.[9] For
analysis of the binding curves one-site and two-site competition
curve fits were used as required. The resulting high and low
affinity binding sites were designated as (1) and (2). (FIG.
2B,D).
[0123] The receptor internalization after binding of
.sup.125I-CPCR4 at the CXCR4 receptor was analyzed after two short
washing steps with an acidic buffer (pH5.0). Thereafter the tracer
was mostly releasable from the receptor (>80%). This indicates
that no receptor internalization occurs as expected from a receptor
antagonist (data not shown).
1.2.2.4 Receptor Functionality
[0124] To determine whether the human CXCR4 is functional in mouse
cells, cells were pre-incubated with human SDF-1.alpha., stained
for surface CXCR4 and subsequently FACS analysis was performed.
54.7% of CMS5/CXCR4 cells stained positive for CXCR4 after
pre-incubation with human SDF-1.alpha. as compared to 79.2% of
control-treated cells, indicating functionality of the human
receptor in murine CMS5 cells. The CXCR4-background staining in
CMS5 cells decreased from 7.9 to 2.7% in the presence of
SDF-1.alpha.. Jurkat cells served as positive control and did not
exhibit a decrease in % positive cells, but a drop in mean
fluorescence intensity from 385.4 to 155.4. In CMS5/CXCR4 cells the
mean fluorescence intensity did drop from 209.0 of mock treated
cells to 80.5 of SFD-1.alpha. treated cells. This indicates that
Jurkat cells do contain more CXCR4 receptors than CMS5/CXCR4
cells.
1.2.2.5 In Vivo Studies
[0125] The biodistribution and tumour accumulation of
.sup.125I-CPCR4 was determined 30, 60 and 120 min post injection in
CMS5 and CMS5/CXCR4 tumour bearing nude mice. Highest tumour
accumulation of .sup.125I-CPCR4 in CMS5/CXCR4 tumours was achieved
after 60 min with 5.5 (.+-.1.5) percent of injected dose per gram
(% ID/g) whereas in parental CMS5 tumours only 0.6 (.+-.0.2)% ID/g
were observed at this time. After 30 min .sup.125I-CPCR4 shows an
accumulation in CMS5/CXCR4 tumours with 4.7 (.+-.1.3)% ID/g and
after 120 min with 3.8 (.+-.1.4)% ID/g. For all time points a
higher tracer accumulation was observed only for liver, intestine
and kidneys. Other organs showed only very low background
accumulation. Whereas in the liver the accumulation of
.sup.125I-CPCR4 decreases with the time from 27.7 (.+-.4.9)% ID/g
after 30 min to 15.0 (.+-.1.8)% ID/g at 120 min, the tracer
accumulation in the intestine slightly increases from 16.0
(.+-.4.7)% ID/g after 30 min to 19.2 (.+-.4.5)% ID/g at 120 min
indicative for the metabolic processes in these organs. The tracer
accumulation in the kidneys shows a peak after 60 min with 12.2
(.+-.2.3)% ID/g and decreases to 8.2 (.+-.1.1)% ID/g after 120 min.
(FIG. 3A,B)
[0126] FIG. 4 shows PET/SPECT results for radioiodinated-CPCR4
distribution in mice bearing both CXCR4-positive (CMS5/CXCR4) and
negative (CMS5 control) tumours. A clear delineation can be
observed due to the difference in CPCR4 uptake of the two types of
tumour. MRI results are shown for comparison. The CXCR4 positive
tumour was recognisable by PET even after 25 hr post injection.
Similar results were obtained using PET with .sup.18F-labeled
radioligand, and using a gamma camera with .sup.1231-labeling.
Similarly, in ex vivo analysis of cryosections of tumours using a
micro-imager, marked differences in radiation could be seen between
positive and negative tumours.
Example 2
Development of Cyclic Peptides for Targeting CXCR4 Chemokine
Receptor Expression
[0127] Several diseases like HIV-1 infection, cancer metastasis,
rheumatoid arthritis and chronic lymphocytic B-cell leukemia are
linked to the interaction of the CXCR4 chemokine receptor to its
natural ligand, the 68 amino acid containing protein stromal
cell-derived factor-1.alpha. (SDF-1.alpha.) [11]. One strategy for
the treatment of these diseases could be to block the interaction
between CXCR4 and SDF-1.alpha. with small CXCR4 antagonists.
Furthermore, radiolabeling of suitable compounds with appropriate
radioisotopes could provide agents for imaging of CXCR4 expression
in vivo via PET.
[0128] Previous studies by Fujii et al. on CXCR4 antagonists led to
the high affinity cyclic pentapeptide CPCR4, having the sequence
cyclo[Gly-D-Tyr-Arg-Arg-NaI][1]. To further improve this structure,
different approaches have been chosen with respect to metabolic
stability, bioavailability, conformational rigidity and chemical
versatility for radiolabeling.
[0129] First, an N-methyl scan of the backbone amides was performed
to influence conformational freedom and to increase metabolic
stability and bioavailability. Ar-methylation of arginine residues
yielded peptides with useful affinity (IC.sub.50 values of 23 nM
(N-Me)Arg.sup.3 and 31 nM (N-Me)Arg.sup.4, respectively, with Arg
residues numbered according to their position in the sequence as
set out in the preceding paragraph) whereas N-methylation of other
amino acids noticeably decreased the affinity (IC.sub.50>100
nM). By substitution of Arg.sup.3 by ornithine, the affinity was
mostly retained [12]. The delta-amino group of Orn can be alkylated
or acylated via radiolabeled groups containing short lived
isotopes. Moreover, the bioavailability should be improved as the
high basicity of the two guanidino groups could be reduced. First
ornithine-acylated derivatives showed IC.sub.50 values between 11
and 35 nM enabling for the first time .sup.18F-radiolabeling of
small CXCR4 antagonists for PET imaging in vivo. The panel below
shows the results obtained with cyclic Orn-containing pentapeptides
in which the Orn is delta-N substituted with FB, FP, Ac and Am,
respectively.
Affinities of Various CXCR4 Antagonists
##STR00002##
[0131] The results of binding assays with
N.sup..alpha.-monomethylated cyclic pentapeptides
(N.sup..alpha.-methyl scan) are shown in Table 1 below (note that
in the following tables, peptides having IC.sub.50 values >250
nM, and thus not falling within the first to third aspects of the
present invention, are included for comparative purposes and are
marked with * after the IC.sub.50 value):
TABLE-US-00004 TABLE 1 IC.sub.50 Calculated Observed Code Sequence
[nM] mass m/z (m + H) CPCR4* cyc[D-Tyr-Arg-Arg-Nal-Gly] 4 (SEQ ID
NO: 1) OD1 cyc[D-Tyr-(Me)Arg-Arg-Nal-Gly] 23 743.39 744.7 (SEQ ID
NO: 2) OD3 cyc[D-Tyr-Arg-(Me)Arg-Nal-Gly] 31 743.39 744.7 (SEQ ID
NO: 3) OD5 cyc[D-Tyr-Arg-Arg-(Me)Nal-Gly] 894* 743.39 744.7 (SEQ ID
NO: 38) OD7 cyc[D-Tyr-Arg-Arg-Nal-(Me)Gly] 136 743.39 744.6 (SEQ ID
NO: 4) OD9 cyc[(Me)D-Tyr-Arg-Arg-Nal-Gly] 247 743.39 744.7 (SEQ ID
NO: 11)
[0132] The structure of OD1 (cyc[D-Tyr-(Me)Arg-Arg-NaI-Gly]) (SEQ
ID NO:2) is as follows:
##STR00003##
From these results, it can be observed that a loss of affinity by a
factor of only 5-10 is obtained when Arg residues are methylated. A
larger loss is obtained when other residues are methylated.
[0133] Corresponding results with N.sup..quadrature.-dimethylated
pentapeptides are shown below (Table 2), indicating a further loss
of affinity from such a modification:
TABLE-US-00005 TABLE 2 IC.sub.50 Calculated Observed Code Sequence
[nM] mass m/z (m + H) OD11 cyc[(Me)Arg-Nal-Gly-(Me)D-Tyr-Arg]
>1000* 757.4 758.7 (SEQ ID NO: 39) OD12
cyc[(Me)Arg-(Me)Nal-Gly-D-Tyr-Arg] >1000* 757.4 758.6 (SEQ ID
NO: 40) OD13 cyc[Arg-Nal-(Me)Gly-(Me)D-Tyr-Arg] >1000* 757.4
758.7 (SEQ ID NO: 41) OD14 cyc[Arg-(Me)Nal-Gly-(Me)D-Tyr-Arg]
>1000* 757.4 758.6 (SEQ ID NO: 42) OD15
cyc[Arg-Nal-(Me)Gly-D-Tyr-(Me)Arg] ~300-400* 757.4 758.8 (SEQ ID
NO: 43) OD16 cyc[Arg-Nal-Gly-(Me)D-Tyr-(Me)Arg] ~1000* 757.4 758.8
(SEQ ID NO: 44) OD18 cyc[Arg-(Me)Nal-(Me)Gly-D-Tyr-Arg] >1000*
757.4 758.8 (SEQ ID NO: 45) OD19 cyc[(Me)Arg-Nal-(Me)Gly-D-Tyr-Arg]
>1000* 757.4 758.9 (SEQ ID NO: 46) OD20
cyc[Arg-(Me)Nal-Gly-D-Tyr-(Me)Arg] 100-200 757.4 758.7 (SEQ ID NO:
47) OD21 cyc[(Me)Arg-Nal-Gly-D-Tyr-(Me)Arg] >1000* 757.4 758.6
(SEQ ID NO: 48)
[0134] The results of binding assays with pentapeptides in which
Arg was substituted with ornithine or citrulline are shown in Table
3 below:
TABLE-US-00006 TABLE 3 IC.sub.50 Calculated Observed Code Sequence
[nM] mass m/z (m + H) OD23 cyc[Nal-Gly-D-Tyr-Orn-Orn] >1000*
645.33 646.5 (SEQ ID NO: 49) OD24 cyc[Nal-Gly-D-Tyr-Arg-Orn] ~1000*
687.35 688.6 (SEQ ID NO: 50) OD25 cyc[Nal-Gly-D-Tyr-Orn-Arg] 9 .+-.
0.1 687.35 688.4 (SEQ ID NO: 51) OD26 cyc[Nal-Gly-D-Tyr-Cit-Cit]
>1000* 731.34 732.6 (SEQ ID NO: 52) OD27
cyc[Nal-Gly-D-Tyr-Cit-Arg] 35 .+-. 7 730.36 731.6 (SEQ ID NO: 53)
OD28 cyc[Nal-Gly-D-Tyr-Arg-Cit] >1000* 730.36 731.7 (SEQ ID NO:
54)
The results of Table 3 indicate that the first Arg residue in
cyclic pentapeptides may be substituted with a cationic residue,
such as ornithine, without dramatic loss of affinity.
[0135] In an evaluation of side chain-acylated ornithine
derivatives for incorporation of .sup.18F-containing prosthetic
groups, it was found that the fluorobenzoylated derivative showed
the highest affinity (11 nM--see panel above). This compound showed
a relatively high lipophilicity (Log P 1.06).
[0136] A number of other Orn-N.sup..delta. and/or
Orn-N.sup..alpha.-modified pentapeptides were also prepared,
including a series of derivatives with N.sup..delta. spacer
moieties. The CXCR4 binding results are shown in Table 4.
TABLE-US-00007 TABLE 4 IC.sub.50 Calculated Observed Sequence [nM]
mass m/z (m + H) cyc[D-Tyr-Orn(Me)-Arg-Nal-Gly] (SEQ ID NO: 22) 105
.+-. 7 701.36 702.7 cyc[D-Tyr-Orn(Bz)-Arg-Nal-Gly] (SEQ ID NO: 23)
155 .+-. 63 777.4 778.6 cyc[D-Tyr-Orn(N1)-Arg-Nal-Gly] (SEQ ID NO:
18) 40 .+-. 3 827.41 828.6 cyc[D-Tyr-Orn(N2)-Arg-Nal-Gly] (SEQ ID
NO: 19) 49 .+-. 1 827.41 828.7 cyc[D-Tyr-Orn(Me, N1)-Arg-Nal-Gly]
(SEQ ID NO: 20) 39.7 841.43 842.7 cyc[D-Tyr-Orn(Me,
N2)-Arg-Nal-Gly] (SEQ ID NO: 21) 34.2 841.43 842.7
cyc[D-Tyr-Orn(FB)-Arg-Nal-Gly] (SEQ ID NO: 13) 11 .+-. 2 809.37
810.6 cyc[D-Tyr-Orn(Bz, FB)-Arg-Nal-Gly] (SEQ 1D NO: 24) 100 899.41
900.7 cyc[D-Tyr-Orn(Me, FB)-Arg-Nal-Gly] (SEQ ID NO: 30) 78 .+-. 25
823.38 824.6 cyc[D-Tyr-Orn(Ahx)-Arg-Nal-Gly] (SEQ ID NO: 25) 70
.+-. 23 800.43 801.7 cyc[D-Tyr-Orn(Ahx.sub.2)-Arg-Nal-Gly] (SEQ 1D
NO: 55) 947* 913.51 914.9 cyc[D-Tyr-Orn(Ahx.sub.3)-Arg-Nal-Gly]
(SEQ ID NO: 26) 227 1026.6 1027.9 cyc[D-Tyr-Orn(TGAS)-Arg-Nal-Gly]
(SEQ ID NO: 27) 125 832.42 833.7
cyc[D-Tyr-Orn(TGAS.sub.2)-Arg-Nal-Gly] (SEQ ID NO: 28) 189 977.49
978.9 cyc[D-Tyr-Orn(TGAS.sub.3)-Arg-Nal-Gly] (SEQ ID NO: 29) 146
1122.57 1123.9 cyc[D-Tyr-Orn(Ac)-Arg-Nal-Gly] (SEQ ID NO: 15) 29
.+-. 11 729.36 730.6 cyc[D-Tyr-Orn(Am)-Arg-Nal-Gly] (SEQ ID NO: 16)
35 .+-. 7 730.36 731.6 cyc[D-Tyr-Orn(FP)-Arg-Nal-Gly] (SEQ ID NO:
14) 35 .+-. 13 761.37 762.6 cyc[D-Tyr-Orn(Palm)-Arg-Nal-Gly] (SEQ
ID NO: 37) >1000* 925.58 926.9
[0137] In addition, a series of pentapeptides containing
derivatives of D-Orn were prepared, together with pentapeptides in
which B is His or Phe. The CXCR4-binding results are shown in Table
5.
TABLE-US-00008 TABLE 5 IC.sub.50 Calculated Observed Sequence [nM]
mass m/z (m .+-. H) cyc[D-Tyr-D-Orn(FB)-Arg-Nal-Gly] 86 809.37
810.6 (SEQ ID NO: 31) cyc[D-Tyr-(Me)D-Orn(FB)-Arg-Nal-Gly] 8.7 .+-.
0.6 823.38 824.6 (SEQ ID NO: 32) cyc[D-Tyr-(Me)D-Orn(Me,
FB)-Arg-Nal-Gly] 59 837.4 838.6 (SEQ ID NO: 33)
cyc[D-Tyr-His-Arg-Nal-Gly] (SEQ ID NO: 34) 30
cyc[D-Tyr-Phe-Arg-Nal-Gly] (SEQ ID NO: 35) 154
[0138] A number of cyclic hexapeptides in which an Ala or similar
residue was inserted in the chain were tested for binding affinity
to CXCR4. The results are shown in Table 6 (note--Dap(FP) is
(N-fluoropropionyl)-diaminopropionic acid):
TABLE-US-00009 TABLE 6 Code Sequence IC.sub.50 [nM] CPCR4*
cyc[D-Tyr-Arg-Arg-Nal-Gly] (SEQ ID NO: 1) 4 BL 36
cyc[D-Tyr-Arg-Arg-Nal-Ala-Gly] (SEQ ID NO: 56) 75 (.+-.7) BL 56
cyc[D-Tyr-Arg-Arg-Nal-D-Ala-Gly] (SEQ ID NO: 57) >1000* BL 58
cyc[D-Tyr-Arg-Arg-Nal-Dap(FP)-Gly] (SEQ ID NO: 58) ~1000* BL 37
cyc[D-Tyr-Arg-Arg-D-Ala-Nal-Gly] (SEQ ID NO: 59) ~1000* BL 38
cyc[D-Tyr-Arg-Arg-nal-Nal(SEQ ID NO: 60) >1000* BL 39
cyc[D-Tyr-Arg-Arg-D-Ala-Ala-Gly] (SEQ ID NO: 61) >1000* BL 40
cyc[D-Tyr-Arg-Arg-nal-Ala-Gly] (SEQ ID NO: 56) ~1000* BL 42
cyc[D-Tyr-Arg-Arg-Nal-Nal-Gly] (SEQ ID NO: 60) >1000* BL130
cyc[D-Tyr-Arg-Arg-Nal-Gly-Gly] (SEQ ID NO: 62) 1000* BL131
cyc[D-Tyr-Arg-Arg-Ala-Nal-Gly] (SEQ ID NO: 63) >1000* BL132
cyc[D-Tyr-Arg-Ala-Arg-Nal-Gly] (SEQ ID NO: 64) >1000* BL133
cyc[D-Tyr-Arg-D-Ala-Arg-Nal-Gly] (SEQ ID NO: 65) >1000* BL134
cyc[D-Tyr-D-Ala-Arg-Arg-Nal-Gly] (SEQ ID NO: 66) >1000* BL135
cyc[D-Tyr-Ala-Arg-Arg-Nal-Gly] (SEQ ID NO: 67) >1000* BL136
cyc[D-Ala-D-Tyr-Arg-Arg-Nal-Gly] (SEQ ID NO: 68) >1000* BL137
cyc[Ala-D-Tyr-Arg-Arg-Nal-Gly] (SEQ ID NO: 69) ~1000* BL158
cyc[D-Tyr-Arg-Arg-Nal-Ala-Ala] (SEQ ID NO: 70) 114
[0139] The results of Table 6 suggest that Ala may be inserted
between NaI and Gly, and/or Gly may be replaced with Ala, with only
moderate loss of affinity. Insertion of other residues in this
position, or insertion of any of the residues studied in Table 6 in
other positions, was not well tolerated.
[0140] A further N.sup..alpha.-methyl scan was conducted with a
series of cyclic hexapeptides (N-mono-, di- and trimethylated), as
reported in Table 7:
TABLE-US-00010 TABLE 7 Code Sequence IC.sub.50 [nM] BL56
cyc[Arg-Nal-D-Ala-Gly-D-Tyr-Arg] (SEQ ID NO: 71) >1000* BL58
cyc[Arg-Nal-Dap(FP)-Gly-D-Tyr-Arg] (SEQ ID NO: 72) ~1000* BL66
cyc[(Me)Arg-Nal-Ala-Gly-D-Tyr-Arg] (SEQ ID NO: 73) >1000* BL67
cyc[Arg-(Me)Nal-Ala-Gly-D-Tyr-Arg] (SEQ ID NO: 74) >1000* BL68
cyc[Arg-Nal-(Me)Ala-Gly-D-Tyr-Arg] (SEQ ID NO: 75) >1000* BL69
cyc[Arg-Nal-Ala-(Me)Gly-D-Tyr-Arg] (SEQ ID NO: 76) >1000* BL70
cyc[Arg-Nal-Ala-Gly-(Me)D-Tyr-Ara] (SEQ ID NO: 77) ~200-300 BL71
cyc[Arg-Nal-Ala-Gly-D-Tyr-(Me)Arg] (SEQ ID NO: 78) ~1000* BL72
cyc[(Me)Arg-Nal-Ala-Gly-D-Tyr-(Me)Arg] (SEQ ID NO: 79) >1000*
BL73 cyc[Arg-(Me)Nal-Ala-Gly-D-Tyr-(Me)Arg] (SEQ ID NO: 80)
>1000* BL74 cyc[Arg-Nal-(Nle)Ala-Gly-D-Tyr-(Me)Arg] (SEQ ID NO:
81) >1000* BL75 cyc[Arg-Nal-Ala-(Me)Gly-D-Tyr-(Me)Arg] (SEQ ID
NO: 82) >1000* BL76 cyc[Arg-Nal-Ala-Gly-(Nle)D-Tyr-(Me)Arg] (SEQ
ID NO: 83) >1000* BL77 cyc[(Me)Arg-Nal-Ala-Gly-(Me)D-Tyr-Arg]
(SEQ ID NO: 84) >1000* BL78
cyc[Arg-(Me)Nal-Ala-Gly-(MOD-Tyr-Arg] (SEQ ID NO: 85) >1000*
BL79 cyc[Arg-Nal-(Me)Ala-Gly-(Me)D-Tyr-Ar] (SEQ ID NO: 86)
>1000* BL80 cyc[Arg-Nal-Ala-(Me)Gly-(MOD-Tyr-Arg] (SEQ ID NO:
87) >1000* BL81 cyc[(Me)Arg-Nal-Ala-(Me)Gly-D-Tyr-Arg] (SEQ ID
NO: 88) >1000* BL82 cyc[Arg-(Me)Nal-Ala-(Me)Gly-D-Tyr-Arg] (SEQ
ID NO: 89) >1000* BL83 cyc[Arg-Nal-(Me)Ala-(Me)Gly-D-Tyr-Ara]
(SEQ ID NO: 90) >1000* BL84
cyc[(Me)Arg-Nal-(Me)Ala-Gly-D-Tyr-Arg] (SEQ ID NO: 91) >1000*
BL85 cycfArg-(Me)Nal-(Me)Ala-Gly-D-Tyr-Arg] (SEQ ID NO: 92)
>1000* BL86 cyc[(Me)Arg-(Me)Nal-Ala-Gly-D-Tyr-Arg] (SEQ ID NO:
93) >1000* BL88 cyc[Arg-Nal-(Me)Ala-Gly-(Me)D-Tyr-(Me)Arg] (SEQ
ID NO: 94) >1000* BL89
cyc[Arg-(Me)Nal-Ala-Gly-(Me)D-Tyr-(Me)Arg] (SEQ ID NO: 95)
>1000* BL92 cyc[Arg-(Me)Nal-Ala-(Me)Gly-D-Tyr-(Me)Arg] (SEQ ID
NO: 96) >1000* BL93 cyc[(Me)Arg-Nal-Ala-(Me)Gly-D-Tyr-(Me)Arg]
(SEQ ID NO: 97) >1000* BL94
cyc[Arg-(Me)Nal-(Me)Ala-Gly-D-Tyr-(Me)Arg] (SEQ ID NO: 98)
>1000* BL96 cyc[Arg-Nal-(Me)Ala-(Me)Gly-(Me)D-Tyr-Arg] (SEQ ID
NO: 99) >1000* BL97 cyc[Arg-(Me)Nal-Ala-(Me)Gly-(Me)D-Tyr-Arg]
(SEQ ID NO: 100) >1000* BL98
cyc[(Me)Arg-Nal-Ala-(Me)Gly-(Me)D-Tyr-Arg] (SEQ ID NO: 101)
>1000* BL99 cyc[Arg-(Me)Nal-(Me)Ala-Gly-(Me)D-Tyr-Arg] (SEQ ID
NO: 102) >1000* BL102 cyc[Arg-(Me)Nal-(Me)Ala-(Me)Gly-D-Tyr-Arg]
(SEQ ID NO: 103) >1000* BL104
cyc[(Me)Arg-(Me)Nal-Ala-(Me)Gly-D-Tyr-Arg] (SEQ ID NO: 104)
>1000*
[0141] These results indicate appreciable loss of binding affinity
after N.sup..alpha.-methylation of cyclic hexapeptides, although
the N-methyl-D-Tyr hexapeptide did not suffer such a significant
loss of affinity as most of the other derivatives.
[0142] In order to allow more flexibility for the attachment of
prosthetic groups for labeling, the introduction of an amino group
was investigated by substitution of the Gly residue in CPCR4 for
Dap. The results (Table 8) indicate only a moderate loss of
affinity following this substitution (note--FP: 2-fluoropropionyl;
FB: 4-fluorobenzoyl).
TABLE-US-00011 TABLE 8 Code Sequence IC.sub.50 [nM] CPCR4
cyc[D-Tyr-Arg-Nal-Gly] (SEQ ID NO: 1) 4 Dap(FP)-8k
cyc[D-Tyr-Arg-Nal-Dap(FP)] (SEQ ID NO: 17) 140 Dap(FB)-8k
cyc[D-Tyr-Arg-Nal-Dap(FB)] (SEQ ID NO: 105) 350*
[0143] Other possible modifications of CPCR4 or the other peptides
described herein include NaI substitutions with other
fluorine-containing aromatic moieties as analogues for the
corresponding .sup.18F-labeled compounds. For example:
##STR00004##
AMS (FB) is an oxime of an aminooxy-serine moiety and
4-fluorobenzaldehyde.
[0144] For the development of fluorescent CXCR4 ligands, it is
possible to substitute NaI with a fluorescent Dap derivative, such
as Dap(NBD) (NBD is 7-nitro-1,2,3-benzoxadiazole). This derivative
showed an affinity which was reduced compared to CPCR4, although
results from FACS analysis suggest that such a ligand may still be
suitable for the investigation of CXCR4 expression by such a
technique.
Example 3
Multimodal Molecular Imaging of CXCR4 Chemokine Receptor Expression
with Peptide-Based PET Probes and Bioluminescence
[0145] A key role in metastasis and organ specific homing of tumor
cells is attributed to the chemokine receptor CXCR4 and its
endogenous ligand SDF-1.alpha.. For targeting of CXCR4 expression
in vivo we developed a radiolabeled cyclic peptide, CPCR4.
.sup.125I-CPCR4 is the first PET imaging probe that binds with high
affinity to CXCR4 (K.sub.D=0.4 nM), shows high accumulation in
CXCR4 expressing tumors in vivo (5.5% ID/g, 1 h post injection),
and allows a clear delineation of CXCR4 positive tumors.
[0146] To allow correlation of tumor development with receptor
expression and to monitor potential therapeutic interventions using
the non-radiolabeled probe by multimodality (bioluminescence and
nuclear) imaging, tumor cells have been transduced with luciferase
(luc). Lentiviral vectors were constructed containing genes of
CXCR4 and luc or otherwise only luc or eGFP as controls. These
vectors were successfully used for stable transduction of murine
CMS5 fibrosarcoma cells. Surface expression of CXCR4 on
CMS5/CXCR4/luc cells was investigated in radioligand binding assays
and FACS studies. High affinity and specificity of CPCR4-binding
and functional expression of luc were ascertained in cell assays.
Transduced cells were injected subcutaneously into nude mice.
Animals were analyzed with .mu.-PET using radiolabeled CPCR4 and
bioluminescence (luc)/fluorescence (eGFP) imaging. Ex vivo analysis
was performed by autoradiography, bioluminescence measurements and
immunohistochemistry. For a better understanding of CPCR4-binding
and to design ligands with improved pharmacokinetics, a newly
proposed CXCR4 receptor model has been developed and is currently
validated by investigating CXCR4 receptor mutants. Based on this
computer model, studies on the structure-activity relationship of
CPCR4-derivatives are performed for tracer optimization and
investigation of other labeling options.
[0147] In conclusion, this approach allows imaging of CXCR4
expression in vivo and allows development of enhanced imaging
probes for the non-invasive investigation of the metastatic
potential of tumors and determination of CXCR4 expression for
individualized therapy.
Example 4
Preparation of a Conjugate Between a CXCR4-Binding Cyclic
Oligopeptide and a Chelating Agent
[0148] The person of ordinary skill in the art would readily be
able to prepare a construct or conjugate consisting of a cyclic
oligopeptide of the present invention, a suitable spacer moiety
(preferably one of the linker moieties described herein), and a
chelator or other moiety suitable for complexation of a radiometal.
Typically, as described in numerous publications in recent years,
DOTA, for example, is coupled to a linker-bearing, fully protected
oligopeptide, either using a tri-protected (e.g.
tri-tert-butyl-protected) DOTA using standard activation
procedures, or using pre-activated species of DOTA, for example
mono-, di-, tri- or tetra N-succinimidyl esters or 4-nitrophenyl
esters of DOTA. Alternatively, standard peptide coupling conditions
can be used to achieve this goal.
[0149] Similarly, other chelators/complexation moieties, such as
TETA or DTPA, can be coupled. DTPA may also be coupled using the
cyclic bis-anhydride. Obviously, the chelator may also be
pre-coupled to the spacer, thus resulting in the formation of the
peptide-spacer bond in the final step.
[0150] This coupling can also be achieved by a person of ordinary
skill in the art using the well-described coupling procedures
established in the radiopharmaceutical field. Other coupling routes
such as oxime or hydrazone formation, as well as other selective
methods, such as the reaction of thiols and maleimides, may be used
to reach similar results.
[0151] The foregoing Examples are intended to illustrate specific
embodiments of the present invention and are not intended to limit
the scope thereof, the scope being defined by the appended claims.
All documents cited herein are incorporated herein by reference in
their entirety.
REFERENCES
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CXCR4-chemokine antagonist using orthogonal combination of
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[0158] 7. Fan, G. H., et al., Hsc/Hsp70 interacting protein (hip)
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Forster, R., et al., Intracellular and surface expression of the
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Hesselgesser, J., et al., Identification and characterization of
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160(2): p. 877-83. [0162] 11. Balkwill, F., Nature Reviews, 2004,
4: p. 540-550 [0163] 12. Tamamura H et al., J Med Chem, 2005, 48:
p. 3280-9 [0164] 13 Fields G B, Noble R L. Solid phase peptide
synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J.
Pept. Protein Res. 1990; 35: p. 161-214. [0165] 14 Biron E,
Chatterjee J, Kessler H. Optimized Selective N-Methylation of
Peptides on Solid Support. J. Peptide Sci. 2006; 12: p. 213-219.
Sequence CWU 1
1
10515PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa =
L-3-(2-naphthyl)alanine 1Tyr Arg Arg Xaa Gly 1 5 25PRTHomo
sapiensMISC_FEATURE(2)..(2)methyl derivative of Arg 2Tyr Arg Arg
Xaa Gly 1 5 35PRTHomo sapiensMISC_FEATURE(3)..(3)methyl derivative
of Arg 3Tyr Arg Arg Xaa Gly 1 5 45PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa = L-3-(2-naphthyl)alanine 4Tyr Arg
Arg Xaa Gly 1 5 55PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
ornithine 5Tyr Xaa Arg Xaa Gly 1 5 65PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = citrulline 6Tyr Xaa Arg Xaa Gly 1
5 76PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa =
L-3-(2-naphthyl)alanine 7Tyr Arg Arg Xaa Ala Gly 1 5 86PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa = L-3-(2-naphthyl)alanine 8Tyr Arg
Arg Xaa Ala Ala 1 5 95PRTHomo sapiensMISC_FEATURE(2)..(2)methyl
derivative of Arg 9Tyr Arg Arg Xaa Gly 1 5 105PRTHomo
sapiensMISC_FEATURE(2)..(2)methyl derivative of Arg 10Tyr Arg Arg
Xaa Gly 1 5 116PRTHomo sapiensMISC_FEATURE(1)..(1)methyl derivative
of Tyr 11Tyr Arg Arg Xaa Ala Gly 1 5 125PRTHomo
sapiensMISC_FEATURE(1)..(1)methyl derivative of Tyr 12Tyr Arg Arg
Xaa Gly 1 5 135PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine
13Tyr Xaa Arg Xaa Gly 1 5 145PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa
= Ornithine 14Tyr Xaa Arg Xaa Gly 1 5 155PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 15Tyr Xaa Arg Xaa Gly 1
5 165PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 16Tyr Xaa
Arg Xaa Gly 1 5 175PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa =
L-3-(2-naphthyl)alanine 17Tyr Arg Arg Xaa Xaa 1 5 185PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 18Tyr Xaa Arg Xaa Gly 1
5 195PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 19Tyr Xaa
Arg Xaa Gly 1 5 205PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
Ornithine 20Tyr Xaa Arg Xaa Gly 1 5 215PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 21Tyr Xaa Arg Xaa Gly 1
5 225PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 22Tyr Xaa
Arg Xaa Gly 1 5 235PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
Ornithine 23Tyr Xaa Arg Xaa Gly 1 5 245PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 24Tyr Xaa Arg Xaa Gly 1
5 255PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 25Tyr Xaa
Arg Xaa Gly 1 5 265PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
Ornithine 26Tyr Xaa Arg Xaa Gly 1 5 275PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 27Tyr Xaa Arg Xaa Gly 1
5 285PRTHomo sapiensMISC_FEATURE(2)..(2)(triethyleneglycolamino
acyl)2 derivative of Xaa 28Tyr Xaa Arg Xaa Gly 1 5 295PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 29Tyr Xaa Arg Xaa Gly 1
5 305PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 30Tyr Xaa
Arg Xaa Gly 1 5 315PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
Ornithine 31Tyr Xaa Arg Xaa Gly 1 5 325PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 32Tyr Xaa Arg Xaa Gly 1
5 335PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 33Tyr Xaa
Arg Xaa Gly 1 5 345PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa =
L-3-(2-naphthyl)alanine 34Tyr His Arg Xaa Gly 1 5 355PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa = L-3-(2-naphthyl)alanine 35Tyr Phe
Arg Xaa Gly 1 5 365PRTHomo sapiensMISC_FEATURE(2)..(2)methyl
derivative of D-Tyr 36Tyr Tyr Arg Arg Xaa 1 5 375PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = Ornithine 37Tyr Xaa Arg Xaa Gly 1
5 385PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa =
L-3-(2-naphthyl)alanine 38Tyr Arg Arg Xaa Gly 1 5 395PRTHomo
sapiensMISC_FEATURE(1)..(1)methyl derivative of Arg 39Arg Xaa Gly
Tyr Arg 1 5 405PRTHomo sapiensMISC_FEATURE(1)..(1)methyl derivative
of Arg 40Arg Xaa Gly Tyr Arg 1 5 415PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 41Arg Xaa
Gly Tyr Arg 1 5 425PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 42Arg Xaa Gly Tyr Arg 1 5 435PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 43Arg Xaa
Gly Tyr Arg 1 5 445PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 44Arg Xaa Gly Tyr Arg 1 5 455PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 45Arg Xaa
Gly Tyr Arg 1 5 465PRTHomo sapiensMISC_FEATURE(1)..(1)methyl
derivative of Arg 46Arg Xaa Gly Tyr Arg 1 5 475PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 47Arg Xaa
Gly Tyr Arg 1 5 485PRTHomo sapiensMISC_FEATURE(1)..(1)methyl
derivative of Arg 48Arg Xaa Gly Tyr Arg 1 5 495PRTHomo
sapiensMISC_FEATURE(1)..(1)Xaa = L-3-(2-naphthyl)alanine 49Xaa Gly
Tyr Xaa Xaa 1 5 505PRTHomo sapiensMISC_FEATURE(1)..(1)Xaa =
L-3-(2-naphthyl)alanine 50Xaa Gly Tyr Arg Xaa 1 5 515PRTHomo
sapiensMISC_FEATURE(1)..(1)Xaa = L-3-(2-naphthyl)alanine 51Xaa Gly
Tyr Xaa Arg 1 5 525PRTHomo sapiensMISC_FEATURE(1)..(1)Xaa =
L-3-(2-naphthyl)alanine 52Xaa Gly Tyr Xaa Xaa 1 5 535PRTHomo
sapiensMISC_FEATURE(1)..(1)Xaa = L-3-(2-naphthyl)alanine 53Xaa Gly
Tyr Xaa Arg 1 5 545PRTHomo sapiensMISC_FEATURE(1)..(1)Xaa =
L-3-(2-naphthyl)alanine 54Xaa Gly Tyr Arg Xaa 1 5 555PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = ornithine 55Tyr Xaa Arg Xaa Gly 1
5 566PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa =
L-3-(2-naphthyl)alanine 56Tyr Arg Arg Xaa Ala Gly 1 5 576PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa = L-3-(2-naphthyl)alanine 57Tyr Arg
Arg Xaa Ala Gly 1 5 586PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa =
L-3-(2-naphthyl)alanine 58Tyr Arg Arg Xaa Xaa Gly 1 5 596PRTHomo
sapiensMISC_FEATURE(5)..(5)Xaa = L-3-(2-naphthyl)alanine 59Tyr Arg
Arg Ala Xaa Gly 1 5 606PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa =
L-3-(2-naphthyl)alanine 60Tyr Arg Arg Xaa Xaa Gly 1 5 616PRTHomo
sapiens 61Tyr Arg Arg Ala Ala Gly 1 5 626PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa = L-3-(2-naphthyl)alanine 62Tyr Arg
Arg Xaa Gly Gly 1 5 636PRTHomo sapiensMISC_FEATURE(5)..(5)Xaa =
L-3-(2-naphthyl)alanine 63Tyr Arg Arg Ala Xaa Gly 1 5 646PRTHomo
sapiensMISC_FEATURE(5)..(5)Xaa = L-3-(2-naphthyl)alanine 64Tyr Arg
Ala Arg Xaa Gly 1 5 656PRTHomo sapiensMISC_FEATURE(3)..(3)Ala =
D-Ala 65Tyr Arg Ala Arg Xaa Gly 1 5 666PRTHomo
sapiensMISC_FEATURE(2)..(2)Ala = D-Ala 66Tyr Ala Arg Arg Xaa Gly 1
5 676PRTHomo sapiensMISC_FEATURE(5)..(5)Xaa =
L-3-(2-naphthyl)alanine 67Tyr Ala Arg Arg Xaa Gly 1 5 686PRTHomo
sapiensMISC_FEATURE(1)..(1)Ala = D-Ala 68Ala Thr Arg Arg Xaa Gly 1
5 696PRTHomo sapiensMISC_FEATURE(5)..(5)Xaa =
L-3-(2-naphthyl)alanine 69Ala Tyr Arg Arg Xaa Gly 1 5 706PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa = L-3-(2-naphthyl)alanine 70Tyr Arg
Arg Xaa Ala Ala 1 5 716PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 71Arg Xaa Ala Gly Tyr Arg 1 5 726PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 72Arg Xaa
Xaa Gly Tyr Arg 1 5 736PRTHomo sapiensMISC_FEATURE(1)..(1)methyl
derivative of Arg 73Arg Xaa Ala Gly Tyr Arg 1 5 746PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 74Arg Xaa
Ala Gly Tyr Arg 1 5 756PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 75Arg Xaa Ala Gly Tyr Arg 1 5 766PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 76Arg Xaa
Ala Gly Tyr Arg 1 5 776PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 77Arg Xaa Ala Gly Tyr Arg 1 5 786PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 78Arg Xaa
Ala Gly Tyr Arg 1 5 796PRTHomo sapiensMISC_FEATURE(1)..(1)methyl
derivative of Arg 79Arg Xaa Ala Gly Tyr Arg 1 5 806PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 80Arg Xaa
Ala Gly Tyr Arg 1 5 816PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 81Arg Xaa Ala Gly Tyr Arg 1 5 826PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 82Arg Xaa
Ala Gly Tyr Arg 1 5 836PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 83Arg Xaa Ala Gly Tyr Arg 1 5 846PRTHomo
sapiensMISC_FEATURE(1)..(1)methyl derivative of Arg 84Arg Xaa Ala
Gly Tyr Arg 1 5 856PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 85Arg Asn Ala Gly Tyr Arg 1 5 866PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 86Arg Xaa
Ala Gly Tyr Arg 1 5 876PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 87Arg Xaa Ala Gly Tyr Arg 1 5 886PRTHomo
sapiensMISC_FEATURE(1)..(1)methyl derivative of Arg 88Arg Xaa Ala
Gly Tyr Arg 1 5 896PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 89Arg Xaa Ala Gly Tyr Arg 1 5 906PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 90Arg Xaa
Ala Gly Tyr Arg 1 5 916PRTHomo sapiensMISC_FEATURE(1)..(1)methyl
derivative of Arg 91Arg Xaa Ala Gly Tyr Arg 1 5 926PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 92Arg Xaa
Ala Gly Tyr Arg 1 5 936PRTHomo sapiensMISC_FEATURE(1)..(1)methyl
derivative of Arg 93Arg Xaa Ala Gly Tyr Arg 1 5 946PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 94Arg Xaa
Ala Gly Tyr Arg 1 5 956PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 95Arg Xaa Ala Gly Tyr Arg 1 5 966PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 96Arg Xaa
Ala Gly Tyr Arg 1 5 976PRTHomo sapiensMISC_FEATURE(1)..(1)methyl
derivative of Arg 97Arg Xaa Ala Gly Tyr Arg 1 5 986PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 98Arg Xaa
Ala Gly Tyr Arg 1 5 996PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 99Arg Xaa Ala Gly Tyr Arg 1 5 1006PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 100Arg Xaa
Ala Gly Tyr Arg 1 5 1016PRTHomo sapiensMISC_FEATURE(1)..(1)methyl
derivative of Arg 101Arg Xaa Ala Gly Tyr Arg 1 5 1026PRTHomo
sapiensMISC_FEATURE(2)..(2)Xaa = L-3-(2-naphthyl)alanine 102Arg Xaa
Ala Gly Tyr Arg 1 5 1036PRTHomo sapiensMISC_FEATURE(2)..(2)Xaa =
L-3-(2-naphthyl)alanine 103Arg Xaa Ala Gly Tyr Arg 1 5 1046PRTHomo
sapiensMISC_FEATURE(1)..(1)methyl derivative of Arg 104Arg Xaa Ala
Gly Tyr Arg 1 5 1055PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa =
L-3-(2-naphthyl)alanine 105Tyr Arg Arg Xaa Xaa 1 5
* * * * *