U.S. patent application number 10/756289 was filed with the patent office on 2004-07-15 for inhibitors of the urokinase receptor.
This patent application is currently assigned to Wilex AG. Invention is credited to Burgle, Markus, Graeff, Heinrich, Kessler, Horst, Magdolen, Viktor, Riemer, Christoph, Schmitt, Manfred, Wilhelm, Olaf G..
Application Number | 20040138110 10/756289 |
Document ID | / |
Family ID | 8226683 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040138110 |
Kind Code |
A1 |
Kessler, Horst ; et
al. |
July 15, 2004 |
Inhibitors of the urokinase receptor
Abstract
The present invention concerns peptides as inhibitors of the
binding of urokinase to the urokinase receptor. The peptides, which
are preferably cyclic, are suitable as pharmaceutical agents for
diseases that are mediated by urokinase and its receptor.
Inventors: |
Kessler, Horst;
(Schwalbach-Limes, DE) ; Graeff, Heinrich;
(Muenchen, DE) ; Schmitt, Manfred; (Muenchen,
DE) ; Magdolen, Viktor; (Kirchheim, DE) ;
Wilhelm, Olaf G.; (Muenchen, DE) ; Riemer,
Christoph; (Muenchen, DE) ; Burgle, Markus;
(Muenchen, DE) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Wilex AG
Muenchen
DE
|
Family ID: |
8226683 |
Appl. No.: |
10/756289 |
Filed: |
January 14, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10756289 |
Jan 14, 2004 |
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09402464 |
Jan 7, 2000 |
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09402464 |
Jan 7, 2000 |
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PCT/EP98/02179 |
Apr 14, 1998 |
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Current U.S.
Class: |
514/14.6 ;
514/19.3 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 9/00 20180101; A61P 43/00 20180101; A61P 19/10 20180101; A61P
35/00 20180101; A61P 9/10 20180101; C12N 9/6462 20130101; A61P
35/04 20180101; C12Y 304/21073 20130101 |
Class at
Publication: |
514/009 |
International
Class: |
A61K 038/12 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 11, 1997 |
EP |
EP 97 106 024.9 |
Claims
1. A method of inhibiting the binding of at least one urokinase
plaminogen activator to at least one urokinase plaminogen activator
receptor in a patient in need of such inhibition comprising:
administering to said patient a peptide comprising monomeric
building blocks and having the general structural formula (I):
2(SEQ ID NO:4 in which X.sup.21 to X.sup.30 each denotes an
aminocarboxylic acid and X.sup.21 and X.sup.29 are bridged
together, Y is a spacer group that can couple the peptide to
carrier substances n and m are each independently 0 or 1, and the
monomeric building blocks are linked by --NR.sup.1CO-- or
--CONR.sup.1-- bonds where R.sup.1 in each case independently
denotes hydrogen, methyl or ethyl, and wherein the amino acid
residues X.sup.21-X.sup.30 each independently have one of the
following meanings: (i) X.sup.21 and X.sup.29 are each
independently an aminocarboxylic acid residue with an SH side chain
or X.sup.21 and X.sup.29 are together two aminocarboxylic acid
residues which are bridged by a thioether bond; (ii) X.sup.22 and
X.sup.27 are each independently an aminocarboxylic acid residue
with an aliphatic side chain; (iii) X.sup.23 is an aminocarboxylic
acid residue with a basic or an aliphatic hydrophilic side chain;
(iv) X.sup.24, X.sup.25 and X.sup.30 are each independently an
aminocarboxylic acid residue with an aromatic side chain, (v)
X.sup.26 is an aminocarboxylic acid residue with an aliphatic side
chain, and (vi) X.sup.28 is an aminocarboxylic acid residue with an
aliphatic side chain; and a pharmaceutically compatible salt or
derivative thereof, wherein said derivative comprises a peptide of
formula I in which reactive groups of a side chain and/or of the
N-terminus or C-terminus have been subjected to one or more
modifications, said modifications being selected from the group
consisting of acylation, amidation and esterification of carboxylic
acid groups.
2. The method of claim 1, wherein X.sup.21 and X.sup.29 are bridged
via a disulfide bond.
3. A peptide comprising monomeric building blocks and having the
general structural formula (II):
X.sup.1--[X.sup.2].sub.n--[X.sup.3].sub.m--X.sup-
.4--K--Y--F--X.sup.5--X.sup.6--I--X.sup.7--W--[X.sup.8].sub.r (II)
wherein, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6,
X.sup.7 and X.sup.8 are each independently an aminocarboxylic acid,
n, m and r are each independently 0 or 1, K is an
.alpha.-aminocarboxylic acid with a lysine side chain, F is an
.alpha.-aminocarboxylic acid with a phenylalanine side chain, I is
an .alpha.-aminocarboxylic acid with an isoleucine side chain, w is
.alpha.-aminocarboxylic acid with a tryptophan side chain, and the
monomeric building blocks are linked by --NR.sup.1CO-- or
--CONR.sup.1-- bonds, wherein R.sup.1 in each case independently
denotes hydrogen, methyl or ethyl, or pharmaceutically compatible
salts and derivatives thereof, wherein at least one of the amino
acid residues X.sup.1, X.sup.2, X.sup.3, X.sup.6, I, X.sup.7, W and
X.sup.8 is a D-amino acid residue.
4. The peptide of claim 3, wherein the monomeric building blocks
X.sup.1 and X.sup.7, or X.sup.1 and X.sup.8 are bridged
together.
5. A pharmaceutical composition comprising at least one peptide of
claim 3 and a pharmaceutical acceptable carrier thereof, and
optionally at least one auxiliary agent and/or diluent.
6. A method of inhibiting the binding of at least one urokinase
plaminogen activator to at least one urokinase plaminogen activator
receptor in a patient in need of such inhibition comprising:
administering to said patient at least one peptide of claim 3 in a
binding of urokinase plaminogen activator to urokinase plaminogen
activator receptor inhibting amount.
7. The peptide or of claim 3, wherein X.sup.1 and X.sup.8 are (a)
each independently an aminocarboxylic acid residue with an SH side
chain or (b) are aminocarboxylic acid residues which are adapted to
be bridged by a thioether bond.
8. The peptide of claim 7, wherein X.sup.1 and X.sup.8 are bridged
via a disulfide bond formed between said SH side chains.
9. The peptide of claim 7, wherein X.sup.1 and X.sup.8 are bridged
via a thioether bond.
10. The peptide of claim 3, wherein X.sup.2 is an aminocarboxylic
acid residue with an aliphatic and uncharged side chain.
11. The peptide of claim 10, wherein said chain is a valine,
leucine or isoleucine side chain.
12. The peptide of claim 3, wherein X.sup.3 and X.sup.5 are each
independently an aminocarboxylic acid residue with an aliphatic
hydrophilic side chain.
13. The peptide of claim 12, wherein said side chain is a serine or
threonine side chain.
14. The peptide of claim 3, wherein X.sup.4 and X.sup.6 are each
independently an aminocarboxylic acid residue with an aliphatic
hydrophilic side chain.
15. The peptide of claim 14, wherein said side chain is an amide
side chain.
16. The peptide of claim 15, wherein said side chain is an
asparagine or glutamine side chain.
17. The peptide of claim 3, wherein X.sup.1 and X.sup.7 are each
independently (a) a basic aminocarboxylic acid residue or (b) an
aminocarboxylic acid residue with a SH side chain.
18. The peptide of claim 17 wherein said basic aminocarboxylic acid
residue is histidine.
19. The peptide of claim 17 wherein said side chain in (b) is a
cysteine side chain.
20. The peptide of claim 19 wherein X.sup.1 and X.sup.7 are bridged
via a disulfide bridge.
21. The method of claim 6, wherein said peptide is an antagonist of
urokinase plaminogen activator.
22. The method of claim 6, wherein said patient suffers from a
disease associated with the expression of urokinase plaminogen
activator receptor.
23. The method of claim 22, wherein said disease is a tumor.
24. The method of claim 6, wherein said at least one peptide is
administered to said patient via a targeting vehicle.
25. A targeting vehicle comprising at least one peptide of claim
3.
26. The targeting vehicle of claim 25, wherein said targeting
vehicle is a viral vector or a liposome.
27. The peptide of claim 2, wherein X.sup.21 and X.sup.29 are
bridged via a disulfide bond.
28. The method of claim 1, wherein said peptide is a cyclic peptide
and acts as an urokinase plaminogen activator antagonist.
29. The method of claim 1, wherein said peptide is administered to
said patient via a targeting vehicle.
30. The method of claim 1, wherein said patients suffers from a
disease associated with the expression of urokinase plaminogen
activator receptor.
31. The method of claim 30, wherein said disease is a tumor.
32. The method of claim 31, wherein said peptide is part of (or is
coupled to) a polypeptide.
33. (New) A targeting vehicle comprising a polypeptide comprising
(or having coupled thereto) at least one peptide having the general
structural formula (I): 3(SEQ ID NO:4 in which X.sup.21 to X.sup.30
each denotes an aminocarboxylic acid and X.sup.21 and X.sup.29 are
bridged together, Y is a spacer group that can couple the peptide
to carrier substances n and m are each independently 0 or 1, and
the monomeric building blocks are linked by --NR.sup.1CO-- or
--CONR.sup.1-- bonds where R.sup.1 in each case independently
denotes hydrogen, methyl or ethyl, and wherein the amino acid
residues X.sup.21--X.sup.30 each independently have one of the
following meanings: (i) X.sup.21 and X.sup.29 are each
independently an aminocarboxylic acid residue with an SH side chain
or X.sup.21 and X.sup.29 are together two aminocarboxylic acid
residues which are bridged by a thioether bond; (ii) X.sup.22 and
X.sup.27 are each independently an aminocarboxylic acid residue
with an aliphatic side chain; (iii) X.sup.23 is an aminocarboxylic
acid residue with a basic or an aliphatic hydrophilic side chain;
(iv) X.sup.24, X.sup.25 and X.sup.30 are each independently an
aminocarboxylic acid residue with an aromatic side chain, (v)
X.sup.26 is an aminocarboxylic acid residue with an aliphatic side
chain, and (vi) X.sup.28 is an aminocarboxylic acid residue with an
aliphatic side chain; and a pharmaceutically compatible salt or
derivative thereof, wherein said derivative comprises a peptide of
formula I in which reactive groups of a side chain and/or of the
N-terminus or C-terminus have been subjected to one or more
modifications, said modifications being selected from the group
consisting of acylation, amidation and esterification of carboxylic
acid groups.
Description
[0001] The present invention concerns peptides as inhibitors of the
binding of urokinase to the urokinase receptor. These peptides
which are preferably cyclic are suitable as pharmaceutical agents
for diseases which are mediated by urokinase and its receptor.
[0002] The serine protease uPA (urokinase-type plasminogen
activator) is responsible for various physiological and
pathological processes such as the proteolytic degradation of
extracellular matrix material which is necessary for the
invasiveness and migration of cells and for tissue remodelling. uPA
binds with high affinity (K.sub.D=10.sup.-10-10.sup.-9M) to the
membrane-based uPA receptor (uPAR) on the cell surface.
[0003] The binding of uPA to its receptor is involved in many
invasive biological processes such as the metastatic spread of
malignant tumours, trophoplast implantation, inflammation and
angiogenesis. Hence antagonists of uPA are able to inhibit the
invasiveness, metastatic spread and angiogenesis of tumours. uPA
antagonists can be used as agents for the treatment of invasive and
metastasising cancer diseases in which uPA and UPAR occur at the
invasive foci of tumours (Dano et al., The receptor for urokinase
plasminogen activator: Stromal cell involvement in extracellular
proteolysis during cancer invasion, in: Proteolysis and Protein
Turnover, Barrett, A. J. and Bond, J., Editor, Portland Press,
London, 1994, 239) e.g. in cancers of the breast, lung, intestine
and ovaries. In addition uPA antagonists can also be used for other
purposes in which it is necessary to inhibit the proteolytic
activation of plasminogen, for example to treat diseases such as
arthritis, inflammation, osteoporosis, retinopathies and for
contraception.
[0004] The uPA receptor is described in WO 90/12091 and in the
publications by Ploug et al., J. Biol. Chem. 268 (1993), 17539 and
Ronne et al., J. Immunol. Methods 167 (1994), 91.
[0005] uPA is synthesized as a single chain molecule (pro-uPA) and
is converted enzymatically into an active two-chain uPA. The uPA
molecule is composed of three structurally independent domains, the
N-terminal growth factor-like domain (GFD, uPA 1-46), a kringle
structure domain (uPA 45-135) and the serine protease domain (uPA
159-411). GFD and the kringle domain together form the so-called
aminoterminal fragment of uPA (ATF, uPA 1-135) which is produced by
further proteolytic cleavage of two-chain uPA. ATF binds to the uPA
receptor with a similar affinity as uPA.
[0006] The receptor-binding region of uPA spans the region of the
amino acids 12 to 32 since a peptide which contains the amino acid
residues 12 to 32 of uPA (in which case cysteine is replaced by
alanine in position 19) competes with ATF for binding to the uPA
receptor (Appella et al., J. Biol. Chem. 262 (1987), 4437-4440). In
this publication it was also shown that this peptide also has an
affinity for the uPA receptor after cyclization by bridging the two
cysteine residues at positions 12 and 32. In an alternative
approach Goodson et al., (Proc. Natl. Acad. USA 91 (1994),
7129-7133) identified antagonistic uPA peptides for the uPAR by
screening a bacteriophage peptide library. These peptides had no
apparent sequence homology to the natural uPAR-binding sequence of
uPA.
[0007] Further investigations of the uPAR-binding region of uPA are
described in recent publications (Rettenberger et al., Biol. Chem.
Hoppe-Seyler 376 (1995), 587-594); Magdolen et al., Eur. J.
Biochem. 237 (1996), 743-751; Goretzki et al., Fibrinolysis and
Proteolysis 11 (1997), 11-19). The residues Cys19, Lys23, Tyr24,
Phe25, Ile28, Trp30 and Cys31 were identified as important
determinants for a uPA/uPAR interaction. In these investigations a
uPA peptide having the amino acids 16 to 32 of uPA was identified
as the most effective inhibitor.
[0008] Magdolen et al., (1996) supra analysed the UPAR binding
region of the uPA molecule using a peptide having the amino acids
14 to 32 of uPA and peptides derived therefrom. However, these
peptides and also peptides used by other research groups (cf. e.g.
Appella et al., (1987) supra) have a relatively low affinity for
UPAR.
[0009] WO-A-94/22646 discloses linear peptides with a length of 6
to 18 amino acids which are derived from the region of the amino
acids 14 to 33 of uPA. It is described that short peptides derived
from uPA (uPA 21-29 and uPA 21-26) are able to influence the growth
of keratinocytes. Although WO-A-94/22646 makes reference to a
potential use of the claimed peptides to block the uPA/uPAR
interaction, no data or information whatsoever are shown on such
binding studies. Moreover, the peptides uPA 21-29 and uPA 21-26
which are said to be preferred linear peptides do not contain the
minimal UPAR binding region of linear uPA peptides which comprises
the sequence region of amino acids 19 to 31. Hence the influence of
the growth of keratinocytes by these short peptides is very
probably not due to a uPA/uPAR interaction.
[0010] However, a disadvantage of the previously known uPA peptide
inhibitors is that their affinity of binding to the uPA receptor is
relatively low and inadequate for a therapeutic application. Thus
there is a great need for new uPA peptide antagonists which have a
higher affinity for the receptor.
[0011] In quantitative investigations it was surprisingly found
that the linear peptide uPA (19-31), cyclic derivatives of this
peptide and sequence-modified peptides from this uPA region have a
considerably improved affinity of binding to the uPA receptor.
[0012] Experimental data demonstrate that the peptides according to
the invention can be used as uPA antagonists which bind with high
affinity to the uPAR. Cyclic peptides are particularly preferred
which are characterized by bridges, especially disulfide bridges,
which do not occur in the native uPA molecule.
[0013] Hence the present invention concerns peptides having the
general structural formula (I): 1
[0014] in which
[0015] X.sup.21 to X.sup.30 each denotes an aminocarboxylic acid,
preferably an .alpha.-aminocarboxylic acid and X.sup.21 and
X.sup.29 are bridged together,
[0016] Y is a spacer
[0017] m and n are each independently 0 or 1,
[0018] and the monomeric building blocks are linked by
--NR.sup.1CO-- or --CONR.sup.1-- bonds where R.sup.1 in each case
independently denotes hydrogen, methyl or ethyl, and
pharmaceutically compatible salts and derivatives thereof.
[0019] The monomeric building blocks X.sup.21 to X.sup.30 have
preferably the following meanings:
[0020] X.sup.21 and X.sup.29 are .alpha.-aminocarboxylic acid
building blocks which can be bridged together and they particularly
preferably have an SH side chain, in particular a cysteine side
chain or a structurally related side chain e.g. a penicillamine
side chain. Alternatively X.sup.21 and X.sup.29 can also be two
.alpha.-aminocarboxylic acid residues linked by a thioether group
e.g. a lanthionine group.
[0021] X.sup.22 and X.sup.27 are each independently
.alpha.-aminocarboxylic acids with an aliphatic side chain,
preferably an aliphatic hydrophilic side chain and in particular an
amide side chain such as asparagine or glutamine, in particular
asparagine.
[0022] X.sup.23 is an .alpha.-aminocarboxylic acid with a basic
side chain e.g. lysine, ornithine or arginine or with an aliphatic
hydrophilic side chain e.g. with an amide side chain such as
glutamine or asparagine. X.sup.23 is particularly preferably
lysine.
[0023] X.sup.24 to X.sup.25 are each independently
.alpha.-aminocarboxylic acids with an aromatic side chain such as
tyrosine, phenylalanine or tryptophan. X.sup.24 is particularly
preferably tyrosine and X.sup.25 is phenylalanine.
[0024] X.sup.26 is an .alpha.-aminocarboxylic acid with an
aliphatic side chain, preferably with an aliphatic hydrophilic side
chain such as hydroxyvaline, homoserine, serine or threonine, in
particular serine. However, X.sup.26 can also have an aliphatic
hydrophobic side chain such as alanine.
[0025] X.sup.28 is an .alpha.-aminocarboxylic acid with an
aliphatic side chain, preferably with an aliphatic hydrophobic side
chain such as valine, norvaline, norleucine, isoleucine, leucine or
alanine. X.sup.28 is particularly preferably isoleucine.
[0026] X.sup.30-- if present--is an .alpha.-aminocarboxylic acid
with an aromatic side chain, preferably with a tryptophan side
chain. The tryptophan side chain can be optionally modified for
example by reduction.
[0027] The peptides according to the invention are preferably
derived from the uPA sequence and contain at least 2 and
particularly preferably at least 3, for example 4 amino acid
residues which also occur at corresponding positions in the native
uPA sequence. At least two of the amino acid residues X.sup.22,
X.sup.23, X.sup.24, X.sup.25, X.sup.26, X.sup.28 and X.sup.30
particularly preferably have a side chain which is identical to an
amino acid at the same position in the native uPA sequence. Most
preferably at least 2 of the amino acid residues X.sup.24,
X.sup.25, X.sup.28 and--if present--X.sup.30 have the same side
chain as in the native uPA sequence.
[0028] Y is a spacer group e.g. a peptidic spacer group composed of
one or several amino acids e.g. poly-Lys or another spacer group
e.g. a polyethylene glycol group. The peptide can be coupled to
carrier substances via the group Y.
[0029] Hence a further subject matter of the present invention are
cyclic peptides with a nine-membered ring of which at least two,
preferably at least 3 and particularly preferably at least 4 of the
amino acids forming the ring have a sequence from the uPA region 22
to 28.
[0030] In addition to peptides having the structural formula (I),
pharmaceutically compatible salts and derivatives thereof are also
suitable as uPA antagonists. Suitable derivatives are in particular
compounds in which the reactive groups of the side chain or/and of
the N-terminus or C-terminus e.g. amino or carboxylic acid groups
have been modified. Examples of such modifications are acylation
e.g. an acetylation of amino groups or/and an amidation or
esterification of carboxylic acid groups.
[0031] Natural amino acids or enantiomers thereof or
non-naturally-occurring amino acids such as .gamma.-aminobutyric
acid, .beta.-alanine can be used as the aminocarboxylic acids that
the building blocks for the peptides according to the
invention.
[0032] The monomeric building blocks are linked by acid amide bonds
NR.sup.1CO or CONR.sup.1 i.e. the direction of the peptide sequence
can also be reversed (retropeptides). As in native polypeptides,
R.sup.1 can denote hydrogen. On the other hand, R.sup.1 can also
denote an alkyl residue e.g. methyl or ethyl and in particular
methyl since N-alkylation of the amide bond often has a major
influence on the activity (cf. e.g. Levian-Teitelbaum et al.,
Biopolymers 28 (1989), 51-64).
[0033] The .alpha.-aminocarboxylic acids can also be used as
monomeric building blocks in the form of L-enantiomers or/and
D-enantiomers. The spatial structure of the peptides according to
the invention can be modified by changing the chirality which can
also influence the activity. Retro-inverso peptides are
particularly preferred i.e. pep tides which are present in a
reversed sequence direction and contain D-amino acids as monomeric
building blocks. In these retro-inverso structures the functional
side chains have a similar spatial orientation to those in the
native peptide sequence, but their biological degradation can be
impaired due to the presence of D-amino acids and they therefore
have advantages as drugs (cf. for example Wermuth et al., J. Am.
Chem. Soc. 119 (1997), 1328-1335 and references cited therein).
[0034] The peptides according to the invention ate preferably
cyclic compounds in which in particular the monomeric building
blocks X.sup.21 and X.sup.29 are bridged together. This bridging
can for example utilize the side chains of the respective
.alpha.-aminocarboxylic acid residues in which case bridging by
means of disulfide bonds e.g. between two cysteine residues is
particularly preferred. Other types of cyclization between amino
acid side chains are, however, also possible e.g. amide bonds
between an amino acid with an amino side group e.g. ornithine or
Lys and an amino acid with a carboxylic acid side group such as Asp
or Glu. In addition the disulfide bridge can also be replaced by an
alkylene bridge in order to increase the chemical stability. In
addition an amino acid side chain may also be linked to the peptide
backbone e.g. an .omega.-amino side group may be linked to the
C-terminal end or a carboxylic acid side group may be linked to the
N-terminal end. A linkage of the N-terminus and C-terminus is also
possible.
[0035] It is particularly preferred when at least one of the amino
acids X.sup.21, X.sup.27, X.sup.29 and X.sup.30 is a D-amino acid.
At least one of the amino acids X.sup.21 to X.sup.30 is
particularly preferably a D-amino acid e.g. D-cysteine.
[0036] Instead of the disulfide bridge it is also possible to use
so-called turn mimetics (Haubner et al., J. Am. Chem. Soc. 118
(1996), 7884-7891) or sugar amino acids (Graf von Rodern et al., J.
Am. Chem. Soc. 118 (1996), 10156-10167).
[0037] The peptides according to the invention can be obtained by
chemical synthesis as elucidated in the examples. Alternatively the
peptides according to the invention can also be components of
recombinant polypeptides.
[0038] Yet a further subject matter of the present invention are
peptides which are derived from the linear peptide uPA (19 to 31)
and cyclic derivatives thereof and carry D-amino acid residues at
selected positions. Such peptides have the general structural
formula (II):
X.sup.1--[X.sup.2].sub.n--[X.sup.3].sub.m--X.sup.4--K--Y--F--X.sup.5--X.su-
p.6--I--X.sup.7--W--[X.sup.8].sub.r (II)
[0039] in which
[0040] X.sup.1 to X.sup.8 each denotes an aminocarboxylic acid
preferably an .alpha.-aminocarboxylic acid and X.sup.1 and X.sup.7
or X.sup.1 and X.sup.8 are optionally bridged together,
[0041] n, m and r are each independently 0 or 1,
[0042] K is defined as X.sup.23 and preferably denotes an
.alpha.-amino-carboxylic acid with a lysine side chain,
[0043] Y is defined as X.sup.24 and preferably denotes an
.alpha.-amino-carboxylic acid with a tyrosine side chain,
[0044] F is defined as X.sup.25 and preferably denotes an
.alpha.-amino-carboxylic acid with a phenylalanine side chain,
[0045] I is defined as X.sup.28 and preferably denotes an
.alpha.-amino-carboxylic acid with an isoleucine side chain,
[0046] W is defined as X.sup.30 and preferably denotes an
.alpha.-amino-carboxylic acid with a tryptophan side chain
[0047] and the monomeric building blocks are linked by
--CONR.sup.1-- or --NRLCO-- bonds where R.sup.1 in each case
independently denotes hydrogen, methyl or ethyl and
pharmaceutically compatible salts and derivatives thereof and in
which at least one of the amino acid residues denotes X.sup.1,
X.sup.2, X.sup.3, X.sup.6, I, X.sup.7, W and X.sup.8 denotes a
D-amino acid residue.
[0048] The monomeric building blocks X.sup.1 to X.sup.8 preferably
have the following meanings:
[0049] X.sup.1 and--if present--X.sup.8 correspond to the meaning
of X.sup.21 and X.sup.29 and are e.g. .alpha.-aminocarboxylic acid
building blocks with an SH side chain, in particular with a
cysteine side chain.
[0050] X.sup.2--if present--is an .alpha.-aminocarboxylic acid with
an aliphatic and uncharged side chain e.g. valine, leucine or
isoleucine, in particular valine.
[0051] X.sup.3 and X.sup.5 correspond to the meaning of X.sup.26
and are e.g. .alpha.-aminocarboxylic acids with an aliphatic
hydrophilic side chain such as serine or threonine, in particular
serine.
[0052] X.sup.4 and X.sup.6 correspond to the meaning of X.sup.22
and X.sup.27 and are e.g. .alpha.-aminocarboxylic acids with an
aliphatic hydrophilic side chain, in particular an amide side chain
such as asparagine or glutamine, in particular asparagine.
[0053] If not bridged with X.sup.1, X.sup.7 is preferably a basic
.alpha.-aminocarboxylic acid, in particular histidine. If it is
bridged with X.sup.1, then X.sup.7 is an .alpha.-aminocarboxylic
acid with an SH side group, in particular cysteine.
[0054] The present invention additionally concerns a pharmaceutical
composition which contains at least one peptide or polypeptide as
defined above as the active substance optionally together with
common pharmaceutical carriers, auxiliary agents or diluents. The
peptides or polypeptides according to the invention are used
especially to produce uPA antagonists which are suitable for
treating diseases associated with the expression of UPAR especially
for treating tumours.
[0055] An additional subject matter of the present invention is the
use of peptides derived from the uPA sequence and in particular of
uPA antagonists such as the above-mentioned peptides and
polypeptides to produce targeting vehicles e.g. liposomes, viral
vectors etc. for UPAR-expressing cells. The targeting can be used
for diagnostic applications to steer the transport of marker groups
e.g. radioactive or non-radioactive marker groups. On the other
hand the targeting can be for therapeutic applications e.g. to
transport pharmaceutical agents and for example also to transport
nucleic acids for gene therapy.
[0056] The pharmaceutical compositions according to the invention
can be present in any form, for example as tablets, as coated
tablets or in the form of solutions or suspensions in aqueous or
non-aqueous solvents. The peptides are preferably administered
orally or parenterally in a liquid or solid form. When they are
administered in a liquid form, water is preferably used as the
carrier medium which optionally contains stabilizers, solubilizers
or/and buffers that are usually used for injection solutions. Such
additives are for example tartrate or borate buffer, ethanol,
dimethyl sulfoxide, complexing agents such as EDTA, polymers such
as liquid polyethylene oxide etc.
[0057] If they are administered in a solid form, then solid carrier
substances can be used such as starch, lactose, mannitol, methyl
cellulose, talcum, highly dispersed silicon dioxide, high molecular
fatty acids such as stearic acid, gelatin, agar, calcium phosphate,
magnesium stearate, animal and vegetable fats or solid high
molecular polymers such as polyethylene glycols. The formulations
can also contain flavourings and sweeteners if desired for oral
administration.
[0058] The therapeutic compositions according to the invention can
also be present in the form of complexes e.g. with cyclodextrins
such as .gamma.-cyclodextrin.
[0059] The administered dose depends on the age, state of health
and weight of the patient, on the type and severity of the disease,
on the type of treatment, the frequency of the administration and
the type of desired effect. The daily dose of the active compound
is usually 0.1 to 50 mg/kilogramme body weight. Normally 0.5 to 40
and preferably 1.0 to 20 mg/kg/day in one or several doses are
adequate to achieve the desired effects.
[0060] The invention is further illustrated by the examples
described in the following and the figures.
[0061] FIG. 1 shows the quantity-dependent inhibition of the
binding of pro-uPA to a cell surface-associated uPAR by synthetic
peptides;
[0062] FIG. 2 shows the competition of synthetic peptides with ATF
for binding to the uPAR;
[0063] FIG. 3A shows the structure of cyclo.sup.19-31 uPA 19-31
(right) compared to the structure of the corresponding domain from
native uPA and
[0064] FIG. 3B shows the structure of the cyclic peptide derivative
cyclo.sup.21,29 [Cys21,29]uPA.sub.21-30.
[0065] FIG. 4 shows the inhibition of the uPA/uPAR interaction by
synthetic peptides and
[0066] FIG. 5 shows the inhibition of tumour growth in naked mice
by administration of synthetic peptides.
EXAMPLES
[0067] 1. Methods
[0068] 1.1 Solid Phase Peptide Synthesis
[0069] Linear peptides were synthesized on a 2-chlorotrityl resin
(Barlos et al., Int. J. Pept. Protein Res. 37 (1991), 513 to 520)
using an Applied Biosystems Model 431 A peptide synthesizer or a
multiple peptide synthesizer model Syro II (MultiSynTech). Using
the orthogonal Fmoc strategy (Carpino and Han, J. Org. Chem. 37
(1972), 3404-3409; Fields and Noble, Int. J. Peptide Protein Res.
35 (1990), 161-214) the amino acid side chains were blocked with
the protecting groups trityl (Asn, Cys, Gln and His),
tert.-butyloxycarbonyl (Lys and Trp), tert.-butyl (Asp, Glu, Ser,
Thr and Tyr), acetamidomethyl (Cys) and
2,2,5,7,8-pentamethylchroman- -6-sulfonyl or
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Arg). The
coupling was carried out at room temperature in dimethylformamide
using a three-fold excess of
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-
-uroniumtetrafluoroborate/1-hydroxybenzotriazole/Fmoc-amino acid
with 2.5 equivalents of N-ethyldiiso-propylamine in
N-methyl-pyrrolidone. The Fmoc group was removed by sequential
treatment of the resins with an excess of 40% or 20% piperidine in
dimethylformamide. The cleavage of the peptides and removal of the
side chain protecting groups was carried out simultaneously by
treatment with 82.5% trifluoroacetic acid/5% phenol/2.5% ethane
dithiol/5% thioanisol/5% H.sub.2O (0.degree. C./1 h; room
temperature/1 h). In the case of Arg groups protected with
2,2,5,5,7,8-pentamethylchroman-6-sulfonyl, the peptides were
incubated for an additional 12 h at room temperature. The crude
peptides were precipitated at -30.degree. C. with diethyl ether,
dissolved in methanol, precipitated as previously described,
dissolved in tert.-butanol and lyophilized. Peptides containing
tryptophan were additionally treated for 2 h with 5% acetic acid
before the lyophilization.
[0070] The peptides were purified by HPLC using a reversed phase
C18 column (Nucleosil 1005-C18) or a YMC pack ODS column. They were
cyclized by forming a disulfide bridge between the cysteine
residues. The oxidation required for this was carried out by taking
0.1 to 0.3 mg/ml of the purified linear peptides up in 80% water
and 20% DMSO (vol/vol) and removing the solvent under reduced
pressure after 10 h. The cyclic peptides were again purified by
HPLC as described above.
[0071] 1.2 Mass Spectroscopy and Amino Acid Analysis
[0072] The purified and desalted peptides were analysed on a HPLC
system 140 B (Applied Biosystems, Foster City, USA). The UV
absorbance was measured with a UVIS 200 detector (Linear
Instruments, Reno, USA) at 206 nm. The chromatography was carried
out on an Aquapore 3.mu. (Applied Biosystems, Foster City, USA)
reversed phase column (1 mm.times.50 mm) at a flow rate of 20
.mu.l/min. The solvent system was 0.1% TFA in water (A) and 0.1%
TFA in acetonitrile (B). The HPLC system was coupled to an
atmospheric pressure ionisation source which was connected to a
tandem quadrupole instrument API III (Sciex, Perkin Elmer,
Thornhill, Canada).
[0073] The quadrupole M/Z scale was calibrated with the ammonium
addition products of polypropylene glycol. The average mass values
were calculated from the M/Z peaks in the charge distribution
profiles of the multiple charged ions (Covey et al., Rapid Commun.
Mass Spectrom. 2 (1988), 249-256; Fenn et al., Science 246 (1989),
64-71).
[0074] The amino acid analysis was carried out according to the
ninhydrin method using the analytical system 6300 (Beckman
Instruments, Fullerton, USA) after hydrolysing the peptides by the
TFA-HCl vapour phase method which allows a quantitative
determination of the peptide concentration (Tsugita et al., J.
Biochem. 102 (1987), 1593-1597).
[0075] 1.3 Flow Cytometry
[0076] The ability of the synthetic peptides to inhibit the
uPA/uPAR interaction was determined by means of flow cytometry on a
FACScan flow cytometer (Becton-Dickinson, Heidelberg, Germany)
using the human promyeloid cell line U937 as a source of cellular
native uPAR (Chuchulowski et al., Fibrinolysis 6, Suppl. 4 (1992),
95-102; Magdolen et al., (1996), supra). The U937 cells were
stimulated with 1 mM phorbol-12-myristate-13-acetate (PMA) for 48
h. After stimulation with PMA the U937 cells expressed considerable
amounts of cell surface-associated uPAR.
[0077] The stimulated cells were treated for 1 min at room
temperature with 50 mM glycine HCl, 0.1 NaCl, pH 3.6 in order to
dissociate endogenous receptor-bound uPA. Subsequently the acidic
buffer was neutralized with 0.5 M HEPES-100 mM NaCl, pH 7.5. The
cells were then immediately washed twice with PBS/0.1% bovine serum
albumin (BSA) and centrifuged for 10 min at room temperature and
300.times.g. The cells were resuspended in PBS/0.1% BSA, adjusted
to a concentration of 10.sup.6 cells per ml and simultaneously
incubated for 45 minutes at room temperature with 16 ng
FITC-conjugated pro-uPA and various amounts of the synthetic
peptides. Before the analysis, propidium iodide, a fluorescent dye
which specifically binds double-stranded DNA, was added to each
sample in order to determine the viability of the analysed U937
cells. Damaged, propidium iodide-labelled cells were excluded from
the analysis.
[0078] 1.4 Solid Phase uPAR/uPA Binding Test
[0079] In addition to the flow cytometric analyses, a solid phase
ATF-ligand binding test was carried out in order to examine the
interactions of synthetic peptides with the uPAR. For this
microtitre plates were coated with recombinant human uPAR from CHO
cells (Wilhelm et al., FEBS Lett. 337 (1994), 131-134; Magdolen et
al., Electrophoresis 16 (1995), 813-816) and the remaining
protein-binding sites were saturated with 2% BSA (weight/vol).
After incubation with the samples (0.6 ng ATF together with 15
.mu.g synthetic peptide per ml) and several wash steps, the amount
of ATF which had bound to the uPAR immobilized on the microtitre
plate was determined using a biotinylated monoclonal mouse antibody
against the kringle domain of ATF (No. 377, American Diagnostics,
Greenwich, Conn., USA) and subsequent addition of avidin-peroxidase
conjugate and 3,3', 5,5'-tetramethylbenzidine/H.sub.2O as a
substrate for the peroxidase. The presence of synthetic peptides
which compete with the ATF binding to UPAR reduces the conversion
of the chromogenic substrate.
[0080] 2. Results
[0081] 2.1 Determination of the uPAR Binding Capacity of Synthetic
Peptides by Quantitative Flow Cytometric Analysis
[0082] A comparison was made of the inhibitory capacity of the
peptides uPA.sub.12-32 [C19A] (Appella et al., (1987), supra) the
so-called clone 20-peptide AEPMPHSLNFSQYLWYT (Goodson et al.,
(1994), supra) which was identified as the most effective peptide
from a phage peptide library and of the synthetic peptide
uPA.sub.16-32 derived from the wild-type uPA sequence.
[0083] For this the purified peptides were analysed by mass
spectroscopy, quantified by amino acid analysis and then tested by
flow cytometry according to the method described in 1.3 for their
ability to inhibit the binding of fluorescent-labelled pro-uPA to
the uPA receptor on U937 cells. It was found that pro-uPA is
displaced in a dose-dependent manner from the cell
surface-associated uPAR by all three synthetic peptides (FIG. 1).
An approximately 15,000 to 12,000 molar excess of uPA.sub.12-32
[C19A] or clone 20 peptide resulted in a 50% inhibition of the
binding of uPA. The peptide uPA.sub.16-32 exhibited a 4- to 5-fold
higher affinity to uPAR compared to the two other peptides: an
approximately 3,000-fold molar excess is sufficient to achieve a
50% inhibition.
[0084] Furthermore it was found that the linear peptide
uPA.sub.19-31 surprisingly has an IC50 value of ca. 0.8 .mu.M
whereas the IC50 value for uPA.sub.16-32 is ca. 3.2 .mu.M.
[0085] 2.2 Determination of the uPAR Binding Capacity of Synthetic
Peptides in a Microtitre Plate Solid Phase Ligand binding Test
[0086] A series of peptides with variable sequence regions from the
receptor binding region of uPA were synthesized and were
increasingly shortened at the amino terminus starting with
uPA.sub.10-32. The microtitre plate solid phase binding test
described in 1.4 was used to determine the inhibitory capacity of
these peptides. The results of this test are shown in FIG. 2.
[0087] It can be seen in FIG. 2A that the peptides uPA.sub.10-32,
uPA.sub.12-31, uPA.sub.14-32 and uPA.sub.16-32 effectively inhibit
the binding of ATF to uPAR. The peptides uPA.sub.17-32 and
uPA.sub.18-34 have considerably reduced uPAR binding capacities.
The peptide uPA.sub.20-34 does not bind at all to the uPAR. In a
further experiment the binding capacity of the peptides
uPA.sub.19-31, uPA.sub.18-30, uPA.sub.20-32 and uPA.sub.20-30 was
tested. The result of this experiment is shown in FIG. 2B.
Surprisingly it was found that uPA.sub.19-31 binds to the uPAR with
higher affinity than the longer peptide uPA.sub.16-32. The other
tested linear peptides had no significant binding capacity.
[0088] The cyclic peptide cyclo.sup.19-31uPA.sub.19-31 which
contains an intramolecular disulfide bond between the cysteine
residues at positions 19 and 31 was surprisingly still able to
inhibit the binding of uPA to the uPA receptor. Furthermore the
binding activity of cyclo.sup.19-31uPA.sub.19-31 was significantly
more stable after long storage in aqueous solution or repeated
freeze/thaw cycles than that of the linear peptide
uPA.sub.19-31.
[0089] 2.3 Systematic Replacement of L-Amino Acids by D-Amino Acids
in Chemically Synthesized Linear and Cyclic Peptides From the
Region uPA.sub.19-31
[0090] The uPAR binding capacity of synthetic linear and cyclic
peptides from the region uPA.sub.19-31 was determined by in each
case replacing one L-amino acid by the corresponding D-amino acid.
The results of this experiment are shown in the following table
1.
1 TABLE 1 D-amino acid Peptide structure Inhibition Trp30
[D-Trp.sup.30]uPA.sub.19-31 ++ Trp30
cyclo[D-Trp.sup.30]uPA.sub.19-31 + His29
[D-His.sup.29]uPA.sub.19-31 ++ His29 cyclo[D-His.sup.29]uPA.sub.1-
9-31 + Asn27 [D-Asn.sup.27]uPA.sub.19-31 ++ Asn27
cyclo[D-Asn.sup.27]uPA.sub.19-31 ++ Ser21 [D-Ser.sup.21]uPA.sub.1-
9-31 ++ Ser21 cyclo[D-Ser.sup.21]uPA.sub.19-31 ++ Val20
[D-Val.sup.20]uPA.sub.19-31 ++ Val20 cyclo[D-Val.sup.20]uPA.sub.1-
9-31 + Cys19 [D-Cys.sup.19]uPA.sub.19-31 +++ Cys19
cyclo[D-Cys.sup.19]uPA.sub.19-31 +++ cyclo19-31
cyclo[19-31]uPA.sub.19-31 +++
[0091] It can be seen from this table that the introduction of
D-amino acids at positions Cys19, Val20, Ser21, Asn27, His29 and
Trp30 in the linear as well as in the cyclic peptides is possible
without loss of the inhibitory effect. Moreover it was found that
in the case of the linear peptides the inhibitory effect is not
lost by introducing D-amino acids at positions Ile28 and Cys31.
[0092] 2.4 Synthesis of Modified Cyclic uPA Peptides
[0093] Using cyclo.sup.19,31uPA.sub.19-31 as the lead structure, a
cyclic peptide was prepared in which certain amino acids were
deleted and/or substituted by other amino acids. The structure of
this new synthetic peptide variant
cyclo.sup.21,29[Cys21,29]uPA.sub.21-30 is shown in FIG. 3. In
contrast to the synthesis method stated in 1.1 this peptide was
prepared on a trityl chloride polystyrene resin.
[0094] FIG. 4 shows the inhibitory effect of this synthetic peptide
variant compared to cyclo.sup.19,31uPA.sub.19-31 and
cyclo.sup.19,31[D-Cys19]uPA.sub.19-31.
[0095] 2.5 In Vivo Effect
[0096] 6.times.10.sup.6 human breast cancer cells MDA-MB-231 (Price
et al., Cancer Res. 50 (1990), 717-721) in a total volume of 300
.mu.l were injected into the right side of 4-6 week old Balbc/3
naked mice. Before injection the cancer cells were mixed with 200
.mu.g of the cyclic UPA peptides cyclo.sup.19,31uPA.sub.19-31 and
cyclo.sup.21,29[Cys21]uPA.sub.2- 1-30 in PBS, pH 7.4. Subsequently
the mice were treated twice weekly intraperitoneally with the
respective peptide at a dose of 10 mg/kg body weight (injection
volume 300 .mu.l). The volume of the primary tumours which occurred
in the mice in cm.sup.3 was determined after 1, 2, 3 and 5 weeks by
measuring the two largest diameters. The control mice were
administered PBS pH 7.4. Each group was composed of 5 mice. The
results for the peptide cyclo.sup.19,31uPA.sub.19-31 are shown in
Tab. 2.
2TABLE 2 Week Control uPA peptide 1 0 0 2 0.34 .+-. 0.3 0.086 .+-.
0.047 3 0.71 .+-. 0.5 0.303 .+-. 0.129 5 2.33 .+-. 0.32 0.62 .+-.
0.21* *p = 0.02
[0097] The volume of the primary tumour after a five week treatment
is shown in FIG. 5. It can be seen that the administration of both
peptides led to a significant reduction of the tumour growth in
vivo.
* * * * *