Inhibitors for urokinase receptor

Details Inhibitors for urokinase receptor Inhibitors for urokinase receptor

2000-01-10

2003-03-04

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

11 to 14 amino acid residues in defined sequence

C530S328000, C514S014800, C514S015800

Reexamination Certificate

active

06528619

ABSTRACT:

FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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
D
=10
−10
−10
−9
M) to the membrane-based uPA receptor (uPAR) on the cell surface.
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.
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.
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 to uPA.
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.
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 Cysl9, 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.
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.
WO-A-94/22464 discloses 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-94/22464 makes reference to a potential use of the claimed peptides to block the uPA/uPAR interaction, no data or information whatsoever is shown on such binding studies. Moreover, the peptides uPA 21-29 and uPA 21-26 which are said to be preferred do not contain the minimal uPAR binding region in the uPA molecule 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.
A disadvantage of the previously known uPA peptide inhibitors is that the affinity of the 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.
SUMMARY OF THE INVENTION
The present invention provides a peptide having the general structural formula (I):
X
1
-[X
2
]
n-X
3
-X
4
-K-Y-F-X
5
-X
6
-I-X
7
-W-[X
8
]
m
  (I)
in which
X
1
, X
2
, X
3
, X
4
, X
5
, X
6
, X
7
and X
8
each denote an aminocarboxylic acid,
n and m are each independently 0 or 1,
K denotes an aminocarboxylic acid with a lysine side chain,
Y denotes an aminocarboxylic acid with a tyrosine side chain,
F denotes an aminocarboxylic acid with a phenyl-alanine side chain,
I denotes an aminocarboxylic acid with an isoleucine side chain,
W denotes an aminocarboxylic acid with a tryptophan side chain,
and the monomeric building blocks are linked by —CONR
1
— or —NR
1
CO— bonds where R
1
in each case independently denotes hydrogen, methyl or ethyl, and pharmaceutically compatible salts and derivatives thereof.
The present invention also provides a pharmaceutical composition comprising at least one peptide of formula I as an active substance.
The present invention further provides the use of a peptide of formula I as a urokinase (uPA) antagonist.
The present invention further provides the treatment of a tumor by the use of a peptide of formula I.
In quantitative investigations it was surprisingly found that the linear peptide uPA (19-31) and cyclic derivatives of this peptide have a considerably improved binding affinity for the uPA receptor.
Experimental data have shown 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.
Hence the present invention concerns peptides having the general structural formula (I):
X
1
-[X
2
]
n
-X
3
-X
4
-K-Y-F-X
5
-X
6
-I-X
7
-W-[X
8
]
m
  (I)
in which
X
1
, X
2
, X
3
, X
4
, X
5
, X
6
, X
7
and X
8
each denote an amino-carboxylic acid, preferably an a-aminocarboxylic acid,
n and m are each independently 0 or 1,
K denotes an aminocarboxylic acid, preferably an &agr;-aminocarboxylic acid with a lysine side chain,
Y denotes an aminocarboxylic acid, preferably an &agr;-aminocarboxylic acid with a tyrosine side chain,
F denotes an aminocarboxylic acid, preferably an &agr;-aminocarboxylic acid with a phenylalanine side chain,
I denotes an aminocarboxylic acid, preferably an &agr;-aminocarboxylic acid with an isoleucine side chain,
W denotes an aminocarboxylic acid, preferably an &agr;-aminocarboxylic acid with a tryptophan side chain,
and the monomeric building blocks are linked by —CONR
1
— or —NR
1
CO— bonds in where R
1
in each case independently denotes hydrogen, methyl or ethyl, and pharmaceutically compatible salts and derivatives thereof.
In addition to peptides which contain a sequence having the structural formula (I), ph

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