U.S. patent application number 10/349543 was filed with the patent office on 2003-09-04 for cyclic peptides that bind to urokinase-type plasminogen activator receptor.
Invention is credited to Haney, David N., Jones, Terence R., Varga, Janos.
Application Number | 20030166514 10/349543 |
Document ID | / |
Family ID | 25007232 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166514 |
Kind Code |
A1 |
Jones, Terence R. ; et
al. |
September 4, 2003 |
Cyclic peptides that bind to urokinase-type plasminogen activator
receptor
Abstract
Cyclic peptide compounds having 11 amino acids joined by a
linking unit L, such that the linear dimension between the
C.sup..alpha. carbon of the first amino acid and the C.sup..alpha.
carbon of eleventh amino acid is between about 4 and 12
.ANG.ngstrom units; are useful for inhibiting the binding of uPA to
the uPAR receptor.: Methods for using the cyclic peptide compounds,
and compositions containing them, for inhibiting the growth or
metastasis of cancerous tumors are also disclosed.
Inventors: |
Jones, Terence R.; (San
Diego, CA) ; Haney, David N.; (Mercer Island, WA)
; Varga, Janos; (Napa, CA) |
Correspondence
Address: |
CAMPBELL & FLORES LLP
4370 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122
US
|
Family ID: |
25007232 |
Appl. No.: |
10/349543 |
Filed: |
January 22, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10349543 |
Jan 22, 2003 |
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09285783 |
Apr 5, 1999 |
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6514710 |
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09285783 |
Apr 5, 1999 |
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08747915 |
Nov 12, 1996 |
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5942492 |
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Current U.S.
Class: |
435/7.2 ;
514/14.6; 514/21.1; 530/317 |
Current CPC
Class: |
A61P 35/04 20180101;
C12N 9/6462 20130101; A61P 19/02 20180101; A61K 38/00 20130101;
A61P 29/00 20180101; A61P 35/00 20180101; C07K 7/54 20130101; A61P
27/02 20180101; C12Y 304/21073 20130101 |
Class at
Publication: |
514/9 ;
530/317 |
International
Class: |
A61K 038/12; C07K
007/64 |
Claims
We claim:
1. A cyclic peptide compound of Formula 1 or Formula 2 4 5wherein,
in Formula 1, all of X.sup.1 through X.sup.11 represent L-series
amino acids and, in Formula 2, all of X.sup.1 through X.sup.11
represent D-series amino acids; X.sup.1 is Val, Pro, or Ala;
X.sup.2 is Ser or Ala; X.sup.3 is Asn or Gln; X.sup.4 is Lys or
His; X.sup.5 is Tyr, Trp, Phe, substituted Phe, di-substituted Phe,
homophenylalamine, .beta.-(3-pyridyl)alanine,
.beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or
.beta.-(2-naphthyl)alanine; X.sup.6 is Tyr, Trp, Phe, substituted
Phe, di-substituted Phe, homophenylalanine,
.beta.-(3-pyridyl)alanine, .beta.-(2-thienyl)alanine,
.beta.-(1-naphthyl)alanine, or .beta.-(2-naphthyl)alanine; X.sup.7
is Ser or Ala; X.sup.8 is Asn or Ala; X.sup.9 is Ile, Leu, or Val;
X.sup.10 is His or Ala; X.sup.11 is Tyr, Trp, Phe, substituted Phe,
di-substituted Phe, homophenylalanine, .beta.-(3-pyridyl)alanine,
.beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or
.beta.-(2-naphthyl)alanine; and L is a linking unit, such that when
X.sup.1 and X.sup.11 are linked, the linear dimension between the
C.sup..alpha. carbon of amino acid X.sup.1 and the C.sup..alpha.
carbon of amino acid X.sup.11 is between about 4 and 12
.ANG.ngstrom units; with the proviso that, when said compound is of
Formula 1, L does not comprise two cysteine units linked by a
disulfide bond.
2. The compound of claim 1 wherein the linear dimension between the
C.sup..alpha. carbon of amino acid X.sup.1 and the C.sup..alpha.
carbon of amino acid X.sup.11 is between about 5 and 10
.ANG.ngstrom units.
3. The compound of claim 1 wherein the linear dimension between the
C.sup..alpha. carbon of amino acid X.sup.1 and the C.sup..alpha.
carbon of amino acid X.sup.11 is between about 6 and 8 .ANG.ngstrom
units.
4. The compound of claim 1 wherein said compound is of Formula 1,
and all of X.sup.1 through X.sup.11 represent L-series natural
amino acids.
5. The compound of claim 4 wherein L is selected from the group
consisting of:
--CO--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(COOH)--NH---
;
--CO--CH(NH.sub.2)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CH(COOH)--NH--,
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--S--CH.sub.2-- -CH.sub.2--CH(COOH)--NH--; and
--CO--CH.sub.2-meta-phenylene--CH.sub.2--NH- --.
6. The compound of claim 1 wherein said compound is of Formula 2,
and wherein all of X.sup.1 through X.sup.11 represent D-series
non-natural amino acids.
7. The compound of claim 6 wherein L is selected from the group
consisting of:
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO---
;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(NH.sub.2)--CO--;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CO--;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH- .sub.2--S--CH.sub.2--CO--; and
--NH--CH.sub.2-meta-phenylene-CH.sub.2--CO-- -.
8. The compound of claim 1 wherein said compound exhibits an
IC.sub.50 value of less than about 10.sup.-5 molar, i.e., less than
about 10 .mu.M.
9. The compound of claim 1 wherein said compound exhibits an
IC.sub.50 value less than about 10.sup.-6 molar.
10. The compound of claim 1 wherein said compound exhibits an
IC.sub.50 value in the range of 10.sup.-7 molar.
11. A method for inhibiting the binding of uPA to uPAR, said method
comprising the step of contacting living cells with a cyclic
peptide compound of Formula 1 or Formula 2 6 7wherein, in Formula
1, all of X.sup.1 through X.sup.11 represent L-series amino acids
and, in Formula 2, all of X.sup.1 through X.sup.11 represent
D-series amino acids; X.sup.1 is Val, Pro, or Ala; X.sup.2 is Ser
or Ala; X.sup.3 is Asn or Gln; X.sup.4 is Lys or His; X.sup.5 is
Tyr, Trp, Phe, substituted Phe, di-substituted Phe,
homophenylalanine, .beta.-(3-pyridyl)alanine,
.beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or
.beta.-(2-naphthyl)alanine; X.sup.6 is Tyr, Trp, Phe, substituted
Phe, di-substituted Phe, homophenylalanine,
.beta.-(3-pyridyl)alanine, .beta.-(2-thienyl)alanine
.beta.-(1-naphthyl)alanine, or .beta.-(2-naphthyl)alanine; X.sup.7
is Ser or Ala; X.sup.8 is Asn or Ala; X.sup.9 is Ile, Leu, or Val;
X.sup.10 is His or Ala; X.sup.11 is Tyr, Trp, Phe, substituted Phe,
di-substituted Phe, homophenylalanine, .beta.-(3-pyridyl)alanine,
.beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or
.beta.-(2-naphthyl)alanine; and L is a linking unit, such that when
X.sup.1 and X.sup.11 are linked, the linear dimension between the
C.sup..alpha. carbon of amino acid X.sup.1 and the C.sup..alpha.
carbon of amino acid X.sup.11 is between about 4 and 12
.ANG.ngstrom units; with the proviso that, when said compound is of
Formula 1, L does not comprise two cysteine units linked by a
disulfide bond.
12. The method of claim 11 wherein the linear dimension between the
C.sup..alpha. carbon of amino acid X.sup.1 and the C.sup..alpha.
carbon of amino acid X.sup.11 is between about 5 and 10
.ANG.ngstrom units.
13. The method of claim 11 wherein the linear dimension between the
C.sup..alpha. carbon of amino acid X.sup.1 and the C.sup..alpha.
carbon of amino acid X.sup.11 is between about 6 and 8 .ANG.ngstrom
units.
14. The method of claim 11 wherein said compound is of Formula 1,
and all of X.sup.1 through X.sup.11 represent L-series natural
amino acids.
15. The method of claim 14 wherein L is selected from the group
consisting of:
--CO--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(COOH)--NH---
;
--CO--CH(NH.sub.2)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CH(COOH)--NH--,
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--S--CH.sub.2-- -CH.sub.2--CH(COOH)--NH--; and
--CO--CH.sub.2-meta-phenylene--CH.sub.2--NH- --.
16. The method of claim 11 wherein said compound is of Formula 2,
and wherein all of X.sup.1 through X.sup.11 represent D-series
non-natural amino acids.
17. The method of claim 16 wherein L is selected from the group
consisting of:
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO---
;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(NH.sub.2)--CO--;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CO--;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH- .sub.2--S--CH.sub.2--CO--; and
--NH--CH.sub.2-meta-phenylene-CH.sub.2--CO-- -.
18. The method of claim 11 wherein said compound exhibits an
IC.sub.50 value of less than about 10.sup.-5 molar.
19. The method of claim 11 wherein said compound exhibits an
IC.sub.50 value of less than about 10.sup.-6 molar.
20. The method of claim 11 wherein said compound exhibits an
IC.sub.50 value of less than about 10.sup.-7 molar.
21. The method of claim 11 wherein said method has the effect of
inhibiting the growth or metastasis of cancerous tumors.
22. A pharmaceutical composition comprising (a) the cyclic peptide
compound of Formula 1 or Formula 2 8 9wherein, in Formula 1, all of
X.sup.1 through X.sup.11 represent L-series amino acids and, in
Formula 2, all of X.sup.1 through X.sup.11 represent D-series amino
acids; X.sup.1 is Val, Pro, or Ala; X.sup.2 is Ser or Ala; X.sup.3
is Asn or Gln; X.sup.4 is Lys or His; X.sup.5 is Tyr, Trp, Phe,
substituted Phe, di-substituted Phe, homophenylalanine,
.beta.-(3-pyridyl)alanine, .beta.-(2-thienyl)alanine,
.beta.-(1-naphthyl)alanine, or .beta.-(2-naphthyl)alanine; X.sup.6
is Tyr, Trp, Phe, substituted Phe, di-substituted Phe,
homophenylalanine, .beta.-(3-pyridyl)alanine,
.beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or
.beta.-(2-naphthyl)alanine; X.sup.7 is Ser or Ala; X.sup.8 is Asn
or Ala; X.sup.9 is Ile, Leu, or Val; X.sup.10 is His or Ala;
X.sup.11 is Tyr, Trp, Phe, substituted Phe, di-substituted Phe,
homophenylalanine, .beta.-(3-pyridyl)alanine,
.beta.-(.sup.2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or
.beta.-(2-naphthyl)alanine; and L is a linking unit, such that when
X.sup.1 and X.sup.11 are linked, the linear dimension between the
C.sup..alpha. carbon of amino acid X.sup.1 and the C.sup..alpha.
carbon of amino acid X.sup.11 is between about 4 and 12
.ANG.ngstrom units; with the proviso that, when said compound is of
Formula 1, L does not comprise two cysteine units linked by a
disulfide bond; and (b) a pharmaceutically acceptable carrier.
23. The composition of claim 22 wherein the linear dimension
between the C.sup..alpha. carbon of amino acid X.sup.1 and the
C.sup..alpha. carbon of amino acid X.sup.11 is between about 5 and
10 .ANG.ngstrom units.
24. The composition of claim 22 wherein the linear dimension
between the C.sup..alpha. carbon of amino acid X.sup.1 and the
C.sup..alpha. carbon of amino acid X.sup.11 is between about 6 and
8 .ANG.ngstrom units.
25. The composition of claim 22 wherein said compound is of Formula
1, and all of X.sup.1 through X.sup.11 represent L-series natural
amino acids.
26. The composition of claim 25 wherein L is selected from the
group consisting of:
--CO--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(COOH)-- -NH--;
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(COOH)-
--NH--;
--CO--CH(NH.sub.2)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--NH--
-; --CO--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CH(COOH)--NH--,
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--S--CH.sub.2-- -CH.sub.2--CH(COOH)--NH--; and
--CO--CH.sub.2-meta-phenylene--CH.sub.2--NH- --.
27. The composition of claim 22 wherein said compound is of Formula
2, and wherein all of X.sup.1 through X.sup.11 represent D-series
non-natural amino acids.
28. The composition of claim 27 wherein L is selected from the
group consisting of:
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2-- -CO--;
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2-
--CO--;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(NH.sub.2)--CO--
-; --NH--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CO--;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH- .sub.2--S--CH.sub.2--CO--; and
--NH--CH.sub.2-meta-phenylene-CH.sub.2--CO-- -.
29. The composition of claim 22 wherein said compound exhibits an
IC.sub.50 value of less than about 10.sup.-5 molar.
30. The composition of claim 22 wherein said compound exhibits an
IC.sub.50 value of less than about 10.sup.-4 molar.
31. The composition of claim 22 wherein said compound exhibits an
IC.sub.50 value of less than about 10.sup.-7 molar.
32. The composition of claim 22 wherein said composition is in a
form suitable for injection.
Description
TECHNICAL FIELD
[0001] The present invention relates to certain cyclic peptides
that bind to the cell surface receptor for urokinase-type
plasminogen activator and, thus, are capable of inhibiting the
binding of urokinase-type plasminogen activator to this cell
surface receptor. The invention also relates to pharmaceutical
compositions containing these peptides and to the use of these
peptides to inhibit the binding of urokinase-type plasminogen
activator to its cell surface receptor. Effects derived from the
inhibition of binding of urokinase-type plasminogen activator to
its cell surface receptor include the inhibition of proteolysis;
the inhibition of programmed gene expression; the inhibition of
cell motility, migration, and morphogenesis; the inhibition of the
activation of certain pro-growth factors to the active form of the
growth factor; the inhibition of angiogenesis; the inhibition of
tumor metastasis; the inhibition of retinal neovascularization in
the treatment of certain forms of blindness; and the inhibition of
tissue remodeling as a treatment for inflammatory diseases, such as
arthritis. The peptides of the invention that are capable of
carrying a suitable radioactive, fluorogenic, chromogenic, or
chemical label can also be used to quantitate urokinase-type
plasminogen activator receptor levels in tissue samples and can be
used, therefore, as diagnostic and prognostic tools in all diseases
where the receptor plays a pathological role, including those
mentioned above.
BACKGROUND OF THE INVENTION
[0002] Urokinase-type plasminogen activator (uPA) has been
identified as the initiator of a major amplified cascade of
extracellular proteolysis. This cascade, when regulated, is vital
to certain normal physiological processes but, when dysregulated,
is strongly linked to pathological processes, such as cell invasion
and metastasis in cancer. Dan.o slashed. et al., Adv. Cancer Res.,
44:139-266 (1985). Cells express uPA as an inactive form, pro-uPA
or single-chain uPA, which then binds to its receptor, uPAR. This
binding event is necessary for activation to two-chain uPA. Ellis
et al., J. Biol. Chem., 264:2185-88 (1989). The amino acid sequence
for human pro-uPA is as follows:
1 Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp 1 10 Cys Leu Asn
Gly Gly Thr Cys Val Ser Asn Lys Tyr 20 Phe Ser Asn Ile His Trp Cys
Asn Cys Pro Lys Lys 30 Phe Gly Gly Gln His Cys Glu Ile 40
[0003] The sequence of amino acids of pro-uPA are represented above
by their standard three-letter abbreviations as follows:
2 Amino Acid Three-letter Symbol Alanine Ala Arginine Arg
Asparagine Asn Aspartic acid Asp Cysteine Cys Glutamine Gln
Glutamic acid Glu Glycine Gly Histidine His Isoleucine Ile Leucine
Leu Lysine Lys Methionine Met Phenylalanine Phe Proline Pro Serine
Ser Threonine Thr Tryptophan Trp Tyrosine Tyr Valine Val
[0004] The structure of pro-uPA is shown in FIG. 1.
[0005] uPA is a three-domain protein comprising (1) an N-terminal
epidermal growth factor-like domain, (2) a kringle domain, and (3)
a C-terminal serine protease domain. The receptor for pro-uPA
(uPAR) is a multi-domain protein anchored by a glycolipid to the
cell membrane, thus ensuring that activation of uPA is a
pericellular event. Behrendt et al., Biol. Chem. Hoppe-Seyler,
376:269-79 (1995). uPA activity is confined to the cell surface by
plasminogen activator inhibitors (PAI-1 and PAI-2), which bind to
and inactivate the bound uPA. This tight control of uPA activity is
necessary because uPA acts upon a substrate, plasminogen, that is
present at a high concentration in plasma. Robbins, Meth. Enzymol.,
19:184-99 (1970). The product of uPA's action upon plasminogen,
plasmin, is a powerful broad spectrum protease that not only
degrades extracellular matrix proteins directly, but also activates
the latent forms of other proteases, including several
metalloproteases. Werb et al., N. Eng. J. Med., 296:1017-1023
(1977); Mignatti et al., Cell, 47:487-98 (1986); He et al., Proc.
Natl. Acad. Sci. USA, 86:2632-36 (1989); and Matrisian, Bioessays,
14:455-63 (1992).
[0006] In tumor biology, the link between extracellular proteolysis
and angiogenesis is clearly evident. Break-up and dissolution of
existing extracellular matrix is necessary in order to create new
space for blood vessels to grow into. The processes of proteolysis
and angiogenesis are highly coordinated. For example, two
pre-eminent angiogenic growth factors, basic fibroblast growth
factor and vascular endothelial growth factor markedly up-regulate
the production of uPA. (Montesano et al., Proc. Natl. Acad. Sci.
USA, 83:7297-7301 (1986); Pepper et al., Biochem. Biophys. Res.
Comm., 181:902-906 (1991)) and the expression of uPAR by
endothelial cells (Mignatti et al., J. Cell Biol., 113:1193-1201
(1991); Mandriota et al., J. Biol. Chem., 270:9709-9716(1995)).
Thus uPA/uPAR has emerged as a new target for developing an
anti-metastatic/anti-angiogenic therapy for cancer, where most
studies have been conducted (Fazioli et al., Trends Pharmacological
Sci., 15:25-29 (1994).
[0007] However, the uPA/uPAR interaction goes far beyond localizing
proteolysis at the cell surface. Independent of all proteolytic
effects, the mere occupation of uPAR by uPA induces, by indirect
means, signal transduction events leading to one or more of the
following effects: mitogenesis (Rabbani et al., J. Biol. Chem.,
267:14151-56 (1992)); expression of the c-fos gene (Dumler et al.,
FEBS Lett. 322:37-40 (1994)); cysteine- and metalloprotease
expression by macrophages (Rao et al., J. Clin. Invest. 96:465-74
(1995)): transfer of mechanical force leading to increased
cytoskeletal stiffness (Wang et al., Am. J. Physiol.
268:C1062-C1066 (1995)); endothelial cell migration (Odekon et al.,
J. Cellul. Physiol., 150:258-63 (1992)); endothelial cell
morphogenesis into tubular structures (Schnaper et al., J. Cellul.
Physiol. 165:107-118 (1995)); and endothelial cell deformability
and motility (Lu et al., FEBS Lett. 380:21-24 (1996)). All of these
phenomena are blocked by blocking the access of uPA to uPAR. An
antagonist of uPAR that prevented the binding of uPA would thus
interfere with proteolytic activity by preempting uPA activation
and, further, would greatly diminish uPAR's capacity for signal
transduction.
[0008] The anti-angiogenic effects accompanying uPAR antagonism
(Min et al., Cancer Res., 56:2428-33 (1996)) should allow a uPAR
antagonist to play a role in other diseases characterized by
inappropriate angiogenesis, e.g. ocular angiogenesis leading to
blindness. Furthermore, it is likely that a uPAR antagonist would
also play a therapeutic role in inflammatory diseases, for example,
rheumatoid arthritis. (Ronday et al., Br. J. Rheum., 35:416-423
(1996).
[0009] One approach to drug therapy is to target uPA itself at its
catalytic serine protease domain. Yang et al., Fibrinolysis, 6
(Suppl. 1):31-34, (1992). Amiloride (Vassalli et al., FEBS Lett.,
214:187-191 (1987); and Kellen et al., Anticancer Res. 8:1373-76
(1988)) and p-aminobenzamidine (Geratz et al., Thrombosis Res.
24:73-83 (1981); and Billstrom et al., Int. J. Cancer, 61:542-47
(1995)) are competitive inhibitors of this site and have
anti-metastatic activity in vivo. Selective inhibition of uPA as
compared with other serine proteases, was evident in
phenylguanidines (Yang et al., J. Med. Chem., 33:2956-61 (1990))
and, even more so, in benzo[b]thiophene-2-carboxamidines (Bridges,
Bioorganic & Medicinal Chemistry, 1:403-410 (1993); Towle et
al., Cancer Res., 53:2553-59 (1993); and Rabbani et al., Int. J.
Cancer, 63:840-45 (1995)).
[0010] Towards defining the binding epitope for the uPA-uPAR
interaction, it was first shown that the amino terminal fragment of
uPA (residues 1-135) that lacked the serine protease domain,
sufficed for high affinity, sub-nanomolar binding. (Stoppelli et
al., Proc. Natl. Acad. Sci. USA 82:4939-43 (1985). Further work
showed that the growth factor domain alone (residues 1-48)
conferred this binding. (Robbiati et al., Fibrinolysis, 4:53-60
(1990); and Stratton-Thomas et al., Protein Engineering 8:463-70
(1995.)) Dan.o slashed. et al., in WO 90/12091 published Oct. 18,
1990, discloses that the binding of uPA to uPAR could be prevented
by administering a substance comprising a sequence identical or
substantially identical to a uPAR binding site of uPA amino
residues 12-32. WO 94/28145, by Rosenberg and Stratton-Thomas, Dec.
8, 1994, discloses the preparation and use of de-fucosylated
HuPA.sub.1-48 that prevents uPA binding to uPAR.
[0011] Earlier studies with peptide fragments within the growth
factor domain had showed that residues 20-30 conferred the
specificity of binding, but that residues 13-19 were needed in
addition for residues 20-30 to attain the proper binding
conformation. Specifically, the peptide [Ala.sup.19]uPA-(12-32),
which contains two cysteines (the third cysteine being replaced by
Ala to avoid undesired disulfide bond formations), in its open
chain form prevented uPA binding to uPAR with an IC.sub.50 of 100
nM. In its oxidized cyclic form, having an intrachain disulfide
bond between Cys.sup.13 and Cys.sup.31, the peptide prevented
binding with an IC.sub.50 of 40 nM. It was proposed that residues
13-19 might act indirectly to provide a scaffold that would help
residues 20-30 attain the correct binding conformation. Appella et
al., J. Biol. Chem., 262:4437-40 (1987).
[0012] These results were partially confirmed when it was reported
that, while the linear peptide 20-30 inhibited the binding of uPA
to uPAR with an IC.sub.50 of 1,000 nM, the longer peptide 17-34 was
significantly more potent, having an IC.sub.50 of 100 nM. It was
also shown that the corresponding longer peptide (17-34) derived
from the mouse sequence inhibited spontaneous metastasis of a
murine Lewis Lung carcinoma in mice, whereas the corresponding
linear shorter peptide (20-30) had no effect. Kobayashi et al.,
Int. J. Cancer, 57:727-33 (1994). WO 94/28014 by Rosenberg and
Doyle, Dec. 8, 1994 discloses the preparation and use of 25 random
peptides displayed on bacteriophage which competed with the
N-terminal fragment of uPA for binding to uPAR with IC.sub.50
values of 15 nM to>50 .mu.M.
[0013] Most recently, Magdolen et al., "Systematic Mutational
Analysis of the Receptor-binding Region of the Human Urokinase-type
Plasminogen Activator", Eur. J. Biochem., 237:743-51 (1996),
describes alanine-scanning mutagenesis of the binding loop of the
amino-terminal fragment of uPA with the finding that Asn22, Lys23,
Tyr24, Phe25, Ile28 and Trp30 are important side chains that should
be kept. Further, Magdolen et al., citing Hansen et al.,
Biochemistry, 33:4847-64 (1994), disclose that the region between
Thr 18 and Asn32 consists of a flexible, seven-residue omega loop
that is forced into a ring-like structure. Although Cys19 and Cys31
are in close proximity to each other (0.61 nm), they do not form a
disulfide bond with each other. Instead Cys19 forms a disulfide
bond with Cys11, and Cys31 forms a bond with Cys13. See FIG. 2.
Accordingly, the uPAR binding site on uPA does not form a simple,
small ring structure.
[0014] Some scientists have explored the possibility of cyclizing
the one or more of the growth factor domains of peptide analogues
to increase their competitive binding activity, but not with any
great success without at least adding some other constraining-type
modifications of the structure. For example, in Chamberlin et al.,
J. Biol. Chem., 270:21062-21067 (1995), peptides constrained by the
introduction of an intramolecular disulfide bond also required the
substitution of another entity for proline in the peptide loop to
achieve any significant activity. Lougheed et al., Protein Sci., 4:
773-80 (1995) found that peptides from the fifth EGF-like domain of
thrombomodulin had very weak biological activity that increased
marginally (two-fold) by cyclization. The additional presence of a
stair of amino acids and the deletion of one of the amino acids
were both found necessary and, even then, the best epptide was-only
weakly active (text of micromolar range). Thus, cyclization per se
conferred no significant activity. Further, others working in the
thrombomodulin field have found that the number of crossing
disulfide bonds in the fifth EGF-like domain is inversely, rather
than directly, related to inhibitory potency. Hunter et al.,
Protein Sci., 4:2129-37 (1995).
[0015] It has now been found by the present inventors that novel
cyclic structures derived from the peptide fragment 20-30, in which
residue 20 is covalently bonded to residue 30, do exhibit the
ability to bind to uPAR and are also antagonists of the binding of
uPA to uPAR. These peptides are shorter than either
[Ala.sup.19]uPA-(12-32) (Appella et al., supra.) or uPA17-34
(Kobayashi et al., supra.), but bind almost as effectively. In
contradiction of what was hitherto thought, it has been discovered
that the eight amino acids N-terminal to Val20 in
[Ala.sup.19]uPA-(12-32) and the four amino acids CO-terminal to
Trp3O in uPA17-34 are not necessary for high binding affinity.
While not wishing to be bound by any particular theory, it now
appears that the minimal binding epitope in urokinase-type
plasminogen activator, which is needed for binding to its receptor,
is a loop of only eleven amino acids.
DISCLOSURE OF THE INVENTION
[0016] The invention is a cyclic peptide compound of Formula 1 or
Formula 2 1 2
[0017] wherein, in Formula 1, all of X.sup.1 through X.sup.11
represent L-series amino acids and, in Formula 2, all of X.sup.1
through X.sup.11 represent D-series amino acids;
[0018] X.sup.1 is Val, Pro, or Ala;
[0019] X.sup.2 is Ser or Ala;
[0020] X.sup.3 is Asn or Gln;
[0021] X.sup.4 is Lys or His;
[0022] X.sup.5 is Tyr, Trp, Phe, substituted Phe, di-substituted
Phe, homophenylalanine, .beta.-(3-pyridyl)alanine,
.beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or
.beta.-(2-naphthyl)alanine;
[0023] X.sup.6 is Tyr, Trp, Phe, substituted Phe, di-substituted
Phe, homophenylalanine, .beta.-(3-pyridyl)alanine,
.beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or
.beta.-(2-naphthyl)alanine;
[0024] X.sup.7 is Ser or Ala;
[0025] X.sup.8 is Asn or Ala;
[0026] X.sup.9 is Ile, Leu, or Val;
[0027] X.sup.10 is His or Ala;
[0028] X.sup.11 is Tyr, Trp, Phe, substituted Phe, di-substituted
Phe, homophenylalanine, .beta.-(3-pyridyl)alanine,
.beta.-(2-thienyl)alanine, .beta.-(1-naphthyl)alanine, or
.beta.-(2-naphthyl)alanine; and
[0029] L is a linking unit, such that when X.sup.1 and X.sup.11 are
linked, the linear dimension between the C.sup..alpha. carbon of
amino acid X.sup.1 and the C.sup..alpha. carbon of amino acid
X.sup.11 is between about 4 and 12 .ANG.ngstrom units;
[0030] with the proviso that, when said compound is of Formula 1, L
does not comprise two cysteine units linked by a disulfide
bond.
[0031] In other embodiments, the compounds of the invention are
used in methods and therapeutic compositions to inhibit the binding
of uPA to uPAR, particularly in the treatment of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a schematic representation of the pro-uPA molecule
and its major cleavage sites.
[0033] FIG. 2 shows the NH-terminal growth factor domain of human
uPA.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The cyclic peptide compounds of the invention can be of
either Formula 1 or the corresponding retro-inverso cyclic peptide
compounds of Formula 2. When the cyclic peptide compounds of the
invention are of Formula 1, all of X.sup.1 through X.sup.11
represent L-series natural amino acids. On the other hand, when the
compounds of the invention are of Formula 2, all of X.sup.1 through
X.sup.11 represent D-series non-natural amino acids. Whether of
Formula 1 or Formula 2, however, when any one of X.sup.5, X.sup.6
or X.sup.11 is a substituted or disubstituted phenylalanine, the
substituent is halo, such as 4-fluoro, 4-chloro, 4-bromo, or
3,4-dichloro; lower alkyl, such as methyl, ethyl, isopropyl,
tertiary butyl or n-pentyl; nitro; or the like.
[0035] The linker moiety L in the compounds of the invention forms
a bridge between X.sup.11 and X.sup.1, thus cyclizing the compound.
The linker L can be almost any divalent group which would set the
linear dimension between the C.sup..alpha. carbon of amino acid
X.sup.1 and the C.sup..alpha. carbon of amino acid X.sup.11 between
about 4 and 12 .ANG.ngstrom units, preferably between about 5 and
10 .ANG.ngstrom units and, even more preferably, between about 6
and 8 .ANG.ngstrom units.
[0036] Compounds of Formula 1
[0037] In Formula 1, the amide bond, CO--NH, which links X.sup.1 to
X.sup.2, is such that the C.dbd.O moiety is from the amino acid
X.sup.1 and the NH moiety is from the amino acid X.sup.2. The same
arrangement applies to the link between X.sup.2 and X.sup.3, and so
on. In other words, the peptide has X.sup.1 as its N-terminus and
X.sup.11 as its C-terminus.
[0038] It should be noted, however, that L cannot comprise two
cysteine units linked by a disulfide bond, be it unsubstituted,
substituted at its N-terminus with a group R.sup.1, substituted at
its C-terminus with a group R.sup.2, or modified at both N- and
C-termini, respectively, with R.sup.1 and R.sup.2, where R.sup.1 is
acetyl and R.sup.2 is amino such that the C-terminus is a primary
carboxamide. Specific examples of such excluded L groups
include:
--CO--CH(NH.sub.2)--CH.sub.2--S--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH(NHR.sup.1)--CH.sub.2--S--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH(NH.sub.2)--CH.sub.2--S--S--CH.sub.2--CH(COR.sup.2)--NH--;
and
--CO--CH(NHR.sup.1)--CH.sub.2--S--S--CH.sub.2--CH(COR.sup.2)--NH--.
[0039] To prepare the compounds of Formula 1, L is chosen so as to
provide, at one of its termini, a functional group that can be
chemically bonded to the carbon of the .alpha.-carboxyl group of
the amino acid X.sup.11 and, at its other terminus, a functional
group that can be chemically bonded to the .alpha.-amino nitrogen
atom of the amino acid X.sup.1.
[0040] Alternatively, the linear peptide
X.sup.1--X.sup.2--X.sup.3--X.sup.-
4--X.sup.5--X.sup.6--X.sup.7--X.sup.8--X.sup.9--X.sup.10--X.sup.11
can be synthesized with an extension at its X.sup.11 terminus
comprising a portion of the final linker group, i.e., L.sub.b, and
later, after synthesis of the desired peptide chain, the X.sup.1
terminus can be extended with a group L., to give the compound
L.sub.a-X.sup.1--X.sup.2---
X.sup.3--X.sup.4--X.sup.5--X.sup.6--X.sup.7--X.sup.8--X.sup.9--X.sup.10--X-
.sup.11-L.sub.b. The free ends of L.sub.a and L.sub.b can then be
chemically bonded to each other. In this way, the linker L can be
formed during the cyclization step from pre-attached fragments
L.sub.a and L.sub.b. In the examples given below for L, the
direction of L, reading left to right, is from to X.sup.1 to
X.sup.11, i.e., the CO-terminus of L is connected to X.sup.1, and
the NH-terminus of L is connected to X.sup.11.
[0041] Typical examples of useful L groups for Formula 1 include
the following:
--CO--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH(NH.sub.2)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--NH--;
--CO--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CH(COOH)--NH--,
--CO--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--S--CH.sub.2--CH(COOH)--NH--;
--CO--CH.sub.2--S--CH.sub.2--CH.sub.2--CH(COOH)--NH--; and
--CO--CH.sub.2-meta-phenylene--CH.sub.2--NH--.
[0042] When L contains a cysteine or a homocysteine residue, the
configuration of the enantiomeric center of such a residue can be
either L- or D-.
[0043] Compounds of Formula 2 (Retro-Inverso Compounds)
[0044] When the cyclic peptide compound of the invention is of
Formula 2, the amino acids X.sup.1-X.sup.11 are non-natural,
"D-series" peptides having an enantiomeric configuration opposite
to that the of L-series natural amino acids described above for
Formula 1. In Formula 2, the amide bond CO--NH, which links
X.sup.11 to X.sup.10, is such that the C.dbd.O moiety is from the
amino acid X.sup.11 and the NH moiety is from the amino acid
X.sup.10. The same arrangement applies to the link between X.sup.10
and X.sup.9 and so on. In other words, the peptide has X.sup.11 as
its N-terminus and X.sup.1 as its C-terminus.
[0045] In the above structure of Formula 2, the moiety L is chosen
so as to provide, at one of its termini, a functional group that
can be chemically bonded to the .alpha.-amino nitrogen atom of the
X.sup.11 residue, rather than the .alpha.-carboxyl group and, at
its other terminus, a functional group that can be chemically
bonded to the carbon of the .alpha.-carboxyl group of the of the
amino acid X.sup.1, rather than the .alpha.-amino nitrogen atom, as
described above.
[0046] Also as described above, the linear peptide
X.sup.1--X.sup.2--X.sup-
.3--X.sup.4--X.sup.5--X.sup.6--X.sup.7--X.sup.8--X.sup.9--X.sup.10--X.sup.-
11 can be synthesized at its X.sup.1 terminus with a group L.sub.b
and, at its X.sup.11 terminus, with a group L, to give the compound
L.sub.b-X.sup.1--X.sup.2--X.sup.3--X.sup.4--X.sup.5--X.sup.6--X.sup.7--X.-
sup.8--X.sup.9--X.sup.10--X.sup.11-L.sub.a, such that the free ends
of L.sub.a and L.sub.b can be chemically bonded to form the cyclic
retro-inverso compounds of the invention.
[0047] In the examples given below for L, the direction of L,
reading left to right, is from to X.sup.1 to X.sup.11, that is, the
NH-terminus of L is covalently bonded to X.sup.1, and the CO
terminus of L is connected to X.sup.11. When these linkers contain
a cysteine or a homocysteine residue, the configuration of the
enantiomeric center of such a residue can be either the D- or
L-form.
[0048] Examples of useful L groups for the retro-inverso peptides
of Formula 2 include:
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH(NH.sub.2)--CO--;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CO--;
--NH--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--S--CH.sub.2--CO--;
--NH--CH(COOH)--CH.sub.2--CH.sub.2--S--CH.sub.2--CO--; and
--NH--CH.sub.2-meta-phenylene-CH.sub.2--CO--.
[0049] General Chemical Synthetic Procedures
[0050] When the peptides of the invention are not prepared using
recombinant, DNA technology, they are preferably prepared using
solid-phase synthesis, such as that generally described by
Merrifield, J. Amer. Chem. Soc., 85:21 49-54 (1963), although other
equivalent chemical syntheses known in the art are also useful.
Solid-phase peptide synthesis may be initiated from the C-terminus
of the peptide by coupling a protected .alpha.-amino acid to a
suitable resin. Such a starting material can be prepared by
attaching an .alpha.-amino-protected amino acid by an ester linkage
to a chloromethylated resin or to a hydroxymethyl resin, or by an
amide bond to a BHA resin or MBHA resin.
[0051] The preparation of the hydroxymethyl resin is described by
Bodansky et al., Chem. Ind., 38:1597-98 (1966). Chloromethylated
resins are commercially available from BioRad Laboratories,
Richmond, Calif. and from Lab. Systems, Inc. The preparation of
such a resin is described by Stewart et al., "Solid Phase Peptide
Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1, 1-6.
BHA and MBHA resin supports are commercially available and are
generally used only when the desired polypeptide being synthesized
has an unsubstituted amide at the C-terminus.
[0052] The amino acids X.sup.1 through X.sup.11 can be coupled to
the growing peptide chain using techniques well known in the art
for the formation of peptide bonds. For example, one method
involves converting the amino acid to a derivative that will render
the carboxyl group of the amino acid more susceptible to reaction
with the free N-terminal amino group of the growing peptide chain.
Specifically, the C-terminal of the protected amino acid can be
converted to a mixed anhydride by the reaction of the C-terminal
with ethyl-chloroformate, phenyl chloroformate, sec-butyl
chloroformate, isobutyl chloroformate, or pivaloyl chloride or the
like acid chlorides. Alternatively, the C-terminal of the amino
acid can be converted to an active ester, such as a
2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, a
pentafluorophenyl ester, a p-nitrophenyl ester, a
N-hydroxysuccinimide ester, or an ester formed from
1-hydroxybenzotriazole.
[0053] Another coupling method involves the use of a suitable
coupling agent, such as N,N'dicyclohexylcarbodiimide or
N,N'-diisopropylcarbodiimi- de. Other appropriate coupling agents,
apparent to those skilled in the art, are disclosed in Gross et
al., The Peptides: Analysis, Structure, Biology, Vol. I., "Major
Methods of Peptide Bond Formation" (Academic Press 1979), the
disclosure of which is hereby incorporated by reference.
[0054] It will be recognized that the .alpha.-amino group of each
amino acid employed in the peptide synthesis must be protected
during the coupling reaction to prevent side reactions involving
their active .alpha.-amino function. It should also be recognized
that certain amino acids contain reactive side-chain functional
groups (e.g. sulfhydryl, amino, carboxyl, and hydroxyl) and that
such functional groups must also be protected with suitable
protecting groups to prevent a chemical reaction from occurring at
either (1) the .alpha.-amino group site or (2) a reactive side
chain site during both the initial and subsequent coupling
steps.
[0055] In the selection of a particular protecting group to be used
in synthesizing the peptides, the following general rules are
typically followed. Specifically, an .alpha.-amino protecting group
(a) should render the .alpha.-amino function inert under the
conditions employed in the coupling reaction, (b) should be readily
removable after the coupling reaction under conditions that will
not remove side-chain protecting groups and will not alter the
structure of the peptide fragment, and (c) should substantially
reduce the possibility of racemization upon activation, immediately
prior to coupling.
[0056] On the other hand, a side-chain protecting group (a) should
render the side chain functional group inert under the conditions
employed in the coupling reaction, (b) should be stable under the
conditions employed in removing the .alpha.-amino protecting group,
and (c) should be readily removable from the desired
fully-assembled peptide under reaction conditions that will not
alter the structure of the peptide chain.
[0057] It will be apparent to those skilled in the art that the
protecting groups known to be useful for peptide synthesis will
vary in reactivity with the agents employed for their removal. For
example, certain protecting groups, such as triphenylmethyl and
2-(p-biphenyl)isopropyloxy- carbonyl, are very labile and can be
cleaved under mild acid conditions. Other protecting groups, such
as t-butyloxycarbonyl (BOC), t-amyloxycarbonyl,
adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, are less
labile and require moderately strong acids for their removal, such
as trifluoroacetic, hydrochloric, or boron trifluoride in acetic
acid. Still other protecting groups, such as benzyloxycarbonyl (CBZ
or Z), halobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl
cycloalkyloxycarbonyl, and isopropyloxycarbonyl, are even less
labile and require even stronger acids, such as hydrogen fluoride,
hydrogen bromide, or boron trifluoroacetate in trifluoroacetic
acid, for their removal. Suitable protecting groups, known in the
art are described in Gross et al., The Peptides: Analysis,
Structure, Biology, Vol. 3: "Protection of Functional Groups in
Peptide Synthesis" (Academic Press 1981).
[0058] Among the classes of amino acid protecting groups useful for
protecting the .alpha.-amino group or for protecting a side chain
group are included the following.
[0059] (1) For an .alpha.-amino group, three typical classes of
protecting groups are: (a) aromatic urethane-type protecting
groups, such as fluorenylmethyloxycarbonyl (FMOC), CBZ, and
substituted CBZ, such as, e.g., p-chlorobenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, and
p-methoxybenzyloxycarbonyl, o-chlorobenzyloxycarbonyl,
2,4-dichlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, and
the like; (b) aliphatic urethane-type protecting groups, such as
BOC, t-amyloxycarbonyl, isopropyloxycarbonyl,
2-(p-biphenyl)-isopropyloxycarbonyl, allyloxycarbonyl and the like;
and (c) cycloalkyl urethane-type protecting groups, such as
cyclopentyloxycarbonyl, adamantyloxycarbonyl, and
cyclohexyloxycarbonyl. The preferred .alpha.-amino protecting
groups are BOC and FMOC.
[0060] (2) For the side chain amino group present in Lys,
protection may be by any of the groups mentioned above in (1) such
as BOC, 2-chlorobenzyloxycarbonyl and the like.
[0061] (3) For the guanidino group of Arg, protection may be
provided by nitro, tosyl, CBZ, adamantyloxycarbonyl,
2,2,5,7,8-pentamethylchroman-6-s- ulfonyl,
2,3,6-trimethyl-4-methoxyphenylsulfonyl, or BOC groups.
[0062] (4) For the hydroxyl group of Ser, Thr, or Tyr, protection
may be, for example, by t-butyl; benzyl (BZL); or substituted BZL,
such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl,
o-chlorobenzyl, and 2,6-dichlorobenzyl.
[0063] (5) For the carboxyl group of Asp or Glu, protection may be,
for example, by esterification using such groups as BZL, t-butyl,
cyclohexyl, cyclopentyl, and the like.
[0064] (6) For the imidazole nitrogen of His, the benzyloxymethyl
(BOM) or tosyl moiety is suitably employed as a protecting
group.
[0065] (7) For the phenolic hydroxyl group of Tyr, a protecting
group such as tetrahydropyranyl, tert-butyl, trityl, BZL,
chlorobenzyl, 4-bromobenzyl, and 2,6-dichlorobenzyl are suitably
employed. The preferred protecting group is
bromobenzyloxycarbonyl.
[0066] (8) For the side chain amino group of Asn or Gln, xanthyl
(Xan) is preferably employed.
[0067] (9) For Met, the amino acid is preferably left
unprotected.
[0068] (10) For the thio group of Cys, p-methoxybenzyl is typically
employed.
[0069] The first C-terminal amino acid of the growing peptide
chain, e.g., Lys, is typically protected at the N-amino position by
an appropriately selected protecting group such as BOC. The
BOC-Lys-(2-chlorobenzyloxycarb- onyl)-OH group can be first coupled
to a benzylhydrylamine resin using isopropylcarbodiimide at about
25.degree. C. for two hours with stirring or to a chloromethylated
resin according to the procedure set forth in Horiki et al.,
Chemistry Letters, 165-168 (1978). Following the coupling of the
BOC-protected amino acid to the resin support, the .alpha.-amino
protecting group is usually removed, typically by using
trifluoroacetic acid (TFA) in methylene chloride or TFA alone. The
.alpha.-amino group de-protection reaction can occur over a wide
range of temperatures, but is usually carried out at a temperature
between about 0.degree. C. and room temperature.
[0070] Other standard .alpha.-amino group de-protecting reagents,
such as HCl in dioxane, and conditions for the removal of specific
.alpha.-amino protecting groups are within the skill of those
working in the art, such as those described in Lubke et al., Chemie
und Biochemie der Aminosauren, Peptide und Proteine I, Chapter
II-1, 102-117 (Georg Thieme Verlag Stuttgart 1975), which is hereby
incorporated by reference. Following the removal of the
.alpha.-amino protecting group, the unprotected .alpha.-amino
group, generally still side-chain protected, can be coupled in a
stepwise manner in the intended sequence.
[0071] An alternative to the stepwise approach is the fragment
condensation method in which pre-formed peptides of short length,
each representing part of the desired sequence, are coupled to a
growing chain of amino acids bound to a solid phase support. For
this stepwise approach, a particularly suitable coupling reagent is
N,N'-dicyclohexylcarbodiimide or diisopropylcarbodiimide. Also, for
the fragment approach, the selection of the coupling reagent, as
well as the choice of the fragmentation pattern needed to couple
fragments of the desired nature and size are important for success
and are known to those skilled in the art.
[0072] Each protected amino acid or amino acid sequence is usually
introduced into the solid-phase reactor in amounts in excess of
stoichiometric quantities, and the coupling is suitably carried out
in an organic solvent, such as dimethylformarnide (DMF),
CH.sub.2Cl.sub.2 or mixtures thereof. If incomplete coupling
occurs, the coupling procedure is customarily repeated before
removal of the N-amino protecting group in preparation for coupling
to the next amino acid. Following the removal of the .alpha.-amino
protecting group, the remaining .alpha.-amino and
side-chain-protected amino acids can be coupled in a stepwise
manner in the intended sequence. The success of the coupling
reaction at each stage of the synthesis may be monitored. A
preferred method of monitoring the synthesis is by the ninhydrin
reaction, as described by Kaiser et al., Anal. Biochem., 34:595
(1970). The coupling reactions can also be performed automatically
using well-known commercial methods and devices, for example, a
Beckman 990 Peptide Synthesizer.
[0073] Upon completion of the desired peptide sequence, the
protected peptide must be cleaved from the resin support, and all
protecting groups must be removed. The cleavage reaction and
removal of the protecting groups is suitably accomplished
concomitantly or consecutively with de-protection reactions. When
the bond anchoring the peptide to the resin is an ester bond, it
can be cleaved by any reagent that is capable of breaking an ester
linkage and of penetrating the resin matrix. One especially useful
method is by treatment with liquid anhydrous hydrogen fluoride.
This reagent will usually not only cleave the peptide from the
resin, but will also remove all acid-labile protecting groups and,
thus, will directly provide the fully de-protected peptide. When
additional protecting groups that are not acid-labile are present,
additional de-protection steps must be carried out. These steps can
be performed either before or after the hydrogen fluoride treatment
described above, according to specific needs and circumstances.
[0074] When a chloromethylated resin is used, the hydrogen fluoride
cleavage/de-protection treatment generally results in the formation
of the free peptide acids. When a benzhydrylamine resin is used,
the hydrogen fluoride treatment generally results in the free
peptide amides. Reaction with hydrogen fluoride in the presence of
anisole and dimethylsulfide at 0.degree. C. for one hour will
typically remove the side-chain protecting groups and,
concomitantly, release the peptide from the resin.
[0075] When it is desired to cleave the peptide without removing
protecting groups, the protected peptide-resin can be subjected to
methanolysis, thus yielding a protected peptide in which the
C-terminal carboxyl group is methylated. This methyl ester can be
subsequently hydrolyzed under mild alkaline conditions to give the
free C-terminal carboxyl group. The protecting groups on the
peptide chain can then be removed by treatment with a strong acid,
such as liquid hydrogen fluoride. A particularly useful technique
for methanolysis is that of Moore et al., Peptides, Proc. Fifth
Amer. Pept. Symp., 518-521 (Goodman et al., eds., 1977), in which
the protected peptide-resin is treated with methanol and potassium
cyanide in the presence of a crown ether.
[0076] Other methods for cleaving a protected peptide from the
resin when a chloromethylated resin is employed include (1)
ammoniolysis and (2) hydrazinolysis. If desired, the resulting
C-terminal amide or hydrazide can be hydrolyzed to the free
C-terminal carboxyl moiety, and the protecting groups can be
removed conventionally. The protecting group present on the
N-terminal .alpha.-amino group may be removed either before, or
after, the protected peptide is cleaved from the support.
[0077] Purification of the polypeptides of the invention is
typically achieved using chromatographic techniques, such as
preparative HPLC (including reverse phase HPLC), gel permeation,
ion exchange, partition chromatography, affinity chromatography
(including monoclonal antibody columns), and the like, or other
conventional techniques such as countercurrent distribution or the
like.
[0078] The compounds of the invention can be easily tested for
their ability to inhibit the binding of pro-uPA to uPAR in a
competitive ligand-binding assay. The assay is preferably a solid
phase immunoassay that uses the recombinant soluble human urokinase
receptor (uPAR) derived from Chinese hamster ovary cells coated
onto the walls of test wells to act as capture molecules. The
compound being tested competes with pro-uPA for the binding site on
uPAR. The amount of pro-uPA that binds to the uPAR on the walls of
the test wells can be detected with a biotinylated monoclonal
anti-uPA antibody that is, in turn, recognized by
streptavidin-horse radish peroxidase. The addition of perborate and
3,3',5,5'-tetramethylbenzidine ("TMB") as a substrate allows the
peroxidase to generate a blue-colored product, thus providing a
colorimetric signal. The sensitivity of the assay is preferably
increased even further by adding sulfuric acid to the test
solution, which provides a yellow color that can be easily read
out.
[0079] In general, the greater the binding of the test compound to
uPAR, the greater the exclusion of pro-uPA from binding, and the
smaller will be the generated optical signal. The specificity of
this capture-type assay is inherent in the purity of the uPAR being
used, which itself may be tested by SDS-PAGE, non-reducing Western
blot analysis (clean, single band at 46 kD, the known molecular
weight of intact uPAR).
[0080] The detection antibody in the assay recognizes an epitope
within the kringle domain of uPA and, thus, is suitable for
measuring the competitive binding of polypeptides that lack the
kringle domain, such as the growth factor domain of uPA or peptide
derivatives representing a part of the growth factor domain, for
example, the compounds of the invention. This assay has been
validated against three different forms of uPA: (1) pro-uPA, also
known as single-chain uPA; (2) high molecular-weight uPA, also
known as two-chain uPA, and (3) low molecular-weight uPA.
[0081] A typical assay procedure is as follows:
[0082] uPAR is coated onto microtest wells in 96-well plates. 0.8
nM (160 ng/mL) pro-uPA is incubated with serially diluted
concentrations of the test compound. Both pro-uPA and the compound
being tested are diluted in phosphate-buffered saline (PBS) with
0.1% Triton X-100 and 1.0% bovine serum albumin (pH 7.4). The total
volume per well of pro-uPA/test compound mixture is 50 .mu.L, and
ligands are allowed to bind approximately 16 hours overnight at
4.degree. C. The wells are washed four times with saline wash
buffer (100 mM trisodium phosphate, 150 mM sodium chloride, pH 7.4,
containing 1% Triton X-100, and 0.025% sodium azide). The detection
antibody solution (50 .mu.L) is added, and the mixture is kept at
room temperature for one hour. The wells are washed with wash
buffer.
[0083] Streptavidin-horse radish peroxidase (50 .mu.L) is next
added and incubated for one hour at room temperature. The wells are
again washed with wash buffer. Finally, the peroxidase substrate
TMB (50 .mu.L) and perborate are added and allowed to react for 20
minutes to generate a blue color. Sulfuric acid (0.5 M, 50 .mu.L)
is added to yield a yellow color. The absorbance of the yellow
color is then read out at 450 nanometers on an MRX microplate
reader made by Dynatech Laboratories. Each test compound is assayed
in triplicate at five different concentrations.
[0084] The inhibition of binding of pro-uPA to uPAR is usually
dose-related, such that the concentration of the test compound
necessary to produce a 50% inhibition of binding (the IC.sub.50
value) is easily determined. In general, the compounds of the
invention have IC.sub.50 values of less than about 10.sup.-5 molar,
i.e., less than about 10 .mu.M. Preferably, the compounds of the
invention have IC.sub.50 values of less than about 10.sup.-6 molar,
i.e., less than about 1 .mu.M and, even more preferably, less than
about 10.sup.-7 molar.
[0085] Administration and Use
[0086] The cyclic peptide compounds of the invention that may be
employed in the pharmaceutical compositions of the invention
include all of those compounds described above, as well as the
pharmaceutically acceptable salts of these compounds.
Pharmaceutically acceptable acid addition salts of the compounds of
the invention containing a basic group are formed where appropriate
with strong or moderately strong, non-toxic, organic or inorganic
acids in the presence of a basic amine by methods known to the art.
Exemplary of the acid addition salts that are included in this
invention are maleate, fumarate, lactate, oxalate,
methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate,
citrate, hydrochloride, hydrobromide, sulfate, phosphate and
nitrate salts.
[0087] Pharmaceutically acceptable base addition salts of compounds
of the invention containing an acidic group are prepared by known
methods from organic and inorganic bases and include, for example,
nontoxic alkali metal and alkaline earth bases, such as calcium,
sodium, potassium and ammonium hydroxide; and nontoxic organic
bases such as triethylamine, butylamine, piperazine, and
tri(hydroxymethyl)methylamine.
[0088] As stated above, the compounds of the invention possess the
ability to inhibit uPA formation, a property that may express
itself in the form of anti-tumor activity. A compound of the
invention may be active per se, or it may be a pro-drug that is
converted in vivo to an active compound.
[0089] The compounds of the invention, as well as the
pharmaceutically acceptable salts thereof, may be incorporated into
convenient dosage forms, such as capsules, tablets or injectable
preparations. Solid or liquid pharmaceutically acceptable carriers
may be employed. Preferably, the compounds of the invention are
administered systemically, e.g., by injection. When used, injection
may be intravenous, subcutaneous, intramuscular, or even
intraperitoneal. Injectables can be prepared in conventional forms,
either as solutions or suspensions, solid forms suitable for
solution or suspension in liquid prior to injection, or as
emulsions.
[0090] Solid carriers include starch, lactose, calcium sulfate
dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin,
acacia, magnesium stearate and stearic acid. Liquid carriers
include syrup, peanut oil, olive oil, saline, water, dextrose,
glycerol and the like. Similarly, the carrier or diluent may
include any prolonged release material, such as glyceryl
monostearate or glyceryl distearate, alone or with a wax. When a
liquid carrier is used, the preparation may be in the form of a
syrup, elixir, emulsion, soft gelatin capsule, sterile injectable
liquid (e.g., a solution), such as an ampoule, or an aqueous or
nonaqueous liquid suspension. A summary of such pharmaceutical
compositions may be found, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton Pa.
(Gennaro 18th ed. 1990).
[0091] The pharmaceutical preparations are made following
conventional techniques of a pharmaceutical chemist involving such
steps as mixing, granulating and compressing, when necessary for
tablet forms, or mixing, filling and dissolving the ingredients, as
appropriate, to give the desired products for oral, parenteral,
topical, transdermal, intravaginal, intranasal, intrabronchial,
intraocular, intraaural and rectal administration. Of course, these
compositions may also contain minor amounts of nontoxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents and so forth.
[0092] The compositions of the invention may further comprise one
or more other compounds that are anti-tumor agents, such as mitotic
inhibitors, e.g., vinblastine; alkylating agents, e.g.,
methotrexate, pritrexim or trimetrexate); antimetabolites, e.g.,
5-fluorouracil and cytosine arabinoside; intercalating antibiotics,
e.g., adriamycin and bleomycin); enzymes, e.g., asparaginase;
topoisomerase inhibitors, e.g., etoposide; or biological response
modifiers, e.g., interferon.
[0093] The composition of the invention may also comprise one or
more other compounds, including antibacterial, anti-fungal,
anti-parasitic, anti-viral, anti-psoriatic and anti-coccidial
agents. Exemplary antibacterial agents include, for example,
sulfonamides such as sulfamethoxazole, sulfadiazine, sulfameter or
sulfadoxine; DHFR inhibitors such as trimethoprim, bromodiaprim or
trimetrexate; penicillins; cephalosporins; aminoglycosides;
bacteriostatic inhibitors of protein synthesis; the
quinolonecarboxylic acids and their fused isothiazole analogs; and
the like.
[0094] Another aspect of the invention relates to a therapeutic
process preventing the binding of uPA to uPAR, which process
comprises administering to a vertebrate host, such as a mammal or
bird, an amount effective to inhibit the binding of uPA to uPAR
according to the present invention. The compounds of the invention
are particularly useful in the treatment of mammalian hosts, such
as human hosts and other animal hosts likely to be provided with a
veterinarian's care. Further, the peptides of the invention that
are capable of carrying a suitable radioactive fluorogenic,
chromogenic, or chemical lable can also be used to quantitate uPAR
levels in tissue samples and has use, therefore, in diseases where
the receptor plays a pathological role.
[0095] Any of the cyclic peptide compounds described above, or
pharmaceutically acceptable salts thereof, may be used in the
therapeutic process of the invention. The compounds may be
administered in the therapeutic process of the invention in the
form of a pharmaceutically acceptable composition comprising a
diluent or carrier, such as those described above. Doses of the
compounds preferably include pharmaceutical dosage units comprising
an efficacious quantity of active compound. By an efficacious
quantity is meant a quantity sufficient to inhibit the binding of
uPA to uPAR and derive the beneficial effects therefrom through
administration of one or more of the pharmaceutical dosage
units.
[0096] The quantity of compound to be administered depends on the
choice of active ingredient, the conditions to be treated, the mode
of administration, the individual subject, and the judgment of the
practitioner. Depending on the specificity of the preparation,
smaller or larger doses may be needed. An exemplary daily dosage
unit for a vertebrate host comprises an amount of up to about 5,000
mg of active compound per square meter of the body area of the
vertebrate host. Dosages in the range of about 0.05-10 mg/kg are
suggested for systemic administration. Dosages in the range of
about 0.01-20% concentration of active ingredient, preferably 1-5%,
are suggested for topical administration. A total daily dosage in
the range of about 10-300 mg is suggested for oral administration.
The foregoing ranges are, however, merely suggestive, as the number
of variables in regard to an individual treatment regime is large,
and considerable excursions from these recommended values are
expected.
[0097] The selected dose may be administered to a warm-blooded
animal or mammal, for example, a human patient, in need of
treatment mediated by inhibition of the binding of uPA to uPAR by
any known method of administration, including topically or
transdermally, e.g., as an ointment, cream or gel; orally;
rectally, e.g., as a suppository; parenterally, by injection or
continuously by infusion; intravaginally; intranasally;
intrabronchially; intra-aurally; or intraocularly.
[0098] The cyclic peptide compounds of the invention may be further
characterized as producing any one or more of inhibitor effect on
uPA binding to uPAR, a proteolysis inhibitor effect, an inhibitory
effect on programmed gene expression; an inhibitory effect on cell
motility, migration and morphogenesis; an effect of slowing the
activation of pro-growth factors to the active form of the growth
factor; an anti-angiogenesis effect; an inhibitory effect on tumor
metastasis; a lessening of retinal neovascularizations; and a
protective effect against inflammatory diseases such as arthritis.
The compounds are especially useful in producing an anti-tumor
effect in a vertebrate host harboring a tumor.
[0099] Further details of the production and purification of the
compounds of the invention are given in the following illustrative
specific example, which is not in any way intended to limit the
scope of the invention.
EXAMPLE 1
[0100] Synthesis of 3
[0101] The starting material was
BOC-L-Cys(S-p-Methoxybenzyl)-O-resin, substituted at a level of
0.84 milli-equivalent per gram of resin. Each of the remaining
L-amino acids was added in sequence in a synthesis cycle consisting
of:
[0102] 1. TFA De-protection
[0103] The BOC protecting group was removed from the .alpha.-amino
nitrogen of the starting material by treating the resin with 50%
trifluoroacetic acid (TFA) in dichloromethane (DCM) (two to three
volumes per resin volume). The mixture was stirred at room
temperature for 30 minutes and then drained. The resin was then
washed once with an equal volume of isopropanol for one minute and
washed twice with an equal volume of methanol, each wash taking one
minute.
[0104] 2. Coupling
[0105] The de-protected resin was washed twice with an equal volume
of 10% triethylamine in DCM, each wash taking one minute, and
washed twice with an equal volume of methanol, each wash taking one
minute, and washed twice with an equal volume of DCM, each wash
taking one minute. A BOC-protected amino acid (three equivalents,
dissolved in DCM or in a mixture of DCM and N,N'-dimethylformamide
(DMF)) and 1-hydroxybenzotriazole (1M solution in DMF, three
equivalents) was added to the resin, and the mixture was stirred
for a few seconds. Dicyclohexylcarbodiimide ("DCC") (IM solution in
DCM, three equivalents) was then added, and the whole mixture was
stirred for 60-120 minutes. The resin was washed twice with an
equal volume of methanol and then washed twice with an equal volume
of DCM. A small sample was taken for a ninhydrin test to assess the
completeness of-coupling. Generally, if incomplete, the coupling
step 2 is repeated. If complete, the synthesis is continued with
the capping step 3.
[0106] In the case of each of the five N-terminal amino acids,
Val-Ser-Asn-Lys-Tyr, coupling was only partially complete and,
therefore, was repeated at least once. The coupling of
4-bromobutyric acid was first attempted with DCC, as described
above for the coupling step 2, but was incomplete. Three manual
repeat couplings were performed using the symmetric anhydride
method, the pentafluorophenylester method and, finally, with
bromobutyryl chloride. A ninhydrin test following these procedures
indicated virtually complete coupling.
[0107] All amino acids were used as .alpha.-BOC derivatives. Side
chain protecting groups were as follows:
3 Cysteine p-Methoxybenzyl Tryptophan N-Formyl Histidine
Benzyloxymethyl Asparagine Xanthyl Serine O-benzyl Tyrosine
2-Bromo-Z Lysine 2-Chloro-Z
[0108] Before coupling with bromobutyric acid, the formyl group of
the tryptophan residue was removed by treatment with 20% piperidine
in DMF at room temperature for 30 minutes.
[0109] 3. Capping
[0110] The resin was stirred with an equal volume of acetic
anhydride (20% solution in DCM) for 5 minutes at room temperature.
The resin was washed twice with an equal volume of methanol and
then washed twice with an equal volume of DCM.
[0111] 4. HF Cleavage
[0112] The resin bearing the desired amino acid sequence (1.0 gram)
was placed in a Teflon reaction vessel, and anhydrous anisole (1
mL) was added. The vessel was cooled with liquid N.sub.2, and
anhydrous HF (10 mL) was distilled into it. The temperature was
raised with ice water to 0.degree. C. The mixture was stirred at
this temperature for one hour, and then the HF was distilled off at
0.degree. C. The residue was washed with anhydrous ether, and the
peptide was extracted with a 1:1 mixture of
CH.sub.3CN:H.sub.2O.
[0113] 5. Cyclization
[0114] The above peptide solution was diluted with H.sub.2O to a
final volume of approximately 1000 mL, and the pH was adjusted with
NaHCO.sub.3 to 8.0. The cyclization was monitored by analytical
HPLC. After 2-3 days, no further shift was observed in the
chromatogram, and the cyclization was judged complete.
[0115] 6. Purification
[0116] The turbid cyclization reaction mixture was filtered through
a 1 .mu.M filter. The filtrate was adjusted to pH 4.0 and loaded
onto a Waters C18 preparative column (2 inches diameter, 15-20
.mu.m particle size, 300 .ANG. pore size). The loaded column was
eluted with a two-component eluent applied as a linear gradient,
starting with 15% of solution A in solution B and finishing with
40% of solution A in solution B. Solution A was 0.1% TFA in
H.sub.2O, and solution B was 0.1% TFA in CH.sub.3CN. Fractions
exhibiting purity equal to or better than that desired were pooled
and lyophilized to render the purified final product as the
trifluoroacetate salt.
Sequence CWU 1
1
* * * * *