Cyclic CRF antagonist peptides

Details Cyclic CRF antagonist peptides Cyclic CRF antagonist peptides

1999-11-17

2001-11-27

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

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Corticotropin ; related peptides

C530S324000, C530S317000, C514S012200, C514S009100

Reexamination Certificate

active

06323312

ABSTRACT:

This invention is generally directed to peptides and to the pharmaceutical treatment of mammals using such peptides. More specifically, the invention relates to cyclic antagonists of the CRF hentetracontapeptides as well as to members of the larger family of CRF-like peptides, to pharmaceutical compositions containing such cyclic CRF antagonists, to methods of treatment of mammals using such cyclic CRF antagonists, and to methods of screening for new drugs using such peptides.
Ovine CRF (oCRF) (SEQ ID NO:1), in 1981, was the first physiologic corticotropin releasing factor characterized; it is disclosed in U.S. Pat. No. 4,415,558 as a 41-residue amidated peptide which lowers blood pressure in mammals when injected peripherally and stimulates the secretion of ACTH and &bgr;-endorphin.
Rat CRF (rCRF) (SEQ ID NO:2) was later isolated, purified and characterized; it is described in U.S. Pat. No. 4,489,163. The amino acid sequence of human CRF was determined to be the same as that of rCRF, so rCRF and hCRF are used interchangeably, as is the designation r/hCRF. These peptide hormones are considered to form a part of a larger family of native CRF-like peptides and analogs which include the mammalian and fish CRFs, the urotensins and sauvagine (SEQ ID NO:3).
A CRF analog having a high alpha-helical forming potential was developed in about early 1984. It is a 41-residue amidated peptide commonly referred to as AHC (alpha-helical CRF) (SEQ ID NO:4) which is described in U.S. Pat. No. 4,594,329 and is considered to be a member of the overall family of CRF-like peptides. Other CRF analogs containing D-isomers of &agr;-amino acids were developed, such as those shown in U.S. Pat. No. 5,278,146. Synthetic r/hCRF, oCRF and AHC all stimulate ACTH and &bgr;-endorphin-like activities (&bgr;-END-Li) in vitro and in vivo and substantially lower blood pressure when injected peripherally. Antagonists of these three peptides and of sauvagine and urotensin are disclosed in U.S. Pat. No. 4,605,642, issued Aug. 12, 1986. Cyclic CRF antagonists exhibiting biopotency are disclosed in U.S. Pat. Nos. 5,493,006 and in 5,510,458 and published international application WO 96/19499, the latter two of which disclose cyclizing bonds between the residues in the 30 and 33 positions. More specifically, the latter document discloses a potent CRF antagonist referred to as Destressin having the formula (cyclo-33) [D-Phe
12
, Nle
21,38
, Glu
30
, D-His
32
, Lys
33
]-r/hCRF(12-41).
Since the foregoing discoveries, the search for still further improved CRF antagonists has continued.
CRF antagonist peptides have now been discovered which exhibit further increased biological activity in comparison to known CRF antagonists, and many exhibit substantially no residual CRF agonist activity. They also exhibit high solubility in neutral aqueous solutions, e.g. at physiological pH, and high receptor affinity.
It is also shown that the various members of the family of CRF-like peptides can be modified to create such highly biopotent CRF antagonists that bind strongly to the known CRF receptors (CRF-R) without significantly activating same and thus block the action of CRF at its receptors. They exhibit an affinity for CRF-R well above that exhibited by oCRF.
In one basic aspect, the invention provides a cyclic CRF antagonist peptide which binds to CRF receptors but has an intrinsic activity with respect to such receptors equal to 20% or less than that of native CRF, which peptide has the formula Y-A-D-Xaa-B-Xaa
c
-Xaa
a
,-Xaa
b
-Xaa
c
-C-NH
2
wherein: Y is an acyl group having up to 15 carbon atoms; A is Asp-Leu-Thr or Asp-Leu-Ser; D-Xaa is D-Phe, D-2Nal or D-Leu; B is a sequence of 17 amino acid residues of a peptide of the CRF family selected from the group of sequences consisting of (a) residues 13-29 of mammalian and fish CRFs and of fish urotensins and (b) residues 12-28 of sauvagine; Xaa
c
n, represent a pair of amino acid residues, the side chains of which are linked in a cyclizing bond; Xaa
a
is a natural &agr;-amino acid residue other than Cys; Xaa
b
is a residue of either (c) a D-isomer amino acid from the group consisting of D-isomers of natural &agr;-amino acids other than Cys and unnatural aromatic &agr;-amino acids, or (d) a natural L-isomer &agr;-amino acid other than Cys; and C is a sequence of the last 8 amino acid residues of the C-terminal portion of a peptide of the CRF family selected from the group of sequences consisting of (e) residues 34-41 of mammalian and fish CRFs and of fish urotensins and (f) residues 33-40 of sauvagine; provided that CML is present as residue-27 in sequence (a), or as residue-26 in sequence (b) and wherein Nle may be substituted for Met in said peptide sequences.
It has been found that the combination of such a cyclizing bond, the presence of CML
27
and the acylation of the N-terminus in a peptide of 33 residues in length creates a molecule of long-acting duration and high biopotency, e.g. (cyclo 30-33)[Ac-Ser
9
, CML
27
, Glu
30
, Lys
33
]-r/hCRF(9-41). The family of CRF-like peptides is considered to encompass those peptides which bind to the CRF receptors and have at least about 45% amino acid structural homology with ovine CRF, the first mammalian CRF isolated and characterized. The CRF-like family includes the following known peptides: ovine CRF (SEQ ID NO:1), rat/human CRF (SEQ ID NO:2), porcine CRF (SEQ ID NO:5), bovine CRF (SEQ ID NO:6), fish CRFs (SEQ ID NO:7), &agr;-helical CRF(AHC) (SEQ ID NO:4), carp urotensin (SEQ ID NO:8), sucker urotensin (SEQ ID NO:9), maggy sole urotensin (SEQ ID NO:10), flounder urotensin (SEQ ID NO:11) and sauvagine (SEQ ID NO:3).
As indicated above, these peptides have a cyclizing bond between the residues in what would be the 30- and 33-positions in mammalian CRF. A second such bond may optionally be provided between the residues in the 20- and 23-positions. Preferably, the cyclizing bond is a lactam bridge (amide bond) between a side chain carboxyl group on the residue in the 30-position, preferably Glu, and a side chain amino group on the 33-position residue, preferably Lys or Orn. Although the naturally occurring residues of the CRF-like family may be present in the position which corresponds to the 32-position of CRF, i.e. His, Gly, Leu, Gin and Ala, it appears that any &agr;-amino acid is tolerated here. Preferably however, a basic and/or aromatic D-isomer residue or an equivalent is present in the 32-position in the region between the residues joined by this lactamn bridge, e.g. D-His, D-Arg, D-Tyr, imBzlD-His, D-Nal D-Pal, D-Trp, D-Dpr(Nic), DAph, D-Agl(Nic), D-Orn, D-Dbu, D-Dpr, D-Orn(Nic) or a comparable D-isomer. However, a wide variety of other residues such as D-Ala, D-Glu, D-Asn, Aib, Asn, Pal, Nal, Phe and Tyr may be present.
As described herein, the lactam linkage between the side chains of the residues in the 30- and 33-positions is preferred; however, biopotency is also increased, but to a somewhat lessor degree, by alternative cyclizing linkages in this same region of the molecule. For example, the side chain of Glu
28
or Glu
29
can be linked respectively to Lys
31
or Lys
32
, or instead respectively to Lys
32
or Lys
33
(creating a one-residue longer span). These somewhat less biopotent alternatives are considered to be equivalents to the 30-33 cyclizing linkage.
These peptides also have the preferred inclusion of D-Phe, D-2Nal or D-Leu or an equivalent D-isomer, e.g., D-Cpa, D-Tyr, D-Trp or D-3Pal, in the 12-position, and they preferably have norleucine substituted for any naturally occurring Met, e.g., in the 21 and 38 positions of r/h CRF. If it is desired to provide a labelled peptide for use in a binding or other assay, as more fully discussed in WO 96/19499, as by adding a radioactive isotope or a fluorescent dye as is well known in this art, D-Tyr, Tyr (e.g. Ac-D-Tyr or Ac-Tyr) or an acyl group having a hydroxy aryl moiety (e.g. desNH
2
-Tyr) may be present at the N-terminus to form equivalents which are suitable for labelling. other optional substitutions may also be made throughout the molec

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