U.S. patent application number 10/004530 was filed with the patent office on 2003-03-13 for octapeptide bombesin analogs.
This patent application is currently assigned to Biomeasure Inc., Massachusetts corporation. Invention is credited to Coy, David H., Kim, Sun Hyuk, Moreau, Jacques-Pierre.
Application Number | 20030050436 10/004530 |
Document ID | / |
Family ID | 27583742 |
Filed Date | 2003-03-13 |
United States Patent
Application |
20030050436 |
Kind Code |
A1 |
Coy, David H. ; et
al. |
March 13, 2003 |
Octapeptide bombesin analogs
Abstract
A linear (i.e., non-cyclic) analog of biologically active
amphibian bombesin, mammalian gastrin-releasing peptide (GRP), or
mammalian growth hormone releasing factor (GRF), having an active
site and a binding site responsible for the binding of the peptide
to a receptor on a target cell. Cleavage of a peptide bond in the
active site of naturally occurring bombesin, GRP, or GRF is
unnecessary for in vivo biological activity. The analog has one of
the following modifications: (a) a deletion of an amino acid
residue within the active site and a modification of an amino acid
residue outside of the active site, (b) a replacement of two amino
acid residues within the active site with a synthetic amino acid, a
.beta.-amino acid, or a .gamma.-amino acid residue, or (c) a
non-peptide bond instead of a peptide bond between an amino acid
residue of the active site and an adjacent amino acid residue.
Inventors: |
Coy, David H.; (New Orleans,
LA) ; Moreau, Jacques-Pierre; (Upton, MA) ;
Kim, Sun Hyuk; (Needham, MA) |
Correspondence
Address: |
Brian R. Morrill, Esq.
Biomeasure, Incorporated
27 Maple Street
Milford
MA
01757
US
|
Assignee: |
Biomeasure Inc., Massachusetts
corporation
|
Family ID: |
27583742 |
Appl. No.: |
10/004530 |
Filed: |
October 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10004530 |
Oct 23, 2001 |
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09260846 |
Mar 2, 1999 |
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6307017 |
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09260846 |
Mar 2, 1999 |
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08337127 |
Nov 10, 1994 |
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5877277 |
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08337127 |
Nov 10, 1994 |
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07779039 |
Oct 18, 1991 |
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07779039 |
Oct 18, 1991 |
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07502438 |
Mar 30, 1990 |
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5084555 |
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07502438 |
Mar 30, 1990 |
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07397169 |
Aug 21, 1989 |
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07397169 |
Aug 21, 1989 |
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07376555 |
Jul 7, 1989 |
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07376555 |
Jul 7, 1989 |
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07317941 |
Mar 2, 1989 |
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07317941 |
Mar 2, 1989 |
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07282328 |
Dec 9, 1988 |
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5162497 |
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07282328 |
Dec 9, 1988 |
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07257998 |
Oct 14, 1988 |
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07257998 |
Oct 14, 1988 |
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07248771 |
Sep 23, 1988 |
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07248771 |
Sep 23, 1988 |
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07207759 |
Jun 16, 1988 |
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07207759 |
Jun 16, 1988 |
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07204171 |
Jun 8, 1988 |
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07204171 |
Jun 8, 1988 |
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07173311 |
Mar 25, 1988 |
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07173311 |
Mar 25, 1988 |
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07100571 |
Sep 24, 1987 |
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Current U.S.
Class: |
530/328 ;
530/329 |
Current CPC
Class: |
C07K 14/57572 20130101;
A61K 38/00 20130101; C07K 7/086 20130101; C07K 7/02 20130101; C07K
7/18 20130101; C07K 14/685 20130101 |
Class at
Publication: |
530/328 ;
530/329 |
International
Class: |
C07K 007/08; C07K
007/06 |
Claims
What is claimed is:
1. A therapeutic peptide comprising between seven and ten amino
acid residues, inclusive, said peptide being an analog of one of
the following naturally occurring peptides terminating at the
carboxy-terminus with a Met residue: (a) litorin; (b) the ten amino
acid carboxy-terminal region of mammalian gastrin releasing
peptide; and (c) the ten amino acid carboxy-terminal region of
amphibian bombesin; said therapeutic peptide being of the formula:
16wherein A.sup.0=Gly, Nle, .alpha.-aminobutyric acid, or the
D-isomer of any of Ala, Val, Gln, Asn, Leu, Ile, Met, p-X-Phe
(where X=F, Cl, Br, NO.sub.2, OH, H or CH.sub.3), Trp, Cys, or
.beta.-Nal, or is deleted; A.sup.1=the D or L-isomer of any of
pGlu, Nle, or .alpha.-amirobutyric acid, or the D-isomer of any of
Ala, Val, Gin, Asn, Leu, Ile, Met, p-X-Phe (where X=F, Cl, Br,
NO.sub.2, OH, H or CH.sub.3), F.sub.5-Phe, Trp, Cys, or .beta.-Nal,
or is deleted; A.sup.2=pGlu, Gly, Ala, Val, Gln, Asn, Leu, Ile,
Met, p-X-Phe (where X=F, Cl, Br, NO.sub.2, OH, H or CH.sub.3), Trp,
Cys, .beta.-Nal, His, 1-methyl-His, or 3-methyl-His; A.sup.4=Ala,
Val, Gln, Asn, Gly, Leu, Ile, Nle, .alpha.-aminobutyric acid, Met,
p-X-Phe (where X=F, Cl, Br, NO.sub.2, OH, H or CH.sub.3), Trp, Cys,
or .beta.-Nal; A.sup.5=Gln, Asn, Gly, Ala, Leu, Ile, Nle,
.alpha.-amincbutyric acid, Met, Val, p-X-Phe (,where X=F, Cl, Br,
OH, H or CH.sub.3) , Trp, Thr, or .beta.-Nal; A.sup.6=Sar, Gly, or
the D-isomer of any of Ala, N-methyl-Ala, Val, GIn, Asn, Leu, Ile,
Met, p-X-Phe (where X=F, Cl, Br, ,NO.sub.2, OH, H or CH.sub.3),
Trp, Cys, or .beta.-Nal; A.sup.7=1-methyl-His, 3-methyl-His, or
His; provided that, if A.sup.0 is present, A.sup.1 cannot be pGlu;
further provided that, if A.sup.0 or A.sup.1 is present, A.sup.2
cannot be pGlu; further provided that, when A.sup.0 is deleted and
A.sup.1 is pGlu, R.sub.1 must be H and R.sub.2 must be the portion
of Glu that forms the imine ring in pGlu; and further provided
that, W can be any one of the following: 17wherein R.sub.3 is
CHR.sub.20--(CH.sub.Z).sub.n1 (where R.sub.20 is either of H or OH;
and n1 is either of 1 or 0), or is deleted, and Z.sub.1 is the
identifying group of any of the amino acids Gly, Ala, Val, Leu,
Ile, Ser, Asp, Asn, Glu, Gln, p-X-Phe (where X=H, F, Cl, Br,
NO.sub.2, OH, or CH.sub.3), F.sub.5-Phe, Trp, Cys, Met, Pro, HyPro,
cyclohexyl-Ala, or .beta.-nal; and V is either OR.sub.4, or 18where
R.sub.4 is any of C.sub.1-20 alkyl, C.sub.3-20 alkenyl, C.sub.3-20
alkinyl, phenyl, naphthyl, or C.sub.7-10 phenylalkyl, and each
R.sub.5, and R.sub.6, independently, is any of H, C.sub.1-12 alkyl,
C.sub.7-10 phenylalkyl, lower acyl, or, 19where R.sub.22 is any of
H, C.sub.1-12 alkyl, C.sub.7-10 phenylalkyl, or lower acyl;
provided that, when one of R.sub.5 or R.sub.6 is --NHR.sub.22, the
other is H; 20wherein Z.sub.1 is the identifying group of any one
of the amino acids Gly, Ala, Val, Leu, Ile, Ser, Asp, Asn, Glu,
.beta.-Nal, Gln, p-X-Phe (where X=H, F, Cl, Br, NO.sub.2, OH or
CH.sub.3), F.sub.5-Phe, Trp, Cys, Met, Pro, or HyPro; and each
Z.sub.2, Z.sub.3, and Z.sub.4, independently, is H, lower alkyl,
lower phenylalkyl, or lower naphthylalkyl; or 21wherein each
Z.sub.20 and Z.sub.30 independently, is H, lower alkyl, lower
phenylalkyl, lower naphthylalkyl; further provided that, when
either of Z.sub.20 or Z.sub.30 is other than H, A.sup.7 is His,
A.sup.6is Gly, A.sup.5 is Val, A.sup.4 is Ala, A.sup.2 is His, and
either of R.sub.1 or R.sub.2 is other than H, A.sup.1 must be other
than deleted; further provided that, for the formulas (I) through
(III), any asymmetric carbon atom can be R, S or a racemic mixture;
and further provided that each R.sub.1 and R.sub.2, independently,
is H, C.sub.1-12 alkyl, C.sub.7-10 phenylalkyl, COE.sub.1 (where
E.sub.1 is C.sub.1-20 alkyl, C.sub.3-20 alkenyl, C.sub.3-20
alkinyl, phenyl, naphthyl, or C.sub.7-10 phenylalkyl) , or lower
acyl, and R.sub.1 and R.sub.2 are bonded to the N-terminal amino
acid of said peptide, and further provided that when one of R.sub.1
or R.sub.2 is COE.sub.1, the other must be H, or a pharmaceutically
acceptable salt thereof.
2. The therapeutic peptide of claim 1 wherein A.sup.0=Gly, D-Phe,
or is deleted; A.sup.1=p-Glu, D-Phe, D-Ala, D-.beta.-Nal, D-Cpa, or
D-Asn; A.sup.2=Gln, His, 1-methyl-His, or 3-methyl-His;
A.sup.4=Ala; A.sup.5=Val; A.sup.6=Sar, Gly, D-Phe, or D-Ala;
A.sup.7=His; and, where W is (I) and R.sub.3 is CH.sub.2 or
CH.sub.2--CH.sub.2, Z.sub.1 is the identifying group of Leu or Phe,
where W is (I) and R.sub.3 is CHOH--CH.sub.2, Z.sub.1 is the
identifying group of Leu, cyclohexyl-Ala, or Phe and each R.sub.5
and R.sub.6 is H; and where W is (I), V is NHR.sub.6, and R.sub.6
is NH.sub.2; where W is (II ), Z.sub.1 is the identifying group of
any one of the amino acids Leu or p-X-Phe (where X=H, F, Cl, Br,
NO.sub.2, OH or CH.sub.3); and each Z.sub.2, Z.sub.3 and Z.sub.4,
independently, is H, lower alkyl, lower phenylalkyl, or lower
naphthylalkyl; and where w is (III), each Z.sub.20 and Z.sub.30, is
H; and each R.sub.1 and R.sub.2, independently, is H, lower alkyl,
or lower acyl.
3. The therapeutic peptide of claim 2 of the formula:
D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ethylamide.
4. The therapeutic peptide of claim 2 of the formula:
p-Glu-Gln-Trp-Ala-val-Gly-His-statine-amide
5. The therapeutic peptide of claim 2 of the formula:
D-Cpa-Gln-Trp-Ala-Val-Gly-His-.beta.-Leu-NH.sub.2.
6. The peptide of claim 1 wherein W is (I), V is OR.sub.4, and
R.sub.4 is any of C.sub.1-20 alkyl, C.sub.3-20 alkenyl, C.sub.3-20
alkinyl, phenyl, naphthyl, or C.sub.7-10 phenylalkyl, and A.sup.6
is N-methyl-D-Ala or A.sup.1 is D-F.sub.5-Phe.
7. The therapeutic peptide of claim 6 of the formula:
D-Phe-Gln-Trp-Ala-Val-N-methyl-D-Ala-His-Leu-methylester.
8. The therapeutic peptide of claim 2 of the formnula:
D-Cpa-Gln-Trp-Ala-Val-D-Ala-His-.beta.-Leu-NH.sub.2.
Description
BACKGROUND OF THE INVENTION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 397,169, filed Aug. 21, 1989, which is a
continuation-in-part of U.S. patent application Ser. No. 376,555,
filed Jul. 7, 1989, which is a continuation-in-part of U.S. patent
application Ser. No. 317,941, filed Mar. 2, 1989, which is a
continuation-in-part of U.S. patent application Ser. No. 282,328,
filed Dec. 9, 1988, which in turn is a continuation-in-part of U.S.
patent application Ser. No. 257,998, filed Oct. 14, 1988, which in
turn is a continuation-in-part of U.S. patent application Ser. No.
248,771, filed Sep. 23, 1988, which in turn is a
continuation-in-part of Coy et al., U.S. patent application Ser.
No. 207,759, filed Jun. 16, 1988, which in turn is a
continuation-in-part of Coy et al., U.S. patent application Ser.
No. 204,171, filed Jun. 8, 1988, which in turn is a
continuation-in-part of Coy et al., U.S. patent application Ser.
No. 173,311, filed Mar. 25, 1988, which in turn is a
continuation-in-part of Coy et al. U.S. patent application Ser. No.
100,571. filed Sep. 24, 1987.
[0002] This invention relates to therapeutic peptides useful, e.g.,
for treatment of benign or malignant proliferation of tissue, for
gastrointestinal disorders, and for diabetes.
[0003] Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2
(Anastasi et al., Experientia 27:166-167 (1971)), is closely
related to the mammalian gastrin-releasing peptides (GRP), e.g.,
the porcine GRP,
H.sub.2N-Ala-Pro-Val-Ser-Val-Gly-Gly-Gly-Thr-Val-Leu-Ala-Lys-Met-Tyr-Pro--
Arg-Gly-Asn-His-Trp-Ala-Val Val-Gly-His-Leu-Met-(NH.sub.2)
(McDonald et al., Biochem. Biophys. Res. Commun. 90:227-233 (1979))
and human GRP,
H.sub.2N-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu-Thr-Lys-Met-Tyr-Pro--
Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met (NH.sub.2). Bombesin
has been found to be a growth factor for a number of human cancer
cell lines, including small-cell lung carcinoma (SCLC), and has
been detected in human breast and prostate cancer (Haveman et al.,
eds. Recent Results in Cancer Research--Peptide Hormones in Lung
Cancer, Springer-Verlag, New York:1986). A number of these cancers
are known to secrete peptide hormones related to GRP or bombesin.
Consequently, antagonists to bombesin have been proposed as agents
for the treatment of these cancers.
[0004] Cuttitta et al. demonstrated that a specific monoclonal
antibody to bombesin inhibited in vivo the growth of a human
small-cell lung cancer cell line xenografted to nude mice (Cuttitta
et al., Cancer Survey 4:707-727 (1985)). In 3T3 murine fibroblasts
which are responsive to the mitotic effect of bombesin, Zachary and
Rozengurt observed that a substance P antagonist (Spantide) acted
as a bombesin antagonist (Zachary et al., Proc. Natl. Acad. Sci.
(USA), 82:7616-7620 (1985)). Heinz-Erian et al. replaced His at
position 12 in bombesin with D-Phe and observed bombesin antagonist
activity in dispersed acini from guinea pig pancreas (Heinz-Erian
et al., Am. J. of Ph. 252:G439-G44 Rivier reported work directed
toward restricting the conformational freedom of the bioactive
C-terminal decapeptide of bombesin by incorporating intramolecular
disulfide bridges; however, Rivier mentioned that, so far, bombesin
analogs with this modification fail to exhibit any antagonist
activity (Rivier et al., "Competitive Antagonists of Peptide
Hormones," in Abstracts of the International Symposium on
Bombesin-Like Peptides in Health and Disease, Rome, Italy (October,
1987).
[0005] Abbreviations (uncommon):
[0006] cyclohexyl-Ala=(cyclohexyl alanine) 1
[0007] identifying group
[0008] pGlu= 2
[0009] (pyroglutamic acid);
[0010] Nle= 3
[0011] (norleucine)
[0012] Pal=3-pyridyl-alanine
[0013] .beta.-leu=.beta.-homoleucine
[0014] .gamma.-leu=gamma-homoleucine
[0015] D-Cpa=D-p-chlorophenylalanine
[0016] HyPro=hydroxyproline
[0017] Nal=naphthylalanine
[0018] Sar=sarcosine
[0019] F-Phe=penta-fluoro-Phenylalanine
[0020] ine)=(3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid, and
has the chemical structure: 4
[0021] identifying group
[0022] AHPPA=(3s,4s)-4-amino-3-hydroxy-5-phenylpentanoic acid, and
has the chemical structure: 5
[0023] identifying group
[0024] ACHPA=(3S, 4S)-4-amino-5-cyclohexyl-3-hydroxypentanoic acid
and has the chemical structure: 6
[0025] identifying group
[0026] R=right (D) configuration; S=left (L) configuration;
racmate=equal mi 1-methyl-His; 3-methyl-His=methyl (CH.sub.3) group
on nitrogen at positions 1 or 3 of Histidine: 7
SUMMARY OF THE INVENTION
[0027] In general, the invention features a linear (i.e.,
non-cyclic) analog of biologically active mammalian
gastrin-releasing peptide (GRP), amphibian bombesin, or mammalian
growth hormone releasing factor (GRF) having an active site and a
binding site responsible for the binding of the peptide to a
receptor on a target cell; cleavage of a peptide bond in the active
site of naturally occurring bombesin, GRP, or GRF is unnecessary
for in vivo biological activity. The analog has one of the
following modifications: (a) a deletion of an amino acid residue
within the active site and a modification of an amino acid residue
outside of the active site, (b) a replacement of two amino acid
residues within the active site with a synthetic amino acid, e.g.,
statine, AHPPA, or ACHPA, a .beta.-amino acid, or a .gamma.-amino
acid residue, or (c) a non-peptide bond instead of a peptide bond
between an amino acid residue of the active site and an adjacent
amino acid residue.
[0028] In preferred embodiments the analog is capable of acting as
a competitive inhibitor of the naturally occurring peptide by
binding to the receptor and, by virtue of one of the modifications,
failing to exhibit the in vivo biological activity of the naturally
occurring peptide.
[0029] The locations of the modifications that give rise to
antagonists are determined by the location of the active site in
the naturally occuring peptide. For non-peptide bond between the
carboxyl terminal and adjacent amino acid residues, or the
replacement of the natural carboxyl terminal and adjacent amino
acid residues with a synthetic, .beta.-, or .gamma.-amino acid
residue, or the deletion ("des") of the C-terminal amino acid
residue are useful in creating or enhancing antagonist activity are
those in which activity is associated with the two C-terminal amino
acid residues of the amino acid chain. Similarly, where the active
site is located in the amino terminal portion of the naturally
occuring peptide, the corresponding analogs of the invention will
possess modifications in their amino terminal portions.
[0030] In preferred embodiments the active site includes at least
one amino acid residue located in the carboxyl terminal half of the
naturally occurring biologically active peptide and that amino acid
residue is located in the carboxyl terminal half of the linear
peptide.
[0031] In preferred embodiments the binding sites includes at least
one amino acid residue located in the amino terminal half of the
naturally occurring biologically active peptide and that amino acid
residue is located in the amino terminal half of the linear
peptide.
[0032] Modifications can be introduced in a region involved in
receptor binding, or in a non-binding region. Preferably, analogs
of the invention are 25% homologous, most preferably, 50%
homologous, with the naturally occurring peptides.
[0033] The analogs of the invention may have one of the
modifications given in the generic formula given bond between an
amino acid residue of the active site and an adjacent amino acid
residue; or a synthetic amino acid, e.g. a statine, an AHPPA, or an
ACHPA, a .beta.-amino acid, or a .gamma.-amino acid residue in
place of two natural amino acid residues; or a deletion of the
C-terminal amino acid residue, accompanied by the addition of a
substituent on the actual C-terminal group and the presence of an
N-terminal residue that is not the natural N-terminal amino acid
residue of the peptides from which the analogs are derived.
(Statine, AHPPA, and ACHPA have the chemical structures defined
above. Where statine is used herein, AHPPA or ACHPA may also be
used.)
[0034] By non-peptide bond is meant that the carbon atom
participating in the bond between two residues is reduced from a
carbonyl carbon to a methylene carbon, i.e., CH.sub.2--NH; or, less
preferably that CO--NH is replaced with any of CH.sub.2--S,
CH.sub.2--O, CH.sub.2--CH.sub.2, CH.sub.2--CO, or CO--CH.sub.2. (A
detailed discussion of the chemistry of non-peptide bonds is given
in Coy et al. (1988) Tetrahedron 44,3:835-841, hereby incorporated
by reference, Tourwe (1985) Janssen Chim. Acta 3:3-15, 17-18,
hereby incorporated by reference, and Spatola (1983) in Chemistry
and Biochemistry of Amino Acids, Peptides, and Proteins, (B.
Weinstein, ed.) M. Dekker, New York and Basel, pp. 267-357, hereby
incorporated by reference.)
[0035] One modification of the naturally occurring peptide to
create an antagonist is of the amino terminal end of the molecule,
such as those described for the amino terminal positions in the
generic formula below; for example, the N-terminal amino acid
residue, which is A.sup.0 or, if A.sup.0 is deleted, is A.sup.1 or,
if A.sup.0 , is below, may be an aromatic D-isomer, or may be an
alkylated amino acid residue. (Where "D" is not designated as the
configuration of an amino acid, L is intended.)
[0036] The therapeutic peptide includes between seven and ten amino
acid residues, inclusive, and is an analog of one of the following
peptides terminating at the carboxcy-terminus with a Met residue:
(a) litorin; (b) the ten amino acid carboxy-terminal region of
mammalian GRP; and (c) the ten amino acid carboxy-terminal region
of amphibian bombesin. The therapeutic peptide is of the following
formula: 8
[0037] wherein
[0038] A.sup.0=pGlu, Gly, Nle, .alpha.-aminobutyric acid, or the
D-isomer of any of Ala, Val, Gln, Asn, Leu, Ile, Met, p-X-Phe
(where X=F, Cl, Br, NO.sub.2, OH, H or CH.sub.3) Trp, Cys, or
.beta.-Nal, or is deleted;
[0039] A.sup.1=the D or L-isomer of any of pGlu, Nle,
.alpha.-aminobutyric acid, or the D-isomer of any of Ala, Val, Gln,
Asn, Leu, Ile, Met, p-X-Phe (where X=F, Cl, Br, NO.sub.2, OH, H or
CH.sub.3) F.sub.5-Phe, Trp, Cys, or .beta.-Nal, or is deleted;
[0040] A.sup.2=pGlu, Gly, Ala, Val, Gln, Asn, Leu, Ile, Met,
p-X-Phe (where X=F, Cl, Br, NO.sub.2, OH, H or CH.sub.3), Trp, Cys,
.beta.-Nal, His, 1-methyl-His, or 3-methyl-His;
[0041] A.sup.4=Ala, Val, Gln, Asn, Gly, Leu, Ile. Nle, yric acid,
et, p--Phe (were X=F, Cl, Br, N.sub.2, OH, H or CH.sub.3), Trp,
Cys, or .beta.-Nal;
[0042] A.sup.5=Gln, Asn, Gly, Ala, Leu, Ile, Nle,
.alpha.-aminobutyric acid, Met, Val, p-X-Phe (where X=F, Cl, Br,
OH, H or CH.sub.3), Trp, Thr, or .beta.-Nal;
[0043] A.sup.6=Sar, Gly, or the D-isomer of any of Ala,
N-methyl-Ala, Val, Gln, Asn, Leu, Ile, Met, p-X-Phe (where X=F, Cl,
Br, NO.sub.2, OH, H or CH.sub.3), Trp, Cys, or .beta.-Nal;
[0044] A.sup.7=1-methyl-His, 3-methyl-His, or His;
[0045] provided that, if A.sup.0 is present, A.sup.1 cannot be
pGlu; further provided that, if A.sup.0 or A.sup.1 is present,
A.sup.2 cannot be pGlu; further provided that, when A.sup.0 is
deleted and A.sup.1 is pGlu, R.sub.1 must be H and R.sub.2 must be
the portion of Glu that forms the imine ring in pGlu; and further
provided that, W can be any one of the following: 9
[0046] wherein R.sub.3 is CHR.sub.20--(CH.sub.2).sub.n1 (where
R.sub.20 is either of H or OH; and n1 is either of 1 or 0), or is
deleted, and Z.sub.1 is the identifying group of any of the amino
acids Gly, Ala, Val, Leu, Ile, Ser, Asp, Asn, Glu, Gln, p-X-Phe
(where X=H, F, Cl, Br, NO.sub.2, OH, or CH.sub.3), F.sub.5-Phe,
Trp, Cys, Met, Pro, HyPro, cyclohexyl-Ala, or .beta.-nal; and V is
either OR.sub.4, or 10
[0047] where R.sub.4 is any of C.sub.1-20 alkyl, C.sub.3-20
alkenyl, C.sub.3-20 alkinyl, phenyl, naphthyl, or C.sub.7-10
phenylalkyl, and each R.sub.5, and R.sub.6, independently, is any
of H, C.sub.1-12 alkyl, C.sub.7-10 phenylalkyl, lower acyl, or,
11
[0048] where R.sub.22 is any of H, C.sub.1-12 alkyl, C.sub.7-10
phenylalkyl, or lower acyl; provided that, when one of R.sub.5 or
R.sub.6 is --NHR.sub.22, the other is H; 12
[0049] wherein R.sub.4 is CH.sub.2--NH, CH.sub.2--S, CH.sub.2--O
CO--CH.sub.2, CH.sub.2--CO, or CH.sub.2--CH.sub.2, and each Z.sub.1
and Z.sub.2, independently, is the identifying group of any one of
the amino acids Gly, Ala, Val, Leu, Ile, Ser, Asp, Asn, Glu, Gln,
.beta.-Nal, p-X-Phe (where X=H, F, Cl, Br, NO.sub.2, OH or
CH.sub.3), F.sub.5-Phe, Trp, Cys, Met, Pro, HyPro, or
cylcohexyl-Ala; and V is either OR.sub.5 or 13
[0050] where each R.sub.3, R.sub.5, R.sub.6, and R.sub.7,
independently, is H, lower alkyl, lower phenylalkyl, or lower
naphthylalkyl; 14
[0051] wherein Z.sub.1 is the identifying group of any one of the
amino acids Gly, Ala, Val, Leu, Ile, Ser, Asp, Asn, Glu,
.beta.-Nal, Gln, p-X-Phe (where X=H, F, Cl, Br, NO.sub.2, OH or
CH.sub.3), F.sub.5-Phe, Trp, Cys, Met, Pro, or HyPro; and each
Z.sub.2, Z.sub.3 and Z.sub.4, independently, is H, lower alkyl,
lower phenylalkyl, or lower naphthylalkyl; or 15
[0052] wherein each Z.sub.20 and Z.sub.30 independently, is H,
lower alkyl, lower phenylalkyl, lower naphthylalkyl; further
provided that, when either of Z.sub.20 or Z.sub.30 is other than H,
A.sup.7 is His, A.sup.6 is Gly, A is Val, A.sup.4 is Ala, A.sup.2
is His, and either of R.sub.1 or R.sub.2 is other than H, A.sup.1
must be other than deleted; further provided that, for the formulas
(I) through (IV), any asymmetric carbon atom can be R, S or a
racemic mixture; independently, is H, C.sub.1-12 alkyl, C.sub.7-10
phenylalkyl, COE.sub.1 (where E.sub.1 is C.sub.1-20 alkyl,
C.sub.3-20 alkenyl, C.sub.3-20 alkinyl, phenyl, naphthyl, or
C.sub.7-10 phenylalkyl), or lower acyl, and R.sub.1 and R.sub.2 are
bonded to the N-terminal amino acid of said peptide, and further
provided that when one of R.sub.1 or R.sub.2 is COE.sub.1, the
other must be H, or a pharmaceutically acceptable salt thereof.
[0053] Preferably, the amino acid sequence of the therapeutic
peptide of the generic formula (id) is at least 25% homologous with
the amino acid sequence of the naturally occurring peptide; most
preferably, this homology is at least 50%.
[0054] More preferably, in the generic formula above,
[0055] A.sup.0=Gly, D-Phe, or is deleted;
[0056] A.sup.1=p-Glu, D-Phe, D-Ala, D-.beta.-Nal, D-Cpa, or
D-Asn;
[0057] A.sup.2=Gin, His, 1-methyl-His, or 3-methyl-His;
[0058] A.sup.4=Ala;
[0059] A.sup.5=Val;
[0060] A.sup.6=Sar, Gly, D-Phe, or D-Ala;
[0061] A.sup.7=His; and
[0062] and, where W is (I) and R.sub.3 is CH.sub.2 or
CH.sub.2--CH.sub.2, Z.sub.1 is the identifying group of Leu or Phe,
where W is (I) and R.sub.3 is CHOH--CH.sub.2, Z.sub.1 is the
identifying group of Leu, cyclohexyl-Ala, or Phe and each R.sub.5
and R.sub.6 is H, and where W is (I), V is NHR.sub.6 and R.sub.6 is
NH.sub.2; where W is (II) and R.sub.4 is CH.sub.2--NH each Z.sub.1
is the identifying group of Leu, or Phe, and Z.sub.2 is the
identifying group of Leu or Phe; where W is (III), Z.sub.1 is the
identifying group of any one of the amino acids Leu or p-X-Phe
(where X=H, F, Cl, Br, NO2# OH or CH.sub.3); and each Z.sub.2,
Z.sub.3 and Z.sub.4, independently, is lower phenylalk, or
naphthylalkyl; and where W is (IV), each Z.sub.20 and Z.sub.30, is
H; and each R.sub.1 and R.sub.2, independently, is H, lower alkyl,
or lower acyl. Preferred peptides include those in which W is (II),
R.sub.4 is CH.sub.2--NH, and the carbon atom bonded to Z.sub.2 is
of the R configuration.
[0063] Examples of preferred bombesin or GRP peptides are:
[0064]
D-.beta.-Nal-Gln-Trp-Ala-Val-Gly-His-Leu.psi.[CH.sub.2NH]Phe-NH.sub-
.2,
[0065] D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ethylamide,
[0066] p-Glu-Gln-Trp-Ala-Val-Gly-His-statine-amide,
[0067]
D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu.psi.(CH.sub.2NH]-D-Phe-NH.sub.2,
[0068] D-Cpa-Gln-Trp-Ala-Val-Gly-His-.beta.-Leu-NH.sub.2,
[0069] D-Cpa-Gln-Trp-Ala-Val-D-Ala-His-.beta.-Leu-NH.sub.2,
[0070]
D-Cpa-Gln-Trp-Ala-Val-Gly-His-Leu.psi.[CH.sub.2NH]-Phe-NH.sub.2,
[0071] In another preferred embodiment, W is (I), V is OR.sub.4,
R.sub.4 can be any substituent provided in (I) of the generic
formula (id), and A.sup.6 is N-methyl-D-Ala, i.e., a preferred
peptide contains both a carboxy terminal ester component in
combination with N-methyl-D-Ala, in position A.sup.6; or
D-F.sub.5-Phe in position A.sup.1.
[0072] Examples of preferred peptides are
D-Phe-Gln-Trp-Ala-Val-N-methyl-D- -Ala-His-Leu-methylester.
D-F.sub.5-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-meth- ylester.
[0073] An example of a preferred GRF peptide of the invention is
Tyr-Ala.sup.2-Asp-Ala-Ile-Phe-Thr-Asn-Ser.psi.[CH.sub.2NH]Tyr-Arg-Lys-Val-
-leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser
-Arg-NH.sub.2; most preferably, the peptide contains, D-Ala,
N-methyl-D-Ala, or a-aminobutyric acid in position 2. (Non-peptide
bonds in which the peptide or ".psi.".)
[0074] Antagonists of the invention are useful for treating
diseases involving the malignant or benign proliferation of tissue,
such as all forms of cancer where bombesin-related, GRP-related, or
GRF-related substances act as autocrine or paracrine mitotic
factors, e.g., cancers of the gastrointestinal tract, pancreatic
cancer, colon cancer, lung cancer, particularly the small cell
subtype, prostate or breast cancer; or for treating
artherosclerosis, and disorders of gastrointestinal tissues related
to gastric and pancreatic secretions and motility; for example, for
causing the suppression of amylase secretion, or for appetite
control. GRF antagonists suppress the release of growth hormone
and, therefore, may be used to slow down the progression of
muscular dystrophy or for treating diabetes, or diabetes-related
retinopathy.
[0075] In the generic formulas given above, when R.sub.1, R.sub.2,
R.sub.4 of I, R.sub.5 of I, R.sub.6 of I, R.sub.22 of I, R.sub.3 of
II, R.sub.5 of II, R.sub.6 of II, R.sub.7 of II, Z.sub.2 of III,
Z.sub.3 of III, Z.sub.4 of III, Z.sub.20 of IV, or Z.sub.30 of IV
is an aromatic, lipophilic group, the in vivo activity can be long
lasting, and delivery of the compounds of the invention to the
target tissue can be facilitated.
[0076] The identifying group of an .alpha.-amino acid is the atom
or group of atoms, other than the .alpha.-carbonyl carbon atom, the
.alpha.-amino nitrogen atom, or the H atom, bound to the asymmetric
.alpha.-carbon atom. To illustrate by examples, the identifying
group of alanine is CH.sub.3, the identifying group of valine is
(CH.sub.3).sub.2CH, the identifying group of lysine is
H.sub.3N.sup.+(CH.sub.2).sub.4 and the identifying group of
phenylalanine is aor .gamma.-amino acid is the analagous atom or
group of atoms bound to respectively, the .beta.-or the
.gamma.-carbon atom. Where the identifying group of an amino acid
is not specified it may be .alpha., .beta., or .gamma..
[0077] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof. and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0078] We first briefly describe the drawings.
DRAWINGS
[0079] FIG. 1 is a graph of tumor growth curves for the small cell
lung cancer (NCI-H69) xenografts.
[0080] FIG. 2 is a series of amino acid sequences of naturally
occurring peptides of which peptides of the invention are
analogs.
[0081] FIG. 3 is a series of amino acid sequence of naturally
occurring peptides of the VIP peptide family, of which GRF peptides
of the invention are analogs.
[0082] FIGS. 4 and 5 are graphs showing the effect of pseudopeptide
bond containing analogs of GRF(1-29)NH.sub.2 on GH secretion from
dispersed rat pituitary cells.
[0083] FIG. 6 is a graph showing the antagonism of GRF stimulated
GH secretion by
Ser.sup.9.psi.[CH.sub.2NH]Tyr.sup.10GRF(1-29)NH.sub.2. We now
describe the structure, synthesis, and use of the preferred
embodiments of the invention.
STRUCTURE
[0084] Peptides of the invention have either a non-peptide bond in
at least one of the indicated positions, or a statine, .beta.-amino
acid, or .gamma.-amino acid substitution, e.g.,
sta.sup.8-des-Met.sup.9 litorin. By non-peptide bond is meant e.g.,
that the carbon atom participating in the bond between two residues
is arbonyl carbon to a methylene carbon. The peptide bond reduction
method which yields this non-peptide bond is described in Coy et
al., U.S. patent application, Ser. No. 879,348, assigned to the
same assignee as the present application, hereby incorporated by
reference. Any one of the amino acids in positions 0, 1, 2, and 9
of the litorin antagonists may be deleted from the peptides, and
the peptides are still active as antagonists.
[0085] The peptides of the invention can be provided in the form of
pharmaceutically acceptable salts. Examples of preferred salts are
those with therapeutically acceptable organic acids, e.g., acetic,
lactic, maleic, citric, malic, ascorbic, succinic, benzoic,
salicylic, methanesulfonic, toluenesulfonic, or pamoic acid, as
well as polymeric acids such as tannic acid or carboxymethyl
cellulose, and salts with inorganic acids such as the hydrohalic
acids, e.g., hydrochloric acid, sulfuric acid, or phosphoric
acid.
Synthesis of Litorin, Bombesin, and GRF Analogs
[0086] The synthesis of the bombesin antagonist
pGlu-Gln-Trp-Ala-Val-Gly-H- is-Leu.psi.[CH.sub.2NH]Leu-NH.sub.2
follows. Other bombesin, GRP, or GRF antagonists can be prepared by
making appropriate modifications of the following synthetic
methods.
[0087] The first step is the preparation of the intermediate
pGlu-Gln-Trp-Ala-Val-Gly-His(benzyloxycarbonyl)-Leu.psi.[CH.sub.2
NH]Leu-benzhydrylamine resin, as follows.
[0088] Benzhydrylamine-polystyrene resin (Vega Biochemicals, Inc.)
(0.97 g, 0.5 mmole) in the chloride ion form is placed in the
reaction vessel of a Beckman 990B peptide ssizer programmed to
perform th following reaction cycle: (a) methylene chloride; (b)
33% trifluoroacetic acid (TFA) in methylene chloride (2 times for 1
and 25 min. each); (c) methylene chloride; (d) ethanol; (e)
methylene chloride; and (f) 10% triethylamine in chloroform.
[0089] The neutralized resin is stirred with
alpha-t-butoxycarbonyl(Boc)-l- eucine and diisopropylcarbodiimide
(1.5 mmole each) in methylene chloride for 1 hour, and the
resulting amino acid resin is then cycled through steps (a) to (f)
in the above wash program. Boc-leucine aldehyde (1.25 mmoles),
prepared by the method of Fehrentz and Castro, Synthesis, p. 676
(1983), is dissolved in 5 ml of dry dimethylformamide (DMF) and
added to the resin TFA salt suspension followed by the addition of
100 mg (2 mmoles) of sodium cyanoborohydride (Sasaki and Coy,
Peptides 8:119-121 (1987); Coy et al., id.). After stirring for 1
hour, the resin mixture is found to be negative to ninhydrin
reaction (1 min.), indicating complete derivatization of the free
amino group.
[0090] The following amino acids (1.5 mmole) are then coupled
successively in the presence diisopropylcarbodiimide (1.5 mmole),
and the resulting amino acid resin is cycled through
washing/deblocking steps (a) to (f) in the same procedure as above:
Boc-His(benzyloxycarbonyl), Boc-Gly (coupled as a 6 M excess of the
p-nitrophenylester), Boc-Val, Boc-Ala, Boc-Trp, Boc-Gln (coupled as
a 6 M excess of the p-nitrophenylester), and pGlu. The completed
resin is then washed with methanol and air dried.
[0091] The resin described above (1.6 g, 0.5 mmole) is mixed with
anisole (5 ml) and anhydrous hydrogen fluoride (35 ml) at 0.degree.
C. and stirred for 45 min. Excess hydrogen fluoride is evaporated
rapidly under a stream or ary nitrogen, anc tree peptide is
precipitated and washed with ether. The crude peptide is dissolved
in a minimum volume of 2 M acetic acid and eluted on a column
(2.5.times.100 mm) of Sephadex G-25 (Pharmacia Fine Chemicals,
Inc.). Fractions containing a major component by uv absorption and
thin layer chromatography (TLC) are then pooled, evaporated to a
small volume and applied to a column (2.5.times.50 cm) of
octadecylsilane-silica (Whatman LRP-1, 15-20 .mu.m mesh size).
[0092] The peptide is eluted with a linear gradient of 0-30%
acetonitrile in 0.1% trifluoroacetic acid in water. Fractions are
examined by TLC and analytical high performance liquid
chromatography (HPLC) and pooled to give maximum purity. Repeated
lyophilization of the solution from water gives 60 mg of the
product as a white, fluffy powder.
[0093] The product is found to be homogeneous by HPLC and TLC.
Amino acid analysis of an acid hydrolysate confirms the composition
of the peptide. The presence of the Leu.psi.[CCH.sub.2--NH]Leu bond
is demonstrated by fast atom bombardment mass spectrometry.
[0094]
pGlu-Gln-Trp-Ala-Val-Gly-His-Phe.psi.[CH.sub.2NH]Leu-NH.sub.2
and
[0095]
pGlu-Gln-Trp-Ala-Val-Gly-His-Leu.psi.[CH.sub.2NH]Leu-NH.sub.2 or
other peptides are prepared in similar yields in an analogous
fashion by appropriately modifying the above procedure.
[0096] Solid phase synthesis ofD-Phe.sup.1,
Leu.sup.8.psi.[CH.sub.2NH]D-Ph- e.sup.9-litorin,
D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu.psi.[CH.sub.2NH]-D-Phe--
NH.sub.2, was carried out as follows:
[0097]
-Val-Gly-His(tosyl)-Leu.psi.[CH.sub.2NH)-D-Phe-benzhydrylamine
resin was synthesized first.
[0098] Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc.)
(1.25 g, 0.5 mmole) in the chloride ion form is placed in the
reaction vessel of an Advanced ChemTech ACT 200 peptide synthesizer
programmed to perform the following reaction cycle: (a) methylene
chloride; (b) 33% trifluoroacetic acid in methylene chloride (2
times for 1 and 25 min each); (c) methylene chloride; (d) ethanol;
(e) methylene chloride; (f) 10% triethylamine in chloroform.
[0099] The neutralized resin is stirred with Boc-D-phenylalanine
and diisopropylcarbodiimide (1.5 mmole each) in methylene chloride
for 1 h and the resulting amino acid resin is then cycled through
steps (a) to (g) in the above wash program. The Boc group is then
removed by TFA treatment. Boc-leucine aldehyde (1.25 mmoles),
prepared by the method of Fehrentz and Castro (1), is dissolved in
5 ml of dry DMF and added to the resin TFA salt suspension followed
by the addition of 100 mg (2 mmoles) of sodium cyanoborohydride
(2,3). After stirring for 1 h, the resin mixure is found to be
negative to ninhydrin reaction (1 min) indicating complete
derivatization of the free amino group.
[0100] The following amino acids (1.5 mmole) are then coupled
successively by the same procedure:
[0101] Boc-His(benzyloxycarbonyl), Boc-Gly, Boc-Val, Boc-Ala,
Boc-Trp, Boc-Gln (coupled in the presence of 1 equiv.
hydroxybenzotriazole), Boc-D-Phe (coupled in the presence of 1
equiv. hydroxybenzotriazole). After drying, the peptide resin
weighed 1.93 g.
[0102] The resin (1.93 g, 0.5 mmole) is mixed with anisole (5 ml)
and anhydrous hydrogen fluoride (35 ml) at 0.degree. C. and stirred
for 45 min. Excess hydrogen fluoride is evaporated rapidly under a
stream of dry nitrogen and free peptide precipitated and washed
with ether. The crude peptide is dissolved in a minimum volume of 2
M acetic acid and eluted on a column (2.5.times.100 mm) of Sephadex
G-25. Fractions containing a major component by uv absorption and
thin layer chromatography are then pooled, evaporated to a small
volume and applied to a column (2.5.times.50 cm) of Vydac
octadecylsilane (10-15 .mu.M). This is eluted with a linear
gradient of 15-45% acetonitrile in 0.1% trifluoroacetic acid in
water. Fractions are examined by thin layer chromatography and
analytical high performance liquid chromatography and pooled to
give maximum purity. Repeated lyophlization of the solution from
water gives 120 mg of the product as a white, fluffy powder.
[0103] The product is found to be homogeneous by hplc and tlc.
Amino acid analysis of an acid hydrolysate confirms the composition
of the octapeptide. The presence of the Leu.psi.[CH.sub.2NH]
peptide bond is demonstrated by fast atom bombardment mass
spectrometry.
[0104] Solid phase synthesis of D-Phe.sup.1-Leu.sup.8des-Met.sup.9
litorin, D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NH.sub.2, was carried
out as follows.
[0105] Step (1): Benzhydrylamine-polystyrene resin (Advanced
ChemTech, Inc. (0.62 gm, 0.25 mmole) in the chloride ion form is
placed in the reacton vessel of an ACT 200 peptide synthesizer
programmed to perform the following reaction cycle: (a) methylene
chloride; (b) 33% trifluoroacetic acid in methylene chloride (2
times for 1 and 25 min each); (c) methylene chloride; (d) ethanol;
(e) methylene chloride; (f) 10% triethylamine in chloroform.
[0106] The neutralized resin is stirred with Boc-leucine and
diisopropylcarbodiimide (1.5 mmole each) in methylene chloride for
1 hr and the resulting amino acid resin is then cycled through
steps (a) to (g) in the above wash program. The following amino
acids (1.5 mmole) are then coupled successively by the same
procedure: Boc-His (benzyloxvcarbonyl), Boc-Gly, Boc-Val, Boc-Ala,
Boc-Trp, Boc-Gln (coupled as a 6M excess of the p-nitrophenylester,
and pGlu (coupled in the presence of hydroxzybenzotriazole). After
drying, the peptide resin weighed 0.92 g.
[0107] Step (2): The resin (0.92 g) is then mixed with anisole (5
ml), dithiothreitol (200 mg) and anhydrous hydrogen fluoride (35
ml) at 0.degree. C. and stirred for 45 min. Excess hydrogen
fluoride is evaporated rapidly under a stream of dry nitrogen and
free peptide precipitated and washed with ether. The crude peptide
is dissolved in a minimum volume of 2 M acetic acid and eluted on a
column (2.5.times.100 cm) of Sephadex G-25. Fractions containing a
major component by UV absorption and thin layer chromatography are
then pooled, evaporated to a small volume and applied to a column
(2.5.times.50 cm) of Vydac octadecylsilane (10-15 microM). The
column is eluted with a linear gradient of 0-30% acetonitrile in
0.1% trifluoroacetic acid in water. Fractions are examined by thin
layer chromatography and pooled to give maximum purity. Repeated
lyophilization of the solution from water gives a white, fluffy
powder; this product is found to be homogeneous by hplc and tlc.
Amino acid analysis of an acid hydrolysate confirms the composition
of the peptide.
[0108] Synthesis of D-Nal-Gln-Trp-Ala-Val-Gly-His-Leu-NH was
accomplished using the same procedure as described above (0.62 g,
0.25 mmole of benzyhydrylamine resin in step (1), and 0.92 g in
step (2)).
[0109] Synthesis of
N-acetyl-D-phe-Gln-Trp-Ala-Val-Gly-His-Leu-NH.sub.2 was
accomplished using the same procedure as that described above,
using 0.62 g (0.25 mmole) of benzhydrylamine resin in step (1), and
mixing 0.92 g of the resin with anisole in step(2), except that the
final Boc group was removed and the resin acetylated with acetic
anhydride in methylene chloride.
[0110] Synthesis of
D-Phe-Gln-Trp-Ala-Val-N-Me-D-Ala-His(Tos)-Leu-O-resin is as
follows:
[0111] Boc-Leu-O-Merrifield resin (1.0 g. 0.5 mmole) is placed in
the reaction vessel of an Advanced ChemTech ACT 200 automatic
peptide synthesizer programmed to perform the following
reaction/wash cycle: (a) methylene chloride; (b) 33%
trifluoroacetic acid in methylene chloride (2 times for 1 and 25
min. each); (c) propanol; (d) dimethylformamide; (e) 10%
triethylamine in dimethylformamide; (f) dimethylformamide.
[0112] The neutralized resin is stirred with
Boc-N.sup.im-tosyl-histidine and diisopropylcarbodiimide (1.5 mmole
each) in methylene chloride for 1 h. and the resulting amino acid
resin is then cycled through steps (a) to (f) in the above wash
program. The Boc group is then removed by TFA treatment. The
following amino acids (1.5 mmole) are then coupled successively by
the same procedure: Boc-N-Me-D-Ala (purchased from Bachem, Inc.,
CA), Boc-Val, Boc-Ala, Boc-Trp, Boc-Gln (coupled in the presence of
1 equiv. hydroxybenzotriazole), and Boc-D-Phe. After the last
coupling was complete, the final Boc group was removed by TFA
treatment as already tide resin weighed 1.28 g.
[0113] Synthesis of D-F.sub.5-Phe-Gln-Trp-Ala-Val-DA
His(Tos)-Leu-O-resin is as follows:
[0114] This analogue is prepared under the same conditions
described above, except that Boc-D-Ala is used in place of
N-Me-D-Ala and Boc-D-F.sub.5Phe in place of D-Phe.
[0115] Synthesis of
D-F.sub.5Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-methyl ester is as
follows.
[0116] This peptide is cleaved from the Merrifield resin described
above under the same conditions, to give 198 mg of the product as a
white, fluffy powder.
[0117] The product is found to be homogeneous by hplc and tlc.
Amino acid analysis of an acid hydrolysate confirms the composition
of the octapeptide and fast atom bombardment mass spectrometry
gives the expected molecular weight for the peptide.
[0118] Synthesis of D-Phe-Gln-Trp-Ala-Val-N-Me-D-Ala-His-Leu-methyl
ester is as follows:
[0119] The Merrifield resin described above (1.28 g, 0.5 mmole) is
suspended in methanol containing 10% triethylamine and stirred at
room temperature for 3 days. After filtration, the solution is
evaporated to an oil which is dissolved in water and eluted on a
column of Vydac octadecylsilane (10-15 .mu.M) with a linear
gradient of 10-40% acetonitrile in 0.1% trifluoroacetic acid in
water. Fractions are examined by thin layer chromatography and
analytical high performance liquid chromatography and pooled to
give lization of the solution from water gives 49 mg of the product
as a white, fluffy powder.
[0120] The product is found to be homogeneous by hplc and tlc.
Amino acid analysis of an acid hydrolysate confirms the composition
of the octapeptide and fast atom bombardment mass spectrometry
gives the expected molecular weight for the peptide.
[0121] The synthesis of Sta.sup.8-des-Met.sup.9 litorin follows. A
statine, AHPPA, or ACHPA residue can be substituted in place of any
two amino acids of the peptide, where the peptide contains only
peptide bonds. For example, sta.sup.8-des Met.sup.9 litorin was
prepared in an analagous fashion by first coupling statine to the
resin and then proceeding with the addition of
Boc-His(benzylocarbonyl). Statine or Boc-statine can be synthesized
according to the method of Rich et al., 1978, J. Organic Chem. 43;
3624; and Rich et al., 1980, J. Med. Chem. 23: 27, and AHPPA and
ACHPA can be synthesized according to the method of Hui et al.,
1987, J. Med. Chem. 30: 1287; Schuda et al., 1988, J. Org. Chem.
53:873; and Rich et al., 1988, J. Org. Chem. 53:869.
[0122] Solid-phase synthesis of the peptide BIM-26120,
pGlu-Gln-Trp-Ala-Val-Gly-His-Sta-NH.sub.2, was accomplished through
the use of the following procedures in which alpha-t-butoxycarbonyl
statine (prepared by the procedure of Rich et al., J. Org. Chem.
1978, 43, 3624) is first coupled to
methylbenzhydrylamine-polystyrene resin. After acetylation, the
intermediate p-Glu-Gln-Gln-Trp-Ala-Val-Gly-His(benzyloxy-
carbonyl)-Sta-methylbenzhydrylamine resin is Drepared. The
synthetic procedure used tor this preparation tollows in
detail:
[0123] 1. Incorporation of alpha-t-butoxycarbonyl statine on
methylbenzhydrylamine resin.
[0124] Methylbenzhydrylamine-polystyrene resin (Vega Biochemicals,
Inc.) (1.0 g, 0.73 mmol) in the chloride ion form is placed in the
reaction vessel of a Vega 250C Coupler peptide synthesizer. The
synthesizer was programmed to perform the following reactions: (a)
methylene chloride; (b) 10% triethylamine in chloroform; (c)
methylene chloride; and (d) dimethylformamide.
[0125] The neutralized resin is mixed for 18 hours with the
preformed active ester made from alpha-t-butoxycarbonyl statine
(1.46 mmol), diisopropyl carbodiimide (2 mmol), and
hydroxybenzotriazole hydrate (1.46 mmol in dimethylformamide at
0.degree. C. for one hour. The resulting amino acid resin is washed
on the synthesizer with dimethylformamide and then methylene
chloride. The resin mixture at this point was found by the Kaiser
ninhydrin test (5 minutes) to have an 84% level of statine
incorporation on the resin.
[0126] Acetylation was performed by mixing the amino-acid resin for
15 minutes with N-acetyl imidazole (5 mmol) in methylene chloride.
Derivatization to the 94-99% level of the free amino groups of the
resin was indicated by the Kaiser ninhydrin test (5 minutes). The
Boc-statine-resin is then washed with methylene chloride.
[0127] 2. Couplings of the Remaining Amino Acids.
[0128] The peptide synthesizer is programmed to perform the
following reaction cycle: (a) methylene chloride; (b) 33%
trifluoroacetic acid (TFA) in methylene chloride (2 times for 5 and
25 min. each); (c) lene chloride: (d) isol alcohol; (e) 10%
triethylamine in chloroform; and (f) methylene chloride.
[0129] The following amino acids (2.19 mmol) are then coupled
successively by diisopropyl carbodiimide (4 mmol) alone or
diisopropyl carbodiimide (4 mmol) plus hydroxybenzotriazole hydrate
(1-47 or 0.73 mmol) and the resulting peptide-resin is washed on
the synthesizer with dimethylformamide and then methylene chloride,
and then cycled through the washing and deblocking steps (a) to (f)
in the procedure described above.
[0130] Boc-His (benzyloxycarbonyl) (coupled in the presence of 2
equivalents hydroxybenzotriazole); Boc-Gly; Boc-Val; Boc-Ala and
Boc-Trp (coupled as the preformed hydroxybenzotriazole active
esters made by reaction at 0.degree. C. for one hour with 1
equivalent hydroxybenzotriazole hydrate); Boc-Gln and pGlu (also
coupled as the preformed active esters of hydroxybenzotriazole made
by reaction at 0.degree. C. for one hour with 1 equivalent
hydroxybenzotriazole hydrate). The completed peptide-resin is then
washed with methanol and air dried.
[0131] The peptide-resin described above (1.60 g, 0.73 mmol) is
mixed with anisole (2.5 mL), dithioerythreitol (50 mg), and
anhydrous hydrogen fluoride (30 mL) at 0.degree. C. for one hour.
Excess hydrogen fluoride is evaporated rapidly under a stream of
dry nitrogen, and the free peptide is precipitated and washed with
ether. The crude peptide is dissolved in 100 mL of 1 M acetic acid
and the solution is then evaporated under reduced pressure. The
crude peptide is dissolved in a minimum volume of methanol/water
1/1 and triturated with 10 volumes of ethyl acetate.
[0132] The triturated peptide is applied to a column (9.4 mm
I.D..times.50 cm) of octadecylsilane-silica (Whatman .is linear
gradient of 20-80% of 20/80 0.1% trifluoroacetic acid/acetonitrile
in 0.1% trifluoroacetic acid in water. Fractions are examined by
TLC and analytical high performance liquid chromatography (HPLC)
and pooled to give maximum purity. Lyophilization of the solution
from water gives 77 mg of the product as a white fluffy powder.
[0133] Other compounds including D-Cpa.sup.1,
.beta.-leu.sup.8.sub.1, desMet.sup.9 Litorin can be prepared as
above and tested for effectiveness as agonists or antagonists in
the test program described below.
[0134] GRF analogues were prepared by solid-phase synthesis on
methylbenzhydrylamine resin. The parent GRF(1-29) peptidyl-resin
was assembled on 4-methylbenzhydrylamine functionalized, 1%
crosslinked polystyrene resin (0.41 mequiv. g.sup.-1), on 2 mmol
scale utilizing an Advanced ChemTech ACT 200 synthesizer, using the
following protocol: deblocking, 33% TFA (1 min, 25 min); DCM wash
cycle; PrOH wash cycle; neutralization, 10% DIEA (2 wash cycles);
DMF wash cycle; coupling of preformed HOBt esters (formed during
deprotection), 45 min in DMF, 15 min DMAP; PrOH wash cycle; DCM
wash cycle. Coupling reactions were monitored qualitatively with
the ninhydrin test (Kaiser et al, Anal. Biochem. 1970, 34, 595).
The peptidylresin was divided into aliquots and the various
analogues then assembled on a 0.25 mmol scale. The reduced peptide
bonds were formed by the reductive alkylation of the deprotected
N.sup..alpha.-amino group with the appropriate protected amino acid
aldehyde (3.0 equiv.) in the presence of NaBH.sub.3CN (10 equiv.)
in DMF (25 mL) containing 1% acetic acid at ambient temperature for
16 .
[0135] The introduction of the reduced peptide bond was
accomplished by the reductive alkylation of the resin-bound peptide
amino terminus with a preformed protected amino acid aldehyde
(Sasaki et al., Peptides 1987, 8, 119; Sasaki et al, J. Med. Chem.
1987, 30, 1162). The protected amino acid aldehydes were prepared
in two steps using a modification of the method of Fehrentz and
Castro: (Fehrentz, Synthesis, 1983, 676) the protected amino acids
were converted to the corresponding N,O-dimethylhydroxamates by
reaction with an excess of N,O-dimethylhydroxylamine hydrochloride
(1.1 equiv.) and dicyclohexylcarbodiimide (1.1 equiv.) in
dichloromethane containing an excess of diisopropylethylamine (4
equiv.) at 0.degree. C. The reaction was allowed to warm up to
ambient temperature over 16 h with stirring. The crude
N,O-dimethylhydroxamates were isolated as oils after washing with 3
M HCl (3.times.30 mL), 3 M NaOH (3.times.30 mL), water (3.times.30
mL), drying over MgSO.sub.4 and evaporation to dryness at reduced
pressure. The N,O-hydroxamates were then reduced with LiAlH.sub.4
in tetrahydrofuran at 0.degree. C. The reaction was followed by TLC
and the crude aldehydes isolated as oils which were briefly stored
at -20.degree. C. until used. The isosteres were formed by the
reductive alkylation of the preformed protected amino-acid aldehyde
(3 equiv.) with an excess of NaBH.sub.3CN in acidified DMF. The
progress of the alkylation was monitored with the qualitative
ninhydrin test and most reactions produced a pink or red colour
which was taken as the end point. The method was shown to be free
of racemization in a model study (Coy et al, Tetrahedron 1988, 3,
835). However, appreciable racemization can occur if the aldehyde
is stored for a prolonged period before use. Peptide assembly was
completed using the same protocol as before. No attempt was made to
block any remaining primary amino groups or to protect the
secondary amino group formed during the alkylation, since previous
work has shown this moiety to be unreactive during subsequent
coupling reactions (Sasaki, Peptides, supra; Hocart et al., J. Med.
Chem. 1988, 31, 1820. The peptides were cleaved from the resin
support with simultaneous side-chain acidolysis using anhydrous
hydrogen fluoride containing anisole (.about.30% v/v) and
dithiothreitol (.about.0.6% w/v) as scavengers for 1 h at 0.degree.
C.
[0136] The peptides were then purified by gel filtration and
RP-HPLC to a final purity of >92.5% as judged by analytical
RP-HPLC. The crude peptides were subjected initially to gel
permeation chromatography on Sephadex G50 (2.5.times.100 cm) with 2
M acetic acid eluent. Final purification was effected by
preparative RP-HPLC on C.sub.18 bonded silica gel (Vydac C.sub.18,
10-15 .mu.m. 1.0.times.45 cm) eluted with a linear acetonitrile
gradient with a constant concentration of trifluoroacetic acid
(0.1% v/v). The linear gradient was generated using a
Chromat-a-Trol Model II (Eldex Laboratories Inc.) gradient maker.
The separations were monitored at 280 nm, by TLC on silica gel
plates (Merck F60) and analytical RP-HPLC. The fractions containing
the product were pooled, concentrated in vacuo and subjected to
filtration. Each peptide was obtained as a fluffy white powder of
constant weight by lyophilisation from aqueous acetic acid. The
purity of the final peptides was assessed by RP-HPLC. Analytical
RP-HPCL's were recorded using a Vdac Csuort (4.6..times.250 mm, 5
.mu.m, 300 angstrom pore size, Liquid Separations Group). A linear
gradient from 30% to 60% acetonitrile over 30 min with a constant
concentration of trifluoroacetic acid (0.1% v/v) was employed for
all the analyses at a flow rate of 1.5 mL min.sup.-1. Column eluent
was monitored at 215 nm. The retention time and purity of each
peptide was assessed by the Rainin Dynamax HPLC Method Manager.
[0137] Amino acid analyses of the peptides was done by hydrolysis
in vacuo (110.degree. C.; 20 h) in 4 M methanesulphonic acid
containing 0.2% 3-(2-aminoethyl)-indole.sup.2,23 (Pierce). Amino
acid analyses were performed on the hydrolysates using an LKB 4150
analyser, equipped with an Ultropac 11 column (6.times.273 mm) and
a Shimadzu C-R3A recording integrator with in-house software. The
buffer sequence pH 3.20 (17.5 min), pH 4.25 (32 min), pH 10.00
(borate; 16 min) and temperature sequence 50.degree. C. (5 min),
55.degree. C. (5 min), 58.degree. C. (39.5 min), 65.degree. C. (7
min), 80.degree. C. (17 min) were used. Standard retention times
were as follows: His, 65.1; Lys, 70.1; NH.sub.74.3; Arg, 77.2 min
respectively. The results are shown in Table 5.
[0138] FAB-MS was conducted by Oneida Research Services, Inc.,
Whitesboro, N.Y. using a Finnigan TSQ-70 equipped with an Ion Tech
FAB gun at 6 kV with a primary current of 0.2 mA whilst scanning
from 2800 to 3500 amu. The samples were dissolved in a "Magic
Bullet" matrix and the results are given in Table 4. The position
of the pseudopeptide bond in each of the synthesized peptides is
shown in Table 6.
[0139] Solid phase synthesis of
Ser.sup.9.psi.[CH.sub.2NH]Tyr.sup.10GRF(1-- 29),
Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser.psi.[CHNH]Tyr-Arg-Lys-Val-Leu-Gly-
-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Gln-Asp-Ile-Met-Ser-Arg-NH.sub.2, was
carried out as follows.
[0140] Methylbenzhydrylamine resin (Advanced ChemTech, Inc.) (0.62
g, 0.25 mmole) in the chloride ion form is placed in the reaction
vessel of an Advanced ChemTech ACT 200 peptide synthesizer
programmed to perform the following reaction cycle: (a) 33%
trifluoroacetic acid in methylene chloride (2 times for 1 and 25
min each); (b) DCM wash; (c) PrOH wash; (d) neutralization; (e) 10%
diisopropyl ethylamine (DIEA) in chloroform (2 washes); (f) 0.75 mM
DMF wash; (g) coupling of preformed activated amino acid (HOBT)
esters, 45 min. in DMF; (h) 15 min. DMAP; (i) PrOH wash; (j) DCM
wash. The pseudopeptide bond was incorporated by racemization-free
reductive alkylation with a preformed protected amino-acid aldehyde
in the presence of NaCNBH.sub.3 in acidified DMF, as described by
Coy et al., 1988, Tetrahedron 44:835 and Hocart et al., 1988, J.
Med. Chem. 31:1820.
[0141] The resin bound peptide was elongated by repeating cycles
(a-j) to give
Boc-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp--
Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn
-Gln-Glu-Arg-Gly-Ala-Arg-Als-Arg-L- eu-methylbenzhydrylamine. The
Boc group is then removed by TFA treatment. Boc-serine aldehyde
(0.75 mmoles), prepared by the method of Fehrentz and Castro (1),
is dissolved in 5 ml of dry DMF and added to the resin TFA salt
suspension followed by the addition of 100 mg (2 mmoles) of sodium
cyanoborohydride (2,3). After stirring for 1 h, the resin mixure is
found to be negative to ninhydrin reaction (1 min) indicating
complete derivatization of the free amino group.
[0142] The following amino acids (1.5 mmole) are then . Boc-Asn,
Boc-Thr (benzyl), Boc-Phe, Boc-Ile, Boc-Ala, Boc-Asp (o-cyclohexyl
ester), Boc-Ala, Boc-Tyr (dichlcrobenzyl). After drying, the
peptide resin weighed 1 g.
[0143] The aldehydes were prepared by reduction of the protected
dimethylhydroxamates with LiAlH.sub.4 (0.75 equiv.) for 180 min. at
0.degree. C. The resin (1.0 g, 0.25 mmole) is mixed with anisole (5
ml), anhydrous hydrogen fluoride (35 ml), and 100 mg dithiothretol
at 0.degree. C. and stirred for 45 min. Excess hydrogen fluoride is
evaporated rapidly under a stream of dry nitrogen and free peptide
precipitated and washed with ether. The crude peptide is dissolved
in a minimum volume of 2 M acetic acid and eluted on a column
(2.5.times.100 mm) of Sephadex G-50. Fractions containing a major
component by uv absorption and thin layer chromatography are then
pooled, evaporated to a small volume and applied to a column
(2.5.times.50 cm) of Vydac octadecylsilane (10-15 .mu.M). This is
eluted with a linear gradient of 0-50% acetonitrile in 0.1%
trifluoroacetic acid in water. Fractions are examined by thin layer
chromatography and analytical high performance liquid
chromatography and pooled to give maximum purity. Repeated
lyophilization of the solution from water gives 8.0 mg of the
product as a white, fluffy powder.
[0144] The product is found to be homogeneous by hplc and tlc.
Amino acid analysis of an acid hydrolysate confirms the composition
of the octapeptide. The presence of the Ser.psi.[CH.sub.2NH]Tyr
psuedo peptide bond is demonstrated by fast atom bombardment mass
spectrometry.
[0145] A statine, AHPPA, ACHPA, .beta.-amino acid, or .gamma.-amino
acid residue is added in the same way as is a natural .alpha.-amino
acid residue, by coupling as a Boc derivative.
[0146] All but one of the peptides shown in Table 4 gave
satisfactory amino acid analyses and the expected FAB-MS value
within the error of the methods (see Table 4 and Table 5). The
analogue [Asn.sup.8.psi.[CH.sub.2N- H]-Ser.sup.9]GRF(1-29)NH.sub.2
could not be synthesised. The reaction of Boc-Asn(Xan)CHO with
Ser(Bzl).about.[resin] was very slow and no appreciable reaction
was noted after several prolonged alkylations. This lack of
reactivity may be due to the steric hindrance of the bulky
side-chain protecting groups since, in a series of Substance P
analogues, Boc-Gln(Xan)CHO was used successfully (Qain et al., J.
Biol. Chem. 1989, 16667). No problems were experienced with the
preparation or used of Boc-Asp(OCHx)CHO and no evidence of over
reduction to homo-Ser was observed.
PHASE 1
3T3 PeDtide Stimulated [.sup.3H] Thymidine Uptake Assay
[0147] Cell Culture. Stock cultures of Swiss 3T3 cells are grown in
Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10%
fetal calf serum in humidified atmosphere of 10% CO.sub.2/90% air
at 37.degree. C. For experimental use, the cells are seeded into
24-well cluster trays and used four days after the last change of
medium. The cells are arrested in the G1/G0 phase of the cell cycle
by changing to serum-free DMEM 24 hours prior to the thymidine
uptake assay.
[0148] Assay of DNA Synthesis. The cells are washed twice with 1 ml
aliquots of DMEM (-serum) then incubated with DMEM (-serum),
0.5.mu.M [methyl-.sup.3H] thymidine initially four concentrations
of the test compounds (1, 10, 100, 1000 nM) in a final volume of
1.0 ml. After 28 hours at 37.degree. C., [methyl-.sup.3H] thymidine
incorporation into acid-insoluble pools is assayed as follows. The
cells are washed twice with ice-cold 0.9% NaCl (1 ml aliquots), and
acid solu.)le radioactivity is removed by a 30 min. (40.degree. C.)
incubation with 5% trichloroacetic acid (TCA). The cultures are
then washed once (1 ml) with 95% ethanol and solubilized by a 30
min. incubation (1 ml) with 0.1N NaOH. The solubilized material is
transferred to vials containing 10 ml ScintA (Packard), and the
radioactivity is determined by liquid scintillation
spectrometry.
PHASE 2
Small Cell Carcinoma (SCLC)-Bombesin Stimulated [.sup.3H] Thymidine
Uptake Assay
[0149] Cell Culture. Cultures of the human cell carcinoma cell line
(NCI-H69) (obtained from the American Type Culture Association) are
maintained in RPMI 1640 medium supplemented with 10% fetal calf
serum in 10% CO.sub.2/90% air at 37.degree. C. Twenty-four hours
prior to assay, the cells are washed with serum-free medium and
seeded in 24-well cluster trays.
[0150] Assay of DNA Synthesis. Bombesin (1 nM), 0.5 .mu.M [methyl-
.sup.3H] thymidine (20 Ci/mmole, New England Nuclear), and four
concentrations of the test compounds (1, 10, 100, 1000 nM) are
added to the cultures to achieve a final volume of 0.5 ml. After a
28 hr incubation at 37.degree. C., the cells are collected onto
GF/B glass fiber filters, and the DNA is precipitated with ice-cold
TCA. [.sup.3H] thymidine incorporation into acid-insoluble
fractions of DNA is determined by liquid scintillation
spectrometry.
PHASE 3
Peptide-Induced Pancreatitis
[0151] Male, Sprague-Dawley rats (250 g) are used for these
experiments. The test compound, or 0.9% NaCl is administered s.c.
15 min. prior to the bombesin injection. Bombesin injections are
given s.c. at a dose of 10 .mu.g/kg, and blood samples are obtained
at 1 hr.30 min., 3 hr. and 6 hr. Plasma amylase concentration are
determined by the Pantrak Amylase test.
PHASE 4
In Vitro Inhibition of [.sup.125I] Gastrin Releasing Peptide (GRP)
Binding to Bombesin Receptors
[0152] Membranes from various tissues (rat brain, rat pancreas, rat
anterior pituitary, SCLC, 3T3 cells) are prepared by homogenization
in 50 mM TrisHCl containing 0.1 bovine serum albumin and 0.1 mg/ml
bacitracin followed by two centrifugations (39,000.times.g.times.15
min., 4.degree. C.) with an intermediate resuspension in fresh
buffer. For assay, aliquots (0.8 ml) are incubated with 0.5 nM
[.sup.125I ]GRP (.about.2000 Ci/mmol, Amersham Corp.) and various
concentrations of the test compounds in a final volume of 0.5 ml.
After a 30 minute incubation at 4.degree. C., the binding reaction
is terminated by rapid filtration through Whatman GF/C filters that
have been pre-soaked in 0.3% aqueous polethyleneimine to reduce the
level of nonspecific binding. The filters and tubes are washed
three times with 4 ml alicuots of ice-cold buffer, and the
radioactivity trapped on the filters is counted by
gamma-spectrometry. Specific binding is defined as the total.
[.sup.125I]GRP bound minus that bound in the presence of 1000 nM
bombesin or a related peptide.
PHASE 5
Inhibition of Gastrin Release
[0153] The stomachs of anesthetized rats are perfused with saline
collected over 15 minute periods via pyloric Capn wta Ai le Be Ho
peptdc i infused thah the femoral vein for periods between 0 and
150 minutes.
PHASE 6
In Vivo Antitumor Activity
[0154] NCI-H69 small cell lung carcinoma cells were transplanted
from in vitro culture by implanting each animal with the equivalent
of 5 confluent 75 cm.sup.2 tissue culture flasks in the right
flank. In vitro NCI-H69 cells grow as a suspension of cellular
aggregates. Therefore, no attempt was made to disaggregate the cell
agglomerates by physical or chemical means. Tumor size was
calculated as the average of two diameters, i.e., (length and
width/2) mm.
PHASE 7
Inhibition of Growth Hormone Release
[0155] Anterior pituitaries from adult male rats, weighing 200-250
g and housed under controlled conditions (lights on from 0500-1900
h), were dispersed using aseptic technique by a previously
described trypsin/DNase method (Heiman et al., Endocrinology, 1985,
116, 410) derived from other methods (Hoefer et al., Mol. Cell.
Endocrinol., 1984, 35, 229).
[0156] The dispersed cells were diluted with sterile-filtered
Dulbecco's modified Eagle medium (MEM) (Gibco Laboratories, Grand
Island, N.Y. (GIBCO)), which was supplemented with fetal calf serum
(2.5%; GIBCO), horse serum (3%; GIBCO), fresh rat serum (10% stored
on ice for no longer than 1 h) from the pituitary donors, M
nonessential amino acids (1%; GIBCO), gentamycin (10/mg/mL; Sigma)
and nystatin (10,000 U/mL; GIBCO). The cells were counted with a
hemacytometer (approximately 2,000,000 cells pprpituitary) and
randomiy plated at a density of 200,000 per well (Co-star cluster
24; Rochester Scientific Co., Rochester, N.Y.). The plated cells
were maintained in the above Dulbecco's medium in a humidified
atmosphere of 95% air and 5% CO.sub.2 at 37.degree. C. for 96
h.
[0157] In preparation for a hormone challenge, the cells were
washed 3.times. with medium 199 (GIBCO) to remove old medium and
floating cells. Each dose of secretagogue (diluted in siliconized
test tubes) was tested in quadruplicate wells in medium 199 (total
volume of 1 mL) containing BSA (1%; fraction V; Sigma Chemical Co.,
St. Louis, Mo.). Cells were pulsed in the presence of somatostatin
(0.1 nM) to maintain control levels within narrow limits and to
increase the ratio of maximally stimulated levels to basal
secretory levels without adding additional growth factors or
glucocorticoids. After 3 h at 37.degree. C. in an air/carbon
dioxide atmosphere (95/5%), the medium was removed and stored at
-20.degree. C. until assayed for hormone content.
[0158] Growth hormone (GH) in plasma and media was measured by a
standard double antibody radioimmunoassay using components supplied
by the NIDDK and the National Hormone and Pituitary Program,
University of Maryland School of Medicine. Data are plotted as the
mean values for a given dose of peptide obtained by pooling the
means from individual experiments done in quadruplicate. The number
of experiments for each analogue is given in Table 6. Potencies and
95% confidence intervals were calculateds by 4-point assay
(Pugsley, Endocrinology, 1946, 39, 161).
Results of Assays of Test Peptides
[0159] A number of analogs of bombesin or GRP, each o0 ContainngLtl
& n-pepide bond ar a tae or ACHPA, .beta.-amino acid, or
.gamma.-amino acid residue, can be synthesized and tested in one or
more of the above-described Phase 1-7 assays; the results of Phase
1 and 2 tests are given in Table 1 attached hereto. Table 1 shows
formulas for the non-peptide analogues and results of in vitro
inhibition of [.sup.125I]GRP binding to 3T3 fibroblast bombesin
receptors, and bombesin-stimulated [.sup.3H]Thymidine uptake by
cultured 3T3 cells. (3T3 GRP receptor and thymidine uptake data are
expressed in IC50 (nM).) Table 1 also gives results for non-peptide
bond-containing analogs of one other naturally-occurring peptide,
Neuromedin C, whose C-terminal seven amino acids are similar to
those of bombesin and GRP. (In Table 1, "Litorin" indicates a 9
residue peptide analog or its derivative, whereas "Neuromedin C"
indicates a 10 residue analog or its derivative.)
[0160] In Table 1, the position of the non-peptide bond is
indicated by the position of the symbol .psi.[CH.sub.2NH]; i.e.,
.psi.[CH.sub.2NH] is always shown preceding the amino acid which,
in that peptide, is bonded to the amino acid N-terminal to it via
the non-peptide bond. Where no amino acid is specified under
"structure", the non-peptide bond links the two peptides
represented by the numbers given as post-scripts.
[0161] In Table 1, it can be seen that a preferred placement of the
non-peptide bond in litorin analogs is at the A.sup.8-A.sup.9
position; two most active analogs (as indicated by a low GRP
receptor IC50 value) are BIM-26100
(Phe.sup.8.psi.[CH.sub.2NH]Leu.sup.9) and BIM-26101
[0162] In addition, as shown in Table 1, BIM-26113 (D-Phe.sup.1,
Leu.sup.8.psi.[CH.sub.2NH]Leu.sup.9) and BIM-26114 (D-Nal.sup.1,
Leu.sup.8.psi.[CH.sub.2NH]Leu.sup.9) are active in the 3T3 GRP
receptor binding and thymidine uptake assays. Most notably,
BIM-26136 (D-Nal.sup.1, Leu.sup.8.psi.[CH.sub.2NH]Phe.sup.9), which
contains amino and carboxy terminal aromatic residues that are
capable of forming a hydrophobic interaction, is the most potent
analog. Finally, when statine or .beta.-leucine replaces the
A.sup.8 and A.sup.9 residues of litorin, the resultant analogs
BIM-26120 and BIM-26182 are also potent antagonists.
[0163] Table 1 also shows that Neuromedin C analogs containing a
non-peptide bond between residues A.sup.9-A.sup.10, e.g.,
BIM-26092, 26105, 26106, and 26107, are antagonists when tested in
the 3T3 GRP receptor and thymidine uptake assays.
[0164] Table 1 also gives negative results for analogs of
Neuromedin C and GRP 19-27, e.g., BIM-26108. Thus the non-peptide
bond placement guidelines given herein should be used in
conjunction with the routine assays described above to select
useful antagonists.
[0165] Bombesin and Bombesin analogs have been shown to inhibit the
effect of interleukin-2 (IL-2) (Fink et al., 1988, Klin.
Wochenschr. 66, Suppl. 13, 273). Since IL-2 causes T lymphocytes to
proliferate, it is possible that litorin antagonists may prevent
the inhibitory effect of Bombesin or its analogs on IL-2. IL-2
stimulated lymphocytes are capable of effectively lysing small cell
lung carcinoma cells in vitro. Although Bombesin antagonists have a
direct antiproliferative effect on neoplastic tissues, they may
also favor proliferation of lymphocytes having lytic activity for
so Small teli lung carcinOmd.
[0166] These observations prompted us to evaluate the effect of
BIM-26100 on the in vivo growth of the SCLC tumor cell line
described in Phase 6. Twenty athymic nude females, 5 to 6 weeks of
age, were implanted on day 0 with the NCI-H69 human SCLC,
individually identified and then randomized into the following
vehicle control and test groups:
1 Group No. Treatment No. Animals 1 Saline vehicle treated control:
10 0.2 ml, s.c. inf., b.i.d., QD1-28 2 BIM-26100: 5 50 .mu.g/inj.,
s.c., b.i.d., QD1-28 3 BIM-26100: 5 50 .mu.g/inj., s.c. inf.,
b.i.d., QD1-28 (s.c. = subcutaneously; inf. = infused around tumor;
inj. = injected; b.i.d. = twice per day; QD1-28 = daily treatment,
on days 1-28.)
[0167] Growth of NCI-H69 xenografts and the tumor growth inhibitory
activity of the bombesin antagonist BIM-26100
(pGlu-Gln-Trp-Ala-Val-Gly-H- is-Phe.psi.[CH.sub.2NH]Leu-NH.sub.2)
are illustrated as tumor growth curves in FIG. 1, and relative
tumor sizes in Table 2. Administration of BIM-26100 as a s.c.
infusion around the tumor significantly inhibited tumor growth. The
effectiveness of the antitumor activity of BIM-26100 is evident in
view of the large inoculum of NCI-H69 tumor cells (..e., the
equivalent of 5 confluent 75 cm.sup.2 cell culture flasks per
animal) and he agglomerated condition of the cells. In confluent
flasks, NCI-H69 agglomerates are macroscopically visible and
together resemble a metastatic tumor colony. Many such tumor
colonies were implanted per animal. The dose of BIM-26100 was
availability and is not optimal. Higher doses of BIM-26100 may be
administered, as indicated by body weight gain (minus tumor weight)
gain during the course of treatment (Table 3). This suggest
BIM-26100 completely lacks local or systemic toxicity and is useful
therapeutically as an anti-growth factor with anti-tumor
effects.
[0168] D-Phe-Gln-Trp-Ala-Val-N-methyl-D-Ala-His-Leu-methylester and
D-F.sub.5-Phe-Gln-Trp-Ala-Val-D-Ala-His-Leu-methylester were
examined for their abilities to displace .sup.125I-labeled bombesin
from rat pancreatic acini cells and to inhibit amylase release from
these cells produced by bombesin itself. The analogues exhibit
potencies in the half-maximal effective dose ranges of 5-10 nM and
are thus potent bombesin receptor antagonists.
Testing of GRF Analoas
[0169] The purified analogs were assayed in a 4-day primary culture
of male rat anterior pituitary cells for growth hormone (GH)
release, as described by Hocart et al. (1988, supra) and Murphy and
Coy (1988, Peptide Research 1:36). Potential antagonists were
retested in the presence of GRF(1-29)NH.sub.2 (1 nM). The results
are shown in FIGS. 4-6, in which different dosages of the analogs
were measured against GRF.
[0170] The incorporation of the reduced peptide bond isostere in
the N-terminal region of GRF(1-29)NH.sub.2 produced very weak
agonists and one antagonist with an IC.sub.50 of approximately 10
.mu.M. Placement of the pseudopeptide bond between the N-terminal
9th and 10th residues produced the analogue
[Ser.sup.9.psi.[CH.sub.2NH]Tyr.sup.10]-GRF(1-29)NH.- sub.2 (peptide
VIII). This analog was found to be inactive in the potency assay,
and was therefore tested for antagonist activity in the presence of
a stimulating dose of GRF(1-29)NH.sub.2 (1 nM). The results are
shown graphically in FIG. 6.
[Ser.sup.9.psi.[CH.sub.2NH]Tyr.sup.10]-GRF (1-29)NH.sub.2 was found
to be an antagonist in the 10 .mu.M range vs 1 nM GRF. Earlier
conventional structure-activity studies with the same peptide had
elucidated a more potent antagonist, namely [N-Ac-Tyr.sup.1,
D-Arg.sup.2]GRF(1-29)NH.sub.2 (Robberecht et al., J. Endocrinology,
1985, 117, 1759). This analog had an IC.sub.50 of approximately 1
.mu.M in an assay for adenylate cyclase activity in rat anterior
pituitary homogenates.
[0171] Placement of the pseudopeptide bond in any one position
between the remaining N-terminal 11 amino acids produced analogs
having less activity than GRF(1-29)NH.sub.2. These results are
presented in Table 6 (n=number of separate experiments in
quadruplicate from which the corresponding curves in the Figures
were calculated). Incorporation of the isosteres
Tyr.sup.1.psi.[CH.sub.2NH]Ala.sup.2 and
Ala.sup.2.psi.[CH.sub.2NH]Asp.sup- .3, gave weak agonists with
.about.0.1 activity of the control (peptide I, Tyr.sup.1.psi.
[CH.sub.2NH]Ala.sup.2, 0.12%, and peptide II,
Ala.sup.2.psi.[CH.sub.2NH]Asp.sup.3, 0.13%). At position 3, the
isostere Asp.sup.3.psi.[CH.sub.2NH]Ala.sup.4 (peptide III) produced
the most potent agonist of the series which retained 1.6% of the
activity of the control. When Ala.sup.4.psi.[CH.sub.2NH]Ile.sup.5
was incorporated at position 4 (peptide IV), the activity dropped
to 0.02% of GRF.(1-29)NH.sub.2. This drop continued at position 5,
where the isostere Ile.sup.5.psi.[CH.sub.2NH]Phe.sup.6 (peptide V)
produced the least active agonist with a potency of (0.01% of that
of the control. Phe.sup.6.psi.[CH.sub.2NH]Thr.sup.7 (peptide VI)
produced a potency to 0.13% of that of GRF(1-29)NH but the isostere
Tilt"*cI4Mi)Asi8 produced a weds agonist (peptide VII, 0.02%
potency). With the isostere Ser.sup.9104 [CH.sub.2NH]Tyr.sup.10 in
the peptide (VIII), all trace of agonist activity was lost at doses
as high as 0.1 mM. Another agonist was produced with
Tyr.sup.10.psi.[CH.sub.2NH]Arg.sup.11 in the peptide although it
too had low potency (IX, 0.39%).
[0172] The loss of potency observed after placement of the
pseudopeptide bond at each position of the first 11 N-terminal
positions of GRF(1-29) was greater than that seen with smaller
peptides, such as somatostatin and bombesin which contain
.beta.-bends in the region of the molecule important for receptor
recognition. Chou-Fasman analysis shows GRF to be predominantly
.alpha.-helical in character in the biologically important
N-terminal portion of the molecule. The replacement of the
naturally-occurring C.dbd.O bond by the C.dbd.H.sub.2 bond of the
pseudopeptide bond has pronounced effects on .alpha.-helical
formation due to a loss of intramolecular H-bonding sites and
increased rotational freedom about the isostere C--N bond. Loss of
intermolecular H-bonding sites might also induce changes in
receptor binding capabilities of both .beta.-bend and
.alpha.-helical peptides. However, given the dramatic loss in
activity of these GRF analogues, as compared to somatostatin and
bombesin, effects on peptide conformation are probably more
important in the larger helical GRF molecule.
Use
[0173] The peptides of the invention may be administered to a
mammal, particularly a human, in one of the traditional modes
(e.g., orally, parenterally, transdermally, or transmucosally), in
a sustained polymer, or by on-site delivery (e.g., in the case of
anti-cancer bombesin to the lungs) using micelles, gels and
liposomes.
[0174] The bombesin antagonists of the invention are suitable for
the treatment of all forms of cancer where bombesin-related
substances act as autocrine or paracrine mitotic agents,
particularly small-cell lung carcinoma. The peptides can also be
used for the inhibition of gastric acid secretion and motility
disorders of the GI tract, the symptomatic relief and/or treatment
of exocrine pancreatic adenocarcinoma, and the restoration of
appetite to cachexic patients. The peptides can be administered to
a human patient in a dosage of 0.5 .mu.g/kg/day to 5 mg/kg/day. For
some forms of cancer, e.g., small cell lung carcinoma, the
preferred dosage for curative treatment is 250 mg/patient/day.
[0175] The compound can be administered to a mammal, e.g., a human,
in the dosages used for growth hormone releasing factor or, because
of their decreased potency, in larger dosages. The compounds can be
administred to a mamimal, e.g., a human, in a dosage of 0.01 to
1000 mcg/kg/day, preferably 0.1 to 100 mcg/kg/day.
2 TABLE 1 3T3 GRP Thym. Receptor Uptake Code Structure IC50 (nM)
IC50 (nM) BIM-26092 Gly-Asn-His-Trp-Ala-Val-Gly- 242 466
His-Leu.sub..psi.[CH.sub.2NH- ]Leu-NH.sub.2 Neuromedin C BIM-26095
pGlu-Gln-Trp-Ala-Val-D-Ala- 2623 1209 His-Leu.sub..psi.[CH.sub.2N-
H]Leu-NH.sub.2 Litorin BIM-26100 pGlu-Gln-Trp-Ala-Val-Gly-H- is- 23
26 Phe.sub..psi.[CH.sub.2NH]Leu-NH.sub.2 Litorin BIM-26101
pGlu-Gln-Trp-Ala-Val-Gly-His- 118 296
Leu.sub..psi.[CH.sub.2NH]Leu-NH.sub.2 Litorin BIM-26105
D-Ala-Asn-His-Trp-Ala-Val- 107 107 D-Ala-His-Leu.sub..psi.[CH.sub-
.2CH]Leu-NH.sub.2 Neuromedin C BIM-26106
desGly-D-Ala-His-Trp-Ala-Val- 401 354 D-Ala-His-Leu.sub..psi.[CH.-
sub.2CH]Leu-NH.sub.2 Neuromedin C BIM-26107
D-Phe-His-Trp-Ala-Val-Gly- 199 154 His-Leu.sub..psi.[CH.sub.2NH]L-
eu-NH.sub.2 Neuromedin C BIM-26108 N-Ac-D-Ala-His-Trp-Ala-V- al-
841 >1000 Gly-His-Leu.sub..psi.[CH.sub.2NH]Leu-NH.sub.2 GRP
(19-27) BIM-26113 D-Phe-Gln-Trp-Ala-Val-Gly- 5.8 9
His-Leu.sub..psi.[CH.sub.2NH]Leu-NH.sub.2 Litorin BIM-26114
D-Nal-Gln-Trp-Ala-Val-Gly- 23.5 28
His-Leu.sub..psi.[CH.sub.2NH]Leu-NH.sub.2 Litorin BIM-26120
pGlu-Gln-Trp-Ala-Val-Gly- 150 165 His-Sta-NH.sub.2 Litorin
BIM-26122 D-Phe-Gln-Trp-Ala-Val-Gly- 5.9 28.6 His-Leu-NH.sub.2
Litorin BIM-26136 D-Nal-Gln-Trp-Ala-Val-G- ly-His- 1.4 3.3
Leu.sub..psi.[CH.sub.2NH]Phe-NH.sub.2 Litorin BIM-26182
D-Cpa-Gln-Trp-Ala-Val-Gly-His- 0.88 4.77 .beta.-Leu-NH.sub.2
Litorin
[0176]
3TABLE II IN VIVO TUMOR INHIBITORY ACTIVITY OF THE BOMBESIN
ANTAGONIST BIM-26100: NCI-H69 HUMAN SCLC Group Tumor Size.sup.1 %
Tumor Size % No. Treatment Day 18 (mm) Test/Control Day 28 (mm)
Test/Control 1 Vehicle treated control. 0.2 ml. s.c. inf., 10.9
.+-. 1.82 15.9 .+-. 2.27 b.i.d., QD1-28 2 BIM-26100. 50 .mu.g/inj.,
s.c., b.i.d., QD1-28 10.1 .+-. 1.47 93 17.3 .+-. 1.96 108 3
BIM-26100. 50 .mu.g/inj., s.c. inf., b.i.d., 7.6 .+-. 1.56** 70
13.7 .+-. 0.67* 86 QD1-28 .sup.1Data reported as means .+-. SD on
10 animals in the control and 5 in test groups. Student's t Test
significance of difference from control: *p < 0.05; **p <
0.01.
[0177]
4TABLE III EFFECT OF TUMOR GROWTH AND BIM-26100 TREATMENT ON BODY
WEIGHT: LACK OF SYSTEMIC TOXICITY Group Body Weight (gm).sup.1 Body
Weight (gm) Body Weight (gm) No. Treatment Day 0 Day 18 Day 28 1
Vehicle treated control, 0.2 ml, s.c. inf., 17.3 19.6 19.7 b.i.d.,
QD1-2 2 BIM-26100, 50 .mu.g/inj., s.c., b.i.d., QD1-28 16.9 19.2
19.1 3 BIM-26100, 50 .mu.g/inj., s.c. inf., b.i.d., QD1-28 17.7
20.4 21.1 .sup.1Body weights are presented as means of 10 animals
in the control and 5 in test groups. Tumor weights calculated from
2 largest diameters in mm converted to mgs using the formula for an
ellipsoid (length .times. width 2/2) mgs. were subtracted from
total body weights.
[0178]
5TABLE 4 Peptide Chromatographic, Purity and Mass Spectral Data
HPLC FAB - MS Peptide Analogue t.sub.R/min Purity/% (M - H.sup.+)
GRF (1-29) NH.sub.2 16.9 99.4 I
Tyr.sup.1.sub..psi.[CH.sub.2NH]Ala.sup.2 16.2 99.4 3345 II
Ala.sup.2.sub..psi.[CH.sub.2NH]Asp.sup.3 14.9 94.1 3345 III
Asp.sup.3.sub..psi.[CH.sub.2NH]Ala.sup.4 14.4 95.5 3345 IV
Ala.sup.4.sub..psi.[CH.sub.2NH]Ile.sup.5 13.6 97.6 3345 V
Ile.sup.5.sub..psi.[CH.sub.2NH]Phe.sup.6 14.3 97.2 3345 VI
Phe.sup.6.sub..psi.[CH.sub.2NH]Thr.sup.7 14.7 95.3 33457 VII
Thr.sup.7.sub..psi.[CH.sub.2NH]Asn.sup.8 14.7 96.7 3345 VIII
Ser.sup.9.sub..psi.[CH.sub.2NH]Tyr.sup.10 15.8 92.6 3343 IX
Tyr.sup.10.sub..psi.[CH.sub.2NH]Arg.sup.11 13.5 97.7 3344
[0179]
6TABLE 5 Amino Acid Analyses GRF- (1-29)NH.sub.2 I II III IV V VI
VII VIII IX Asp 3.22 3.02 1.95 1.88 2.89 2.37 3.00 1.90 2.39 3.25
Thr 1.06 1.00 0.87 0.87 1.08 0.68 0.98 0.97 Ser 2.98 2.82 2.62 2.32
3.19 2.75 2.95 2.94 1.85 2.81 Glu 2.25 2.20 2.50 1.81 2.24 2.19
2.25 2.09 2.25 2.10 Gly 1.12 1.10 1.04 1.01 1.03 1.13 0.95 0.97
1.08 1.00 Ala 3.30 2.19 2.23 2.03 2.20 1.80 3.03 3.14 3.13 3.07 Val
0.75 0.97 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Met 0.99 1.13
1.00 0.91 0.98 1.00 1.01 0.96 0.89 0.97 Ile 1.77 1.87 1.83 1.59
1.08 1.06 1.11 1.57 1.90 1.88 Leu 4.00 4.00 3.75 2.92 4.12 4.04
4.05 3.38 4.08 4.06 Tyr 1.92 1.11 1.65 1.73 2.04 1.23 2.02 2.00
0.94 0.92 Phe 0.70 0.91 0.94 0.86 0.90 0.93 0.94 1.07 Lys 2.02 1.81
1.63 2.37 2.15 2.08 1.97 2.01 2.24 1.82 Arg 3.16 2.91 2.90 2.89
3.11 3.14 3.10 3.07 3.40 2.14
[0180]
7TABLE 6 In Vitro Biological Potencies of Reduced Peptide Bond GRF
(1-29) NH.sub.2 Analogues 95% Potency Confidence Peptide Analogue
(%) Interval n GRF (1-29) NH.sub.2 100 -- 28 I
Tyr.sup.1.sub..psi.[CH.sub.2NH]Al- a.sup.2 0.12 0.07-0.22 7 II
Ala.sup.2.sub..psi.[CH.sub.2NH]Asp.sup.- 3 0.13 0.08-0.24 7 III
Asp.sup.3.sub..psi.[CH.sub.2NH]Ala.sup.4 1.6 0.9-3.0 4 IV
Ala.sup.4.sub..psi.[CH.sub.2NH]Ile.sup.5 0.02 0.01-0.05 4 V
Ile.sup.5.sub..psi.[CH.sub.2NH]Phe.sup.6 <0.01 n.d. 7 VI
Phe.sup.6.sub..psi.[CH.sub.2NH]Thr.sup.7 0.13 0.07-0.25 7 VII
Thr.sup.7.sub..psi.[CH.sub.2NH]Asn.sup.8 0.02 0.01-0.03 4 VIII
Ser.sup.9.sub..psi.[CH.sub.2NH]Tyr.sup.10 -- -- 5 IX
Tyr.sup.10.sub..psi.[CH.sub.2NH]Arg.sup.11 0.39 0.23-0.67 6
[0181] Other embodiments are within the following claims.
* * * * *