U.S. patent application number 11/357320 was filed with the patent office on 2006-07-13 for sstr1-selective analogs.
This patent application is currently assigned to The Salk Institute for Biological Studies. Invention is credited to Jean Claude Reubi, Jean E.F. Rivier.
Application Number | 20060155107 11/357320 |
Document ID | / |
Family ID | 26795830 |
Filed Date | 2006-07-13 |
United States Patent
Application |
20060155107 |
Kind Code |
A1 |
Rivier; Jean E.F. ; et
al. |
July 13, 2006 |
SSTR1-selective analogs
Abstract
Analogs of SRIF which are selective for SSTR1 in contrast to the
other cloned SRIF receptors. These analogs are useful in
determining the tissue and cellular expression of the receptor
SSTR1 and its biological role in the endocrine, exocrine and
nervous system, as well as in regulating tumor growth. SRIF analog
peptides, such as des-AA.sup.1,2,5[D-Trp.sup.8, IAmp.sup.9,
Tyr.sup.11]-SRIF and counterparts incorporating Cbm at the
N-terminus, as well as radioiodinated versions thereof, inhibit the
binding of a universal SRIF radioligand to the cloned human
receptor SSTR1, but they do not bind with significant affinity to
human SSTR2, SSTR3, SSTR4 or SSTR5. By incorporating an iodinated
tyrosine in position-2 or in position-11 in these SSTR1-selective
SRIF analogs, a labeled compound useful in drug-screening methods
is provided. The N-terminus accommodates bulky moieties without
loss of selectivity, and a carbamoyl moiety or a conjugating agent
that will accept a radioactive nuclide or will link to a cytotoxin
may be present at the N-terminus.
Inventors: |
Rivier; Jean E.F.; (La
Jolla, CA) ; Reubi; Jean Claude; (Berne, CH) |
Correspondence
Address: |
James J. Schumann;Fitch, Even,Tabin & Flannery
Suite 1600
120 South LaSalle Street
Chicago
IL
60603-3406
US
|
Assignee: |
The Salk Institute for Biological
Studies
La Jolla
CA
|
Family ID: |
26795830 |
Appl. No.: |
11/357320 |
Filed: |
February 17, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10099240 |
Mar 15, 2002 |
7019109 |
|
|
11357320 |
Feb 17, 2006 |
|
|
|
60276871 |
Mar 16, 2001 |
|
|
|
Current U.S.
Class: |
530/311 |
Current CPC
Class: |
A61K 51/088 20130101;
C07K 14/6555 20130101; A61K 51/083 20130101 |
Class at
Publication: |
530/311 ;
514/009 |
International
Class: |
A61K 38/31 20060101
A61K038/31 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. 5R01 DK50124 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1. A cyclic somatostatin (SRIF) analog peptide which selectively
binds the SRIF receptor SSTR1, which peptide has the amino acid
sequence: (cyclo
3-14)H-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-X-Thr-Ser-Cys-OH, where X is
Tyr or ITyr.
2. The peptide of claim 1 wherein X is Tyr.
3. The peptide of claim 1 wherein X is ITyr and I is radioactive
iodine.
4. A method for detecting the presence of cells having SSTR1 by
administering an effective amount of the peptide according to claim
3 so as to selectively bind to such cells and provide a detectable
signal at the location thereof.
5. A method for screening for ligands that bind with high affinity
to SSTR1, which method comprises carrying out a competitive binding
assay with SSTR1, the peptide according to claim 3 and a candidate
ligand, and determining the ability of the said candidate ligand to
displace said labeled peptide.
6. A pharmaceutical composition comprising a mixture of the peptide
according to claim 1 and at least one pharmaceutically acceptable
carrier.
7. A method of treating IH or another SSTR1-mediated
physiopathology, which method comprises administering an amount of
the composition according to claim 6, which amount is effective to
reach tissue affected thereby having SSTR1 receptors and activate
said receptors.
8. A cyclic somatostatin (SRIF) analog peptide which selectively
binds the SRIF receptor SSTR1, which peptide has the amino acid
sequence: (cyclo
3-14)X.sub.1-X.sub.2-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-X.sub.11-Thr-S-
er-Cys-OH wherein X.sub.1 is H, Ala, D-Ala, Cbm, Biu, (lower
alkyl)Cbm, L-Hor, an acyl group having up to 20 carbon atoms, lower
alkyl or a conjugating/complexing agent; X.sub.2 is Gly or
des-X.sub.2; and X.sub.11 is Tyr, or ITyr.
9. The peptide according to claim 8 wherein X.sub.1 is a
conjugating/complexing agent capable of linking to a cytotoxin or
completing to a radioactive nuclide.
10. The peptide according to claim 9 wherein X.sub.1 is a
polyaminopolycarboxylic conjugating agent.
11. The peptide according to claim 9 wherein said
conjugating/complexing agent is DOTA, DTPA, HYNIC or
P.sub.2S.sub.2--COOH.
12. A pharmaceutical composition comprising a mixture of the
peptide according to claim 9 and at least one pharmaceutically
acceptable carrier.
13. A method for destroying SSTR1-containing cells, which method
comprises administering thereto an amount of the peptide according
to claim 12 which includes a radioactive nuclide or a cytotoxin,
which amount is effective to destroy such cells.
Description
[0001] This application is a division of U.S. Ser. No. 10/099,240,
filed Mar. 15, 2002, which claims priority from U.S. Ser. No.
60/276,871, filed Mar. 16, 2001, the subject matter of both
applications is incorporated herein by reference.
[0003] This invention is directed to peptides related to
somatostatin and to methods for pharmaceutical treatment of mammals
using such peptides. More specifically, the invention relates to
shortened receptor-selective somatostatin analogs and the inclusion
of amino acid substitutions in such analogs and optional
modification of the N-terminus that confer thereto
receptor-selectivity and/or increased affinity to the receptor, to
pharmaceutical compositions containing such peptides, to such
peptides complexed with radioactive nuclides or conjugated to
cytotoxins, to methods of diagnostic and therapeutic treatment of
mammals using such peptides and their conjugates, particularly
peptides that are chelated or otherwise labeled, and to methods for
screening for more effective drugs using such peptides.
BACKGROUND OF THE INVENTION
[0004] The cyclic tetradecapeptide somatostatin-14 (SRIF) was
originally isolated from the hypothalamus and characterized as a
physiological inhibitor of growth hormone release from the anterior
pituitary. SRIF is localized throughout the central nervous system,
where it acts as a neurotransmitter and has been shown to both
positively and negatively regulate neuronal firing, to affect the
release of other neurotransmitters, and to modulate motor activity
and cognitive processes.
[0005] Somatostatin and many analogs of somatostatin exhibit
activity in respect to the inhibition of growth hormone (GH)
secretion from cultured, dispersed rat anterior pituitary cells in
vitro; they also inhibit GH, insulin and glucagon secretion in vivo
in the rat and in other mammals. One such analog is
[D-Trp.sup.8]-SRIF which has the amino acid sequence: (cyclo
3-14)H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-Cys-O-
H, which is disclosed in U.S. Pat. No. 4,372,884 (Feb. 8, 1983).
Somatostatin has also been found to inhibit the secretion of
gastrin and secretin by acting directly upon the secretory elements
of the stomach and pancreas, respectively, and somatostatin is
being sold commercially in Europe for the treatment of ulcer
patients. SRIF is also known to inhibit the growth of certain
tumors.
[0006] SRIF induces its biological effects by interacting with a
family of membrane-bound structurally similar receptors. Five SRIF
receptors have been cloned and are referred to as SSTR1-5. All five
receptors bind SRIF and SRIF-28 (an N-terminally extended version)
with high affinity; however, studies have now shown that different
receptor subtypes mediate distinct functions of SRIF in the
body.
[0007] A cyclic SRIF analog, variously termed SMS-201-995 and
Octreotide, i.e. D-Phe-c[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr-ol is being
used clinically to inhibit certain tumor growth; analogs complexed
with .sup.111In or the like are also used as diagnostic agents to
detect SRIF receptors expressed in cancers. Two similar octapeptide
analogs having 6-membered rings, i.e. Lanreotide and Vapreotide,
have also been developed, see Smith-Jones et al., Endocrinology,
140, 5136-5148 (1999). A number of versions of these somatostatin
analogs have been developed for use in radioimaging or as
radiopharmaceuticals in radionuclide therapy; for radioimaging, for
example, labeling with .sup.125I can be used. Proteins have been
previously radiolabeled through the use of chelating agents, and
there are various examples of complexing somatostatin analogs with
.sup.99Tc, .sup.90Y or .sup.111In. A variety of complexing agents
have been used including DTPA (Virgolini, et al., European Journal
of Nuclear Medicine, 23:1388-1399, October 1996); (Stabin, et al.,
J. Nuc. Med., 38:1919-1922, December 1997); (Vallabhajosula, et
al., J. Nuc. Med., 37:1016-1022, June 1996); DOTA (De Jong, et al.,
Int. J. Cancer, 75:406-411, 1998); (Froidevaux, et al., Peptide
Science-Present and Future, 670-673, 1999); HYNIC (Decristoforo, et
al. Eur. J. Nuc. Med., 26:869-876); (Krois, et al., Liebigs Ann.,
1463-1469, 1996); and P.sub.2S.sub.2--COOH (Karra, et al.,
Bioconjugate Chem., 10:254-260, 1999. Another such
complexing/conjugating agent sometimes used is succinimidyl
6-hydrazinium nicotinate hydrochloride (SHNH). A wide variety of
such complexing/conjugating agents are disclosed and discussed in
U.S. Pat. No. 5,972,308 (Oct. 26, 1999).
[0008] Interventional treatment to the arterial system, such as
angioplasty or bypass surgery, can be damaging to the vessel wall.
Injuries to the vessel wall lead to a complex cascade of reparative
responses starting with mural thrombi formation. Death of medial
smooth muscle cells (SMC) from vessel wall injury also initiates
the release of growth factors, such as basic fibroblast growth
factor (bFGF) and platelet-derived growth factor from platelets,
macrophages, and endothelial cells. These factors subsequently
stimulate the proliferation and migration of medial SMC into the
intima. Prolifering SMC synthesize and secrete a wide variety of
mitogenic growth factors to promote further SMC proliferation and
elaboration of extracellular matrix. This reparative process
terminates when the damaged endothelium has been restored. However,
if cell proliferation and matrix deposition continue, they lead to
a pathological condition known as intimal hyperplasia (IH).
[0009] Clinically, IH causes renarrowing, or restenosis, of treated
arteries in 30-50% of coronary angioplasties within six months and
in .about.20% of bypass procedures within two years after
treatment. Apart from intravascular stents and anticoagulation,
which appear effective in limiting restenosis in the short term, no
other interventions have been successful in halting the development
of IH; however, there is now evidence that SRIF may have an effect
upon IH.
[0010] Recent evidence indicates that a somatostatin (SRIF) analog,
angiopeptin (BIM-23014), is effective in inhibiting IH after
arterial injury in animal models. Angiopeptin inhibits the release
of insulin-like growth factor-1 and bFGF from endothelial cells,
thus preventing SMC proliferation and migration. Clinical trials
using angiopeptin to inhibit IH-causing restenosis, however, have
been inconclusive. Angiopeptin is selective for SSTR2, SSTR3 and
SSTR5, and IH appears to be mediated by SSTR1, which may explain
the inconclusive results.
[0011] Octreotide, angiopeptin and other clinically used SRIF
analogs interact significantly with three of the receptor subtypes,
i.e. SSTR2, SSTR3 and SSTR5. SSTR2 and SSTR5 have recently been
reported to mediate antiproliferative effects of SRIF on tumor cell
growth; therefore, they may mediate the clinical effects of
Octreotide in humans. A recent comprehensive review of SRIF and its
receptors is found in Patel, Y. C. "Somatostatin and its receptor
family", Front. Neuroendocrinol, 1999, 20, 157-198. Pending U.S.
patent application Ser. No. 09/607,546, filed Jun. 29, 2000,
discloses SSTR3-selective synthetic analogs of SRIF. U.S. Pat. No.
5,750,499 (May 12, 1998) discloses SRIF analogs which are selective
for SSTR1, and since that discovery, efforts have been made to
discover analogs with even greater selectivity and/or greater
binding strength.
[0012] Nonpeptide SRIF agonists have been identified using
combinatorial chemistry which exhibit selectivity for each of SSTR1
to SSTR5, Rohrer, S. P. et al., Science, 282, 737-740, 23 Oct.
1998. However, improved peptide ligands continue to be sought
because only peptide ligands can be satisfactorily derivatized to
incorporate complexing agents for radionuclides. Additionally,
peptides generally exhibit fewer undesirable side effects, such as
toxicity or cross reactivity with unrelated receptors.
SUMMARY OF THE INVENTION
[0013] Certain modifications have now been discovered which are
effective to create peptide analogs of SRIF that are more selective
for SSTR1 in contrast to the other cloned human SRIF receptors
and/or have greater binding strength than those disclosed in the
'499 patent. The basic original modification substituted an
optionally alkylated amino-methyl Phe into the 9-position of a SRIF
analog that otherwise binds to SSTR1, and it has now been found
that the selectivity and/or the binding strength of such analogs
can be enhanced by modifying the N-terminus and/or the residue in
the 11-position and also by N.sup..alpha.methylating the alkylated
9-position residue. As a result, peptides have now been created
that bind strongly and selectively to cloned SSTR1. Analogs of
these peptides can be iodinated or otherwise radiolabeled while not
only retaining their desirable biological properties, but certain
iodinated Tyr-containing analogs show further increased binding
strength. These novel peptides are useful in determining the tissue
and cellular expression of the receptor SSTR1 and its biological
role in the endocrine, exocrine and nervous system, as well as in
regulating certain pharmacological functions without the
accompanying side effects heretofore characteristic of
administering SRIF. These long-acting SRIF analog peptides, when
radiolabeled, can be used in scintigraphy in order to locate, i.e.
localize, tumors expressing these receptors, either in vitro or in
vivo; other labeling as well known in this art, e.g. fluorescent,
can alternatively be used. With an appropriate chelated
radioligand, these analogs can be turned into radiopharmaceuticals
which are suitable for radionuclide therapy in treatment of such
tumors; alternatively, they can be covalently joined to a cytotoxic
moiety using an appropriate covalent conjugating agent, e.g.
glutaraldehyde or one which binds via a disulfide linkage.
[0014] The SRIF analog peptides of the invention inhibit the
binding of 125I-[Tyr.sup.11] SRIF and
.sup.125I-[Leu.sup.8,D-Trp.sup.22, Tyr.sup.25] SRIF-28 to the
cloned human receptor SSTR1, but they do not bind with high
affinity to SSTR2, SSTR3, SSTR4 or SSTR5. As such, these SSTR1
specific analogs may be used to treat conditions mediated by SSTR1,
such as IH and other such SSTR1-mediated physiopathologies.
Additional of these SRIF analogs which incorporate an iodinated
tyrosine in position-2 of the native molecule also do not bind to
SSTR2, 3, 4 or 5 but still bind potently and saturably to SSTR1.
This is also true for analogs to which .sup.99Tc, .sup.111In or
.sup.90Y, for example, has been chelated by linkers, such as DOTA
or DTPA, or to which other complexing or conjugating agents are
linked to the N-terminus for the purpose of attaching moieties,
e.g. cytotoxins, useful for diagnostic or therapeutic purposes.
Conjugating agent is used herein to broadly refer to this class of
well known chelating, complexing or otherwise covalently binding
agents that serve to link desired moieties to peptides.
[0015] These SRIF analogs not only selectively bind to SSTR1, but
they bind thereto with high affinity. By selectively binding is
meant that they exhibit a K.sub.D or an IC.sub.50 with SSTR1 which
is at least about one-tenth or less of that with respect to at
least 3 of the other five SRIF receptors. The greater the
differential, the more preferred it is. It is believed the four
residues located centrally within the ring structure, i.e. at
positions 7-10 of the native molecule, are primarily responsible
for receptor binding and biological activity; however, as herein
shown both the N-terminus and the 11-position residue, along with
these four residues, can have a significant effect thereupon. These
SRIF analogs can also be readily labeled and effectively used in
drug screening methods and in radionuclide and cytotoxic therapy.
For example, these analogs are useful in localizing such receptor
in the body and in diagnosing the locations of tumors, particularly
prostate cancers, sarcomas and neuroendocrine tumors. As
radionuclide therapeutic agents, they are considered to be
particularly useful in destroying tumors expressing SSTR1
receptors.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0016] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which this invention belongs. The
nomenclature used to define the peptides is that specified by
Schroder & Lubke, "The Peptides", Academic Press (1965)
wherein, in accordance with conventional representation, the amino
group appears to the left and the carboxyl group to the right. The
standard 3-letter abbreviations to identify the alpha-amino acid
residues, and where the amino acid residue has isomeric forms, it
is the L-form of the amino acid that is represented unless
otherwise expressly indicated, e.g. Ser=L-serine.
[0017] SRIF analog peptides are provided having a selective
affinity for the SRIF receptor SSTR1; and also have a high affinity
for SSTR1, i.e. equal to a K.sub.D of about 35 nanomolar or less
and preferably less than 10 nM. These peptides broadly encompass
known analogs of SRIF, or obvious variations thereof, which have
the 5-position residue deleted, have a D-isomer amino acid having
an aromatic side chain in the position corresponding to the
8-position of the native peptide, and have an L-isomer
aminomethylphenylalanine(Amp), preferably Amp(isopropyl), i.e.
IAmp, in the adjacent position that corresponds to the 9-position
of the native peptide. Improved binding strength has been found to
result from the inclusion of one or more of the following
modifications in the analogs described in the '499 patent;
Tyr.sup.11 or ITyr.sup.11 is present, and/or the N-terminus is
modified to create a urea group, with the 9-position residue being
optionally N.sup..alpha.methylated, e.g. N.sup..alpha.MeIAmp. So
long as the basic analog being modified exhibits SRIF properties by
binding generally to SRIF receptors, insertion of
N.sub..alpha.MeIAmp or IAmp in the corresponding 9-position, (the
insertion of a D-isomer amino acid in the 8-position if one should
not already be present and the deletion of any residue in the
5-position should one be present), and the insertion of Tyr in the
11-position will create a molecule which is highly selective for
the SSTR1 receptor and have good binding strength. Further
improvements in binding strength will result from the optional
modification at the N-terminus and the iodination of Tyr, i.e.
ITyr, in the 11-position residue. The 1- and/or 2-position residues
may be deleted to increase binding affinity to SSTR1 or may be
replaced by Tyr or D-Tyr and/or a carbamoyl functionality may be
included.
[0018] Since the characterization of SRIF, a large number of SRIF
analogs have been synthesized having increased potency in some
respect. Numerous U.S. patents have been issued disclosing such
more potent SRIF analogs, and these analogs which include residues
3-4 and 6-14 can be rendered selective for the SSTR1 receptor by
the incorporation of the modification of the present invention.
[0019] Examples of representative peptides exhibiting the desired
specificity for SSTR1 are provided by the following amino acid
sequence, which is based upon a numbering system consistent with
the 14-residue sequence of native mammalian SRIF, but in which the
residue at position 5 has been eliminated: (cyclo
3-14)Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Lys-Phe-Xaa.sub.7-D-Xaa.sub.8-Xaa.sub.-
9-Thr-Xaa.sub.11-Thr-Xaa.sub.13-Cys wherein Xaa.sub.1 is Ala,
D-Ala, Cbm, Biu, (lower alkyl)Cbm, L-Hor, an acyl group having up
to 20 carbon atoms, preferably 7 or less, e.g. 4-hydroxybenzoyl,
lower alkyl or a conjugating agent, such as DOTA; Xaa.sub.2 is Tyr,
ITyr, D-Tyr, D-ITyr, Gly or des-Xaa; Xaa.sub.3 is Cys or D-Cys;
Xaa.sub.7 is (A)Phe wherein A is H, Cl, F, Br, NO.sub.2, Me or
NH(Q) where Q is H, Cbm or L-Hor; and D-Xaa.sub.8 is a D-isomer
amino acid having an aromatic side chain; Xaa.sub.9 is an
aminomethyl Phe which is (C.sub.2 to C.sub.5)alkylated and which is
optionally N.sup..alpha.methylated; Xaa.sub.11 is Phe, Tyr or ITyr;
and Xaa.sub.13 is Ser, N.sup..alpha.MeSer or D-Ser; provided,
however, that at least one of the following is present: (a)
Xaa.sub.11 is Tyr or ITyr; or (b) Xaa.sub.1 is Cbm, Biu or (lower
alkyl)Cbm, with Xaa.sub.9 being optionally N.sup..alpha.methylated.
ITyr represents iodinated tyrosine, and when present at either
position 2 or 11, its presence increases binding affinity whether
radioiodinated or not. Of course, when radioiodinated, it acts as a
tracer. As indicated, a conjugating/complexing agent can be linked
to the .alpha.-amino group at the N-terminus of any of these
peptide analogs, which agent is capable of joining thereto a
radioactive nuclide or a cytotoxin. These conjugating/complexing
agents may be any of those presently used which covalently bond to
the .alpha.-amino group at the N-terminus of the analog. They may
be designed to link, as by chelation, to a radioactive metal or to
covalently bind to a cytotoxin, such as saporin, gelonin, ricin A
chain, etc.
[0020] One preferred subgenus of SRIF analogs has the amino acid
sequence: (cyclo
3-14)Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Lys-Phe-Xaa.sub.7-D-Xaa.sub.8-IAmp-Thr-
-Xaa.sub.11-Thr-Xaa.sub.13-Cys wherein Xaa.sub.1 is H, Cbm, Biu or
(lower alkyl)Cbm; Xaa.sub.2 is Tyr, ITyr, D-Tyr or des-Xaa;
D-Xaa.sub.8 is either D-Trp or a substituted D-Ala wherein one
hydrogen on the .beta.-carbon is replaced by (a) a carbocyclic
aryl-containing moiety selected from the group consisting of phenyl
substituted with three or more straight chain lower alkyl groups,
naphthyl, pyridyl, anthryl, fluorenyl, phenanthryl, biphenylyl and
benzhydryl; or (b) a saturated carbocyclic moiety selected from the
group consisting of cycloalkyl substituted with three or more
straight chain lower alkyl groups, perhydronaphthyl,
perhydrobiphenylyl, perhydro-2,2-diphenylmethyl, and adamantyl; and
Xaa.sub.11 is Tyr or ITyr. When not stated, the remaining variables
should be understood to be as defined hereinbefore.
[0021] Another preferred subgenus of SRIF agonist peptides has the
amino acid sequence: (cyclo
3-14)Xaa.sub.1-Xaa.sub.2-Cys-Lys-Phe-Phe-D-Xaa.sub.8-N.sup..alpha.MeIAmp--
Thr-Tyr-Thr-Xaa.sub.13-Cys wherein Xaa.sub.1 is H; Xaa.sub.2 is
des-Xaa; and D-Xaa.sub.8 is either D-Trp or a D-Ala wherein one
hydrogen on the .beta.-carbon is replaced by(a) a carbocyclic
aryl-containing radical selected from the group consisting of
phenyl substituted with three or more straight chain lower alkyl
groups, naphthyl, pyridyl, anthryl, fluorenyl, phenanthryl,
biphenylyl and benzhydryl; or (b) a saturated carbocyclic radical
selected from the group consisting of cycloalkyl substituted with
three or more straight chain lower alkyl groups, perhydronaphthyl,
perhydrobiphenylyl, perhydro-2,2-diphenylmethyl, and adamantyl;
Xaa.sub.9 is a (C.sub.3 or C.sub.4)alkylated aminomethyl Phe; and
Xaa.sub.11 is Tyr or ITyr.
[0022] An additional preferred subgenus of SRIF agonist peptides
has the amino acid sequence: (cyclo
3-14)Xaa.sub.1-Xaa.sub.2-Cys-Lys-Phe-Phe-D-Xaa.sub.8-N.sup..alpha.MeIAmp--
Thr-Tyr-Thr-Xaa.sub.13-Cys wherein Xaa.sub.1 is Cbm, Biu, (lower
alkyl)Cbm or H; Xaa.sub.2 is des-Xaa; and D-Xaa.sub.8 is (A)D-Trp
where A is H or NO.sub.2, or (B)D-Ala or (B)D,L-Ala where B is
naphthyl, pyridyl, fluorenyl, adamantyl, anthryl, biphenyl,
tri(lower alkyl)phenyl, pentamethylphenyl, phenanthryl,
trialkylcyclohexyl, perhydronaphthyl or perhydrobiphenyl.
[0023] Still another preferred subgenus of SRIF agonist peptides
has the amino acid sequence: TABLE-US-00001 (cyclo 3-14)
Xaa.sub.1-Xaa.sub.2-Cys-Lys-Phe-Phe-D-Xaa.sub.8-N.sup..alpha.MeIAmp-Thr-
Xaa.sub.11-Thr-Xaa.sub.13-Cys wherein Xaa.sub.1 is Cbm; Xaa.sub.2
is Tyr, ITyr or des-Xaa; D-Xaa.sub.8 is D-Trp, D-2Nal, D-Me5-Phe,
D-TMP, D-BIA, D-TBA, D-anthryl-Ala, D-fluorenyl-Ala or
D-adamantyl-Ala; and Xaa.sub.11 is Tyr or ITyr.
[0024] Another preferred subgenus of SRIF analogs has the amino
acid sequence: (cyclo
3-14)Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Lys-Phe-Xaa.sub.7-D-Xaa.sub.8-N.sup..a-
lpha.MeIAmp-Thr-Xaa.sub.11-Thr-Xaa.sub.13-Cys wherein Xaa.sub.1 is
Cbm, a chelating agent, or H; Xaa.sub.2 is des-Xaa; and D-Xaa.sub.8
is D-Trp or D-Nal.
[0025] An additional preferred subgenus of SRIF analogs has the
amino acid sequence: (cyclo
3-14)D-Cys-Lys-Phe-Xaa.sub.7-D-Xaa.sub.8-IAmp-Thr-Xaa.sub.11-Thr-Xaa.sub.-
13-Cys wherein Xaa.sub.7 is Phe or 4NO.sub.2Phe; D-Xaa.sub.8 is
D-Trp; Xaa.sub.11 is Tyr or ITyr and IAmp is optionally
N.sup..alpha.methylated.
[0026] Still another preferred subgenus of SRIF analogs has the
amino acid sequence: TABLE-US-00002 (cyclo 3-14)
Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Lys-Phe-Xaa.sub.7-D-Xaa.sub.8-N.sup..alpha.M-
eIAmp-Thr- Xaa.sub.11-Thr-Xaa.sub.13-Cys wherein Xaa.sub.1 is Cbm
or H; Xaa.sub.2 is des-Xaa; D-Xaa.sub.8 is D-Trp or D-2Nal;
Xaa.sub.11 is Tyr or ITyr; and Xaa.sub.13 is
N.sup..alpha.MeSer.
[0027] By D-Nal is meant the D-isomer of alanine which is
substituted by naphthyl on the .beta.-carbon atom. D-2Nal, wherein
the attachment to naphthalene is at the 2-position on the ring
structure, is preferable, and D-1Nal is less desirable. Cbm stands
for carbamoyl and is preferred; however, lower alkyl carbamoyl,
e.g. methyl, isopropyl, butyl, etc., are considered to be
equivalents. Biu represents biuret which is carbamoyl urea
NH(CONH.sub.2).sub.2. Pal represents alanine which is substituted
by pyridyl on the .beta.-carbon atom; preferably the linkage is to
the 3-position on the pyridine ring. When substituted D-Trp is
employed, single substitutions for hydrogen are preferably made in
either the 5- or 6-position, which are selected from chloro,
fluoro, bromo and nitro, with chloro, fluoro and nitro being
preferred. Alternatively, the indole nitrogen may be acylated with
formyl (N.sup.inFor- or 1For-). By Amp is meant
(aminomethyl)phenylalanine; unless otherwise specified, the methyl
group with its amino substitution should be understood to be in the
4- or para-position on the phenyl ring. By IAmp is meant
(N-isopropyl-aminomethyl)phenylalanine, where the 4-aminomethyl
group is alkylated with an isopropyl group; in EAmp, the alkylation
is with an ethyl group. By D-Me5Phe is meant
3-(2,3,4,5,6-pentamethylphenyl)-D-Ala. By D-TMP is meant
3-(2,4,6-trimethylphenyl)-D-Ala. By D-BIA is meant
3-(benzimidazol-2-yl)-D-Ala. By D-TBA is meant
3-(4,5,6,7-tetrahydrobenzimidazol-2-yl)-D-Ala.
[0028] As used herein, the term "lower alkyl" refers to a straight
or branched chain, saturated hydrocarbon group having from 1 to 6
carbon atoms such as, for example, methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, isopentyl,
n-pentyl and n-hexyl; the term "cycloalkyl group" refers to a
cyclic saturated hydrocarbon group having from 4 to 6 carbon atoms,
e.g. cyclobutyl, cyclopentyl and cyclohexyl. As used herein,
"naphthyl" is inclusive of 1- and 2-naphthyl; "anthryl" is
inclusive of 1-, 2- and 9-anthryl; "fluorenyl" is inclusive of
2-,3-,4- and 9-fluorenyl; "phenanthryl" is inclusive of 2-,3- and
9-phenanthryl; and "adamantyl" is inclusive of 1- and 2-adamantyl.
By Me is meant methyl. By Bzl is meant benzyl, and by Bz is meant
benzoyl. By Ac is meant acetyl, and by Np is meant naphthoyl. As
used herein, naphthoyl is inclusive of 1- and 2-naphthoyl, with
2-naphthoyl being preferred. By Hor is meant the L-isomer of
hydroorotic acid. By SRIF is meant the 14-residue cyclic peptide,
somatostatin.
[0029] The C-terminus is usually free acid, although an equivalent,
e.g. OMe or NH.sub.2, might be used. The N-terminus may be modified
in various ways without significantly adversely effecting the
binding affinity, all of which modifications in these cyclic
peptides are considered to be included as a part of the peptides of
the overall invention. For example, a variety of additions may be
made to the N-terminal amino acid in the form of conjugating agents
which can be then used to link a desired moiety to the peptide. For
example, chelating agents, such as DTPA, DOTA, HYNIC and
P.sub.2S.sub.2--COOH may be attached; alternatively, a cytotoxin
may be covalently linked thereto via a suitable linker well known
in this art if desired. When Tyr or D-Tyr appears at the
N-terminus, it may be radioiodinated or otherwise labeled, and
iodination of Tyr.sup.11 has been shown to increase binding
affinity. Acyl groups having not more than about 20 carbon atoms,
but preferably having not more than 7, e.g. 4-hydroxybenzyl, may
also be present at the N-terminus, as bulky moieties appear to be
accommodated without loss of affinity and selectivity.
Alternatively, the N-terminus may be alkylated with lower alkyl.
Moreover, the addition of Cbm, lower alkyl Cbm or Biu is considered
to increase binding strength.
[0030] Although SSTR1 was the first somatostatin receptor cloned,
identification of its biological and pharmacological properties has
lagged somewhat behind the other SRIF receptors because of the lack
of ligands which are significantly selective for SSTR1. The
peptides of the invention are believed to be improvements over
those disclosed in the '499 patent and they should be very helpful
in determining the many functional roles of this receptor and in
selectively binding only this SRIF receptor and not the others.
They should be valuable in treating IH, and they will be
particularly valuable in SRIF receptor-targeted scintigraphy and
radionuclide therapy. For a number of reasons it is considered
advantageous to have peptide, rather than nonpeptide, ligands of
this character.
[0031] Selectivity for binding of the analog peptides of the
invention to SSTR1 is demonstrated by testing their interaction
with the five different cloned human SRIF receptors as described in
great detail hereinafter. Generally, recombinant cells expressing
the receptor are washed and homogenized to prepare a crude protein
homogenate that is frozen, embedded and then cut in 10-20 .mu.m
thin sections, as known in this art. Candidate substances, such as
potential SRIF agonists and antagonists, are incubated with the
tissue sections, and the interaction between the candidate
substance and the receptor polypeptide is monitored. The peptides
of the invention bind substantially only to SSTR1, and their
binding exhibits high affinity.
[0032] Receptor binding assays are performed on cloned SRIF
receptors as generally set forth in Reubi, J. C. et al., J. Clin.
Endocrinol. Metab.,63, 433-438 (1986) and in Reubi, J. C. et al.,
Eur. J. Nucl. Med. 2000, 27, 273-282. Using such assays, one can
generate K.sub.D values which are indicative of the concentration
of a ligand necessary to occupy one-half (50%) of the binding sites
on a selected amount of a receptor or the like, or alternatively,
competitive assays can generate IC.sub.50 values which are
indicative of the concentration of a competitive ligand necessary
to displace a saturation concentration of a target ligand being
measured from 50% of binding sites. The peptide
des-AA.sup.1,2,5-[D-Trp.sup.8, N.sup..alpha.MeIAmp.sup.9,
Tyr.sup.11]SRIF inhibits the binding to SSTR1 of an iodinated
SRIF-28 ligand that has strong affinity for all five receptors.
Testing shows that it binds to the cloned human SSTR1 with an
IC.sub.50 of about 6.1 nM, while this SRIF analog peptide does not
bind strongly to human SSTR3 or SSTR4 and does not bind to SSTR2 or
SSTR5 at a concentration below 10,000 nM.
[0033] When such a SRIF analog is modified to have its tyrosine
residue in position-11 radioiodinated, the .sup.125I-Tyr.sup.11
analog likewise does not bind any more strongly to SSTR2, 3, 4 or
5, but surprisingly it now binds even more strongly to SSTR1. The
similar analog, des-AA.sup.1,2,5-[D-2Nal.sup.8,
N.sup..alpha.MeIAmp.sup.9, Tyr.sup.11]-SRIF inhibits binding of the
same iodinated SRIF-28 ligand to SSTR1 and binds itself with an
IC.sub.50 of about 21 nM, while not binding to receptors SSTR2,
SSTR4 or SSTR5 at concentrations below 10,000 nM or to SSTR3 at a
concentration below 1,000 nM. These SRIF analogs that selectively
show high affinity to SSTR1 are considered to be particularly
useful in the treatment of IH and other SSTR1-mediated
physiopathologies and in combating tumors by carrying radionuclides
or cytotoxins to the sites of these receptors but not to other SRIF
receptors.
[0034] As hereinbefore indicated, SSTR1 mRNA has been detected in a
variety of tumors. However, it is presently not known whether SSTR1
plays a major role in tumor growth regulation and, if it does,
whether it mediates stimulation or inhibition. Therefore, it is
difficult to foretell whether a selective SSTR1 antagonist would
have a beneficial role for long-term treatment of tumors. However,
the use of SRIF analogs selective for SSTR1 that bind strongly
thereto, and that are long-acting can be effectively used to kill
such tumors via radionuclide or cytotoxic therapy. To date the use
of Octreotide in the treatment of such tumors has not been
considered to be satisfactorily effective particularly because of
its selectivity to SSTR2, 3 and 5.
[0035] Although the binding of the SRIF analogs of the invention to
SSTR1 is potently inhibited by native SRIF and by SRIF-28, it is
not inhibited by many other synthetic SRIF analogs. Moreover, the
lack of strong binding of the peptides of the invention to SSTR4 is
of particular interest because SSTR1 and SSTR4 have approximately
68% amino acid sequence identity, which constitutes higher sequence
similarity than for any other two SRIF receptors. Furthermore,
SSTR1 and SSTR4 both exhibit very low affinity for a large number
of other synthetic analogs of SRIF which bind potently to SSTR2,
SSTR3 and SSTR5. The high amino acid sequence similarity and
similar ligand binding characteristics would usually have predicted
that these two SRIF receptors would share some common structural
similarities in ligand-binding domains; however, the fact that the
peptides of the invention selectively bind only to SSTR1 indicates
that there may be considerable differences in ligand-binding
properties between these two otherwise structurally similar
receptors.
[0036] SSTR1 has been reported to couple to a tyrosine phosphatase,
and stimulation of this enzyme is believed to mediate
anti-proliferative effects of SRIF via activation of this receptor.
SSTR1 mRNA has been detected in a number of tumors. The ability of
SSTR1 to mediate anti-proliferative effects of SRIF renders
SSTR1-selective SRIF agonists effective as therapeutic treatment
agents for treating those cancers, such as prostrate cancers and
sarcomas wherein the malignant tissues express this receptor.
[0037] A particularly important advantage of SSTR1-selective
agonists as anti-cancer agents may be their continued effectiveness
after prolonged use. Continuous use of SMS-201-995 in the treatment
of tumors is considered to be hindered by rapid desensitization of
SSTR2, SSTR3 and SSTR5, the receptors this peptide can interact
with; in fact, all 3 of these receptors have been reported to
rapidly desensitize. In contrast, studies suggest that SSTR1 may be
more resistant to agonist-induced regulation than the other
receptors. As a result, the SSTR1-selective peptide agonists of the
invention are considered to have prolonged anti-proliferative
actions and should therefore exhibit improved effectiveness in
treating SSTR1-mediated cancers, compared to the commercially
available SRIF analogs presently used as anti-cancer agents that
have low affinity for SSTR1.
[0038] Furthermore, an analog of SMS-201-995 has recently been
employed to detect human tumors having high expression of SRIF
receptors through the use of positron-emission tomography. This
SRIF analog does not distinguish among SSTR2, SSTR3 or SSTR5, and
it is unlikely to be able to detect SSTR1 expression. Labelled SRIF
analogs of the present invention can be employed for similar
purposes and are considered to be specifically useful in
identifying tumors expressing SSTR1, which tumors would then be
therapeutic targets for treatment with SSTR1-selective ligands.
[0039] While SSTR1 has the typical structure of other G
protein-linked receptors, controversy exists over whether this
receptor truly associates with G proteins and effectively couples
to adenylyl cyclase. Some investigators have reported that SRIF
binding to this receptor is not affected by GTP analogs or
pertussis toxin and does not effectively couple to adenylyl
cyclase, whereas others have reported opposite findings.
Furthermore, some investigators have failed to show GTP analog
regulation of agonist binding to the receptor but have found that
receptor mediates the inhibition of cAMP formation by SRIF;
however, this may have been a function of the particular cell
systems that were used.
[0040] The SRIF analogs of the present invention are considered to
be useful in combating cancers which express SSTR1 and in combating
IH and other SSTR1-mediated physiopathologies. They are also
considered to be most useful in scintigraphy to determine the
distribution of cells and tissues expressing this receptor in the
brain and in the endocrine and exocrine systems, and also in
identifying selective functions of this receptor in the body.
[0041] Labeled SRIF analogs of the invention are also considered to
be useful in drug-screening assays to screen for new effective
peptide and nonpeptide agents which will bind with high affinity to
SSTR1 and which may be either highly effective agonists or
antagonists for treating GI track motility. Once a known ligand for
the receptor SSTR1 is in hand, one can obtain a baseline activity
for the recombinantly produced receptor. Then, to test for
inhibitors or modifiers, i.e. antagonists of the receptor function,
one can incorporate into a test mixture a candidate substance to
test its effect on the receptor. By comparing reactions which are
carried out in the presence or absence of the candidate substance,
one can then obtain information regarding the effect of the
candidate substance on the normal function of the receptor. The
cyclic SRIF analogs described in the following examples are
agonists which can be employed to selectively stimulate the
inhibitory activity of somatostatin at SSTR1.
[0042] The peptides of the present invention can be synthesized by
classical solution synthesis, but they are preferably synthesized
by solid-phase technique, as described in U.S. Pat. No. 5,750,499
issued May 12, 1998, the disclosure of which is incorporated herein
by reference.
[0043] The SRIF analogs of the invention are generally effective at
levels of less than 100 micrograms per kilogram of body weight. For
prolonged action, it may be desirable to use dosage levels of about
0.1 to about 2.5 milligrams per kilogram of body weight. These
analogs are soluble in water and thus can be prepared as relatively
concentrated solutions for administration.
[0044] The following Examples illustrate the syntheses of a number
of SRIF analog peptides embodying various features of the
invention, together with the syntheses of protected amino acids for
use in such peptide syntheses. All of these peptides include at
least one D-isomer amino acid residue, and although the preferred
SRIF analogs do not include all 14 amino acid residues of the
native SRIF, to permit ready comparison with the native SRIF
sequence, the peptide analogs are described by making reference to
the comparable positions in the native SRIF sequence having
positions 1 through 14, as is commonly done when naming analogs of
a native compound. In each peptide, "cyclo" indicates that the two
cysteine residues are joined by a cyclizing disulfide bond.
EXAMPLE 1
Synthesis of N.sup..alpha.MeBoc-4aminomethylPhe(ipr)
[0045] The synthesis of
L-N-MeBoc-N.sup.4-Cbz-(4-isopropylaminomethyl)phenylalanine, which
is referred to by the shorthand nomenclature as
Boc-N.sup..alpha.MeIAmp(Z), is carried out as follows:
L-N.sup..alpha.-Boc-(4-aminomethyl)phenylalanine (Boc-Amp) (15.3 g,
52 mmol) is dissolved in acetone (200 mL), and molecular sieves
(6.0 g, 4 .ANG.) are added to the solution in a 500 mL Parr
hydrogenation vessel. The mixture is purged with N.sub.2 for 10
minutes; then Pd/C 10% (600 mg) is added. A reductive alkylation
reaction occurs and is monitored by HPLC; it is carried out for
about 26 hours. After filtration to remove the catalyst and
molecular sieves and evaporation of the solvent, the desired
intermediate
L-N.sup..alpha.-Boc-(4-isopropylaminomethyl)phenylalanine is
obtained as a viscous liquid.
[0046] The product is then Cbz-protected using benzyl chloroformate
(Z-Cl, 8.6 mL, 60 mmol) in a mixture of THF/H.sub.2O (1:1,200 mL)
at pH=9.5. A good yield of
L-N-Boc-N.sup.4-Cbz-(4-isopropylaminomethyl)phenylalanine is
obtained as a foam: 17.5 g (37 mmol, 71.4%); m.p. 39-42.degree. C.;
[.alpha.].sub.D=+5.2.degree. (c=1, MeOH, t=20.degree. C.).
[0047] The product is the N.sup..alpha.methylated by dissolution in
a suitable solvent, e.g., THF, and treated with methyl iodide and
sodium hydride (NaH). For example, 0.152 mole of the product is
dissolved in 100 milliliters of tetrahydrofuran (THF) and added
dropwise to 13.58 grams of NaH(0.452 mole) (3eq) which was
dissolved in 500 milliliters of THF at 0.degree. C. Thereafter,
42.6 grams of methyl iodide (2eq), dissolved in THF, are added
dropwise over 30 minutes, and stirring in a beaker is continued for
about 36 hours at room temperature. The reaction is stopped by
placing the beaker in an ice bath and by the slow addition of 20
milliliters of glacial acetic acid and 20 milliliters of distilled
water. The THF is removed by rotary evaporation, and the remaining
oil is diluted with 300 milliliters of water. After adjusting to a
pH of about 8 with sodium carbonate, the mixture is extracted
twice, using 200 milliliters of ethyl ether each time. Following
extraction, the remainder is acidified to a pH of about 2 with
sulfuric acid, and it is then extracted twice with 300 milliliters
of ethylacetate. After drying over anhydrous magnesium sulfate,
solvent removal is effected by evaporation, giving about 40 grams
of an oil which crystallizes to crystals of
Boc-N.sup..alpha.MeIAmp(Z).
EXAMPLE 2
[0048] The somatostatin agonist des-AA.sup.1,2,5[D-Trp.sup.8,
N.sup..alpha.MeIAmp.sup.9, Tyr.sup.11]-SRIF having the structure:
(cyclo)H-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys-OH
is synthesized by the solid phase methodology in a stepwise manner
on a chloromethylated resin generally as described in Example 2 of
the '499 patent. For the 9-position residue, the monomer
synthesized in Example 1, i.e.
N.sup..alpha.(Boc)Me-4-isopropylaminomethyl Phe(Z) is coupled into
the chain. This synthesis creates the intermediate:
Boc-Cys(Mob)-Lys(2-ClZ)-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp(Z)-Thr(Bzl)-Tyr-
(2BrZ)-Thr(Bzl)-Ser(Bzl)-Cys(Mob)-O-CH.sub.2-resin support.
[0049] Cleavage of the peptide from the resin and deprotection of
the side chain protecting groups are performed in hydrofluoric
acid(HF) (25 ml) in the presence of 10% of anisole and 5% of
methylsulfide for 1.5 hours at 0.degree. C. after 20 minutes at
ambient temperature. After elimination of hydrofluoric acid under
high vacuum, the resin-peptide is washed with anhydrous diethyl
ether.
[0050] The resin is immediately extracted with 75% acetic acid (200
ml). The extract is filtered into a 500 milliliter round-bottom
flask and is then oxidized to create the disulfide cyclic linkage
by stirring vigorously while rapidly adding a 10 weight percent
solution of iodine in methanol until the resultant solution remains
orange-colored. It is then stirred for 40 additional minutes and
quenched with 10% ascorbic acid in water until the yellow color is
gone. Concentration under vacuum is carried out to reduce the
volume to about 50 milliliters, followed by dilution to about 300
milliliters with water. The solution is then applied to a 4
centimeter by 7 centimeter pad of C.sub.18 silica in a
coarse-fritted funnel that was previously equilibrated with 5%
CH.sub.3CN in 0.1% trifluoroacetic acid (TFA) in water. Following
vacuum filtration, the eluate is diluted to 500 milliliters and
reapplied to the pad. The eluate is collected and diluted to about
800 ml, and filtration is repeated. Thereafter, the pad is washed
with about 300 milliliters of 5% acetonitrile in 0.1% TFA, and the
peptide is eluted using about 250 milliliters of 60% CH.sub.3CN in
water. The resultant solution is diluted to about 500 milliliters
with distilled water, frozen and lyophilized.
[0051] The lyophilized crude peptides were purified by preparative
RP-HPLC using a linear gradient 1% B per 3 min. increase from the
baseline % B (Eluent A=0.25 N TEAP pH 2.25, eluent B=60%
CH.sub.3CN, 40% A) at a flow rate of 100 mL/min. Peaks are located
which are then individually purified using buffer systems as
disclosed in Hoeger et al., Biochromatography, 2, 134-142 (1987).
Purification in TEAP pH 2.25 was followed by a rechromatography in
a 0.1% TFA solution and acetonitrile on the same cartridge
(gradient of 1% acetonitrile/min.). The separations were monitored
by analytical RP-HPLC at 215 nm. The fractions containing the pure
product were pooled and lyophilized to obtain a fluffy white
powder. The desired cyclic peptide
(cyclo)H-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys-OH
is obtained which appears to be greater than 98% pure on capillary
zone electrophoresis.
[0052] MS analysis shows a mass of 1515.6 Da which compares
favorably to the calculated mass of 1514.7 Da. The peptide is
hereinafter referred to as Peptide No. 2.
EXAMPLES 2A and 2B
[0053] The synthesis described in Example 2 is repeated with one
change; instead of using
N.sup..alpha.(Boc)Me-4-isopropylaminomethyl Phe(Z) for the
9-position residue, Boc-4-isopropylamino Phe(Z), which is also
referred to as Boc-IAmp(Z), is used. Cleavage, deprotection,
cyclization and purification are carried out as in Example 2. A
purified cyclic peptide having the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Tyr-Thr-Ser-Cys-OH is
obtained which appears to be greater than 98% pure on capillary
zone electrophoresis. It is referred to as Peptide 2A. MS analysis
shows a mass of 1501.5 Da which compares favorably with the
calculated mass of 1500.66 Da.
[0054] A portion of the product Peptide 2A above is taken and
subjected to iodination as well known in this art to iodinate the
Tyr.sup.11. The iodinated compound is thereafter referred to as
Peptide 2B. Radioactive iodination is instead used when desired to
create a tracer. MS analysis shows a mass of 1627.5 Da which
compares favorably to the calculated mass of 1626.55 Da.
EXAMPLE 3
[0055] The initial synthesis described in Example 2 is repeated
with one change;
N.sup..alpha.Boc-N.sup.4-Fmoc-(4-aminomethyl)phenylalanine, i.e.
Boc-Amp(Fmoc), is used to provide the 9-position residue. The Boc
group is removed, and the .alpha.-amino group of the Amp residue is
N-alkylated on the resin as described in Kaljuste and Unden, Int.
J. Peptide Protein Res., 42, 118-124 (1993). Further elongation of
the chain then proceeds as in Example 2, until the final coupling
with Boc-Cys(Mob) is effected.
[0056] With the N.sup..alpha.Boc group still in place at the
N-terminus, the side chain of Amp is alkylated. The Fmoc protecting
group is selectively removed by treatment with 20 percent
piperidine in NMP or DMF(10 ml.) for about 15 minutes; the
intermediate is preferably washed with NMP and then treated with
more piperidine/NMP for another 15 minutes. After preferably
washing the peptidoresin with NMP, the newly freed aminomethyl
groups are treated with MeO-phenyl).sub.2-CHCl, which is referred
to as Dod-Cl, in NMP plus diisopropyl ethylamine (DIEA) for 1-2
hours. After washing, the peptidoresin (about 1-2 g) is treated for
20 minutes with 15 mls of a solution of 40% acetaldehyde in 1%
acetic acid in NMP, plus 0.35 gm. of cyanoborohydride and repeated
once. This reaction, details of which reaction can be found in
Kaljuste, supra, adds a single ethyl group to the aminomethylPhe
side chain. The usual washing is carried out, and then the Dod-Cl
protecting group and the Boc group at the N-terminus are removed by
treatment with 60% TFA in DCM for 20 minutes. Cleavage,
deprotection, cyclization and purification are then carried out as
in Example 2. The purified cyclic peptide has the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeEAmp-Thr-Tyr-Thr-Ser-Cys-OH
and is referred to as Peptide No. 3.
EXAMPLE 4
[0057] The synthesis described in Example 2 is used to produce
about 4 grams of product with two changes.
N.sup..alpha.Boc-D-Cys(Mob) is used to provide the 3-position
residue, and an alternative cyclization step is used. Following
removal of the Boc group at the N-terminus, cleavage and
deprotection are carried out as in Example 2. Cyclization is then
effected by dissolving four grams of the deprotected linear
somatostatin analog in 2.500 ml of five percent acetic acid having
a pH of 3. Two and a half grams of potassium ferricyanide is
dissolved in one and a half liter of water containing five grams of
ammonium acetate and having a pH of 6.9. The peptide solution is
added dropwise to the ferricyanide solution at a rate of 140 ml per
hour while the reaction mixture is constantly stirred under
nitrogen.
[0058] A pH stat is used to monitor the pH of the reaction mixture,
and the pH is maintained in the range of 6.8-7 throughout the
reaction by the addition of a 10 percent ammonium hydroxide
solution, as required. After addition of the last increment of the
peptide solution, the reaction is continued for 2 hours under
conditions of constant stirring.
[0059] The pH is then reduced to 5 with acetic acid, and the
yellowish, greenish slightly turbid solution is filtered over
celite. The filtered solution is then applied onto and filtered
through a weakly basic anion exchange resin (Bio Rad AG3.times.4A
column, 100-200 mesh, chloride form --300 ml) under mild suction.
All of the ferro and ferricyanide anion is removed in this step.
The clear solution (4-5 liters, including the different washes used
in the celite filtration and the anion exchange transfer) is
applied onto a weakly acidic carboxylic cation exchange resin (Bio
Rex 70 column, 200 ml--acidic form) which runs overnight. The
following morning 90 percent of the peptide has been retained on
the cation exchange column. The retained peptide is washed with 200
ml of 5 percent acetic acid. The peptide is then eluted from the
cation exchange column by washing the column with 500 ml of 50
percent acetic acid. The peptide containing fractions (about 200
ml) are concentrated on a rotary evaporator to a volume of 30 ml.
The peptide is then lyophylized after dilution with water.
Purification is then effected as in Example 2. The purified cyclic
peptide has the formula:
(cyclo)H-D-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys--
OH and is referred to as Peptide No. 4.
EXAMPLE 5
[0060] The synthesis described in Example 2 is repeated with one
change. Boc-4NO.sub.2-Phe is used instead of N.sup..alpha.Boc-Phe
to provide the 7-position residue. Further elongation of
deprotection, cyclization and purification are carried out as in
Example 2. The purified, cyclic peptide has the formula:
(cyclo)H-Cys-Lys-Phe-4NO.sub.2Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-S-
er-Cys-OH and is referred to as Peptide No. 5.
EXAMPLE 5A
[0061] The synthesis described in Example 5 is repeated with two
changes. Boc-IAmp(Z) is used as the 9-position residue, and
N.sup..alpha.Boc-D-2Nal is used as the 8-position residue.
Cleavage, deprotection, cyclization and purification are carried
out as in Example 2, and the purified cyclic peptide has the
formula:
(cyclo)H-Cys-Lys-Phe-4NO.sub.2Phe-D-2Nal-IAmp-Thr-Tyr-Thr-Ser-Cys-OH.
MS analysis shows a mass of 1541.6 Da which compares favorably to
the calculated mass of 1540.65 Da. The peptide is hereinafter
referred to as Peptide No. 5A.
EXAMPLE 6
[0062] The synthesis described in Example 2 is repeated with one
change. N.sup..alpha.Boc-4Cl-phenylalanine, i.e. Boc-4ClPhe, is
used to provide the 7-position residue. Further elongation of the
chain then proceeds as in Example 2, and cleavage, deprotection,
cyclization, and purification are carried out as in Example 2. The
purified, cyclic peptide has the formula: and is referred to as
Peptide No. 6.
EXAMPLE 7
[0063] The synthesis described in Example 5 is repeated with one
change. N.sup..alpha.Boc-D-Cys(Mob) is used to provide the
3-position residue. Following removal of the Boc group at the
N-terminus, cleavage, deprotection, cyclization, and purification
are carried out as in Example 2. The purified, cyclic peptide has
the formula:
(cyclo)H-D-Cys-Lys-Phe-4NO.sub.2Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-
-Ser-Cys-OH and is referred to as Peptide No. 7.
EXAMPLE 8
[0064] The synthesis described in Example 4 is repeated with one
change. N.sup..alpha.Boc-Phe is used to provide the 11-position
residue. Following removal of the Boc group at the N-terminus,
cleavage, deprotection, cyclization, and purification are carried
out as in Example 2. The purified cyclic peptide has the formula:
(cyclo)H-D-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Phe-Thr-Ser-Cys--
OH and is referred to as Peptide No. 8.
EXAMPLE 9
[0065] The synthesis described in Example 2 is repeated with one
change. Instead of coupling Boc-D-Trp as what is referred to as the
8-position residue, N.sup..alpha.Boc-D-2Nal is used. Further
elongation of the chain then proceeds as in Example 2.
[0066] Cleavage, deprotection, cyclization and purification are
carried out as in Example 2. Amino acid analysis shows the expected
ratio for the different amino acids. The purified cyclic peptide
has the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-2Nal-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys-O-
H and is referred to as Peptide No. 9A. MS analysis shows a mass of
1526.6 Da, which compares favorably to the calculated value
1525.7.
[0067] The synthesis is repeated, substituting
N.sup..alpha.Boc-D-1Nal for N.sup..alpha.Boc-D-2Nal to obtain the
following purified cyclic peptide, which is referred to as Peptide
No. 9B:
(cyclo)H-Cys-Lys-Phe-Phe-D-1Nal-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys-O-
H.
EXAMPLE 10
[0068] The synthesis described in Example 2 is repeated with one
change. Instead of coupling Boc-Ser(Bzl) as what is referred to as
the 13-position residue, N.sup..alpha.Boc-D-Ser(Bzl) is used.
Elongation of the chain is then carried out as in Example 2.
[0069] Cleavage, deprotection, cyclization and purification are
carried out as in Example 2. A purified cyclic peptide having the
formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-D-Ser-Cys--
OH and is referred to as Peptide No. 10.
EXAMPLE 11
[0070] The synthesis described in Example 2 is repeated with one
change. Instead of coupling Boc-D-Trp as what is referred to as the
8-position residue, N.sup..alpha.Boc-D-3-Pal is used. Elongation of
the chain is then carried out as in Example 2.
[0071] Cleavage, deprotection, cyclization and purification are
carried out as in Example 2. The purified cyclic peptide has the
formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-3Pal-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys-O-
H and is referred to as Peptide No. 11.
EXAMPLE 12
[0072] The synthesis described in Example 2 is repeated with one
change. Following removal of the Boc group at the N-terminus, a
reaction is carried out to add a carbamoyl moiety and thus create
an urea group at the N-terminus.
[0073] After deblocking the .alpha.-amino group at the N-terminus
using trifluoroacetic acid (TFA), a reaction is carried out with
about 1 gm of the peptidoresin and 100 mg of sodium cyanate (NaOCN)
and acetic acid (3 ml) for 30 minutes at 22.degree. C. in NMP (4
ml). This reaction results in the addition of the carbamoyl moiety
at the N-terminus. Thereafter, cleavage of the peptide from the
resin and deprotection of the side chain protecting groups,
followed by cyclization and purification, are carried out as in
Example 2. The purified cyclic peptide has the formula:
(cyclo)CbmCys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys-O-
H and is referred to as Peptide No. 12. MS analysis shows a mass of
1558.7 Da, which compares favorably to the calculated value of
1557.7.
EXAMPLES 12A and 12B
[0074] The synthesis set forth in Example 12 is repeated
substituting Boc-IAmp(Z) for the 9-position residue. Cleavage,
deprotection, cyclization and purification of the peptide are
carried out as in Example 2. The purified cyclic peptide has the
formula:
(cyclo)CbmCys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Tyr-Thr-Ser-Cys-OH. The
peptide is hereinafter referred to as Peptide No. 12A. MS analysis
shows a mass of 1544.6 Da, which compares favorably with the
calculated value of 1543.66 Da. A portion of the peptide
synthesized is then subjected to iodination in the normal manner so
as to iodinate the Tyr.sup.11. MS analysis shows a mass of 1670.6
Da which compares favorably to the calculated value of 1669.57 Da
for a nonradioiodinated peptide. To provide a tracer, .sup.125I may
be used. This peptide is referred to hereinafter as Peptide No.
12B.
EXAMPLE 12C
[0075] The synthesis set forth in Example 12A above is repeated
with one change. N.sup..alpha.Boc-D-Cys(Mob) is used to provide the
3-position residue. Cleavage, deprotection, cyclization and
purification are carried out as in Example 2, and the purified
cyclic peptide has the formula:
(cyclo)CbmD-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Tyr-Thr-Ser-Cys-OH. The
peptide is hereinafter referred to as Peptide No. 12C. MS analysis
shows a mass of 1544.6 Da, which compares favorably to the
calculated value of 1543.66 Da.
EXAMPLE 12D
[0076] The synthesis set forth in Example 12 is repeated with one
change. N.sup..alpha.Boc-ITyr(2BrZ) is used to provide the
11-position residue. Cleavage, deprotection, cyclization and
purification are carried out as in Example 2, and the purified
cyclic peptide has the formula:
(cyclo)CbmCys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-ITyr-Thr-Ser-Cys--
OH. The peptide is hereinafter referred to as Peptide No. 12D. MS
analysis shows a mass of 1684.6 Da, which compares favorably to the
calculated value of 1684.59 Da. If desired for use as a tracer,
radioactive ITyr is used.
[0077] Peptides 2-12D bind strongly and selectively to human
SSTR1.
EXAMPLE 13
[0078] The synthesis described in Example 2 is repeated but this
time N.sup..alpha.Boc-Phe is used instead of
N.sup..alpha.Boc-Tyr(2BrZ) for the 11-position to produce the
11-residue peptide. Cleavage, deprotection, cyclization, and
purification are carried out as in Example 2. The purified cyclic
peptide has the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Phe-Thr-Ser-Cys-OH
and is referred to as Peptide No. 13. MS analysis shows a mass of
1499.8 Da, which compares favorably to the calculated value of
1498.7.
EXAMPLE 14
[0079] The synthesis described in Example 13 is repeated with one
change. Following removal of the Boc group at the N-terminus, a
reaction is carried out to add a carbamoyl moiety and thus create
an urea group at the N-terminus.
[0080] After deblocking the .alpha.-amino group at the N-terminus
using trifluoroacetic acid (TFA), a reaction as described in
Example 12 is carried out and results in the addition of the
carbamoyl moiety at the N-terminus. Thereafter, cleavage of the
peptide from the resin and deprotection of the side chain
protecting groups, followed by cyclization and purification, are
carried out as in Example 2. The purified cyclic peptide has the
formula:
(cyclo)CbmCys-Lys-Phe-Phe-D-Trp-n.sup..alpha.MeIAmp-Thr-Phe-Thr-Ser-Cys-O-
H and is referred to as Peptide No. 14.
EXAMPLES 14A and 14B
[0081] The synthesis described in Example 14 is repeated with two
changes. Boc-IAmp(Z) is used to provide the 9-position residue, and
subsequent to the coupling of the Cys residue in the 3-position,
the chain is elongated by one residue by coupling
N.sup..alpha.Boc-Tyr(2BrZ) at the N-terminus. Following removal of
the Boc group after the coupling of the Tyr residue, a reaction is
carried out to add a carbamoyl moiety and thus create an urea group
at the N-terminus as in Example 14. Cleavage, deprotection,
cyclization and purification are carried out as in Example 2. The
purified cyclic peptide has the formula:
(cyclo)CbmTyr-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Phe-Thr-Ser-Cys-OH. MS
analysis shows a mass of about 1691.8 Da which compares favorably
with the calculated mass of 1690.73 Da. The peptide is referred to
hereinafter as Peptide No. 14A.
[0082] A portion of the peptide is then subjected to standard
iodination so as to iodinate the tyrosine in the 2-position. MS
analysis shows a mass of about 1817.8 Da which compares favorably
with the calculated mass of 1816.64 Da. The iodinated peptide is
hereinafter referred to as Peptide No. 14B. Of course .sup.125I may
be used to provide a tracer.
EXAMPLE 14C
[0083] The synthesis as described with respect to Example 14A is
repeated with one change. Boc-D-Cys(Mob) is used to provide the
3-position residue. Following the reaction to create the carbamoyl
moiety at the N-terminus, cleavage, deprotection, cyclization and
purification are carried out as in Example 2. The purified peptide
has the formula:
(cyclo)CbmTyr-D-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Phe-Thr-Ser-Cys-OH
and is referred to as Peptide No. 14C. MS analysis shows a mass of
1691.6 Da which compares favorably with the calculated mass of
1690.73 Da.
EXAMPLE 15
[0084] The synthesis described in Example 9A is repeated but this
time N.sup..alpha.Boc-Phe is used instead of
N.sup..alpha.Boc-Tyr(2BrZ) for the 11-position to produce the
11-residue peptide. Cleavage, deprotection, cyclization, and
purification are carried out as in Example 2. The purified cyclic
peptide has the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-2Nal-N.sup..alpha.MeIAmp-Thr-Phe-Thr-Ser-Cys-O-
H and is referred to as Peptide No. 15. MS analysis shows a mass of
1510.8 Da, which compares favorably to the calculated value
1509.7.
EXAMPLE 16
[0085] The synthesis described in Example 9A is again repeated with
one change. Following removal of the Boc group at the N-terminus, a
reaction is carried out to add a carbamoyl moiety and thus create
an urea group at the N-terminus.
[0086] After deblocking the .alpha.-amino group at the N-terminus
using trifluoroacetic acid (TFA), a reaction as described in
Example 12 is carried out which results in the addition of the
carbamoyl moiety at the N-terminus. Thereafter, cleavage of the
peptide from the resin and deprotection of the side chain
protecting groups, followed by cyclization and purification, are
carried out as in Example 2. The purified cyclic peptide has the
formula:
(cyclo)CbmCys-Lys-Phe-Phe-D-2Nal-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys--
OH and is referred to as Peptide No. 16. MS analysis shows a mass
of 1569.6 Da, which compares favorably to the calculated value of
1568.7.
EXAMPLE 17
[0087] The synthesis described in Example 2 is repeated with one
change. Following removal of the Boc group at the N-terminus, a
reaction is carried out with an alkyl isocyanate to add a methyl
carbamoyl moiety at the N-terminus.
[0088] After deblocking the .alpha.-amino group at the N-terminus
using trifluoroacetic acid (TFA), a reaction is carried out with
about 1 gm of the peptidoresin and 100 mg of methyl isocyanate in
the presence of 2.7 mmol of DIEA for 2 hours at 22.degree. C. in
DMF (10 ml). This reaction results in the addition of the carbamoyl
moiety at the N-terminus. Thereafter, cleavage of the peptide from
the resin and deprotection of the side chain protecting groups,
followed by cyclization and purification, are carried out as in
Example 2. The purified cyclic peptide has the formula: (cyclo
3-14)MeCbm-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys--
OH and is referred to as Peptide No. 17.
EXAMPLE 18
[0089] The synthesis described in Example 12 is repeated with one
change. Following removal of the Boc group at the N-terminus, the
reaction that is carried out to add a carbamoyl moiety employs a
large (i.e. 6-7.times.) excess of NaOCN and thus creates a
significant portion of the end product that has a biuret group at
the N-terminus.
[0090] After deblocking the .alpha.-amino group at the N-terminus
using trifluoroacetic acid (TFA), a reaction is carried out with
about 1 gm of the peptidoresin about 650 mg of NaOCN and 5 ml of
acetic acid for 30 min. at 22.degree. C. in 10 ml of NMP. The
reaction results in the addition of a carbamoyl moiety to the urea
group that is initially formed at the N-terminus. Thereafter,
cleavage of the peptide from the resin and deprotection of the side
chain protecting groups, followed by cyclization and purification,
are carried out as in Example 2. A substantial portion of the
purified cyclic peptide has the formula:
(cyclo)BiuCys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cys-O-
H and is referred to as Peptide No. 18.
[0091] Peptides 17 and 18 bind strongly and selectively to
SSTR1.
EXAMPLE 19
[0092] The synthesis described in Example 9A is repeated with one
change. N.sup..alpha.MeBoc-Ser(Bzl) is used to provide the
13-position residue. Further elongation of the chain then proceeds
as in Example 15, and cleavage, deprotection, cyclization, and
purification are carried out as in Example 2. The purified, cyclic
peptide has the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-2Nal-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-N.sup..al-
pha.MeSer-Cys-OH and is referred to as Peptide No. 19.
EXAMPLE 19A
[0093] The synthesis described in Example 19 is repeated with two
changes. Boc-Phe is used to provide the 11-position residue, and
Boc-IAmp(Z) is used to provide the 9-position residue. Cleavage,
deprotection, cyclization, and purification are carried out as in
Example 2. The purified, cyclic peptide has the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-2Nal-IAmp-Thr-Phe-Thr-N.sup..alpha.MeSer-Cys-O-
H and is referred to as Peptide No. 19A. MS analysis shows a mass
of 1510.5 Da, which compares favorably to the calculated value of
1510.69 Da.
EXAMPLE 20
[0094] The synthesis described in Example 2 is repeated, this time
using N.sup..alpha.MeBoc-Ser(Bzl) to provide the 13-position
residue. Following removal of the Boc group, further elongation of
the chain, as in Example 2 is carried out, followed by cleavage,
deprotection, cyclization, and purification as in Example 2. The
purified, cyclic peptide has the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-N-
.sup..alpha.MeSer-Cys-OH and is referred to as Peptide No. 20.
EXAMPLE 21
[0095] The synthesis described in Example 14 is repeated with one
change. N.sup..alpha.MeBoc-Ser(Bzl) is used for the 13-position
residue. Elongation is then carried out as in Example 14, including
a reaction to add a carbamoyl moiety and thus create a urea group
at the N-terminus.
[0096] Thereafter, cleavage of the peptide from the resin and
deprotection of the side chain protecting groups, followed by
cyclization and purification, are carried out as in Example 2. The
purified cyclic peptide has the formula:
(cyclo)CbmCys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-N.sup..al-
pha.MeSer-Cys-OH and is referred to as Peptide No. 21.
EXAMPLE 21A
[0097] The synthesis described in Example 21 is repeated with one
change. Boc-IAmp(Z) is used for the 9-position residue. Elongation
is then carried out as in Example 14, including a reaction to add a
carbamoyl moiety and thus create a urea group at the N-terminus.
Cleavage of the peptide from the resin and deprotection of the side
chain protecting groups, followed by cyclization and purification,
are carried out as in Example 2. The purified cyclic peptide has
the formula:
(cyclo)CbmCys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Tyr-Thr-N.sup..alpha.MeSer-Cys-O-
H and is referred to as Peptide No. 21A.
[0098] Peptides 19 to 21A bind strongly and selectively to
SSTR1.
EXAMPLE 22
[0099] The synthesis described in Example 13 is repeated with one
change: N.sup..alpha.MeBoc-Ser(Bzl) is used to provide the
13-position residue.
[0100] Cleavage, deprotection and cyclization are then carried out
as in Example 2. The cyclized peptide is then purified by
subjection to analytical HPLC on a C.sub.18 column. The purified
cyclic peptide has the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Phe-Thr-N-
.sup..alpha.MeSer-Cys-OH and is referred to as Peptide No. 22.
EXAMPLE 22A
[0101] The synthesis described in Example 22 is repeated with one
change: Boc-IAmp(Z) is used to provide the 9-position residue.
Cleavage, deprotection and cyclization are then carried out as in
Example 2. The cyclized peptide is then purified by subjection to
analytical HPLC on a C.sub.18 column. The purified cyclic peptide
has the formula:
(cyclo)H-Cys-Lys-Phe-Phe-D-Trp-IAmp-Thr-Phe-Thr-N.sup..alpha.MeSer-Cys-OH
and is referred to as Peptide No. 22A. MS analysis shows a mass of
1499.8 Da, which compares favorably to the calculated value of
1498.68 Da.
EXAMPLE 23
[0102] The synthesis described in Example 22 is repeated with one
change. N.sup..alpha.Boc-D-Cys(Mob) is used instead of
N.sup..alpha.Boc-Cys(Mob). Cleavage, deprotection, cyclization, and
purification are carried out as in Example 2. The purified, cyclic
peptide has the formula:
(cyclo)H-D-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Phe-Thr-N.sup..a-
lpha.MeSer-Cys-OH and is referred to as Peptide No. 23.
EXAMPLE 24
[0103] The synthesis described in Example 15 is repeated with one
change. Following removal of the Boc group at the N-terminus, a
reaction is carried out to add a carbamoyl moiety and thus create
an urea group at the N-terminus.
[0104] After deblocking the .alpha.-amino group at the N-terminus
using trifluoroacetic acid (TFA), a reaction as described in
Example 12 is carried out and results in the addition of the
carbamoyl moiety at the N-terminus. Thereafter, cleavage of the
peptide from the resin and deprotection of the side chain
protecting groups, followed by cyclization and purification, are
carried out as in Example 2. The purified cyclic peptide has the
formula:
(cyclo)CbmCys-Lys-Phe-Phe-D-2Nal-N.sup..alpha.MeIAmp-Thr-Phe-Thr-Ser-Cys--
OH and is referred to as Peptide No. 24.
[0105] Peptides Nos. 22 to 24 bind selectively and strongly to
human SSTR1.
EXAMPLE 25
[0106] The synthesis described in Example 2 is repeated with one
change. Following removal of the Boc group at the N-terminus, a
reaction is carried out to add DOTA (1,4,7,10-tetraazacyclododecane
N, N', N'', N'''-tetraacetic acid) and thus provide a
polyaminopolycarboxylic chelant at the N-terminus.
N-hydroxysuccinimide ester is prepared by modification of a
procedure described by Lewis et al., Bioconjugate Chem. 5: 565-76
(1994). Freshly prepared EDC in H.sub.2O (12.25 mg in 40 .mu.l, 64
.mu.mol) is added to a solution of 60 mg (128 .mu.mol) of trisodium
DOTA (Parish Chemicals) and 27.7 mg (128 .mu.mol) of sulfo-NHS in
960 .mu.L of H.sub.2O at 4.degree. C. The reaction mixture is
stirred at 4.degree. C. for 30 minutes. The theoretical
concentration of active ester in the reaction mixture is 64 mM.
[0107] After deblocking the .alpha.-amino group at the N-terminus
using trifluoroacetic acid (TFA), a reaction is carried out with
the active ester solution at a pH of about 8.5. The mixture is
stirred for 18 hours at 4.degree. C. This reaction results in the
addition of the DOTA moiety at the N-terminus. Thereafter, cleavage
of the peptide from the resin and deprotection of the side chain
protecting groups, followed by cyclization and purification, are
carried out as in Example 2. The purified cyclic peptide has the
formula:
(cyclo)(DOTA)Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cy-
s-OH and is referred to as Peptide No. 25.
EXAMPLE 26
[0108] The synthesis described in Example 25 is repeated with one
change. The reaction at the N-terminus is carried out so as to add
a different polyaminopolycarboxylic chelant moiety, namely DPTA
(diaminetrimethylenepentaacetic acid). A solution of 40 g DTPA
(0.102 mol) in 1.2 1 of dry dimethylsulfoxide (DMSO) is prepared by
heating and stirring, then it is cooled at room temperature and
added with a solution of 11.73 g NHS (0.102 mol) in 300 ml DMSO,
then, drop by drop, with a solution of 19.6 g of
N,N'-dicyclohexylcarbodiimide (0.097 mol) in 400 ml DMSO. The
mixture is stirred for 16 hours, then filtered, and the filtrate is
concentrated by evaporation at 50.degree. C. and 5 Pa to a thick
oil of an about 160 ml volume.
[0109] After deblocking the .alpha.-amino group at the N-terminus
using trifluoroacetic acid (TFA), a reaction is carried out with
about 1 gm of the peptidoresin and the oil (added in portions) 0.1
M borate buffer pH 8, and 0.1 M NaCl, keeping pH at 8 by the
simultaneous addition of 2 N NaOH. At the end of the additions, the
mixture is stirred for 16 hours, then filtered and purified from
by-products and excess reagents by chromatographic desalting on a
Sephadex G-25 column. This reaction results in the addition of the
DPTA moiety at the N-terminus. Thereafter, cleavage of the peptide
from the resin and deprotection of the side chain protecting
groups, followed by cyclization and purification, are carried out
as in Example 2. The purified cyclic peptide has the formula:
(cyclo)(DPTA)Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Tyr-Thr-Ser-Cy-
s-OH and is referred to as Peptide No. 26.
[0110] Peptides 25 and 26 continue to bind strongly and selectively
to SSTR1 despite the presence of the chelants at each
N-terminus.
EXAMPLE 27
[0111] The synthesis described in Example 13 is repeated with one
change. Elongation of the chain by one residue is carried out by
coupling N.sup..alpha.Boc-Tyr(2Brz) at the N-terminus.
[0112] Cleavage, deprotection, cyclization and purification are
carried out as in Example 2. The purified, cyclic peptide has the
formula:
(cyclo)H-Tyr-Cys-Lys-Phe-Phe-D-Trp-N.sup..alpha.MeIAmp-Thr-Phe-Thr-Ser-Cy-
s-OH and is referred to as Peptide No. 27. This peptide and
peptides which include Tyr.sup.11 are readily radioiodinated with
.sup.125I to provide ligands for use in competitive drug screening
assays. Following radioiodination, Peptide No. 27 continues to bind
strongly to SSTR1 as did Peptide 2A.
[0113] In vitro Bioassay: The various somatostatin analogs are
tested in vitro for their ability to bind to isolated cloned
receptors expressed on CHO--K1 cells and CCL39 cells. CHO--K1 cells
are grown in Ham's F-12 medium, and CCL39 cells are grown in
Dulbecco's modified Eagle's medium/Ham's F-12(1:1) mix,
supplemented with 10% fetal bovine serum, 100 U/ml penicillin and
100 .mu.g/ml streptomycin, in humidified air containing 5% CO.sub.2
at 37.degree. C.
[0114] The molecular cloning of the genes encoding multiple
somatostatin receptor subtypes permits the individual expression of
these receptors in mammalian cells and the characterization of
their respective pharmacological profiles. Five such receptor
subtypes, termed SSTR1 through SSTR5, have been cloned and are
reported and described in Raynor et al., Molecular Pharmacology,
43, 838-844 (1993) and in Raynor et al., Molecular Pharmacology,
44, 385-392 (1993). These references describe binding assays that
can be used to determine whether particular SRIF analogs bind
selectively to one or more of the 5 receptor types and also whether
they bind to such receptor types with high or low affinity. Because
these receptor types have now generally been characterized with
regard to their pharmacological profiles, knowledge of the results
of such binding studies, along with knowledge of the unique
patterns of distribution of these receptors in the body indicate
that each receptor subtype may mediate distinct but overlapping
physiological effects of SRIF. As a result, compounds which bind
selectively to receptors SSTR1, for example, can be used to
modulate a particular physiological function of SRIF without
potentially having an undesired effect resulting from another
physiological function of SRIF which is mediated by other SRIF
receptors.
[0115] Cells are washed and prepared to produce homogenates as
described in detail in Reubi, J. C. et al, Eur. J. Nucl. Med. 2000,
27, 273-282.
[0116] Modulation of forskolin-stimulated adenylate cyclase
activity was determined using a radioimmunoassay measuring
intracellular cAMP levels by competition binding. SSTR1-expressing
cells were subcultured in 96-well culture plates at
2.times.10.sup.4 cells/well and grown for 24 hours. Culture medium
was removed from the wells, and 100 .mu.l of fresh medium
containing 0.5 mM 3-isobutyl-I-methylxanthine (IBMX) were added to
each well. Cells were incubated for 30 min. at 37.degree. C. Medium
was then removed and replaced with fresh medium containing 0.5 mM
IBMX, with or without 3 .mu.M forskolin and various concentrations
of peptides. Cells were incubated for 30 min. at 37.degree. C.
After removal of medium, cells were lysed, and cAMP accumulation
was determined using a commercially available cAMP scintillation
proximity assay (SPA) system (RPA 538), according to the
instructions of the manufacturer (Amersham, Aylesbury, UK). In
these studies, basal levels of cAMP production were 0.25.+-.0.02
pmol cAMP/well, rising to 3.2.+-.0.2 pmol cAMP/well in the presence
of 3 .mu.M forskolin representing a 12.8.+-.1.2 fold
stimulation.
[0117] cAMP data are expressed as percentage of stimulation over
the non-stimulated level. Values of EC.sub.50 (the agonist
concentration causing 50% of its maximal effect) are derived from
concentration-response curves.
[0118] Receptor autoradiography is performed on 20 .mu.m thick
cryostat sections of the membrane pellets, mounted on microscope
slides, and then stored at -20.degree. C. For each of the tested
compounds, complete displacement experiments are performed with the
universal somatostatin ligand radioligand .sup.125I
-[Leu.sup.8,D-Trp.sup.22,Tyr.sup.25]-somatostatin-28 that binds
with strong affinity to all five receptors. Increasing
concentrations of the unlabeled peptide are used ranging from
0.1-1000 nM. Unlabeled somatostatin-28 is run in parallel using the
same increasing concentrations, as a control. IC.sub.50 values are
calculated after quantification of the data using a
computer-assisted image processing system as known in this art. At
concentrations of 100 nM, Peptide No. 2 had minimal effects on the
binding of the SRIF-28 radioligand to human SSTR2, SSTR3, SSTR4 and
SSTR5 and Peptides 2A and 2B have even less effect. In contrast,
Peptide No. 2 is selectively bound to SSTR1, displacing the binding
of the radioligand to human SSTR1 with an IC.sub.50 value of about
6.1 nM, and Peptides Nos. 2B and 2A showed binding affinities of
3.6 and 17.1 nM respectively. Peptide No. 12 with the urea group at
the N-terminus shows good selectivity with good binding strength,
and Peptides Nos. 12B and 12A also show strong binding affinities
of 2.5 and 8.1 nM respectively. Peptide No. 9A containing the
D-2Nal.sup.8 modification selectively binds to SSTR1 and exhibits
an IC.sub.50 of about 21 nM. Peptide No. 15, the analog with the
D-2Nal.sup.8 modification and with Phe.sup.11, also selectively
binds to SSTR1, exhibiting an IC.sub.50 of about 32.5. The addition
of a carbamoyl to the N-terminus of Peptide 9A produces Peptide 16
having an IC.sub.50 of about 32, whereas the carbamoyl addition to
a peptide-extended with Tyr at the N-terminus shows a high binding
affinity of 15 in an analog (No. 14A) that can be radioiodinated
while remaining SSTR1-selective. Peptide No. 13 with D-Trp.sup.8
modification and with Phe.sup.11 likewise selectively binds SSTR1;
it exhibits an IC.sub.50 of about 8.7 nM.
[0119] To confirm that an iodinated version of one of these analogs
may serve as a selective SSTR1 radioligand, Peptide No. 27, i.e.
the N-terminus-extended Tyr.sup.2 analog of Peptide No. 13, is
synthesized, iodinated and tested for binding to the five cloned
SRIF receptors. No specific binding of Peptide No. 27 to SSTR2, 3,
4 and 5 is detectable. In contrast, Peptide No. 27 effectively
binds to SSTR1 to about the same extent as Peptide No. 13.
Moreover, it is believed that its binding strength and/or
selectivity can be improved by acylation of the Tyr (with its
phenolic hydroxyl protected) with carbamoyl (see Peptide No. 14B)
or that the alternative substitution and iodination of an analog
containing Tyr.sup.11 (see Peptide No. 2B) may provide improved
binding compared to the comparable Tyr.sup.2 analog.
[0120] Screening assays, as are well known in the art which employ
the receptor polypeptide SSTR1 directly from the recombinant host,
can be used to identify agents useful in blocking or mimicking
certain aspects of somatostatin as desired while eliminating the
undesirable aspects of the hormone which may arise from activation
or blocking of other receptors.
[0121] The potencies of certain SRIF analogs to inhibit radioligand
binding of .sup.125I-(Leu.sup.8,D-Trp.sup.22,Tyr.sup.24]SRIF-28 to
the various cloned SRIF receptors are shown in the following table
wherein the IC.sub.50 values are given in nanomolar concentration.
TABLE-US-00003 TABLE IC.sub.50 (nM) Compound mSSTR1 mSSTR2 mSSTR3
mSSTR4 mSSTR5 Peptide 7.2 >10,000 >1,000 >1,000 >10,000
No. 2 Peptide 17.1 >10,000 >1,000 >10,000 >1,000 No. 2A
Peptide 3.6 >10,000 >1,000 >1,000 >1,000 No. 2B Peptide
35 >1,000 975 >1,000 >1,000 No. 5A Peptide 24.5 >10,000
>1,000 >1,000 >10,000 No. 9A Peptide 43 >10,000
>1,000 >1,000 >10,000 No. 12 Peptide 8.1 >10,000
>1,000 >1,000 >1,000 No. 12A Peptide 2.5 >10,000 618
>1,000 >1,000 No. 12B Peptide 17.4 >10,000 >1,000
>10,000 >1,000 No. 12C Peptide 8.5 >1,000 >1,000 700
>1,000 No. 12D Peptide 8.7 >10,000 610 200 >10,000 No. 13
Peptide 15 >10,000 500 >1,000 >1,000 No. 14A Peptide 34.7
>1,000 290 >1,000 >1,000 No. 14B Peptide 33 >10,000
>1,000 487 >1,000 No. 14C Peptide 43 >1,000 >1,000
>1,000 >10,000 No. 15 Peptide 32 >10,000 >1,000
>10,000 >10,000 No. 16 Peptide 25 >10,000 600 >1,000
>1,000 No. 22A Peptide 25 >10,000 >1,000 300 >10,000
No. 27
[0122] The peptides of the invention not only provide more
selective ligands for binding SSTR1 but the use of labeled
peptides, for example, a radioiodinated version of one of Peptide
Nos. 2B, 12B, 12D, 14B or 27, facilitates drug screening for even
more effective antagonists. Competitive binding assays with
candidate compounds would first be carried out in this manner with
SSTR1 to search for high binding affinity; then by screening the
multiple SRIF receptors, it could be confirmed whether there was
selective binding to only this receptor, as is desired.
[0123] Because, as shown above, additions to the N-terminus of the
SRIF analog do not appear to adversely affect the selective
binding, it should be clear that these compounds can be complexed
with a cytotoxic or a radioactive agent for the purpose of carrying
that agent to a tumor or other tissue for which degradation is
desired. For example, a dialdehyde linker such as glutaraldehyde
may be used to link the SRIF analog to saporin or gelonin.
Likewise, linkers such as DOTA or DTPA or other suitable chelating
agents can be used to complex the SRIF analog with a highly
radioactive element as indicated hereinbefore. If desired, the
solubility of the SRIF analogs can be improved by acylation of the
N-terminal amino group using a hydrophilic compound, such as
hydroorotic acid or the like, or by reaction with a suitable
isocyanate, such as methylisocyanate or isopropylisocyanate, to
create a urea moiety at the N-terminus. Other agents can also be
N-terminally linked that will increase the duration of action of
the SRIF analog as known in this art.
[0124] These SRIF analogs or nontoxic salts thereof, combined with
a pharmaceutically or veterinarily acceptable carrier to form a
pharmaceutical composition, may be administered to animals,
including humans and other mammals, either intravenously,
subcutaneously, intramuscularly, percutaneously, e.g. intranasally,
intracerebrospinally or orally. The peptides should be at least
about 90% pure and preferably should have a purity of at least
about 98%; however, lower purities are effective and may well be
used with mammals other than humans. This purity means that the
intended peptide constitutes the stated weight % of all like
peptides and peptide fragments present. Administration to humans
should be under the direction of a physician to combat specific
tumors and cancers or to mediate other conditions where the SSTR3
receptors exert a control function, such as coupling to a tyrosine
phosphatase so that stimulation of this enzyme can be carried out
to mediate the anti-proliferative effects of SRIF. The required
dosage will vary with the particular condition being treated, with
the severity of the condition and with the duration of desired
treatment.
[0125] Such peptides are often administered in the form of
pharmaceutically or veterinarily acceptable nontoxic salts, such as
acid addition salts or metal complexes, e.g., with zinc, iron,
calcium, barium, magnesium, aluminum or the like. Illustrative of
such nontoxic salts are hydrochloride, hydrobromide, sulphate,
phosphate, tannate, oxalate, fumarate, gluconate, alginate,
maleate, acetate, citrate, benzoate, succinate, malate, ascorbate,
tartrate and the like. If the active ingredient is to be
administered in tablet form, the tablet may contain a binder, such
as tragacanth, corn starch or gelatin; a disintegrating agent, such
as alginic acid; and a lubricant, such as magnesium stearate. If
administration in liquid form is desired, sweetening and/or
flavoring may be used, and intravenous administration in isotonic
saline, phosphate buffer solutions or the like may be effected.
[0126] It may also be desirable to deliver these SRIF analogs over
prolonged periods of time, for example, for periods of one week to
one year from a single administration, and slow release, depot or
implant dosage forms may be utilized as well known in this art. For
example, a dosage form may contain a pharmaceutically acceptable
non-toxic salt of the compound which has a low degree of solubility
in body fluids, for example, an acid addition salt with a polybasic
acid; a salt with a polyvalent metal cation; or combination of the
two salts. A relatively insoluble salt may also be formulated in a
gel, for example, an aluminum stearate gel. A suitable,
slow-release depot formulation for injection may also contain an
SRIF analog or a salt thereof dispersed or encapsulated in a slow
degrading, non-toxic or non-antigenic polymer such as a polylactic
acid/polyglycolic acid polymer, for example, as described in U.S.
Pat. No. 3,773,919.
[0127] Therapeutically effective amounts of the peptides should be
administered under the guidance of a physician, and pharmaceutical
compositions will usually contain the peptide in conjunction with a
conventional, pharmaceutically or veterinarily-acceptable carrier.
A therapeutically effective amount is considered to be a
predetermined amount calculated to achieve the desired effect. The
required dosage will vary with the particular treatment and with
the duration of desired treatment; however, it is anticipated that
dosages between about 10 micrograms and about 1 milligram per
kilogram of body weight per day will be used for therapeutic
treatment. It may be particularly advantageous to administer such
compounds in depot or long-lasting form as earlier described. A
therapeutically effective amount is typically an amount of an SRIF
analog that, when administered peripherally, e.g. intravenously, in
a physiologically acceptable composition, is sufficient to achieve
a plasma concentration thereof from about 0.1 .mu.g/ml to about 100
.mu.g/ml, preferably from about 1 .mu.g/ml to about 50 .mu.g/ml,
more preferably at least about 2 .mu.g/ml and usually 5 to 10
.mu.g/ml. In these amounts, they may be used for the prevention of
IH, or in appropriate treatments for cardiovascular diseases and
other SSTR1-mediated physiopathologies.
[0128] Although the invention has been described with regard to its
preferred embodiments, which constitute the best mode presently
known to the inventors, it should be understood that various
changes and modifications as would be obvious to one having the
ordinary skill in this art may be made without departing from the
scope of the invention which is set forth in the claims appended
hereto. Although the claims variously define the invention in terms
of a peptide sequence, it should be understood that such is
intended to include nontoxic salts thereof which are well known to
be the full equivalent thereof and which are most frequently
administered. Instead of the simple free acid at the C-terminus, a
lower alkyl ester or amide may be incorporated as well known in the
peptide art. Cyclic peptides having an amino acid residue sequence
substantially identical to the sequence of the SRIF analogs
specifically shown herein, in which one or more residues have been
conservatively substituted with a functionally similar amino acid
residue, are considered to be equivalents so long as they
selectively bind to SSTR1.
[0129] As previously indicated, these specified modifications can
be incorporated in previously disclosed SRIF analogs to create
SSTR1-selectivity. The inclusion of residues in the 1- and
2-positions is optional, but except for Tyr, D-Tyr or D-Ala, such
elongation is not considered worthwhile unless it would favorably
influence solubility and/or resistance to aminopeptidases. Often a
carbamoyl moiety or a conjugating agent will be linked to the
.alpha.-amino group of the residue at the N-terminus of these
peptides, and these moieties are included in the definition of
Xaa.sub.1. Broadly it is considered that cyclic somatostatin analog
peptides having specific high affinity for the SRIF receptor SSTR1
can be created by modifying the amino acid sequence of existing
SRIF analogs which are known in the art to exhibit SRIF biological
activity. The modified peptide should contain a Cys-Cys disulfide
bond with a sequence of at least 9 residues located between such
Cys residues as a ring structure with the 5-position residue being
deleted; it should contain
Lys-Phe-Phe-D-Xaa.sub.8-N.sup..alpha.MeIAmp-Thr-Tyr or its
equivalent adjacent the N-terminal Cys of the ring structure. Such
cyclic SRIF analogs may also be modified by N-methylation of the
.alpha.-amino group on another residue in the ring sequence to
create resulting SRIF analogs that retain their high specificity
for SSTR1. Such peptides and salts thereof are considered as being
within the scope of the claimed invention. The inclusion of a
carbamoyl moiety or a lower alkyl carbamoyl, preferably methyl,
ethyl or isopropyl, or biuret at the N-terminus of any of these
analogs should not detract from selectivity and is expected to
increase the duration of biological activity following
administration.
[0130] As used herein, all temperatures are .degree. C. and all
ratios are by volume. Percentages of liquid materials are also by
volume. The disclosures of all U.S. patents and patent applications
and publications set forth hereinbefore are expressly incorporated
by reference.
[0131] Various features of the invention are emphasized in the
claims which follow.
* * * * *