Note: Descriptions are shown in the official language in which they were submitted.
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-1-
BOMBESIN ANALOGS FOR TREATMENT OF CANCER
FIELD OF INVENTION
The present invention encompasses novel peptides that are antagonists
to bombesin and bombesin like peptides and are useful in the treatment of
cancer.
The invention particularly relates to the design and synthesis of the novel
peptides
incorporating a,a-amino acids in a site specific manner. The invention encom-
passes methods for the generation of these peptides, compositions containing
the
peptides and the pharmacological applications of these peptides especially in
the
treatment and prevention of cancer.
BACKGROUND OF THE INVENTION
Bombesin is a 14 amino acid peptide which was first isolated from
the skin of the frog Bombina bombina (Anastasi et al., Experientia, 1971, 27,
166)
and has the sequence:
pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-
Leu-Met-NH2 (SEQ ID NO: 1)
Gastrin releasing peptide (GRP) is a 27 amino acid peptide isolated
from the porcine gut. The last ten amino acids at the C-terminus of gastrin
releasing peptide correspond with one amino acid alteration (3) to the last
ten amino
acids of bombesin, viz:
H-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH2
(SEQ ID NO:2).
It has been reported (J. H. Walsh and J. R. Reeve, Peptides 6, (3),
63-68, (1985)) that bombesin and bombesin-like peptides such as gastrin
releasing
peptide (GRP) are secreted by human small-cell lung cancer (SCLC) cells. It
has
been postulated (P. J. Woll and E. Rozengurt, PNAS 85, 1859-1863, (1988)) that
gastrin releasing factor antagonists would bind competitively to bombesin
receptors
in animals and would therefore be of use in the treatment of SCLC and/or in
the
control of clinical symptoms associated with this disease and due to
hypersecretion
of this peptide hormone. Analogues of bombesin / GRP have been shown to
inhibit
the binding of gastrin releasing peptide to a SCLC cell line and to inhibit
the
growth of SCLC cells in-vitro and in-vivo (S. Mahmoud et al., Cancer Research,
1991, 51, 1798; Moody TW et al., Life Sci., 1995, 56, 521; Moody TW et al.,
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-2-
PF ,tiles, 1996, 17, 1337). After Bombesin/GRP cell receptors were established
on
SCLC cells, receptors were also found to be present on human prostate cells.
Reile
H et al., (Prostate, 1994, 25: 29-38) showed that the PC-3 and DU-145 human
prostate cancer cell lines possess specific high-affinity receptors for
bombesin/GRP
and are suitable models for the evaluation of anti-neoplastic activity of new
bombesin/GRP antagonists in the treatment of androgen-dependent prostate
cancer.
Bombesin also increased the penetration of the two human prostatic carcinoma
cell
lines, the relatively indolent LNCaP cells and the aggressively growing and
invasive
PC-3 cells, in an in vitro invasion of reconstituted basement membrane
(Matrigel)
(Hoosein NM et al., J Urol, 149(5): 1209-1213). High-affinity binding sites
for
GRP were found on human colorectal cancer tissue (Preston, SR. et al, Br. J.
Can.,
1995, 71, 1087), suggesting that bombesin-like peptides may have a role in the
pathogenesis of colorectal cancer, and bombesin receptor antagonists may be of
value in the treatment of receptor-positive tumours. Inhibitory effects of
bombesin/
GRP antagonist RC-3095 and somatostatin analogue RC-160 were also seen on
growth of HT-29 human colon cancer xenografts in nude mice (Radulovic S et
al.,
Acta Oncol, 1994, 33(6): 693-701).
Studies with the anti-bombesin/GRP antibodies lead to the hypothesis
that it may be possible to disrupt the autocrine growth cycle of bombesin/GRP
using
designed peptide receptor antagonists. Since then several types of Bombesin
anta-
gonists have been reported. These antagonists have been defined by type and
position of the substitutions of the natural sequence. Early receptor
antagonists
suffered from low potency, lack of specificity, and toxicity, which presented
serious
problems with their scientific and therapeutic use.
More recent work has concentrated on modification of the carboxy
terminal (C-terminal) region of these peptides to interrupt the receptor
interaction
utilizing a variety of different types of C-terminal modified analogs. These
have
included incorporation of D-amino acids, non-peptide bonds for example (psi.
CH2NH), amide, and ester modifications. These alterations gave rise to certain
peptides having improved characteristics (Staley J et al., Peptides, 1991,
12(1): 145-
9; Coy DH et al., J Natl Cancer Inst Monogr, 1992, 13: 133-9). Other patents
that
describes bombesin and related analogs are:
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-3-
USP5,834,433 (1998)
USP 5,723,578 (1998)
USP 5,620,959 (1997)
USP 5,620,955 (1997)
USP 5,428,019 (1995)
USP 5,369,094 (1994)
USP 5,084,555 (1992)
A Bombesin/GRP antagonist (RC-3940-II) was found to inhibit the
proliferation of SW-1990 human pancreatic adenocarcinoma cells in vivo and in
vitro (Qin, Y. et al., 1995, Int. J. Cancer, 63, 257). Similar effect was seen
with
bombesin/GRP antagonist RC-3095 on the growth of CFPAC-1 human pancreatic
cancer cells transplanted to nude mice or cultured in vitro (Qin Y et al., Can
Res,
1994, 54(4): 1035-41).
As reported earlier, the autocrine growth cycle of bombesin/GRP in
SCLC can be disrupted by bombesin/GRP antagonists such as [Psi 13,14]
bombesin.
Several bombesin analogues were solid phase synthesized and incubated with
intact
SCLC cells at 37 C in RPMI medium in a time course fashion (0-1080 minutes) to
determine enzymatic stability. The proteolytic stability of the compounds was
determined by subsequent HPLC analysis. [Psi 13, 14] Bombesin was found to be
very stable to metabolic enzymes (Tl/2= 646 min.) and inhibited SCLC xenograft
formation in vivo in a dose-dependent manner (Davis TP et al., Peptides, 1992,
13(2): 401-7).
Female athymic nude mice bearing xenografts of the MCF-7 MIII
human breast cancer cell line were treated for 7 weeks with bombesin/GRP
antagonist (DTpi6, Leul3 psi[CH2NH]-Leul4) bombesin (6-14)(RC-3095) injected
subcutaneously daily at a dose of 20 g and LHRH antagonist SB-75 (Cetrorelix)
administered biweekly in the form of microgranules releasing 45 g/ day. After
2
weeks of treatment, a significant inhibition of tumor volume was observed in
the
groups treated with RC-3095 alone or in combination with SB-75 (Yano T et al.,
Cancer, 1994, 73(4): 1229-38).
Pinski J et al., (Int. J. Cancer, 1994, 57(4): 574-580), demonstrated
for the first time that the growth of gastrin-responsive human gastric
carcinoma
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-4-
MKN45 cell line xenografts in nude mice could be inhibited not only by
somatostatin analogues, but also by administration of modem bombesin/GRP
antagonists, such as RC-3095, or. a combination of these. RC-3095 also
effectively
inhibited tumor growth in nude mice bearing xenografts of the human gastric
cancer
cell line Hs746T (Qin Y et al., J Cancer Res Clin Oncol, 1994,120(9):519-528).
This invention describes the preparation and use of peptide analogs of
bombesin/GRP using constrained amino acids and their use for cancer therapy,
alone, or in combination or as an adjunct to or with other chemotherapeutic
agents
and compounds.
The design of conformationally constrained bioactive peptide
derivatives has been one of the widely used approaches for the development of
peptide-based therapeutic agents. Non-standard amino acids with strong
conformational preferences may be used to direct the course of polypeptide
chain
folding, by imposing local stereochemical constraints, in de novo approaches
to
peptide design. The conformational characteristics of a,a-dialkylated amino
acids'
have been well studied. The incorporation of these amino acids restricts the
rotation
of 4), lP angles, within the molecule, thereby stabilizing a desired peptide
conformation. The prototypic member of a,a-dialkylated aminoacids, a-
aminoisobutyric acid (Aib) or a,a-dimethylglycine has been shown to induce ((3-
turn or helical conformation when incorporated in a peptide sequence (Prasad
and
Balaram, (1984); CRC Crit. Rev. Biochem. 16, 307-347; Karle and Balaram (1990)
Biochemistry 29, 6747-6756). The conformational properties of the higher
homologs of a,a-dialkylated amino acids such as diethylglycine (Deg), di-n-
propylglycine (Dpg) and di-n-butylglycine (Dbg) as well as the cyclic side
chain
analogs of a,a-dialkylated amino acids such as 1-aminocyclopentane carboxylic
acid
(Ac5c), 1-aminocyclohexane carboxylic acid (Ac6c), 1-aminocycloheptane
carboxylic acid (Ac7c) and 1-aminocyclooctane carboxylic acid (Ac8c) have also
been shown to induce folded conformation (Prasad et al., (1995), Biopolymers
35,
11-20; Karle et al., (1995); J. Amer. Chem. Soc. 117, 9632-9637). a,a-
dialkylated
amino acids have been used in the design of highly potent chemotactic peptide
analogs (Prasad et al., (1996) Int. J. Peptide Proteins RCS. 48, 312-318).
The present invention exploits the conformational properties of a,a-
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-5-
dialkylated amino acids for the design of biologically active peptide
derivatives,
taking bombesin as the model system under consideration. Furthermore, it has
been
shown that lipophilization of bioactive peptides improves their stability,
bioavailability and the ability to permeate biomembranes (Dasgupta, P et al;
1999,
Pharmaceutical Res. 16, 1047-1053; Gozes, I, et al 1996, Proc. Natl. Acad.
Sci.
USA, 93, 427-432). In the present invention, we have also synthesized peptide
derivatives having N-terminal alkanoyl groups from C2-C16 carbon atoms, which
retain anticancer activity.
The present invention exploits the conformational properties of a,a-
dialkylated amino acids for the design of biologically active peptide
derivatives,
taking bombesin as the model system under consideration. Furthermore, it has
been
shown that lipophilization of bioactive peptides improves their stability,
bioavailability and the ability to permeate biomembranes (Dasgupta, P et al;
1999,
Pharmaceutical Res. 16, 1047-1053; Goes, L, et al., 1996, Proc. Natl. Acad.
Sci.
USA, 93, 427-432).
Throughout the specification and claims the amino acid residues are
designated by their standard abbreviations. Amino acids denote L-configuration
unless otherwise indicated by D or DL appearing before the symbol and
separated
from it by a hypen. Throughout the specification and claims, the following
abbreviations are used with the following meanings:
BOP: Benzotriazole-l-yl-oxy-tris-(dimethylamino)-
phosphonium hexfluorophosphate
PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexofluorophospate
TBTU: 2-(1H-Benzotriazole-lyl)-1,1,3,3-tetramethyluronium
tetrafluroborate
HBTU: O-B enzotriazole-N,N,N',N' -tetramethyl-uronium-
hexofluoro-phosphate
HOBt: 1-Hydroxy Benzotriazole
DCC: Dicyclohexyl carbodiimide
DIPCDI: Diisopropyl carbodiimide
DIEA: Diisopropyl ethylamine
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-6-
DMF: Dimethyl formamide
DCM: Dichloromethane
NMP: N-Methyl-2-pyrrolidinone
TFA: trifluoroacetic acid
SUMMARY OF INVENTION
The present invention provides novel polypeptides of the following
general formula,
X-D-Phe- Gln-Rl -R2-Val-R3 -His-R4-NH2
wherein X is acetyl or straight, branched, or cyclic alkanoyl group from 3-16
carbon
atoms, or X is deleted,
Rl is Trp or D-Trp,
R2 is Ala, Aib or Deg,
R3 is Gly, Aib, Deg, Dpg or Ac5c,
R4 is Leu or Ile or a hydrolyzable carboxy protecting group;
or a pharmaceutically acceptable salt of the polypeptide. At least one of R2
or R3
is a non-standard amino acid. The invention also encompasses methods for
making
the peptides, compositions containing the peptides and use of the peptides.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel polypeptides of the following
general formula,
X-D-Phe-Gln-Rl -R2-V al-R3-His-R4-NH2
wherein X is acetyl or straight, branched, or cyclic alkanoyl group from 3-16
carbon
atoms, or X is deleted,
R1 is Trp or D-Trp,
R2 is Ala, Aib or Deg,
R3 is Gly, Aib, Deg, Dpg or AcSc,
R4 is Leu or Ile or a hydrolyzable carboxy protecting group;
or a pharmaceutically acceptable salt of the polypeptide. At least one of R2
or R3
is a non-standard amino acid.
A hydrolyzable carboxy protecting group are those groups which on
hydrolysis converts to carboxylic group such as -COONH2, -COOMe, etc.
The preferred alkanoyl groups are acetyl, n-butanoyl, n-hexanoyl, n-
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-7-
octanoyl, lauroyl, myristoyl, palmitoyl, isohexanoyl, cyclohexanoyl,
cyclopentyl-
carbonyl, n-heptanoyl, n-decanoyl, n-undecanoyl and 3,7-dimethyloctanoyl.
Salts encompassed within the term "pharmaceutically acceptable salts"
refer to non-toxic salts of the compounds of this invention. Representative
salts and
esters include:
acetate, ascorbate, benzenesulfonate, benzoate, bicarbonate, bisulfate,
bitartrate, borate, camsylate, carbonate, citrate, dihydrochloride,
methanesulfonate,
ethanesulfonate, p-toluenesulfonate, cyclohexylsulfamate, quinate, edetate,
edisylate,
estolate, esylate, fumaxate, gluconate, glutamate, glycerophophates,
hydrobromide, 5
hydrochloride, hydroxynaphthoate, lactate, lactobionate, laurate, malate,
maleate,
mandelate, mesylate, mucate, napsylate, nitrate, n-methylglucamine, oleate,
oxalate,
palmoates, pamoate (embonate), palmitate, pantothenate, perchlorates,
phosphate/diphosphate, polygalacturonate, salicylates, stearate, succinates,
sulfate,
sulfamate, subacetate, succinate, tannate, tartrate, trifluoroacetate,
tosylate and
valerate.
Other salts include Ca, Li, Mg, Na and K salts; salts of amino acids
such lysine or arginine; guanidine, diethanolamine or choline; ammonium,
substituted ammonium salts or aluminum salts.
The salts can be prepared by standard techniques.
Preferred peptides of this invention are:
D-Phe-Gln-Trp-Ala-Val-Aib-His-Leu-NH2 (SEQ ID NO:3)
D-Phe-Gln-Trp-Aib-Val-Gly -His-Leu-NH2 (SEQ ID NO:4)
D-Phe-Gln-D-Trp-Ala-Val-Aib-His-Leu-NH2 (SEQ ID NO:5)
D-Phe-Gln-Trp-Aib-Val-Gly-His-Ile-NH2 (SEQ ID NO:6)
D-Phe-Gln-Trp-Ala-Val-Aib-His-Ile-NH2 (SEQ ID NO:7)
D-Phe-Gln-D-Trp-Ala-Val-Dpg-His-Leu-NH2 (SEQ ID NO:8)
D-Phe-Gln-Trp-Deg-Val-Gly-His-Leu-NH2 (SEQ ID NO:9)
D-Phe-Gln-Trp-Ala-Val-Ac5c-His-Leu-NH2 (SEQ ID NO: 10)
Butanoyl-D-Phe-Gln-Trp-Ala-Val-Aib-His-Leu-NH2 (SEQ ID NO:
11)
Octanoyl-D-Phe-Gln-Trp-Ala-Val-Aib-His-Leu-NH2 (SEQ ID NO:
12)
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-8-
The present invention also envisages methods of prevention and
treatment of cancer using the polypeptides of the present invention,
pharmaceutical
compositions comprising such polypeptides and processes for their preparation.
These peptides possess antagonist properties against bombesin and bombesin-
like
peptides and are useful in the prevention and treatment of malignant diseases.
Suitable routes for administration of the peptides are those known in
the art and include oral, rectal, transdermal, vaginal, transmucosal, or
intestinal
administration; parenteral delivery, including intramuscular, subcutaneous,
intradedullary injections, as well as intrathecal, direct intraventricular,
intravenous,
intraperitoneal, intranasal, or intraocular injections.
Pharmaceutical compositions suitable for use in present invention
include compositions wherein the active ingredients are contained in an
effective
amount to achieve its intended purpose. In addition to the active ingredients,
these
pharmaceutical compositions may contain suitable pharmaceutically acceptable
carriers, excipients, diluents, solvents, flavorings, colorants etc. The
preparations
may be formulated in any form including but not limited to tablets, dragees,
capsules, powders, syrups, suspensions, slurries, time released formulations,
sustained release formulations, pills, granules, emulsions, patches,
injections,
solutions, liposomes or nanoparticles.
The exact formulation, route of administration and dosage can be
chosen by the individual physician in view of the patient's condition.
The term "an effective amount" means that amount of a drug or
pharmaceutical agent that will elicit the biological or medical response of a
tissue,
system, animal or human that is being sought.
Toxicity and therapeutic efficacy of the peptides of this invention can
be determined by standard pharmaceutical procedures in cell cultures or
experimental animals.
The novel peptide analogs embodied in the present invention contain
amino acids, namely a,a-dialkylated amino acids, which have been known to
induce highly specific constraints in the peptide backbone. The a,a-
dialkylated
amino acids, used in the present invention are synthesized from the
corresponding
ketones. In a preferred embodiment of the invention, the ketones are first
converted
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-9-
into the corresponding hydantoins which are hydrolyzed using a strong acid or
base,
preferably H2SO4, HC1, NaOH or Na22CO3 to yield the aforesaid amino acids. In
a
preferred embodiment of the present invention, 60% sulphuric acid has been
employed as the hydrolyzing agent.
The present invention also provides a solid phase synthesis process
for the preparation of peptide analogs of the general formula (I):
X-D-Phe-Gln-RI-R2-Val-R3-His-R4-NH2
wherein X is acetyl or straight, branched, or cyclic alkanoyl group from 3-16
carbon
atoms or X is deleted,
R1 is Trp or D-Trp,
R2 is Ala, Aib or Deg,
R3 is Gly, Aib, Deg, Dpg or Ac5c,
R4 is Leu or Ile
which comprises sequentially loading the corresponding protected a,a-
dialkylated
amino acids in sequential cycles to the amino terminus of a solid phase resin,
coupling the amino acids in the presence of conventional solvents and reagents
to
assemble a peptide-resin assembly, removing the protecting groups and cleaving
the
peptide from the resin to obtain a crude peptide analog.
The novel peptides in the present invention have been generated by
using solid phase techniques or by a combination of solution phase procedures
and
solid phase techniques or by fragment condensation. These methods for the
chemical synthesis of polypeptides are well known in the art (Stewart and
Young,
1969, Solid Phase Peptide Synthesis, W.H. Freeman & Co.).
In a preferred embodiment of the present invention the peptides were
synthesized using the Fmoc strategy, on a semi automatic peptide synthesizer
(CS
Bio, Model 536), using optimum side chain protection. The peptides were
assembled
from C-terminus to N-terminus. Peptides amidated at the carboxy-terminus were
synthesized using the Rink Amide resin. The loading of the first Fmoc
protected
amino acid was achieved via an amide bond formation with the solid support,
mediated by Diiopropylcarbodiimide (DIPCDI) and HOBt. Substitution levels for
automated synthesis were preferably between 0.2 and 0.6 mmole amino acid per
gram resin.
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
- 10-
The resin employed for the synthesis of carboxy-terminal amidated
peptide analogs was 4-(2', 4'-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxymethyl
derivatized polystyrene 1% divinylbenzene (Rink Amide) resin (100-200 mesh),
procured from Calbioichem-Novabiochem Corp., La Jolla, U.S.A., (0.47
milliequivalent NH2/g resin).
The N-terminal amino group was protected by 9-fluorenyhnethoxy-
carbonyl (Fmoc) group. Trityl (trt) or t-butyloxycarbonyl (Boc) were the
preferred
protecting groups for imadazole group of Histidine residue. The hydroxyl
groups of
Serine, Threonine and Tyrosine were preferably protected by t-butyl group
(tBu)
2,2,5,7,8-pentamethyl-chroman-6-sulfonyl (Pmc) or 2,2,4,7,-pentamethyl-dihydro-
benzenofuran-5 5-sulfonyl (Pbf) were the preferred protecting groups for the
guandino group of Arginine. Trityl was the preferred protecting group for
Asparagine and Glutamine and tertiary butyl group (tBu) was the preferred
protecting group for Aspartic acid and Glutamic acid. The tryptophan residue
was
either left unprotected or used with Boc protection. The side chain amino
group of
Lysine was protected using Boc group preferably.
In a preferred embodiment of the invention, 2-8 equivalents of Fmoc
protected amino acid per resin nitrogen equivalent were used. The activating
reagents used for coupling amino acids to the resin, in solid phase peptide
synthesis,
are well known in the art. These include DCC, DIPCDI, DIEA, BOP, PyBOP,
HBTU, TBTU, or HOBt. Preferably, DCC, DIPCDI/HOBt or HBTU/HOBT and
DIEA were used as activating reagents in the coupling reactions.
The protected amino acids were either activated in situ or added in
the form of preactivated esters known in the art such as NHS esters, Opfp
esters etc.
Atherton, E. et. al, 1988, J. Chem. Soc., Perkin Trans.I, 2887; Bodansky, M.
in
"The Peptides, Analysis, Synthesis and Biology (E. Gross, J, Meienhofer, eds)
Vol.
I, Academic Press, New York, 1979, 106.
The coupling reaction was carried out in DMF, DCM or NMP or a
mixture of these solvents and was monitored by Kaiser test (Kaiser et al.,
Anal.
Biochem., 34, 595-598 (1970)). In case of a positive Kaiser test, the
appropriate
amino acid was re-coupled using freshly prepared activated reagents.
After the assembly of the peptide was completed, the amino-terminal
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-11-
Fmoc group was removed and then the peptide-resin was washed with methanol and
dried. The peptides were then deprotected and cleaved from the resin support
by
treatment with trifluoroacetic acid, crystalline phenol, ethanedithiol,
thioanisole and
de-ionized water for 1.5 to 5 hours at room temperature. The crude peptide was
obtained by precipitation with cold dry ether, filtered, dissolved, and
lyophilized.
The resulting crude peptide was purified by preperative high
performance liquid chromatography (HPLC) using a LiChroCART C,8 (250.
Times. 10) reverse phase column (Merck, Darmstadt, Germany) on a Preparative
HPLC system (Shimadzu Corporation, Japan) using a gradient of 0.1 % TFA in
acetonitrile and water. The eluted fractions were reanalyzed on Analytical
HPLC
system (Shimadzu Corporation, Japan) using a C18 LiChrospherg , WP-300 (300 X
4) reverse- phase column. Acetonitrile was evaporated and the fractions were
lyophilized to obtain the pure peptide. The identity of each peptide was
confirmed
by electron-spray mass spectroscopy.
.15 Synthesis Of Peptides
A peptide of the present invention can be made by exclusively solid
phase techniques, by partial solid phase/solution phase techniques and/or
fragment
condensation. Preferred, semi-automated, stepwise solid phase methods for
synthesis of peptides of the invention are provided in the examples discussed
in the
subsequent section of this document.
The present invention will be further described in detail with
reference to the following examples, as will be appreciated by a person
skilled in
the art are merely illustrative and should not be construed as limiting.
Various
other modifications of the invention will be possible without departing from
the
spirit and scope of the present invention.
EXAMPLE I
First loading on Rink Amide Resin
A typical preparation of the Fmoc-Leu-Rink Amide Resin was carried
out using 0.5g of 4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)phenoxymethyl
derivatized polystyrene 1% divinylbenzene (Rink Amide) resin (0.7 mM/g) (100-
200
mesh), procured from Advanced Chemtech, Louisville, KY, U.S.A., (0.7
milliequivalent NH2 resin). Swelling of the resin was typically carried out in
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
- 12-
dichloromethane measuring to volumes 10-40 ml/g resin. The resin was allowed
to
swell in methylene chloride (2 X 25 ml, for 10 min.). It was washed once in
dimethylformamide (DMF) for 1 min. All solvents in the protocol were added in
20 ml portions per cycle. The Fmoc-protecting group on the resin was removed
by
following steps 3-7 in the protocol. The deprotection of the Fmoc group was
checked by the presence of blue beads in Kaiser test. For loading of the first
amino
acid on the free amino (NH2) group of the resin, the first amino acid, Fmoc-
Leu-
OH, was weighed in three to six fold excess, along with a similar fold excess
of
HOBt, in the amino acid vessel of the peptide synthesizer. These were
dissolved in
dimethylformamide (A.C.S. grade) (J.T.Baker, Phillipsburg, New Jersey, U.S.A.)
and activated with DIPCDI, just prior to the addition to the resin in the
reaction
vessel of the peptide synthesizer. HOBt was added in all coupling reactions,
especially in the case of Gln and His. The coupling reaction was carried out
for a
period ranging from 1-3 hours. The loading of the amino acid on the resin was
confirmed by the presence of colorless beads in the Kaiser Test. The loading
efficiency was ascertained by the increase of weight of the resin after the
addition
of the amino acid.
EXAMPLE 2
Synthesis of D-Phe-Gln-Trp-Ala-Val-Aib-His-Leu-NH2 (SEQ ID NO: 3)
The synthesis of SEQ ID NO: 3, amidated at the carboxy- terminus,
was initiated by using all of the resin loaded with Fmoc-Leu-OH as prepared in
Example 1 above. This was subjected to stepwise deprotection and coupling
steps
as in steps 1-10 of the synthesis cycle. In each coupling reaction, a two to
six fold
excess of amino acid, DIPCDI and HOBt were used. Upon completion of synthesis
and removal of the N-terminal Fmoc protecting group (steps 1-6 of the
synthesis
cycle), the peptide- resin was washed twice with methanol, dried and weighed
to
obtain 0.649g. This was subjected to cleavage in a cleavage mixture consisting
of
trifluoroacetic acid and scavengers, ethanedithol, crystalline phenol and
thioanisole
and water for a period of 1.5 to 5 hours at room temperature with continuous
stirring. The peptide was precipitated using cold dry ether to obtain - 330 mg
of
crude peptide. The crude peptide was purified on a C18 preperative reverse
phase
HPLC column (250 X 10) on a gradient system comprising acetonitrile and water
in
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-13-
0.1 % TFA as described previously in the art. The prominent peaks were
collected
and lyophilized, reanalyzed on analytical HPLC and subjected to mass
spectrometry.
There was a good agreement between the observed molecular weight and
calculated
molecular weight (Calculated Mass - 983; Observed Mass - 984.2 ). The pure
peptide was then used for bioassays.
EXAMPLE 3
Synthesis of D-Phe-Gln-TM-Aib-Val-Gly-His-Leu-NH,, (SEQ ID NO:4)
The synthesis, cleavage and lyophilization steps were carried out as in
the Example 2 above using the appropriate amino acids. The calculated mass was
- 969 and the observed mass was 970.4.
EXAMPLE 4
Synthesis of D-Phe-Gln-D-Trp-Ala-Val-Aib-His-Leu-NH. (SEQ ID NO:5)
The synthesis, cleavage and lyophilization steps were carried out as in
the Example 2 above using the appropriate amino acids. The calculated mass was
- 983 and the observed mass was 984.30.
EXAMPLE 5
Synthesis of D-Phe-Gln-TM-Aib-Val-Gly-His-Ile-NH, (SEQ ID NO:6)
The synthesis, cleavage and lyophilization steps were carried out as in
the Example 2 above using the appropriate amino acids. The calculated mass was
- 969 and the observed mass was 970.2.
EXAMPLE 6
Synthesis of D-Phe-Gln-Trp-Ala-Val-Aib-His-Ile-NH2 (SEQ ID NO:7)
The synthesis, cleavage and lyophilization steps were carried out as in
the Example 2 above using the appropriate amino acids. The calculated mass was
- 983 and the observed mass was 984.2.
EXAMPLE 7
Synthesis of D-Phe-Gln-D-Trp-Ala-Val-Dpg -His-Leu-NH, (SEQ ID NO:8)
The synthesis, cleavage and lyophilization steps were carried out as in
the Example 2 above using the appropriate amino acids. The calculated mass was
- 1039 and the 25 observed mass was 1040.4.
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-14-
EXAMPLE 8
Synthesis D-Phe-Gln-Trp-Deg-Val-Gly-His-Leu-NH, (SEQ ID NO:9)
The synthesis, cleavage and lyophilization steps were carried out as in
the Example 2 above using the appropriate amino acids. The calculated mass was
- 997 and the observed mass was 998.5.
EXAMPLE 9
Synthesis of D-Phe-Gln-!W-Ala-Val-Ac5c-His-Leu-NH, (SEO ID NO: 10)
The synthesis, cleavage and lyophilization steps were carried out as in
the Example 2 above using the appropriate amino acids. The calculated mass was
- 1009 and the observed mass was 1010.4.
EXAMPLE 10
Synthesis of Butanoyl-D-Phe-Gln-Trp-Ala-Val-Aib-His-Leu-NHZ (SEQ ID NO: 11)
The conjugation of the butanoyl group at the N-terminal position was
done on solid phase. The above peptide sequence was synthesized on resin as
described in Example 2. After the deprotection of D-Arg amino acid it was
further
coupled with butanoic acid in DMF using DIPCDI and HOBT. The cleavage and
purification was further carried out following the standard protocol as
described in
Example 2. The final peptide was further analyzed by mass spectroscopy. The
calculated mass and observed were in good agreement. (calculated mass = 1053,
observed mass = 1054.2).
EXAMPLE 11
Synthesis of Octanoyl-D-Phe-Gln-TM-Ala-Val-Aib-His-Leu-NH, (SEQ ID NO: 12)
The conjugation of the octanoyl group at the N-terminal position after
the peptide synthesized as described in Example 2 was done on solid phase
using
octanoic acid in DMF using DIPCDI and HOBT. The cleavage and purification
was further carried out following the standard protocol as described in
Example 2.
The final purified peptide was further analyzed by mass spectroscopy. The
calculated mass and observed were in good agreement. (calculated mass = 1109,
observed mass = 1110.5).
BIOLOGICAL ACTIVITY OF PEPTIDES
The cytoxicity of the peptide analog was carried out by two day MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. MTT
assay
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-15-
is based on the principle of uptake of MTT, a tetrazolium salt, by
metabolically
active cells where it is metabolized by active mitochondria into a blue
colored
formazon product, which can be read spectrometrically (J. of Immunological
Methods 65: 55-63, 1983). To prepare the MTT stock solution needed, MTT was
dissolved in phosphate buffered saline with a pH of 7.4 to obtain an MTT
concentration of 5 mg/ml; the resulting mixture was filtered through a 0.22
micron
filter to sterilize and remove a small amount of insoluble residue. This
filtered
mixture was the MTT stock solution.
Briefly, for each tumor type, 10,000 cells were seeded in 96-well
tissue culture plate and incubated with each peptide concentration
individually in a
CO2 incubator for 48 hrs. The peptide analog at different concentrations was
added
once every 24 hrs during the incubation period. Control cultures, which were
not
treated with the peptide was similarly incubated. The assay was terminated by
adding 100 g (20 l) of MTT to each well, incubating for three hours, decanting
supernatant and finally adding 150 l of dimethylsulphoxide to each well to
dissolve
the formazon. The plates were incubated for 15 minutes at 37 C and read
spectro-
photometrically at 540 nm; and cytotoxicity percentage was calculated by
following
formula:
Cytotoxicity Percentage = 100x [1-X/Rl],
where X= (absorbance of the treated sample at 540 nm-absorbance of
a blank at 540 nn) and
R1 = (absorbance of the untreated control at 540nm) - (absorbance of
the blank at 540nm).
Thus in each of the MTT cytotoxicity assay the percentage was
calculated according to the above formula and was based on the proliferation
of the
untreated controls, the value of which was considered as 100%.
EXAMPLE 12
The biological activity of synthesized peptide SEQ ID NO:3 was
tested on different human tumor cell lines such as HT-29 & PTC (colon), A549
(non small lung cell), KB (oral squamous cell), MCF7 & MDA.MB.453 (Breast),
HuTu8O (duodenum), PA-1 (ovary), MOLT-4 (leukemia) and MIAPaCa2 (Pancreas)
at various molar concentrations. The percentage cytotoxicity induced by
different
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
- 16-
concentrations of the peptide SEQ ID NO: 3 is summarized in the following
table.
Percentage cytotoxicity at different concentrations
Cell Line l M 100n M 10 nM 1M 100pM l0pM
MCF Nil Nil 24.35 5 30.68 6 38.95 4.5 39.33 2.6
APaCa2 33.3 4.5 30.3 4.2 33.2 6.7 36.4 0.5 28.2 4.5 27.4 4.5
uTu8O 12.2 4.0 15.5 4.7 14.3 3.5 13.3 4.0 14.7 4.2 10.3 3.5
32.1 5.0 31.6 6.5 30.9 5.5 30.4 6.5 26.4 4.5 40.9 5.5
A549 30.7 6.5 23.6 4.5 32.2 5.5 32.4 4.5 25.2 3.5 30.5 3.5
T29 25.4 5.5 17.8 4.5 11.8 5.0 20.3 4.5 19.9 5.5 18.7 4.5
TC 17.9 2.5 27.7 2.8 27.7 3.6 23.8 2.8 26.5 3.8 80.0 7.1
A.MB.453 5.6 3.5 11.2 3.1 Nil 9.6 1.9 25.5 2.9 49.5 4.2
A-1 31.2 5.134.2 5.8 25.4 4.2 36.1 6.1 40.1 6.2 37.7 3.9
MOLT-4 9.0 1.2 1.4 1.0 Nil 1.0 0.4 15.9 3.0 49.9 4.1
EXAMPLE: 13
The cytotoxic activity of other synthesized bombesin analogs was
tested on eight human tumor cell lines namely HT-29, SW620, PTC (all colon),
PA-
1 (ovary), A549 (lung), HBL100 (breast), MOLT-4 (leukemia) and DU145
(prostate). The tumor cells were collected at exponential growth phase and
resuspended in medium (1.5 x 106) cells/ml in RPMI 1640 containing 10% FBS).
150 l of medium was added to the wells of a 96-well tissue culture plate
(Nunc,
Denmark) followed by 30 l of cell suspension. The plate was left in incubator
(37 C, 5% CO2 overnight. 20 l of the peptide (10"' x 10"10 M concentration)
was
added to marked wells of the 96-well plate. Each concentration was plated in
triplicates. 20 l of medium alone was added to control wells while wells
without
cells served as blanks. A total volume of 200 1 was ensured in each well and
plate
was left in incubator (37 C, 5% CO2). After 72 hours of incubation an MTT
assay
was performed and percentage cytotoxicity was calculated
with respect to control cells. Following tables show the cytotoxicity achieved
on
various cell lines at different concentrations.
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-17-
PA-1
Percent Cytotoxicity
S.No
100 nM lOnM 1 nM 100 PM
SEQ ID:4 2.3 2.9 4.3 0.2 16.2 2.9 12.6 2.9
SEQ ID:5 8.8 1.9 20.9 5.3 16.0 3.9 25.6 6.3
SEQ ID:6 9.2 1.0 8.7 1.9 7.4 1.0 11.1 2.9
SEQ ID:7 9.6 4.1 22.7 3.4 25.6 2.9 24.5 4.2
SEQ ID:8 10.4 3.7 20.4 3.0 23.8 4.2 23.3 5.5
PTC
Percent Cytotoxicity
S.No
100 nM lOnM 1 nM 100 pM
SEQ ID:4 9.8 1.7 2.1 0.2 8.7 1.5 14.9 1.1
SEQ ID:5 20.4 4.2 15.9 2.4 23.0 4.2 13.9 2.2
SEQ ID:6 24.7 5.2 10.4 0.8 9.1 0.7 10.1 0.6
SEQ ID:7 9.3 1.8 7.6 0.7 12.4 2.1 8.2 0.9
SEQID:8 8.7 2.1 5.4 1.7 12.5 1.7 12.3 1.9
DU145
Percent Cytotoxicity
S.No
100 nM lOnM 1 nM. 100 pM
SEQ ID:4 24.9 3.2 23.4 3.3 22.8 4.1 23.2 3.7
SEQ ID:5 32.3 3.8 22.0 3.4 10.6 0.9 29.3 2.9
SEQ ID:6 13.7 0.9 16.6 3.9 5.2 12.1 0.8
SEQ ID:7 NIL NIL ND ND
SEQ ID:8 19.1 2.1 22.5 2-2 21.4 6.2 28.1 3.5
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
- 18 -
SW620
S.No. Percent Cytotoxity
100 nM 10nM 1 nM 100 PM
SEQ ID: 4 34.3 4.2 23.2 2.0 27.8 2.8 30.4 3.2
SEQ ID: 5 25.6 4.2 30.1 4.0 29.7 4.2 38.0 3.8
SEQ ID: 6 23.5 5.1 38.1 7.3 33.5 5.2 24.8 4.2
SEQ ID: 7 25.4 2.9 20.8 1.9 32.0 5.8 33.6 5.8
SEQ ID: 8 29.4 2.9 33.0 3.8 20.6 3.9 20.6 3.9
HT29
Percent Cytotoxicity
S.No
100 nM lOnM 1 nM 100 PM
SEQ ID: 4 38.6 5.3 38.9 7.3 39.6 4.3 43.3 4.4
SEQ ID: 5 35.7 2.8 44.4 4.0 27.9 2.9 42.0 2.0
SEQ ID: 6 NIL 6.8 0.7 26.7 4.2 16.8 0.5
SEQ ID: 7 15.5 1.9 28.2 2.8 ND ND
SEQ ID: 8 34.8 4.2 18.9 4.2 34.7 3.3 21.4 3.1
MOLT4
Percent Cytotoxicity
S.No
100 nM lOnM 1 nM 100 PM
SEQ ID: 4 16.2 0.6 28.7 4.2 19.3 1.8 28.5 4.8
SEQ ID: 5 NIL 4.3 0.6 6.4 0.2 8.7 0.6
SEQ ID: 6 NIL 20.4 4.3 0.8 0.1 11.0 0.6
SEQ ID: 7 13.1 0.3 NIL NIL ND
SEQ ID: 8 2.6 0.1 12.8 3.3 9.3 0.2 16.6 3.1
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
- 19-
HBL
Percent Cytotoxicity
S.No
100 nM 10nM 1 nM 100 PM
SEQ ID: 4 25.0 3.1 33.2 5.2 30.6 4.2 33.0 3.6
SEQ ID: 5 19.4 4.5 16.7 3.6 31.6 5.3 19.3 2.7
SEQ ID: 6 17.0 0.5 6.0 0.4 1.2 0.3 NIL
SEQ ID: 7 16.1 3.9 7.0 0.7 12.0 0.7 4.0 0.6
SEQ ID: 8 11.9 2.1 14.4 2.1 12.2 1.9 12.1 1.9
A549
Percent Cytotoxicity
S.No
100 nM 10nM 1 nM 100 PM
SEQ ID: 4 20.0 2.2 20.6 1.9 22.7 2.9 20.7 4.2
SEQ ID: 5 30.3 4.3 22.2 3.1 20.2 4.2 25.2 5.6
SEQ ID:6 1.9 0.6 3.2 0.1 13.0 0.8 12.4 0.7
SEQ ID:7 6.7 2.0 17.9 0.9 ND ND
SEQ ID:8 21.7 3.3 20.7 2.2 19.7 3.1 17.0 2.7
EXAMPLE 14
The cytotoxic effect of peptide sequences SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, were studied by MTT assay which
is based on the principle of uptake of MTT[3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide], a tetrazolium salt by the metabolically active
cells
where it is metabolized by active mitochondria into a blue colored formazan
product
which can be read spectrophotometrically. Tumor cells KB (oral squamous),
HuTu8O (Stomach), PTC and SW620 (colon), U87MG (Glioblastoma), HBL 100
(Breast), HeP2 (laryngeal) and L132 (Lung) were incubated with the peptide
analogs for 48 hours at 37 C in a 96-well culture plate, followed by the
addition of
100 g MTT and further incubation of 1 hour. The formazan crystals formed
inside
the cells were dissolved with a detergent comprising 10% Sodium dodecyl
sulfate
and 0.01 N HCl and optical density read on a multiscan ELISA reader. The
optical
CA 02405704 2002-10-08
WO 01/62777 PCT/US00/20873
-20-
density was directly proportional to the number of proliferating and
metabolically
active cells. Percent cytotoxicity of peptide analogs is shown in the
following Table.
SEQ ID: 9
Cell Percentage cytotoxicity at different concentrations
l M loon M 10 nM 1nM 100pM 10p M
KB 10.4 1.6 20.8 1.7 23.0 2.1 32.6 3.7 26.9 2.9 20.6 4.1
HuTu80 14.2 0.6 13.5 2.1 23.5 2.9 28.0 1.8 23.8 2.8 19.5 0.4
PTC 10.3 0.9 19.5 4.1 26.8 3.8 25.6 5.1 24.5 3.9 22.4 2.2
U87MG 10.0 0.0 21.4 0.1 20.0 0.0 21.8 0.1 11.9 4.1 0.0 0.0
SW620 21.6 2.1 25.8 2.8 33.2 2.9 30.8 0.6 28.9 0.2 15.1 0.3
HBL100 17.2 0.4 22.4 1.7 28.1 0.6 34.1 1.8 28.6 2.2 17.2 0.1
HeP2 21.6 1.8 17.8 0.3 28.5 3.1 21.3 2.2 14.6 0.6 0.0 0.0
L132 18.3 2.9 25.9 2.6 27.2 3.1 30.5 4.1 22.4 0.8 0.0 0.0
SEQ ID: 10
Cell Percentage cytotoxicity at different concentrations
1 M 100n M 10 nM InM 100pM 10p M
KB 16.5 0.2 22.0 1.1 27.3 2.7 31.1 4.1 25.0 6.3 19.2 2.9
HuTu80 17.2 1.1 21.0 2.0 20.6 1.7 23.3 2.8 22.9 0.2 13.5 0.8
PTC 28.4 3.6 29.3 3.2 32.5 5.1 29.4 2.9 21.6 3.1 22.2 4.9
U87MG 10.0 0.0 15.0 0.5 20.0 0.0 25.6 2.1 16.5 0.5 11.6 1.7
SW620 22.2 2.1 19.4 1.8 25.5 2.8 22.4 1.7 20.9 0.6 16.7 0.2
HBL100 18.5 1.7 21.2 1.7 32.9 0.7 23.3 1.6 16.6 0.1 21.1 0.7
HeP2 19.9 1.5 26.3 1.7 27.5 2.8 27.2 2.6 19.1 0.6 1.7 0.1
L132 22.4 1.8 27.8 2.1 27.5 2.8 29.5 2.8 29.4 1.9 1.9 0.2
CA 02405704 2008-11-26
-21-
SE ID: 11
Cell Percentage cytotoxicity at different concentrations
1 M 100n M 10 nM InM 100pM lOp M
KB 24.2 1.2 31.9 2.1 31.9 _3.1 33.1 2.1 26.7 5.1 21.6 3.7
HuTu8O 14.2 0.1 20.0 3.1 27.3 2.7 30.5 4.1 22.6 3.9 17.6 1.6
PTC 18.6 1.5 25.8 2.5 25.7 4.1 28.5 2.8 28.3 0.8 19.7 0.6
U87MG 1.0 0.1 15.5 0.6 20.0 0.0 24.2 1.7 26.5 2.6 21.9 2.1
SW620 23.7 1.4 21.0 1.5 31.5 2.6 35.1 2.2 25.9 3.8 20.4 0.3
HBL100 24.5 0.8 22.7 0.5 29.9 0.3 24.3 1.6 15.4 4.1 18.2 1.1
HeP2 21.9 2.1 23.9 1.1 34.6 2.2 37.1 3.3 20.1 0.0 15.1 0.3
L132 1.4 1.1 20.4 1.5 30.4 0.4 29.4 0.4 18.3 0.9 10.5 0.
SBO ID: 12
Cell Percentage cytotoxicity at different concentrations
1 pM 100n M 10 nM 1nm 100pM lOp m
KB 12.4 1.2 11.1 3.1 18.6 2.1 26.6 4.9 19.4 2.9 19.3 2.9
HuTu8O 20.0 3.9 21.8 2.1 23.4 0.5 33.1 4.8 13.0 0.7 8.3 1.1
PTC 14.4 2.7 16.1 2.5 20.7 3.8 30.1 4.1 18.6 2.4 19:5 0.8
U87MG 15.4 3.1 13.1 2.3 27.5 2.9 28.3 1.9 22.1 3.8 13.1 2.2
SW620 22.6 1.1 25.3 0.6 36.1 1.9 32.2 2.6 38.4 2.8 34.8 0.4
HBLIOO 11.8 1.1 23.6 2.7 27.7 1.5 29.6 0.4 34.7 2.8 29.0 3.8
HeP2 28.7 0.8 25.6 0.4 29.2 1.1 28.9 0.5 24.4 0.1 10.0 0.0
L132 22.2 0.2 22.0 0.1 26.4 0.3 26.7 0.4 23.1 0.7 0.0 0.0
CA 02405704 2003-02-13
22
SEQUENCE LISTING
<110> BURMAN C, ANAND
PRASAD, SUDHANHAND
MUKHERJEE, RAMA
JAGGI, MANU
SINGH T, ANU
MATHUR, ARCHNA
<120> BOMBESIN ANALOGS FOR TREATMENT OF CANCER
<130> 13906-8-np
<140> PCT/USOO/20873
<141> 2000-07-31
<150> IN 147/DEL/2000
<151> 2000-02-24
<160> 12
<170> Patentln Ver. 2.0
<210> 1
<211> 14
<212> PRT
<213> Bombina bombina
<400> 1
Glu Gin Arg Leu Gly Asn Gln Trp Ala Val Gly His Leu Met
1 5 10
<210> 2
<211> 10
<212> PRT
<213> Sus barbatus
<400> 2
Gly Asn His Trp Ala Val Gly His Leu Met
1 5 10
<210> 3
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated
<220>
<221> MODRES
<222> (1)
<223> /product = D-phenylalanine/label = D-Phe
<220>
<221> MOD_RES
CA 02405704 2003-02-13
23
<222> (6)
<223> /product = alpha-aminoisobutyric acid/label = Aib
<400> 3
Xaa Gln Trp Ala Val Xaa His Leu
1 5
<210> 4
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated
<220>
<221> MODRES
<222> (1)
<223> /product = D-phenylalanine/label = D-Phe
<220>
<221> MODRES
<222> (4)
<223> /product = alpha-aminoisobutyric acid/label = Aib
<400> 4
Xaa Gln Trp Xaa Val Gly His Leu
1 5
<210> 5
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated
<220>
<221> MODRES
<222> (1)
<223> /product = D-phenylalanine/label = D-Phe
<220>
<221> MODRES
<222> (3)
<223> /product = D-tryptophan/label = D-Trp
<220>
<221> MODRES
<222> (6)
<223> /product = alpha-aminoisobutyric acid/label = Aib
<400> 5
Xaa Gln Xaa Ala Val Xaa His Leu
1 5
CA 02405704 2003-02-13
24
<210> 6
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD RES
<222> (1)
<223> /product = D-phenylalanine/label = D-Phe
<220>
<221> MODRES
<222> (4)
<223> /product = alpha-aminoisobutyric acid/label = Aib
<400> 6
Xaa Gln Trp Xaa Val Gly His Ile
1 5
<210> 7
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD_RES
<222> (1)
<223> /product = D-phenylalanine/label = D-Phe
<220>
<221> MODRES
<222> (6)
<223> /product = alpha-aminoisobutyric acid/label =Aib
<400> 7
Xaa Gln Trp Ala Val Xaa His Ile
1 5
<210> 8
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MOD_RES
CA 02405704 2003-02-13
<222> (1)
<223> /product = D-phenylalanine/label = D-Phe
<220>
<221> MODRES
<222> (3)
<223> /product = D-tryptophan/label = D-Trp
<220>
<221> MODRES
<222> (6)
<223> /product = alpha,alpha-di-n-propylglycine/label =
Dpg
<400> 8
Xaa Gln Xaa Ala Val Xaa His Leu
1 5
<210> 9
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MODRES
<222> (1)
<223> /product = D-phenylalanine/label = D-Phe
<220>
<221> MODRES
<222> (4)
<223> /product = alpha,alpha-di-ethyl glycine = Deg
<400> 9
Xaa Gln Trp Xaa Val Gly His Leu
1 5
<210> 10
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MODRES
<222> (1)
<223> /product = D-phenylalanine/label D-Phe
<220>
<221> MODRES
<222> (6)
CA 02405704 2003-02-13
26
<223> /product = 1-Aminocyclopentane caboxylic
acid/label = Ac5c
<400> 10
Xaa Gln Trp Ala Val Xaa His Leu
1 5
<210> 11
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated.
<220>
<221> MODRES
<222> (1)
<223> /product = Butanoyl-D-phenylalanine/label =
Butanoyl-D-Phe
<220>
<221> MODRES
<222> (6)
<223> /product = alpha-aminoisobutyric acid/label = Aib
<400> 11
Xaa Gin Trp Ala Val Xaa His Leu
1 5
<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: This peptide
was synthetically generated
<220>
<221> MODRES
<222> (1)
<223> /product = Octanoyl-D-phenylalanine/label =
Octanoyl-D-Phe
<220>
<221> MODRES
<222> (6)
<223> /product = alpha-aminoisobutyric acid/label = Aib
<400> 12
Xaa Gin Trp Ala Val Xaa His Leu
1 5
