Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
Long lasting natriuretic peptide derivatives
FIELD OF THE INVENTION
This invention relates to natriuretic peptide (NP) derivatives. In particular,
this
invention relates to NP derivatives having an extended irr. vivo half life,
for the treatment of
cardio-vascular diseases and disorders such as acute decompensated congestive
heart failure
(CHF) and chronic CHF, renal disorders and other diseases and disorders.
,_
1 o BACKGROUND OF THE INVENTION
The natriuretic peptide family includes four structurally related polypeptide
hormones: Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP), C-
type
Natriuretic Peptide (CNP) and, recently discovered, Dendroaspis Natriuretic
Peptide (DNP),
(Yandle, 1994; Wilhins et al. 1997; Stein and Levin, 1998).
ANP and BNP mediate natriuresis, diuresis, vasodilatation, antihypertension,
renin
inhibition, antimitogenesis, and lusitropic properties (increase in the
heart's rate relaxation).
CNP lacks natriuretic actions but possesses vasodilating and growth inhibiting
activity (Chen
and Burnett, 2000). Collectively, the natriuretic peptide family
counterbalances the effects of
2 o the renin-angiotensin-aldosterone system (Espiner 1994,Wilkins et al.
1997, Levin et al.
1998). ANP and BNP have been shown to be physiological antagonists of the
effects of
angiotensin II (Ang II) on vascular tone, aldosterone secretion, renal-tubule
sodium
reabsorption, and vascular cell growth (Harris et al. 1987, Itoh et al. 1990,
Wilkins et al.
1997, Levin et al. 1998). In addition, secretion of vasopressin (Obana et al.
1985) and
a 5 endothelin-1 (ET-1) (Saijomnaa et al. 1990) are decreased by ANP.
ANP and BNP do not cross the brain-blood barner (BBB) but they do reach areas
near the central nervous system (i.e. subfornical organ and hypothalamus). The
actions of NPs
in the brain reinforce those in the periphery. Natriuretic peptide receptors
are present in areas
3o adjacent to the third ventricle that are not separated from the blood by
the BBB, a position
that allows binding of circulating ANP as well as locally produced peptide
(Langub et al.,
1995 in Kelly R. and Struthers A.D., 2001).
Biological effects of natriuretic peptides are mediated through the binding
and the
35 activation of cell membrane receptors leading to cyclic GMP production in
target cells. These
include cGMP-dependent protein kinases (PKG), cGMP-gated ion channels and cGMP-
regulated phosphodiesterases (Lincoln & Cornwell 1993, de Bold et al. 1996).
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Three subtypes of natriuretic peptide receptors have been described: NPR-A,
NPR-
B and NPR-C. NPR-A and NPR-B are guanylyl cyclases through which the ligands
induce
the production of cyclic guanosine monophosphate (cGMP) (for review see Maack
1992,
Anand-Srivastava & Trachte 1993). NPR-A is thought to mediate many of the
effects of ANP
s and BNP (Maack 1992, Davidson & Struthers 1994) while CNP acts via NPR-B
receptors
(Koller et al. 1991, Chen & Burnett 1998). NPR-C is a clearance receptor for
all three
natriuretic peptides, which may signal through alternative pathways (Anand-
Srivastava et al.
1990, Levin 1993).
s o ANP is a 28 amino acid peptide having a 17-amino acid loop formed by an
intramolecular disulphide linkage between two cysteine residues, an amino tail
of 6 amino
acids and a carboxy tail of 5 amino acids. The structure of ANP, the first
member of the
family to be identified, was first described in 1984 (Kangawa et al. 1984).
The atria exhibit
the highest levels of ANP gene expression - 1% of the total mRNA codes for
ANP. ANP
i5 mRNA is also found in the ventricle at 1% of the atrial level. Non-cardiac
sites that contain
ANP include the brain, anterior lobe of the pituitary gland, the lung, and the
kidney (Stein and
Levin, 1998).
BNP is a 32 amino acid peptide having a 17-amino acid loop formed by an
~ o intramolecular disulphide linkage between two cysteine residues, an amino-
terminal tail of
9 amino acids and a carboxy-terminal tail of 6 amino acids. BNP, the second
member of the
NP family, was first detected in 1988 in extracts of porcine brain as it names
suggests (Sudoh
et al., 1988). However, it was subsequently shown, similarly to ANP, to be
expressed
primarily in the ventricular myocardium (Minamino et al., 1988; Hosoda et al.,
1991) as well
a s as in the brain and amnion (Stein and Levin, 1998). Like ANP, BNP is
released into the
circulation when the heart is stretched (Kinnunen et al., 1993). Direct
studies of BNP
secretion from isolated perfused heart (Ogawa et al., Circ. Res. 1991), and
from ifa-vivo and
tissue studies in humans (Mukoyama et al., J. Clin. Invest. 1991), showed that
60-80% of
cardiac BNP secretion arises from the ventricle.
ANP is shown to have several therapeutic applications such as for hypertension
and
pulmonary hypertension (Veale et al.), asthma, renal failure, cardiac failure
and radiodiagnostic
(Riboghene Inc., Press Release 1998).
3 5 BNP is shown to have several therapeutic applications such as for
hypertension,
asthma and inflammatory-related diseases (Ivax Corp., 2001),
hypercholesterolemia
(BioNumerik Pharmaceuticals Inc, 2000), emesis (BioNumerik Pharmaceuticals
Inc, 1996),
erectile dysfunction (Ivax Corp., 1998), renal failure (Abraham et al., 1995),
cardiac failure and
diagnostic of such (Marcus et al., 1995; Miller et al., 1994), solid tumor
treatment (BioNumerik
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Pharmaceuticals Inc, 1999) and protection of common and serious toxicity with
placlitaxel in
metastatic breast cancer (Hausheer et al., 1998, BioNumerik Pharmaceuticals
Inc, 2001).
One the major problem to overcome for the administration of ANP and BNP is
their
rapid blood circulation clearance. Human ANP has an ira vivo half life of 1 to
5 miii (Woods,
1988; Tonolo et al., 1988; Tang et al., 1984); and human BNP has an ira vivo
half life of 12.7
min (Smith et al., 2000). Three independent mechanisms are responsible for the
rapid clearance
of ANP and BNP: 1) binding to NPR-C with subsequent internalization and
lysosomal
proteolysis; 2) proteolytic cleavage by endopeptidases such as DPP IV, NEP,
APA, APP and
to ACE; and 3) renal secretion. It has been noted that urodilatin, a
natriuretic peptide found to be
an amino-terminal extended form of ANP, shows that the sole presence of the
four additional
residues at the N-terminal renders it much more resistant to enzymatic
degradation (Kenny et al.
1993). Nevertheless, urodilatin has only an i~z vivo half life of
approximately 6 min (Carstens et
al., 1998).
Several derivatives, analogs, truncations, elongations or constructs of ANP
are
proposed and/or patented for improving the efficiency and/or the half life of
the native form of
ANP; and the related prior art references are listed herein below.
2o First, native human ANP is disclosed and claimed in US patent 5,354,900.
Peptides
with longer or shorter amino-terminal or carboxy-terminal tails of the native
ANP sequence are
disclosed in US patent 4,607,023, US patent 4,952,561, US patent 4,496,544 and
US patent
6,013,630. Fragments of the native ANP comprising the carboxy-terminal tail
and a part of the
loop are disclosed in US patent 4,673,732. Dimers of ANP are proposed in US
a5 patent4,656,158 and JP application 62,283,996. Different ANP constructs are
proposed in JP
application 04,077,499, US patent 5,248,764 and application WO 02/10195.
ANP sequences with truncation of the amino-terminal tail, the carboxy-terminal
tail
or the loop, elongation of the tails, addition of alkyl group at one of the
tails, amino acid
3 o substitutions in the tails or in the loop and/or substitution of the
cysteine by another bridging
group are proposed in US patent 4,935,492, US patent 4,757,048, US patent
4,618,600, US
patent 4,764,504, US patent 5,212,286, US patent 5,258,368, US patent
5,665,704, US patent
5,846,932, EP application 0,271,041, EP application 0,341,603, application WO
90/14362, US
patent 5,095,004, US patent 5,376,635, EP application 0,350,318, EP
application 0,269,299, US
35 patent 5,204,328, US patent 5,057,603, EP application 0,244,169, US patent
4,816,443, CA
patent 1,267,086, EP application 0,303,243, US patent 4,861,755; US patent
5,340,920, JP
application 05,286,997, US patent 4,670,540, and US patent 5,159,061. Linear
peptides having
a portion thereof that has some similarities with the loop section of ANP are
disclosed in US
patent 5,047,397, US patent 4,804,650 and US patent 5,449,662.
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Also, several number of derivatives, analogs, truncations, elongations and
constructs
of BNP are proposed and/or patented for improving the efficiency and/or the
half life of the
native form of BNP; and the related prior art references are listed herein
below.
Native human BNP, amino and carboxy truncations thereof, and amino elongated
sequences thereof are disclosed and claimed is 5,674,710.
Several groups have proposed different modifications of the native human BNP
1 o sequences for preventing it from enzymatic degradation or for increasing
its activity. These
modifications include one or more of the following modifications: truncation
of the amino tail;
truncation of the carboxy tail; elongation of the amino tail with the prepro
sequence or a
fragment thereof; addition of an alkyl group at the amino tail or the carboxy
tail; and amino acid
substitutions in the tails or in the loop; as disclosed in US patent
5,114,923, US patent
5,948,761, US patent 6,028,055, US patent 4,904,763, application JP 07,228,598
and
application WO 98/45329.
All of the above ANP and BNP sequences have a rapid clearance. There is a need
for
a long lasting natriuretic peptide having an half life superior than the
native form of ANP and
z o BNP and the modified forms of the ANP and BNP sequences disclosed in the
prior art.
SUI.VllVMY OF THE INVENTION
In accordance with the present invention, there is now provided a NP
derivative
having an extended in vivo half life when compared with the ones of native ANP
or native
2 ~ BNP. More specifically, the present NP derivative comprises a NP peptide
having a reactive
entity coupled thereto and capable of reacting with available functionalities
on a blood
component, either in vivo or ex vivo, to form a stable covalent bond and
provide a NP peptide-
blood component conjugate. Being conjugated to a blood component, the NP
peptide is
prevented from undesirable cleavage by endogenous enzymes such as NEP and most
likely also
s o prevents binding to the NPR-C receptor which is responsible for a large
amount of the blood
clearance, thereby extending its in vivo half life and activity. The covalent
bonding formed
between the NP derivative and the blood component also substantially prevents
renal excretion
of the NP peptide until the blood component is degraded, thereby also
contributing to extend its
in vivo half life to a period of time closer to the half life of the blood
component which can
3 s represent an increase of 1 000 to 10 000 times. The reactive entity may be
on the N-terminal or
the C-terminal of the NP peptide, or on any other available site along the
peptidic chain.
Optionally, a lysine residue may be added or substituted at the site of the
peptidic chain where
the reactive entity is attached.
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The NP peptide for derivatization according to the present invention is
defined by the
following formula, where it should be understood that a peptidic bond links
Argls and llel9 and
the line between Cysl1 and Cys27 represents a direct disulfide bridge:
Rl_Xl_X2_X3_X4_XS_X6_X7_X8_X9_X10_Cysl l_X12_X13_X14_X15_X16_ASpl7-~'g18-
11e19-X20-X21-X22-Ser2g-X24-Leu25-X2g-Cys27-X2g- X2g-X3p-X31-X32-X33-R2
s wherein
Xl is Thr or absent;
X2 is Ser, Thr, Ala or absent;
X3 is Pro, Hpr, Val, or absent;
X4 is Lys, D-Lys, Arg, D-Arg, Asn, Gln or absent;
Zo XS is Met, Leu, Ile, an oxidatively stable Met-replacement amino acid, Ser,
Thr or absent;
X6 is Val, Ile, Leu, Met, Phe, Ala, D-Ala, Me or absent;
X~ is Gln, Asn, Arg, D-Arg, Asp, Lys, D-Lys or absent;
Xs is Gly, Pro, Ala, D-Ala, Arg, D-Arg, Asp, Lys, D-Lys, Gln, Asn or absent;
X9 is Ser, Thr or absent;
15 Xlo is Gly, Pro, Ala, D-Ala, Ser, Thr or absent;
X12 is Phe, Tyr, Leu, Val, Ile, Ala, D-Ala, Phe with an isosteric replacement
of its amide
bond selected from the group consisting of N-~c-methyl, methyl amino, hydroxyl
ethyl,
hydrazino, ethylene, sulfonamide and N-alkyl-(3-aminopropionic acid, ,or a Phe-
replacement
amino acid conferring on said analog resistance to NEP enzyme;
2 o X13 is Gly, Ala, D-Ala or Pro;
X14 is Arg, Lys, D-Lys, Asp, Gly, Ala, D-Ala or Pro;
X15 is Lys, D-Lys, Arg, D-Arg, Asn, Gln or Asp;
X16 is Met, Leu, Ile or an oxidatively stable Met-replacement amino acid;
X2o is Ser, Gly, Ala, D-Ala or Pro;
25 X21 is Ser, Gly, Ala, D-Ala, Pro, Val, Leu, or Ile;
X22 is Ser, Gly, Ala, D-Ala, Pro, Gln or Asn;
X24 1S Gly, Ala, D-Ala or Pro;
X26 is Gly, Ala, D-Ala or Pro;
X2g is Lys, D-Lys, Arg, D-Arg, Asn, Gln, His or absent;
3 o X2~ is Val, Ile, Leu, Met, Phe, Ala, D-Ala, Nle, Ser, Thr or absent;
X3o is Leu, Nle, lle, Val, Met, Ala, D-Ala, Phe, Tyr or absent;
X31 is Arg, D-Arg, Asp, Lys, D-Lys or absent;
X32 is Arg, D-Arg, Asp, Lys, D-Lys, Tyr, Phe, Trp, Thr, Ser or absent;
X33 1S His, Asn, Gln, Lys, D-Lys, Arg, D-Arg or absent;
3 5 Rl is NH2 or a N-terminal blocking group;
R2 is COOH, CONH2 or a C-terminal blocking group.
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Preferred blood components comprise proteins such as immunoglobulins,
including
IgG and IgM, serum albumin, ferritin, steroid binding proteins, transfernn,
thyroxin binding
protein, oc-2-macroglobulin, haptoglobin etc.; serum albumin and IgG being
more preferred; and
serum albumin being the most preferred.
Reactive entities are capable of forming a covalent bond with the blood
component
by reacting with amino groups, hydroxy groups, phenol groups or thiol groups
present thereon,
either izz vivo or iyz vitro. The expressions "in vit>~o" and "ex vivo" are
used in alternance in the
specification and means the same in the context of the present invention since
what takes place
i o outside the body is performed ifz vitz°o. In a preferred
embodiment, the functionality on the
protein will be a thiol group and the reactive entity will be a Michael
acceptor, such as acrolein
derivatives, a,,(3-unsaturated ketones, a,(3-unsaturated esters, a,[3-
unsaturated amides, a,(3-
unsaturated thioesters, acrylamide, acrylic ester, vinyl benzoate, cimiamate,
maleimide or
maleimido-containing group such as y-maleimide-butyrylamide (GMBA) or
maleimidopropionic acid (MPA), and the like. The reactive entity can also be
iodo methyl
benzoate, haloacetates, haloacetamides or the like. MPA is the most preferred
reactive entity.
In another aspect of the invention, there is provided a pharmaceutical
composition
comprising the NP derivative in combination with a pharmaceutically acceptable
carrier. Such
a o composition is useful for the treatment of congestive heart failure such
as acute decompensated
congestive heart failure of NYHA Class II, III and IV and chronic congestive
heart failure of
NYHA Class III and IV. The composition may also be used for the treatment of
one of the
following disorders or conditions: renal disorder, hypertension, asthma,
hypercholesterolemia,
inflammatory-related diseases, erectile dysfunction and for protection for
toxicity of anti-cancer
z 5 drugs. Finally, the present NP derivative may also be used for diagnostic
or radiodiagnostic
purposes.
In a further aspect of the present invention, there is provided a conjugate
comprising
the present NP derivative covalently bonded to a blood component. The covalent
bond between
s o the NP derivative and the blood component may be performed in vivo or ex
vivo.
In an embodiment of the present invention, there is provided a method for the
treatment of congestive heart failure such as acute decompensated congestive
heart failure of
NYHA Class II, III and IV and chronic congestive heart failure of N-YHA Class
III and IV. The
35 method comprises administering to a subject, preferably a mammal, animal or
human, an
effective amount of the NP derivative or the conjugate thereof, alone or in
combination with a
pharmaceutically acceptable carrier.
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In others embodiment of the present invention, there is provided a method for
the
treatment of renal disorder, a method for the treatment of hypertension and a
method for the
treatment of asthma. These methods comprise administering to a subject,
preferably a mammal,
animal or human, an effective amount of the NP derivative or the conjugate
thereof, alone or in
combination with a pharmaceutically acceptable carrier.
In a further embodiment of the present invention, there is provided a method
for
extending the ire vivo half life of a NP peptide in a subject, the method
comprising coupling to
the NP peptide a reactive group which is capable of forming a covalent bond
with a blood
i o component, and covalently bonding the NP derivative to a blood component.
The covalent
bonding may take place in vivo or i~2 vitf°o.
According to the present invention, the NP peptide or fragment thereof
possesses
natriuretic, diuretic, vasorelaxant and/or renin-angiotensin-aldosterone
system modulating
activity. Details of the sequences of these peptides and fragments are
illustrated below.
In another embodiment of the present invention, the reactive entity is coupled
to the
NP peptide via a linking group. In this case, the linking group is preferably
defined as, without
limitation, a straight or branched C1_lo alkyl; a straight or branched C1_lo
alkyl partly or
a o perfluorinated; a C1_io alkyl or fluoroalkyl wherein one or more carbon
atom is replaced with O,
N or S to form an ether or a thioether; o-, m- or p-disubstituted phenyl
wherein the substituents
are the same or different and are CH2, O, S, NH, NR wherein R is H, Ci-to
alkyl or C1_lo acyl; or
disubstituted heterocycles such as furan, thiophene, pyran, oxazole, or
thiazole. The linking
group can be stable or releasable so as to free the NP peptide if desired.
DESCRIPTION OF THE DRAWINGS
Figure 1 shows the superposition of the LC/MS profiles of a NP peptide before
and
after cyclisation performed with the iodine method.
Figure 2 shows the binding activity of commercial human ANP (hANP),
3 o synthesized human ANP (native ANP) and four NP conjugates to guinea pig
adrenal gland
membranes by displacement of lasI_rANP.
Figure 3 shows the binding activity of synthesized human BNP (native BNP) and
four NP conjugates to guinea pig adrenal gland membranes by displacement of
l2sI_rANP.
Figure 4 and 5 show the increase of cGMP production in human HELA cells being
s 5 incubated with in-house synthetized human ANP (native ANP), five NP
conjugates and two NP
peptides.
Figure 6 shows the increase of cGMP production in human HELA cells being
incubated with in-house synthetized human BNP (native BNP) and four NP
conjugates.
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Figure 7 shows izz vitro degradation in human plasma of hANP versus two
corresponding NP conjugates.
Figure 8 illustrates the site of cleavage of NEP enzyme along the hANP
sequence.
Figure 9 shows ifz vitro degradation by NEP enzyme of hANP versus a
corresponding
s NP conjugate, and capped human serum albumin as reference. Figure 10 shows
the pharmacokinetic in rats of hANP (of commercial source and being
synthetized in-house)
versus two corresponding NP conjugates.
DESCRIPTION OF THE TABLES
1 o Table 1 shows the three-letter code and one-letter code of amino acids.
Table 2 shows the retention times of NP peptides and NP derivatives according
to the
present invention.
Tables 3, 4 and 5 show three different gradients of elution of HPLC used for
the
analysis of NP peptide and NP derivatives of the present invention.
15 Tables 6 and 7 compare the predicted and measured molecular weight of NP
peptides, NP derivatives and NP conjugates.
Table 8 shows the concentrations of 50% inhibition (EC50) and the inhibition
constants (KI) calculated from the data used to draft Figure 2 i.e. binding
activity of
commercial human ANP (hANP), synthesized human ANP (native ANP) and four NP
2 o conjugates to guinea pig adrenal gland membranes by displacement of
l2sl_rANP.
Table 9 shows the concentrations of 50% inhibition (EC50) and the inhibition
constants (KIJ calculated from the data used to draft Figure 3 i.e. binding
activity of synthetized
human BNP (native BNP) and four NP conjugates to guinea pig adrenal gland
membranes by
displacement of l2sI-rANP.
Table 10 lists the concentration of 50% inhibition (EC50) calculated from the
data
used to draft Figures 4, 5 and 6 i.e. the increase of cGMP production in human
HELA cells
being incubated with in-house synthetized human ANP (native ANP); in-house
synthetized
human BNP (native BNP); nine NP conjugates; and two NP peptides.
Tables 11 and 12 show the gradients of elution of HPLC respectively used for
the
3 0 analysis of NP peptides and NP derivatives of the present invention.
Tables 13 and 14 show the izz vivo effect of the injection of an NP derivative
in SHR
rats and Winstar-Kyoto rats respectively, on the increase of urine secretion
and the increase of
cGMP expression.
3 5 DETAILED DESCRIPTION OF THE INVENTION
Izz vivo bioconjugation is the process of covalently bonding a molecule, such
as the
NP derivative according to the present invention, within the body, to the
targeted blood
component, preferably a blood protein, in a manner that permits the
substantial retention, or
increase in some instances, of the biological activity of the original
unmodified NP peptide in
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the conjugate form, while providing an extended duration of the biological
activity though
giving the NP peptide the biophysical parameters of the targeted blood
component.
According to the invention, the present NP derivative comprise a NP peptide
that has
been chemically modified by coupling thereto a reactive entity, either
directly or via a linking
group wluch is a stable or releasable linking group. The reactive entity is
capable of forming a
covalent bond with a blood component, preferably a blood protein. The reactive
entity must be
stable in an aqueous environment. The covalent bond is generally formed
between the reactive
entity and an amino group, a hydroxyl group, or a thiol group on the blood
component. The
s o amino group preferably forms a covalent bond with reactive entities like
carboxy, phosphoryl or
acyl; the hydroxyl group preferably forms a covalent bond with reactive
entities like activated
esters; and the thiol group preferably forms a covalent bond with reactive
entities like esters or
mixed anhydrides. The preferred blood components are mobile blood components
like serum
albumin, immunoglobulins, or combinations thereof, and the preferred reactive
entity comprises
anhydrides like maleimide or maleimido-containing groups. In a most preferred
embodiment,
the blood component is serum albumin and the reactive group is a maleimide-
containing group.
Protective groups may be required during the synthesis process (which is
described in
detail below) to avoid interreaction between the reactive entity and the
functional groups of the
2 o NP peptide itself. These protective groups are conventional in the field
of peptide synthesis, and
can be generically described as chemical moieties capable of protecting the
peptide derivative
from reacting with other functional groups. Various protective groups are
available
commercially, and examples thereof can be found in US 5,493,007 which is
hereby
incorporated by reference. Typical examples of suitable protective groups
include acetyl,
a 5 fluorenylmethyloxycarbonyl (FMOC), t-butyloxycarbonyl (BOC),
benzyloxycarbonyl (CBZ),
etc.
As above-mentioned, conjugation to a blood component definively plays a major
role in
preventing the NP peptide from degradation by endogenous enzymes such as NEP
and
3 o preventing binding to the NPR-C receptor which the most important factor
for the elimination
of the natriuretic peptide from blood circulation. Conjugation to a blood
component also
overcomes renal excretion of the NP peptide as long as the blood component
itself is being
degraded. Therefore, the intrinsec half life of the blood component selected
for conjugation is
the major determinant for the half life of the conjugated NP peptide.
The blood components are preferably mobile, which means that they do not have
a
fixed situs for any extended period of time, generally not exceeding 5
minutes, and more usually
one minute. These blood components are not membrane-associated and are present
in the blood
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for extended periods. Preferred mobile blood components include serum albumin,
transfernn,
ferritin, heptoglobin types 1-1, 2-1, 2-2 and imrnunoglobulins such as IgM,
IgA and IgG.
In greater details, the present invention is directed to the modification of
NP peptides
s and fragments thereof to improve their bioavailability, extend their ifa
vivo half life and
distribution through selective conjugation to a blood component while
substantially maintaining
or improving their remarkable therapeutic properties.
According to the invention, NP peptide is a peptide having at least one of the
1 o physiologic activities of a native ANP or BNP, and particularly of human
ANP and BNP. More
particularly, NP peptide has natriuretic, diuretic, vasorelaxant and/or renin-
angiotensin-
aldosterone system modulating activity.
Table 1 provides the three-letter code and one-letter code for natural amino
acids and
15 the three-letter code for non-natural amino acids.
TABLE 1
NOMENCLATURE FOR
AMINO ACIDS
Name 3-letter code 1-letter code
Alanine Ala A
Ar mine Ar R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamic acid Glu E
Glutamine Gln Q
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan T W
Tyrosine Tyr Y
Valine Val V
Norleucine Nle
Ornithine Orn
The design of the NP peptide for derivatization according to the present
invention is
2 o based on the sequence of native human ANP and BNP. Their sequences share
very high
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silnilarities. Substitution by analogous amino acids are proposed for residues
that seem less
involved in the pharmaceutical activity according to our structural activity
analysis. Therefore,
the NP peptide according to the present invention corresponds to the sequence
of the following
formula, where it. should be understood that a peptidic bond links Argl$ and
llel9 and the line
s between Cysl1 and Cys2~ represents a direct disulfide bridge that forms a
loop in the sequence:
Rl_Xl_X2-X3-X4_XS_X6_X~_X8-X9_Xlo_C ~sll_X12_X13_X14_Xls_X16_A5p17-~'g18-
11c19-X20-X21-X22-scr23-X24-Lcu25-X26-Cys27-X28- X29-X30-X31'X32-X33-R2
wherein
io Xl is Thr or absent;
X2 is Ser, Thr, Ala or absent;
X3 is Pro, Hpr, Val, or absent;
X4 is Lys, D-Lys, Arg, D-Arg, Asn, Gln or absent;
XS is Met, Leu, Ile, an oxidatively stable Met-replacement amino acid, Ser,
Thr or absent;
15 X6 is Val, lle, Leu, Met, Phe, Ala, D-Ala, Nle or absent;
X~ is Gln, Asn, Arg, D-Arg, Asp, Lys, D-Lys or absent; '
X8 is Gly, Pro, Ala, D-Ala, Arg, D-Arg, Asp, Lys, D-Lys, Gln, Asn or absent;
X9 is Ser, Thr or absent;
Xlo is Gly, Pro, Ala, D-Ala, Ser, Thr or absent;
2 o X12 is Phe, Tyr, Leu, Val, Ile, Ala, D-Ala, Phe with an isosteric
replacement of its amide
bond selected from the group consisting of N-oc-methyl, methyl amino, hydroxyl
ethyl,
hydrazino, ethylene, sulfonamide and N-alkyl-(3-aminopropionic acid, or a Phe-
replacement
amino acid conferring on said analog resistance to NEP enzyme;
X13 is Gly, Ala, D-Ala or Pro;
2 s X14 is Arg, Lys, D-Lys, Asp, Gly, Ala, D-Ala or Pro;
X15 is Lys, D-Lys, Arg, D-Arg, Asn, Gln or Asp;
X16 is Met, Leu, Ile or an oxidatively stable Met-replacement amino acid;
X2o is Ser, Gly, Ala, D-Ala or Pro;
X21 is Ser, Gly, Ala, D-Ala, Pro, Val, Leu, or Ile;
3 o X22 is Ser, Gly, Ala, D-Ala, Pro, Gln or Asn;
X24 is Gly, Ala, D-Ala or Pro;
X26 is Gly, Ala, D-Ala or Pro;
X28 is Lys, D-Lys, Arg, D-Arg, Asn, Gln, His or absent;
X29 is Val, Ile, Leu, Met, Phe, Ala, D-Ala, Nle, Ser, Thr or absent;
3 5 X3o is Leu, Nle, lle, Val, Met, Ala, D-Ala, Phe, Tyr or absent;
X31 is Arg, D-Arg, Asp, Lys, D-Lys or absent;
X32 is Arg, D-Arg, Asp, Lys, D-Lys, Tyr, Phe, Trp, Thr, Ser or absent;
SUBSTITUTE SHEET (RULE 26)
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X33 is His, Asn, Gln, Lys, D-Lys, Arg, D-Arg or absent;
Rl is NHZ or a N-terminal blocking group;
R2 is COOH, CONHZ or a C-terminal blocking group.
s According to a first preferred embodiment of the invention,
Xl is Thr or absent;
XZ is Ala or absent;
X3 is Pro or absent;
X4 is Arg or absent;
1 o XS is Ser, Thr or absent;
X6 is Leu, Ile, Nle, Met, Val, Ala, Phe or absent;
X~ is Arg, D-Arg, Asp, Lys, D-Lys, Gln, Asn or absent;
X8 is Arg, D-Arg, Asp, Lys, D-Lys, Gln, Asn or absent;
X9 is Ser, Thr or absent;
s5 Xlo is Ser, Thr or absent;
X12 is Phe, Tyr, Leu, Val, Ile, Ala, D-Ala, Phe with an isosteric replacement
of its amide
bond selected from the group consisting of N-oc-methyl, methyl amino, hydroxyl
ethyl,
hydrazino, ethylene, sulfonamide and N-alkyl-(3-aminopropionic acid, or a Phe-
replacement
amino acid conferring on said analog resistance to NEP enzyme;
z o X13 is Gly, Ala, D-Ala or Pro;
X14 is Gly, Ala, D-Ala or Pro;
X15 is Arg, Lys, D-Lys, or Asp;
X16 is Met, Leu, Ile or an oxidatively stable Met-replacement amino acid;
XZO is Gly, Ala, D-Ala or Pro;
z 5 X21 is Ala, D-Ala, Val, Leu, or lle;
X22 is Gln or Asn;
X24 is Gly, Ala, D-Ala or Pro;
X26 is Gly, Ala, D-Ala or Pro;
X28 is Asn, Gln, His, Lys, D-Lys, Arg, D-Arg or absent;
3 o X29 is Ser, Thr or absent;
X3o is Phe, Tyr, Leu, Val, Ile, Ala or absent;
X31 is Arg, D-Arg, Asp, Lys, D-Lys or absent;
X32 is Tyr, Phe, Trp, Thr, Ser or absent;
X33 is absent;
3 5 Rl is NHZ or a N-terminal blocking group;
RZ is COOH, CONHZ or a C-terminal blocking group
According to the first preferred embodiment of the invention, the following
residues
are more preferred:
SUBSTITUTE SHEET (RULE 26)
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Xl is Thr or absent;
X2 is Ala or absent;
X3 is Pro or absent;
X4 is Arg or absent;
-13-
XS is Ser or absent;
X6 is Leu or absent;
X~ is Arg, Asp or absent;
X8 is Arg, Asp or absent;
X9 is Ser or absent;
to Xlo is Ser or absent;
X12 is Phe or Phe with an isosteric replacement of its amide bond selected
from the group
consisting of N-oc-methyl, methyl amino, hydroxyl ethyl, hydrazino, ethylene,
sulfonamide and
N-alkyl-(3-aminopropionic acid;
X13 is Gly;
X14 iS Gly;
Xls is Arg or Asp;
X16 is Met or Ile;
X2o is Gly;
X21 is Ala;
z o X2z is Gln;
X24 is Gly;
X26 is Gly;
X28 is Asn or absent;
X29 is Ser or absent;
X3o is Phe or absent;
X31 is Arg, Asp or absent;
X32 is Tyr or absent;
X33 is absent;
Rl is NHZ or a N-terminal blocking group;
3 o R2 is COOH, CONH2 or a C-terminal blocking group.
Native human ANP is among the NP peptides in accordance with first embodiment
of the present invention. Further preferred NP peptides iii accordance with
the first embodiment
of the present invention are SEQ m NO: 1, SEQ m NO: 8, SEQ m NO: 12, SEQ m NO:
13,
3 5 SEQ m NO: 15, SEQ m NO: 17 and SEQ m NO: 19. Preferred NP derivatives,
comprising
NP peptides according to the first embodiment of the present invention, are
SEQ m NO: 2,
SEQ m NO: 3, SEQ m NO: 4, SEQ m NO: 5, SEQ m NO: 6, SEQ m NO: 7, SEQ m NO: 9,
SEQ m NO: 10, SEQ m NO:11, SEQ m NO: 14, SEQ m NO: 16, SEQ m N0:18 and SEQ
m NO: 20.
SUBSTITUTE SHEET (RULE 26)
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According to a second preferred embodiment of the invention
Xl is absent;
X2 is Ser, Thr or absent;
s X3 is Pro, Hpr, Val or absent;
X4 is Lys, D-Lys, Arg, D-Arg, Asn, Gln or absent;
Xs is Met, Leu, Ile, an oxidatively stable Met-replacement amino acid or
absent;
X6 is Val, Ile, Leu, Met, Phe, Ala, D-Ala, Nle or absent;
X~ is Gln, Asn or absent;
1 o Xg is Gly, Pro, Ala, D-Ala or absent;
X9 is Ser, Thr or absent;
Xlo is Gly, Pro, Ala, D-Ala or absent;
X12 is Phe, Tyr, Leu, Val, Ile, Ala, D-Ala, Phe with an isosteric replacement
of its amide
bond selected from the group consisting of N-oc-methyl, methyl amino, hydroxyl
ethyl,
15 hydrazino, ethylene, sulfonamide and N-alkyl-(3-aminopropionic acid, or a
Phe-replacement
amino acid conferring on said analog resistance to NEP enzyme;
X13 is Gly, Ala, D-Ala or Pro;
X14 is Arg, Lys, D-Lys, or Asp;
Xls is Lys, D-Lys, Arg, D-Arg, Asn or Gln;
a o X16 is Met, Leu, Ile or an oxidatively stable Met-replacement amino acid;
X2o is Ser, Gly, Ala, D-Ala or Pro;
X21 is Ser, Gly, Ala, D-Ala or Pro;
X22 is Ser, Gly, Ala, D-Ala or Pro;
X24 is Gly, Ala, D-Ala or Pro;
X26 is Gly, Ala, D-Ala or Pro;
X2$ is Lys, D-Lys, Arg, D-Arg, Asn, Gln or absent;
X29 is Val, Ile, Leu, Met, Phe, Ala, D-Ala, Nle or absent;
X3o is Leu, Nle, Ile, Val, Met, Ala, D-Ala, Phe or absent;
X31 is Arg, D-Arg, Asp, Lys, D-Lys or absent;
3 o X32 is Arg, D-Arg, Asp, Lys, D-Lys or absent;
X33 is His, Asn, Gln, Lys, D-Lys, Arg, D-Arg or absent;
Rl is NH2 or a N-terminal blocking group;
R2 is COOH, CONH2 or a C-terminal blocking group.
3 5 According to the second preferred embodiment of the invention, the
following
residues are more preferred:
Xl is absent;
X2 is Ser or absent;
X3 is Pro or absent;
SUBSTITUTE SHEET (RULE 26)
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X4 is Lys or absent;
X5 is Met, Ile or absent;
X6 is Val or absent;
X~ is Gln or absent;
X8 is Gly or absent;
X9 is Ser or absent;
Xlo is Gly or absent;
X12 is Phe or Phe with an isosteric replacement of its amide bond selected
from the group
consisting of N-oc-methyl, methyl amino, hydroxyl ethyl, hydrazino, ethylene,
sulfonamide and
1 o N-alkyl-(3-aminopropionic acid;
X13 is Gly;
X14 is Arg or Asp;
X15 is Lys or Arg;
X16 is Met or Ile;
X2o is Ser;
X21 is Ser;
X22 is Ser;
X24 is Gly;
X26 is Gly;
2 o X2$ is Lys, Arg or absent;
X29 1S Val or absent;
X3o is Leu or absent;
X31 is Arg, Asp or absent;
X32 is Arg, Asp or absent;
X33 1S His or absent.
.Native human BNP is among the NP peptides in accordance with second
embodiment of the present invention. Further preferred NP peptides in
accordance with the
second embodiment of the present invention are SEQ ff~ NO: 21, SEQ ID NO: 22,
SEQ m NO:
23, SEQ 117 NO: 25, SEQ m NO: 28, SEQ lD NO: 31, SEQ 1D NO: 34, SEQ B7 NO: 37,
SEQ
ID NO: 39, SEQ ID NO: 42, SEQ m NO: 45, SEQ ID NO: 48 and SEQ ID NO: 51.
Preferred
NP derivatives, comprising NP peptides according to the second embodiment of
the present
invention, are SEQ m NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ 1D NO: 29, SEQ
ID NO:
30, SEQ m NO: 32, SEQ m NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38,
SEQ
3 5 m NO: 40, SEQ 1D NO: 41, SEQ >I7 NO: 43, SEQ m NO: 44, SEQ ID NO: 46, SEQ
ID NO:
47, SEQ ID NO: 49, SEQ m NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54,
SEQ
m NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57.
SUBSTITUTE SHEET (RULE 26)
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The amino acids of the sequences of the NP peptides given in the present
application
may be D-amino acids or L-amino acids or combinations thereof, unless
otherwise specified. L-
amino acids are generally preferred.
s In a preferred embodiment of the invention, the functionality on the protein
will be
a thiol group and the reactive entity will be a maleimide or maleimido-
containing group such
as y-maleimide-butyrylamide (GMBA) and maleimidopropionic acid (MPA). The
reactive
entity can be linked to the NP peptide via a stable or releasable linking
group. The linking
group corresponds is represented by formula V-W where V is a functional group
reacting
1 o with the NP peptide and W is a chain moiety attached to the reactive
entity. V is an ether, a
thioether, a secondary or tertiary amine, a secondary or tertiary amide, an
ester, a thioester, an
imine, an hydrazone, a semicarbazone, an acetal, an hemi-acetal, a ketal, an
hemi-ketal, an
aminal, an hemi-aminal, an sulfonate, a sulphate, a sulfonamide, a
sulfonamidate, a
phosphate, a phophoramide, a phosphonate or a phosphonamidate, and preferably
a primary
is amide. W is any alkyl chain C1_io, any fluoroalkyl C1_lo or any combination
of
fluorosubstituted alkyl chain C1_lo, any ether or thioether containing alkyl
or fluoroalkyl
chains such as -(Z-CH2CH2-Z)n , -(Z-CF2CH2-Z)ri , -(Z-CHZCFZ-Z)ri , where ri=1-
4 and Z is
either O or S, ortho, mete or pare disubstituted benzene with structure like -
Y-C6H4-, -Y-
C6H4-Y-, where Y is any combination of CHZ, O, S, NH, NR [R=H, alkyl, acyl],
disubstituted
~ o heterocycles such as furan, thiophene, pyran, oxazole, or thiazole,
preferably an alkyl chain
C1_g.
The linking group is preferably selected in the group consisting of
hydroxyethyl
motifs such as (2-amino) ethoxy acetic acid (AEA), ethylenediamine (EDA), 2-[2-
(2-
25 amino)ethoxy)] ethoxy acetic acid (AEEA); one or more alkyl chains (Cl-C10)
motifs such
as glycine, 3-aminopropionic acid (APA), 8-aminooctanoic acid (AOA), 4-
aminobenzoic acid
(APhA). Preferred linking groups are (2-amino) ethoxy acetic acid (AEA),
ethylenediamine
(EDA), and 2-[2-(2-amino)ethoxy)] ethoxy acetic acid (AEEA). Examples of
combinations
of linking group and reactive entity include, without limitations, (AEEA-EDA)-
MPA;
3 0 (AEEA-AEEA)-MPA, (AEA-AEEA)-MPA and the like.
It is also contemplated that one or more additional amino acids may be added
or
substituted to the peptide at the site of coupling the reactive entity, via a
linking group or not,
prior to performing such coupling on the added ox substituted amino acid, in
order to facilitate
3 5 the coupling procedure. The addition or substitution of amino acids) may
be made at the N-
terminal, the C-terminal, or therebetween. It is preferred to substitute an
amino acid of the
sequence of the NP peptide with Lys, D-Lys, Orn, D-Orn or 2,4-diaminobutanoic
acid (DABA)
and couple the reactive group on it, optionally via a linking group. To do so,
lysine is the most
preferred.
SUBSTITUTE SHEET (RULE 26)
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Maleimide groups are most selective for sulfhydryl groups on peptides when the
pH
of the reaction mixture is kept between 6.5 and 7.4. At pH 7.0, the rate of
reaction of
maleimido groups with sulfhydryls is 1000-fold faster than with amines. When a
stable
s thioether linkage between the maleimido group and the sulflrydryl is formed,
it cannot be
cleaved under physiological conditions.
The NP derivatives of the invention can provide specific labeling of blood
components. The specific labeling, particularly with a maleimide, offers
several advantages.
Z o Free thiol groups are less abundant in vivo than amino groups, and as a
result, maleimide
derivatives covalently bond to fewer proteins. For example, in serum albumin,
there is only one
free thiol group per molecule. Thus, a NP peptide - maleimide - albumin
conjugate will tend to
comprise a 1:1 molar ratio of peptide : albumin. In addition to albumin, IgG
molecules (class II)
also have free thiols. Since IgG molecules and serum albumin make up the
majority of soluble
Z5 proteins in the blood, i.e., about 80-85%, they also make up the majority
of the free thiol groups
available to covalently bond to a NP derivative having a maleimido-containing
group. .
Further, even among free thiol-containing blood proteins, specific labeling
with a
maleimide leads to the preferential formation of peptide-maleimide-albumin
conjugates, due to
a o the unique characteristics of albumin itself. The single free thiol group
of albumin, highly
conserved among species, is located at amino acid residue Cys34. It has been
demonstrated
recently that the Cys34 of albumin has an increased reactivity relative to
free thiols on other free
thiol-containing proteins and also compared to thiols on low molecular weight
molecules. This
is due in part to the unusual pK value of 5.5 for the Cys34 of albumin. This
is much lower than
25 typical pI~ values for cysteine xesidues in general, which are typically
about 8-10. Due to this
low pK, under normal physiological conditions, Cys34 of albumin is
predominantly in the
anionic form, which dramatically increases its reactivity. In addition to the
low pI~ value of
Cys34, another factor which enhances the reactivity of Cys34 is its location,
which is in a
hydrophobic pocket close to the surface of one loop of region V of albumin.
This location
a o makes Cys34 accessible to ligands of all kinds, and is an important factor
in Cys34 s biological
role as free radical trap and free thiol scavenger. As a result, the reaction
rate acceleration can
be as much as 1000-fold relative to rates of reaction of peptide-maleimides
with other free-thiol
containing proteins and with free thiols containing low molecular weight
molecules.
35 Another advantage of peptide-maleimide-albumin conjugates is the
reproducibility
associated with the I:1 loading of peptide to albumin specifically at Cys34.
Conventional
activation techniques, such as with glutaraldehyde, DCC, EDC and other
chemical activators of,
for example, free amines, lack this selectivity. For example, human albumin
contains 59 lysine
residues, 25-30 of which are located on the surface of albumin and accessible
for conjugation.
SUBSTITUTE SHEET (RULE 26)
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Activating these lysine residues, or alternatively modifying a peptide to
couple through these
lysine residues, results in a heterogeneous population of conjugates. Even if
an equimolar ratio
peptide:albumin (i.e., 1:1) is employed, the end result is the production of
random conjugation
products, some containing an indefinite number of peptides linked to each
molecule of albumin,
s and each conjugate having peptides randomly coupled at any one of the 25-30
available lysine
sites. Consequently, characterization of the exact composition is virtually
impossible, not to
mention the absence of reproducibility. Additionally, while it would seem that
conjugation
through lysine residues of albumin would at least have the advantage of
delivering more
therapeutic agent per albumin molecule, studies have shown that a 1:1 ratio of
therapeutic agent
s o to albumin is preferred. In an article by Stehle, et al. in Azzti-
Cahcef° D3°ugS, 1997, 8, 677-685,
which is incorporated herein in its entirety, it is reported that a 1:1 ratio
of the anti-cancer
methotrexate to albumin conjugated via glutaraldehyde gave the most promising
results. These
conjugates were taken up by tumor cells, whereas conjugates bearing 5:1 to
20:1 methotrexate
molecules had altered HPLC profiles and were quickly taken up by the liver izz
vivo. It would
15 therefore seems that at higher ratios, the effectiveness of albumin as a
Garner for a therapeutic
agent is diminished.
Through controlled administration of the present NP derivative, and
particularly the
ones with a maleimide reactive entity, specific irz vivo labeling or bonding
of albumin and IgG
a o can be controlled. In typical intravenous administrations, it has been
shown that 80-90% of the
administered peptide derivative bonds to albumin and less than 5% bonds to
IgG. Trace
bonding of free thiols present, such as glutathione and cysteine, also occurs.
Such specific
bonding is preferred for izz vivo use as it permits an accurate calculation of
the estimated half life
of the NP peptide administered. The present invention also relates to NP
derivatives being
a s capable of selectively covalently bonding with one functionality on a
targeted blood component
whith a degree of selectivity of 80% or more. Preferably, the degree of
selectivity is 90% or
more, and more preferably, 95% or more.
As stated above, the desired conjugates of NP derivatives to blood components
may
3 o be prepared in vivo by administration of the derivatives directly to the
subject, which may be an
animal or a human. The administration may be done in the form of a bolus, or
introduced
slowly over time by infusion using metered flow or the like.
Alternately, the conjugate may also be prepared ex vivo or izz vitz°o
by combining
3 5 blood samples or purified blood components with the NP derivatives,
allowing covalent
bonding of the NP derivatives to the functionalities on blood components, and
the resulting
blood solution or the resulting purified blood component conjugates may be
administered to the
subject, animal or human. The purified blood components can be of commercial
source,
SUBSTITUTE SHEET (RULE 26)
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prepared by recombinant techniques or purified from blood samples. The blood
may be treated
to prevent coagulation during handling ex vivo.
The invention is also directed to the therapeutic uses and other related uses
of NP
s derivatives and fragments thereof having an extended half life izz vivo, and
one or more of the
following ANP-associated properties and BNP-associated properties:
- hypertension reduction;
- diuresis inducement;
- natriuresis inducement;
- vascular conduct dilatation or relaxation;
- natriuretic peptide receptors (such as NPR-A) binding;
- liberation suppression of norepinephrine through suppression of sympatic
nerve;
- renin secretion suppression from kidney;
- aldostrerone secretion suppresion from adrenal gland;
- treatment of cardiovascular disease and disorder;
- reducing, stopping or reversing cardiac remodling process in congestive
heart failure;
- treatment of renal disease and disorder; and
- treatment asthma.
a o According to the present invention, the NP derivatives or NP conjugates
can be
administered to patients that would benefit from inducing natriuresis,
diuresis and
vasodilatation. The NP derivatives and conjugates of the present invention are
particularly
useful to treat cardiac failure such as congestive heart failure (CHF) and
more particularly acute
decompensated CHF of NI'IIA Class II, III and IV and chronic CHF of NYHA Class
III and IV.
NP derivatives or NP conjugates can be administered in a single dose in acute
CHF or
following a long term medication for chronic CHF. Also, NP derivatives or NP
conjugates can
be administered alone or in combination with one or more of the following
types of compounds:
ACE inhibitors, beta blockers, diuretics, spironolactone, digoxin,
anticoagulation and
antiplatelet agents, and angiotensin receptor blockers.
Other diseases or conditions can be treated with the administration of NP
derivatives
and NP conjugates of the present invention and include renal disorders and
diseases, asthma,
hypertension and pulmonary hypertension. More particularly for the NP
derivatives and
conjugates based on formula II, the following diseases and conditions can also
be treated:
3 s inflammatory-related diseases, erectile dysfunction and
hypercholesterolemia; and also be used
as protectant for toxicity of anti-cancer drugs.
Two or more NP derivatives or conjugates of the present invention may be used
in
combination to optimize their therapeutic effects. They can be administered in
a physiologically
SUBSTITUTE SHEET (RULE 26)
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acceptable medium, e.g. deionized water, phosphate buffered saline (PBS),
saline, aqueous
ethanol or other alcohol, plasma, proteinaceous solutions, mannitol, aqueous
glucose, alcohol,
vegetable oil, or the like. Other additives which may be included include
buffers, where the
media are generally buffered at a pH in the range of about 5 to 10, where the
buffer will
s generally range in concentration from about 50 to 250 mM, salt, where the
concentration of salt
will generally range from about 5 to 500 mM, physiologically acceptable
stabilizers, and the
like. The compositions may be lyophilized for convenient storage and
transport.
The NP derivatives and conjugates of the present invention may be administered
l o orally, pulmonary, parenterally, such as intravascularly (I~,
intraarterially (IA), intramuscularly
(IM), subcutaneously (SC), or the like. Administration by transfusion may be
appropriate in
some situations. In some cases, administration may be oral, nasal, rectal,
transdermal or by
aerosol. It can be suitable to employ a single dose or multiple daily doses so
as to build the
desired systemic dosage. In the case of chronic use, the inverval of
administration are
15 established in relation with subject's needs. The NP derivative or
conjugate may be
administered by any convenient means, including syringe, trocar, catheter, or
the like. The
particular manner of administration will vary depending upon the amount to be
administered,
whether a single bolus or continuous administration, or the like.
a o The blood of the mammalian host may be monitored for the activity of NP
peptides
and/or presence of the NP derivatives or conjugates. By taking a blood sample
from the host at
different times, one may determine whether the NP peptide has become bonded to
the long-
lived blood components in sufficient amount to be therapeutically active and,
thereafter,
determine the level of NP peptide in the blood. If desired, one may also
determine to which of
2 s the blood components the NP peptide is covalently bonded. Monitoring may
also take place by
using assays of peptide activity, HPLC-MS, antibodies directed to peptides, or
fluorescent-
labeled or radiolabeled derivatives.
Another aspect of this invention relates to methods for deternining the
concentration
3 0 of the NP peptide or its conjugate in biological samples (such as blood)
using antibodies
specific to the NP peptide and to the use of such antibodies as a treatment
for toxicity potentially
associated with such NP peptide or conjugate. This is advantageous because the
increased
stability and life of the NP peptide in the patient might lead to novel
problems during treatment,
including increased possibility for toxicity. The use of anti-NP antibodies,
either monoclonal or
3 5 polyclonal, having specificity for NP, can assist in mediating any such
problem. The antibody
may be generated or derived from a host immunized with the particular NP
derivative, or with
an immunogenic fragment of the NP peptide, or a synthesized immunogen
corresponding to an
antigenic determinant of the NP peptide. Preferred antibodies will have high
specificity and
SUBSTITUTE SHEET (RULE 26)
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affinity any of the NP peptide, the derivatized form thereof and the
conjugated form thereof.
Such antibodies can also be labeled with enzymes, fluorochromes, or
radiolabels.
Antibodies specific for a particular NP derivative may be produced by using
purified
NP peptides for the induction of derivatized NP-specific antibodies. By
induction of antibodies,
it is intended not only the stimulation of an immune response by injection
into animals, but
analogous steps in the production of synthetic antibodies or other specific
binding molecules
such as screening of recombinant immunoglobulin libraries. Both monoclonal and
polyclonal
antibodies can be produced by procedures well known in the art.
The antibodies may also be used to monitor the presence of the NP peptide in
the
blood stream. Blood and/or serum samples may be analyzed by SDS-PAGE and
western
blotting. Such techniques allow determination of the level of conjugation of
the NPderivative.
The anti-NP antibodies may also be used to treat toxicity induced by
administration
of the NP derivative, and may be used ~ vivo or izz vivo. Ex vivo methods
would include
irnmuno-dialysis treatment for toxicity employing anti-therapeutic agent
antibodies fixed to
solid supports. In vivo methods include administration of anti-NP antibodies
in amounts
effective to induce clearance of antibody-agent complexes.
The antibodies may be used to remove the NP derivatives and conjugates
thereof,
from a patient's blood ex vivo by contacting the .blood with the antibodies
under sterile
conditions. For example, the antibodies can be fixed or otherwise immobilized
on a column
matrix and the patient's blood can be removed from the patient and passed over
the matrix. The
z 5 NP derivatives will bind to the antibodies and the blood containing a low
concentration of NP,
then may be returned to the patient's circulatory system. The amount of NP
derivative removed
can be controlled by adjusting the pressure and flow rate. Preferential
removal of the NP
derivative from the serum component of a patient's blood can be effected, for
example, by the
use of a semipermeable membrane, or by otherwise first separating the serum
component from
3 o the cellular component by ways known in the art prior to passing the serum
component over a
matrix containing the anti-therapeutic antibodies. Alternatively the
preferential removal of NP-
conjugated blood cells, including red blood cells, can be effected by
collecting and
concentrating the blood cells in the patient's blood and contacting those
cells with fixed anti-NP
antibodies to the exclusion of the serum component of the patient's blood.
The anti-NP antibodies can be administered iyz vivo, parenterally, to a
patient that has
received the NP derivative or conjugates for treatment. The antibodies will
bind the NP
derivative and conjugates. Once bound, the NP activity will be hindered if not
completely
blocked thereby reducing the biologically effective concentration of NP
derivatives in the
SUBSTITUTE SHEET (RULE 26)
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- 22 -
patient's bloodstream and minimizing harmful side effects if any. In addition,
the bound
antibody-NP complex will facilitate clearance of the NP derivative and
conjugates from the
patient's blood stream.
s Direct attachment of the reactive entity
The reactive entity (via a linking group or not), such as MPA, is activated as
a
succinate ester for example (one skilled in the art can use haloacyl or p-
nitrophenyl or others)
and reacted with an amino group of NP peptide or derivative thereof produced
by Solid Phase
Synthesis or by recombinants means (see Example 2). In order to perform such
direct
i o attaclnnent of the reactive entity, the amino group is selected from the
group consisting of the
amino group of the C-terminal residue, the amino group of the N-terminal
residue, or the amino
group of the lateral chain of an amino acid such as Lys, D-Lys, Orn, D-Orn and
DABA.
Peptide derivative synthesis
15 NP peptides may be synthesized by standard methods of solid phase peptide
chemistry
well known to any one of ordinary skill in the art. For example, the peptide
may be synthesized
by solid phase chemistry techniques following the procedures described by
Steward et al. in
Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford,
Ill., (1984) using
a Rainin PTI SymphonyT"" synthesizer. Similarly, peptides fragments may be
synthesized and
2 o subsequently combined or linked together to form a larger peptide (segment
condensation).
These synthetic peptide fragments can also be made with amino acid
substitutions and/or
deletion at specific locations.
For solid phase peptide synthesis, a summary of the many techniques may be
found
25 in Stewart et al. in "Solid P7Zase Peptide Synthesis", W. H. Freeman Co.
(San Francisco), 1963
and Meienhofer, Hof°monal Proteins arad Peptides, 1973, 2 46. For
classical solution synthesis,
see for example Schroder et al. in "The Peptides", volume l, Acacernic Press
(New York). In
general, such method comprises the sequential addition of one or more amino
acids or suitably
protected amino acids to a growing peptide chain on a polymer. Normally,
either the amino or
s o carboxyl group of the first amino acid is protected by a suitable
protecting group. The protected
and/or derivatized amino acid is then either attached to an inert solid
support or utilized in
solution by adding the next amino acid in the sequence having the
complimentary (amino or
carboxyl) group suitably protected and under conditions suitable for forming
the amide linkage.
The protecting group is then removed from this newly added amino acid residue
and the next
3 s amino acid (suitably protected) is added, and so forth.
After all the desired amino acids have been linked in the proper sequence, any
remaining protecting groups (and any solid support) are cleaved sequentially
or concurrently to
afford the final peptide. By simple modification of this general procedure, it
is possible to add
SUBSTITUTE SHEET (RULE 26)
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more than one amino acid at a time to a growing chain, for example, by
coupling (under
conditions which do not racemize chiral centers) a protected tripeptide with a
properly protected
dipeptide to form, after deprotection, a pentapeptide (segment condensation).
s The particularly preferred method of preparing the present NP derivatives of
the
present invention is solid phase peptide synthesis where the amino acid a-N-
terminal is
protected by an acid or base sensitive group. Such protecting groups should
have the properties
of being stable to the conditions of peptide linkage formation while being
readily removable
without destruction of the growing peptide chain or racemization of any of the
chiral centers
1 o contained therein. Examples of N-protecting groups and carboxy-protecting
groups are
disclosed in Greene, "Protective Groups In Organic Synthesis," (John Wiley ~Z
Sons, New York
pp. 152-186 (1981)), which is hereby incorporated by reference. Examples of N-
protecting
groups comprise, without limitation, loweralkanoyl groups such as formyl,
acetyl ("Ac"),
propionyl, pivaloyl, t-butylacetyl and the like; other acyl groups include 2-
chloroacetyl, 2-
15 bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-
nitrophenoxyacetyl, -chlorobutyryl,
benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like;
sulfonyl groups such as
benzenesulfonyl, p-toluenesulfonyl, o-nitrophenylsulfonyl, 2,2,5,7,8-
pentamethylchroman-6-
sulfonyl (pmc), and the like; carbamate forming groups such as t-
amyloxycarbonyh
benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-
a o nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-
bromobenzyloxycarbonyl, 3,4-
dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-
dimethoxybenzyloxycarbonyl, 4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-
dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-
biphenylyl)-1-
methylethoxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
z s benzhydryloxycarbonyl, t-butyloxycarbonyl (boc),
diisopropylmethoxycarbonyl,
isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,
2,2,2,-
trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-
methoxycarbonyl, isobornyloxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl,
cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; arylalkyl groups such
as benzyl,
3 o biphenylisopropyloxycarbonyl, triphenylmethyl, benzyloxymethyl, 9-
fluorenylmethyloxycarbonyl (Fmoc) and the like and silyl groups such as
trimethylsilyl and the
like. Preferred oc-N-protecting group are o-nitrophenylsulfenyl; 9-
fluorenylmethyloxycarbonyl;
t-butyloxycarbonyl (boc), isobornyloxycarbonyl; 3,5-
dimethoxybenzyloxycarbonyl; t-
amyloxycarbonyl; 2-cyano-t-butyloxycarbonyl, and the like, 9-fluorenyl-
rnethyloxycarbonyl
35 (Fmoc) being more preferred, while preferred side chain N-protecting groups
comprise
2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-
methoxybenzene-
sulfonyl, Cbz, Boc, and adamantyloxycarbonyl for side chain amino groups like
lysine and
arginine; benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-
butyl (t-Bu),
cyclohexyl, cyclopenyl and acetyl (Ac) for tyrosine; t-butyl, benzyl and
tetrahydropyranyl for
SUBSTITUTE SHEET (RULE 26)
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serine; trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl for
histidine; formyl for
tryptophan; benzyl and t-butyl for asparticacid and glutamic acid; and
triphenylmethyl (trityl) for
cysteine.
s A carboxy-protecting group conventionally refers to a carboxylic acid
protecting ester
or amide group. Such carboxy protecting groups are well known to those skilled
in the art,
having been extensively used in the protection of carboxyl groups in the
penicillin and
cephalosporin fields as described in US 3,840,556 and 3,719,667, the
disclosures of which are
hereby incorporated herein by reference. Representative carboxy protecting
groups comprise,
so without limitation, Cl-C$ loweralkyl; arylalkyl such as phenethyl or benzyl
and substituted
derivatives thereof such as alkoxybenzyl or nitrobenzyl groups; arylalkenyl
such as
phenylethenyl; aryl and substituted derivatives thereof such as 5-indanyl;
dialkylaminoalkyl
such as dimethylaminoethyl; alkanoyloxyalkyl groups such as acetoxymethyl,
butyryloxymethyl, valeryloxymethyl, isobutyryloxyrnethyl, isovaleryloxymethyl,
1-
15 (propionyloxy)-1-ethyl, 1-(pivaloyloxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-
1-ethyl,
pivaloyloxymethyl, propionyloxymethyl; cycloalkanoyloxyalkyl groups such as
cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl,
cyclopentylcarbonyloxymethyl,
cyclohexylcarbonyloxy-methyl; aroyloxyalkyl such as benzoyloxymethyl,
benzoyloxyethyl;
arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl, 2-
benzylcarbonyloxyethyl;
~ o alkoxycarbonylalkyl or cycloalkyloxycarbonylalkyl such as
methoxycarbonylmethyl,
cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl; alkoxycarbonyloxyalkyl
or
cycloalkyloxycarbonyloxyalkyl such as methoxycarbonyloxymethyl, t-
butyloxycarbonyl-
oxymethyl, 1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl;
aryloxy-
carbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl, 2-(5-
indanyloxycarbonyloxy)-ethyl;
alkoxyalkylcarbonyloxyalkyl such as 2-(1-methoxy-2-methylpropan-2-oyloxy)-
ethyl;
arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl;
arylalkenyloxycarbonyloxyalkyl such as 2-(3-phenylpropen-2-
yloxycarbonyloxy)ethyl;
alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminornethyl;
alkylaminocarbonyl-
aminoalkyl such as methylaminocarbonylaminomethyl; alkanoylaminoalkyl such as
s o acetylaminomethyl; heterocycliccarbonyloxyalkyl such as 4-
methylpiperazinyl-
carbonyloxymethyl; dialkylaminocarbonylalkyl such as
dirnethylaminocarbonylmethyl,
diethylaminocarbonylmethyl; (5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such
as (5-t-butyl-
2-oxo-1,3-dioxolen-4-yl)methyl; and (5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl
such as (5-
phenyl-2-oxo-1,3-dioxolen-4-yl)methyl. Representative amide carboxy protecting
groups
3 5 comprise, without limitation, aminocarbonyl and loweralkylaminocarbonyl
groups. Of the
above carboxy-protecting groups, loweralkyl, cycloalkyl or arylalkyl ester,
for example, methyl
ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, sec-butyl
ester, isobutyl ester, amyl
ester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl ester and the
like or an
SUBSTITUTE SHEET (RULE 26)
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alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroyloxyallyl or an
arylalkylcarbonyloxyallcyl ester
are preferred. Preferred amide carboxy protecting groups are
loweralkylaminocarbonyl groups.
In the solid phase peptide synthesis method, the a-C-terminal amino acid is
attached
to a suitable solid support or resin. Suitable solid supports useful for the
above synthesis are
those materials that are inert to the reagents and reaction conditions of the
stepwise
condensation-deprotection reactions, as well as being insoluble in the media
used. The preferred
solid support for synthesis of a-C-terminal carboxy peptides is a Ramage Amide
Linker
Resin (R. Ramage et al., THL, 34, p. 6599 (1993)). The preferred solid support
for a-C-terminal
1 o amide peptides Fmoc-protected Ramage Amide Linkers Resin.
When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy-
acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine,
preferably piperidine,
prior to coupling with the a-C-terminal amino acid as described above. The
preferred method
for coupling to the deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-
aminomethyl)phenoxy
acetamidoethyl resin is O-benzotriazol-1-yl-N,N,N',N'-
tetramethyluroniumhexafluoro
phosphate (HBTLT, 5 equiv.), diisopropylethylamine (DIEA, 5 equiv.), and
optionally 1
hydroxybenzotriazole (HOBT, 5 equiv.), in DMF. The coupling of successive
protected amino
acids can be carried out in an automatic polypeptide synthesizer in a
conventional manner as is
2 o well known in the art.
The removal of the Fmoc protecting group from the cc-N-terminal side of the
growing
peptide is accomplished conventionally, for example, by treatment with a
secondary amine,
preferably piperidine. Each protected amino acid is then introduced in about 6-
fold molar
a s excess, and the coupling is preferably carried out in DMF. The coupling
agent is normally O-
benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluoro-phosphate (HBTU, 5
equiv.),
diisopropylethylamine (DIEA, 5 equiv.), and optionally 1-hydroxybenzotriazole
(HOBT, 5
equiv.).
3 o At the end of the solid phase synthesis, the peptide is removed from the
resin and
deprotected, either in successive operations or in a single operation. Removal
of the
polypeptide and deprotection can be accomplished conventionally in a single
operation by
treating the resin-bound polypeptide with a cleavage reagent comprising
thioanisole,
triisopropylsilane, phenol, and trifluoroacetic acid. In cases wherein the a-C-
terminal of the
3 5 polypeptide is an alkylamide, the resin is cleaved by aminolysis with an
alkylamine.
Alternatively, the peptide may be removed by transesterification, e.g. with
methanol, followed
by aminolysis or by direct transamidation. The protected peptide may be
purified at this point or
taken to the next step directly. The removal of the side chain protecting
groups is accomplished
SUBSTITUTE SHEET (RULE 26)
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-26-
using the cleavage mixture described above. The fully deprotected peptide can
be purified by a
sequence of chromatographic steps employing any or all of the following types:
ion exchange on
a weakly basic resin (acetate form); hydrophobic adsorption chromatography on
underivatized
polystyrene-divinylbenzene (such as Amberlite XADT""); silica gel adsorption
chromatography;
s ion exchange chromatography on carboxymethylcellulose; partition
chromatography, e.g. on
Sephadex G-25T"~, LH-20T"~ or countercurrent distribution; high performance
liquid
chromatography (HPLC), especially reverse-phase HPLC on octyl- or
octadecylsilyl-silica
bonded phase column packing. Anyone of ordinary skill in the art will be able
to determine
easily what would be the preferred chromatographic steps or sequences required
to obtain
1 o acceptable purification of the NP peptide.
NP peptides and derivatives are cyclic. For the cyclisation, the thiol groups
of the
peptide can be reduced by a tallium, iodine or by the sulphoxide method. The
iodine method is
exemplified herein below in Example 1 and the sulphoxide method is exemplified
herein below
is in Examples 3, 5, 21 and 24. When the peptide has a reactive entity, and
more particularly when
the reactive entity is MPA, the cyclisation is preferably made with the
sulphoxide method.
After the cyclisation step, a final purification is performed on the cyclised
product.
The preferred method of purification is by HPLC.
Molecular weights of these peptides are determined using Quadrupole Electro
Spray
mass spectroscopy.
The synthesis process for the production of the NP derivatives of the present
invention will vary widely, depending upon the nature of the various elements,
i.e., the
sequence of the NP peptide, the linking group and the reactive entity,
comprised in the NP
derivative. The synthetic procedures are selected to ensure simplicity, high
yields and
repetitivity, as well as to allow for a highly purified product. Normally, the
chemically reactive
entity will be coupled at the last stage of the synthesis, for example, with a
carboxyl group,
3 o esterification to form an active ester. Specific methods for the
production of the embodiment of
NP derivatives of the present invention are described below.
It is imperative that the chemically reactive entity be placed at a site to
allow the
peptide to covalently bond to the blood component while retaining a
substantial proportion, if
3 5 not all, activity and/or beneficial effects of the corresponding NP
peptide.
It is preferred to attach the reactive group at a site along the peptidic
sequence of the
NP peptide selected so as to not interfere with the binding activity and the
pharmacologic
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
activity of the NP peptide. In vitro assays may be used to select the best
site to attach the
reactive group.
The following examples are provided to illustrate preferred embodiments of the
s invention and shall by no means be construed as limiting its scope. Unless
indicated otherwise,
optically active protected amino acids in the L-configuration were used.
Peptide derivative synthesis examples
The synthesis of the present natriuretic peptides and derivatives thereof was
1 o performed using an automated solid-phase procedure on a SymphonyTM peptide
synthesizer with
manual intervention during the generation of the Natriuretic derivatives. The
synthesis was
performed on Fmoc-protected Ramage Amide LinkerT"~ resin using Fmoc-protected
amino
acids. Coupling was achieved by using O-benzotriazol-1-yl-N,N,N',N'-
tetramethyl-uronium
hexafluorophosphate (HBTU) as activator in N,N-dimethylformamide (DMF)
solution and
15 diisopropylethylamine (DIEA) as base. The Fmoc protective group was removed
using 20%
piperidine/DMF. When needed, a Boc-protected amino acid was used at the N-
terminus in
order to generate the free Na terminus after the peptide was cleaved from the
resin. All amino
acids used during the synthesis possessed the L-stereochemistry unless
otherwise stated. Glass
reaction vessels were Sigmacoted~ and used during the synthesis.
In order to make easier the relation between the examples and the formula, it
can be
noted that the NP peptides and NP derivatives prepared in Examples 1 to 20
comprise NP
peptides in accordance with the first preferred embodiment of the present
invention, and ones
prepared in Examples 21 to 57 comprise NP peptides in accordance with the
second preferred
a s embodiment of the present invention. It should be understood that a
peptidic bond links the last
amino acid on the first line and the first amino acid on the second line for
each sequence given
in the examples. It should also be understood that the line between the two
cysteines in each
sequence illustrated in the present application represents a direct disulfide
bridge that forms a
loop in the sequence.
Example 1
Ser-Leu-Arg-Arg-Ser-Ser-C Is-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-
Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONHZ
SEQ )m N0 : 1
Step 1: Solid phase peptide synthesis was carried out on a 100 ,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(pb~-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-
Gly-
SUBSTITUTE SHEET (RULE 26)
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OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ghi(Trt)-OH, Fmoc-Ala-OH,
Fmoc-Gly-OH, Fmoc-lle-OH, Fmoc-Arg(Pbfj-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pb~-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH,
s Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF) and,
according to
the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronum
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the
Fmoc
protecting group was achieved using a solution of 20%
('V/~ piperidine in N,N-
dimethylformamide (DMF) for 20 minutes.
1 o Step 2: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5%
phenol, followed by precipitation by dry-ice cold (0-4°C) Et20. The
crude peptide was collected
on a polypropylene sintered fiumel, dried, redissolved in a 20% mixture of
acetonitrile in water
(0.1 % TFA) and lyophilized to generate the corresponding crude material used
in the
purification process.
1 s Step 3: The resulting peptide fully deprotected and was purified according
to the standard
purification procedure detailed herein below. The desired fractions were
collected pooled
together and lyophilised.
Step 4: The lyophilate of step 3 was placed in 2.5 mL AcOH/H20 (l:l). Then
iodine (I2) (6 eq.)
was added and.followed by mass spectrometry (LC/MS) to monitor the reaction.
The solution
2 o was stirred at room temperature for 12 hours. After the elapsed time, a
solution of vitamine C
(ascorbic acid 1M) was added. The precipitate was filtered out and the
filtrate was lyophilized.
Step 5: The lyophilate of Step 4 was purified using standard purification
procedure (detailed
herein below).
2 s Example 2
MPA-AEEA-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-
Ala-Gln-Ser-Gly-Leu-Gly-Cys-As~er- he-Arg-Tyr-COOH
SEQ >D NO : 2
Step 1: Native Atrial Natriuretic peptide (provided by Phoenix Pharmaceuticals
Inc., Belmont,
CA, USA, catalog number 005-06) was placed in DMF. To the solution was added
MPA-
3 o AEEA-COO(Su) and N-Methyl Morpholine. The solution was stirred for 6 hours
and then the
solution was diluted (1:1) with water and it was purified according to the
standard methodology.
Example 3
MPA-AEEA-Ser-Leu-Arg-Arg-Ser-Ser- ~ ys-Phe-Gly-Gly-Arg-Met-Asp-
Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONH2
3 5 SEQ ID NO : 3
SUBSTITUTE SHEET (RULE 26)
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Step 1: Solid phase peptide synthesis was carried out on a 100 .mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,
s Fmoc-Gly-OH, Fmoc-Tle-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-
OH,
Fmoc-Ser(tBu)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-
1 o N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/~
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5%
phenol, followed by precipitation by dry-ice cold (0-4°C) Et20. The
crude peptide was collected
15 on a polypropylene sintered funnel, dried, redissolved in a 20% mixture of
acetonitrile in. water
(0.1% TFA) and lyophilized to generate the corresponding crude material used
in the
purification process.
Step 3: The resulting peptide fully deprotected, except for the Acm groups
which remained
attached to the thiol portion of the cysteine, and was purified according to
the standard
a o purification procedure detailed herein below. The desired fractions were
collected pooled
together and lyophilised.
Step 4: The lyophilate of step 3 was placed in neat TFA (trifluoroacetic acid)
(lmg/mL). Then
anisole (100 eq.) was added followed by methyltrichlorosilane (l0eq.) and
finally by
diphenylsulphoxide (100 eq.). The solution was stirred at room temperature for
18 hours. After
25 the elapsed time, the solution was placed in a separatory funnel with 2N
Acetic acid (1mL/mg
of peptide) and cold ether (SmL/mL of TFA). After multiple extractions, the
desired cyclised
peptides, present in the aqueous solution, were collected, combined together
and lyophilised.
Step 5: The lyophilate of Step 4 was purified using standard purification
procedure (detailed
herein below).
Example 4
MPA-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-
Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Clys-Asn-Ser-Phe-Arg-Tyr-CONH 2
SEQ ID NO : 4
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
3 5 protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH,
Fmoc-Arg(Pbf)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Frnoc-Asn(Trt)-OH, Frnoc-Cys(Acm)-OH, Fmoc-
Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,
SUBSTITUTE SHEET (RULE 26)
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Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pb~-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-
OH,
Fmoc-Ser(tBu)-OH, MPA-OH. They were dissolved in N,N-dimethylformamide (DMF)
and,
s according to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-
tetxamethyl-
uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal
of the
Fmoc protecting group was achieved using a solution of 20% (V/~ piperidine in
N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5: The steps were performed in the same mamier as Example 3.
to
Example 5
Ser-Leu-Arg-Arg-Ser-Ser-C ~s-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-
Ala-Gln-Ser-Gly Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-Lys(AEEA-MPA)-CONH2
SEQ ID NO : S
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
15 protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-Tyr(tBu)
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc
Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbi]-OH, Fmoc
Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly OH, Fmoc-Phe
2 o OH, Fmoc-Cys(Acm)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, accoxding to the sequence, activated using O-
benzotriazol-1-yl-
N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/~
z ~ piperidine in N,N-dimethylformarnide (DMF) for 20 minutes.
Step 2: The selective deprotection of the Lys (Aloe) group was performed
manually and
accomplished by treating the resin with a solution of 3 eq of Pd(PPh3)4
dissolved in 5 mL of
C6H6 :CHC13 (1:1) : 2.5% NMM (v:v): 5% AcOH (v:v) for 2 h. The resin is then
washed with
CHC13 (6 x 5 mL), 20% AcOH in DCM (6 x 5 rnL), DCM (6 x 5 mL), and DMF (6 x 5
mL).
a o Step 3: The synthesis was then re-automated for the addition of the Fmoc-
AEEA-OH. After
coupling the Fmoc protecting group was removed using 20% piperidine. Finally,
3-
maleimidopropionic acid was coupled to the peptide on resin using standard
coupling
conditions. Between every coupling, the resin was washed 3 times with N,N
dimethylformamide (DMF) and 3 times with isopropanol.
35 Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5%
phenol, followed by precipitation by dry-ice cold (0-4°C) Et20. The
crude peptide was collected
on a polypropylene sintered funnel, dried, redissolved in a 40% mixture of
acetonitrile in water
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
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(0.1% TFA) and lyophilized to generate the corresponding crude material used
in the
purification process.
Step 5: The resulting peptide fully deprotected, except for the Acm groups
which remained
attached to the thiol portion of the cysteine, was purified according to the
standard purification
s procedure. The desired fractions were collected pooled together and
lyoplulised.
Step 6: The lyophilate of step 3 was placed in neat TFA (trifluoroacetic acid)
(lmg/mL). Then
anisole (100 eq.) was added followed by methyltrichlorosilane (l0eq.) and
finally by
diphenylsulphoxide (100eq.). The solution was stirred at room temperature for
18 hours. After
the elapsed time, the solution was placed in a separatory funnel with 2N
Acetic acid (1mL/mg
of peptide) and cold ether (SmLhnL of TFA). After multiple extractions the
aqueous solution
were collected, combined toghether and lyophilised.
Step 7: The lyophilate of Step 4 was purified using standard purification
methodology.
Example 6
Ser-Leu-Arg-Arg-Ser-Se~ ys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Lys(AEEA-MPA)
Gln-Ser-Gly-Leu-Gly-Cys Asn-Ser-Phe-Arg-Tyr-CONH2
SEQmN0:6
Step 1: Solid phase peptide synthesis was carried out on a 100 ,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
a o Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-
Lys(Aloc)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH,
Fmoc-Met-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Leu-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uroniurn hexafluorophosphate (HBTLT) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2-7 The steps were performed in the same manner as Example 5.
Example 7
Ser-Leu-Arg-Arg-Ser-Ser- i ys-Phe-Gly-Gly-Arg-Lys(AEEA-MPA)-Asp-Arg-Ile-Gly-
Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONH2
SEQ ID NO : 7
Step l: Solid phase peptide synthesis was carried out on a 100 p,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
-32-
Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-
OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbfj-OH, Fmoc-Asp(tBu)-OH, Fmoc-
Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pb~-OH, Fmoc-
Arg(Pb~-OH, Fmoc-Leu-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
i o Step 2-7 The steps were performed in the same manner as Example 5.
Example 8
Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-~Ser-Cy~s-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-
Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONH2
SEQ )D N0 : 8
15 Step 1: Solid phase peptide synthesis was carried out on a 100 .mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-
OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
~ o Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH,
Frnoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pb~-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Boc-
Thr(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF) and, according
to the
sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
2 s hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of
the Fmoc
protecting group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 3.
3 o Example 9
MPA-AEEA-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-~ys-Phe-Gly-Gly-Arg-
Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-COOH
SEQ ID NO : 9
Step 1 : Same as Step 1 in example 2 using urodilatin as starting material.
Urodilatin is
provided by Bachem, Torance, CA, USA, catalog number H-3046.1000.
Example 10
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
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MPA-AEEA-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-~Cys-Phe-Gly-Gly-Arg-
Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONH2
SEQ ID NO : 10
Step 1: Solid phase peptide synthesis was carned out on a 100 .mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-
OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
lo Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-
Thr(tBu)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-
dimethylformamide
(DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,
N, N', N'-
tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine
(DIEA).
Removal of the Fmoc protecting group was achieved using a solution of 20%
(V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were perfornied in the same manner as Example 3.
Example 11
MPA-AEEA-Ser-Leu-Asp-Asp-Ser-Ser-C s-Phe-Gly-Gly-Asp-Met-Asp-Asp-Ile-Gly-
Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Asp-Tyr-CONH2
a o SEQ ID NO : 11
Step 1: Solid phase peptide synthesis was carried out on a 100 ,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Asp(tBu)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-
a5 OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Frnoc-Gly-OH, Fmoc-Phe-OH, Frnoc-Cys(Acm)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-
Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-
3 o N, N, N', N'-tetramethyl-uronium hexafluorophosphate (IiBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2-5: The steps were performed in the same manner as Example 3.
3 5 Example 12
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
-34-
Ser-Leu-Asp-Asp-Ser-Ser-Cys-Phe-Gly-Gly-Asp-Met-Asp-Arg-Ile-Gly-
Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Asp-Tyr-CONH2
SEQ H) NO : 12
Step 1: Solid phase peptide synthesis was carried out on a 100 p,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Asp(tBu)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-
OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
Fmoc-Asp(tBu)-OH, Frnoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-
io Leu-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF)
and,
according to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-
tetramethyl-
uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal
of the
Fmoc protecting group was aclueved using a solution of 20% (V/~ piperidine in
N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5: The steps were performed in the same manner as Example 3.
Example 13
Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-
Cy -Asn-Ser-Phe-Arg-Tyr-CONH2
SEQ ID NO : 13
2 o Step 1: Solid phase peptide synthesis was carried out on a 100 p.mole
scale. The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,
Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH,
Fmoc-
2 5 Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Cys(Acm)-OH. They
were
dissolved in N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU)
and
diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved using a
solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
s o Step 2-5: The steps were performed in the same manner as Example 3.
Example 14
MPA-AEEA-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-
Gly-Leu-Gly-C Is-Asn-Ser-Phe-Arg-Tyr-CONH2
SEQ ID NO : 14
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
- 35 -
Step l: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,
s Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH,
Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-
AEEA-OH, MPA-OH. They were dissolved in N,N-dimethylformamide (DMF) and,
according
to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium
hexafluorophosphate (HBTL)] and diisopropylethylamine (DIEA). Removal of the
Fmoc
io protecting group was achieved using a solution of 20% (V/V) piperidine in
N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5: The steps were performed in the same manner as Example 3.
Example 15
Ser-Ser-Cyl -Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-
15 Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONH2
SEQ ID NO : 15
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-
2 o OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-
OH,
Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pb~-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Ser(tBu)-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide
(DMF) and,
according to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-
tetramethyl-
z s uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA).
Removal of the
Fmoc protecting group was achieved using a solution of 20% (V/V) piperidine in
N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5: The steps were performed in the same manner as Example 3.
3 o Example 16
MPA-AEEA-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-
Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONH2
SEQ ID NO : 16
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
35 OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
-36-
Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Ser(tBu)-OH, Boc-Ser(tBu)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-
N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2-5: The steps were performed in the same manner as Example 3.
1 o Example 17
i s-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-
Cys-Asn-Ser-Phe-Arg-Tyr-CONHZ
SEQ ID NO : 17
Step 1: Solid phase peptide synthesis was carried out on a 100 pmole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
Z5 OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Frrioc-Ala-
OH,
Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Cys(Acm)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the sequence,
activated using O-
~ o benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and
diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved using a
solution of 20% (V/~ piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2-5: The steps were performed in the same manner as Example 3.
~ s Example 18
MPA-AEEA-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-
Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONHZ
SEQ ID NO : 18
Step 1: Solid phase peptide synthesis was carried out on a 100 ,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
s o OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH,
Fmoc-Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,
Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-
AEEA-OH, MPA-OH. They were dissolved in N,N-dimethylformamide (DMF) and,
according
35 to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-
tetramethyl-uronium
hexafluorophosphate (I-~TLJ) and diisopropylethylamine (D1EA). Removal of the
Fmoc
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
-37-
protecting group was achieved using a solution of 20% (V/V) piperidine in N,N-
dirnethylformamide (DMF) for 20 minutes.
Step 2-5: The steps were performed in the same manner as Example 3.
s Example 19
Ser-Leu-Arg-Arg-Ser-Ser- ~ys-(N-Methyl-Phe)-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-
Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONH2
SEQ ID NO : 19
Step 1: Solid phase peptide synthesis was carried out on a 100 .mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
lo OH, Fmoc-Phe-OH, Frnoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH,
Fmoc-Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,
Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-(N-Methyl)-Phe-OH, Fmoc-Cys(Acm)-
OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-
15 Leu-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF)
and,
according to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-
tetramethyl-
uronium hexafluorophosphate (HBTU) and diisopropylethylamine (D1EA). Removal
of the
Fmoc protecting group was achieved using a solution of 20% (V/~ piperidine in
N,N-
dimethylformamide (DMF) for 20 minutes.
2 o Step 2-5: The steps were performed in the same manner as Example 3.
Example 20
MPA-AEEA-Ser-Leu-Arg-Arg-Ser-Ser-C~s-(N-Methyl-Phe)-Gly-Gly-Arg-Met-Asp-
Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-CONHZ
SEQ ID NO : 20
25 Step 1: Solid phase peptide synthesis was carried out on a 100 .mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Tyr(tBu)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH,
Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH,
Fmoc-
3 o Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-(N-Methyl)Phe-OH, Fmoc-
Cys(Acm)-
OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pb~-OH,
Fmoc-
Leu-OH, Foc-Ser(tBu)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-
N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
3 5 (DIEA). Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/~
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
-38-
Step 2-5: The steps were performed in the same manner as Example 3.
Example 21
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-C s-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-
Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 21
Step 1: Solid phase peptide synthesis was carried out on a 100 .mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Frnoc-
Cys(Acm)-OH, Frnoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
lo Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,
Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF) and,
according to
the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the
Fmoc
protecting group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5%
z o phenol, followed by precipitation by dry-ice cold (0-4°C) Et20. The
crude peptide was collected
on a polypropylene sintered funnel, dried, redissolved in a 40% mixture of
acetonitrile in water
(0.1% TFA) and lyophilized to generate the corresponding crude material used
in the
purification process.
Step 3: The resulting peptide fully deprotected, except for the Acm groups
which remained
a 5 attached to the thiol portion of the cysteine, and was purified according
to the standard
purification procedure detailed herein below. The desired fractions were
collected pooled
together and lyophilised.
Step 4: The lyophilate of step 3 was placed in neat TFA (trifluoroacetic acid)
(lmg/mL). Then
anisole (100 eq.) was added followed by methyltrichlorosilane (l0eq.) and
finally by
s o diphenylsulphoxide (100 eq.). The solution was stirred at room temperature
for 18 hours. After
the elapsed time, the solution was placed in a separatory funnel with 2N
Acetic acid (1mL/mg
of peptide) and cold ether (SmL/rnL of TFA). After multiple extractions the
aqueous solution
were collected, combined together and lyophilised.
Step 5: The lyophilate of Step 4 was purified using standard purification
procedure (detailed
3 5 herein below).
Example 22
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
-39-
Ser-Gly-C Is-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-
Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 22
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Frnoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Boc-Ser(tBu)-OH. They were
s o dissolved in N,N-dimethylformamide (DMF) and, according to the sequence,
activated using
O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and
diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved using a
solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 23
Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly=
Cy -Asn-Ser-Phe-Arg-Tyr-CONH2
SEQ ID NO : 23
Step 1: Solid phase peptide synthesis was carned out on a 100 .mole scale. The
following
2 o protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH,
Fmoc-Arg(Pbf)
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-AEEA
OH, MPA-OH. They were dissolved in N,N-dimethylformamide (DMF) and, according
to the
sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the
Fmoc
protecting group was achieved using a solution of 20% (V/V) piperidine in N,N
3 o dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 24
Ser-Gly-Cyl -Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-
Leu-Gly-Cys-Lys-Val-Leu-Arg Arg-His-Lys(AEEA-MPA)-CONH2
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
SEQ H) NO : 24
- 40 -
Step 1: Solid phase peptide synthesis was carried out on a 100~,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
His(Trt)-OH, Frnoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
s Fmoc-Lys(Boc)-OH, Frnoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ile-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Boc-
Ser(tBu)-OH,. They were dissolved in N,N-dimethylformamide (DMF) and,
according to the
so sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the
Fmoc
protecting group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2: The selective deprotection of the Lys (Aloc) group was performed
manually and
i5 accomplished by treating the resin with a solution of 3 eq of Pd(PPh3)4
dissolved in 5 mL of
C6H6 :CHCl3 (1:1) : 2.5% NMM (v:v): 5% AcOH (v:v) for 2 h. The resin is then
washed with
CHC13 (6 x 5 mL), 20% AcOH in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5
mL).
Step 3: The synthesis was then re-automated for the addition of the Fmoc-AEEA-
OH. After
coupling the Fmoc protecting group was removed using 20% piperidine. Finally,
3-
2 o maleimidopropionic acid was coupled to the peptide on resin using standard
coupling
conditions. Between every coupling, the resin was washed 3 times with N,N
dimethylformamide (DMF) and 3 times with isopropanol.
Step 4: The peptide was cleaved from the resin using 85% TFA/5% TIS/5%
thioanisole and 5%
phenol, followed by precipitation by dry-ice cold (0-4°C) Et20. The
crude peptide was collected
2 s on a polypropylene sintered funnel, dried, redissolved in a 40% mixture of
acetonitrile in water
(0.1 % TFA) and lyophilized to generate the corresponding crude material used
in the
purification process.
Step 5: The resulting peptide fully deprotected, except for the Acm groups
which remained
attached to the thiol portion of the cysteine, was purified according to the
standard purification
3 o procedure. The desired fractions were collected pooled together and
lyophilised.
Step 6: The lyophilate of step 3 was placed in neat TFA (trifluoroacetic acid)
(lmg/mL). Then
anisole (100 eq.) was added followed by methyltrichlorosilane (l0eq.) and
finally by
diphenylsulphoxide (100eq.). The solution was stirred at room temperature for
18 hours. After
the elapsed time, the solution was placed in a separatory funnel with 2N
Acetic acid (1mL/mg
3 s of peptide) and cold ether (SmL/mL of TFA). After multiple extractions the
aqueous solution
were collected, combined toghether and lyophilised.
Step 7: The lyophilate of Step 4 was purified using standard purification
methodology.
Example 25
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
-41 -
Ser-Gly-Cys-Phe-Gly-Arg-Lys-Ile-Asp-Arg-lle-Ser-
Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 25
Step 1: Solid phase peptide synthesis was carned out on a 100 .mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pb~-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Boc-Ser(tBu)-OH. They were
1 o dissolved in N,N-dimethylformamide (DMF) and, according to the sequence,
activated using
O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and
diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved using a
solution of 20% (VlV) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 26
MPA-AEEA-Ser-Gly-Cys Phe-Gly-Arg-Lys-lle-Asp-Arg-Ile-Ser-
Ser-Ser-Ser-Gly-Leu-Gly-C~ys-Lys-Val-Leu-Arg-Arg-His-CONHZ
SEQ ID NO : 26
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
2 o protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH,
Fmoc-Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tle-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
~ 5 OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
AEEA
OH, MPA-OH. They were dissolved in N,N-dimethylformarnide (DMF) and, according
to the
sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the
Fmoc
protecting group was achieved using a solution of 20% (V/V) piperidine in N,N
s o dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 27
Ser-Gly-C s-Phe-Gly-Arg-Lys-Ile-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-
Leu-Gly- ys-Lys-Val-Leu-Arg-Arg-His-Lys(AEEA-MPA)-CONH2
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
SEQ H) NO : 27
-42-
Step 1: Solid phase peptide synthesis was carned out on a 100 pmole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Frnoc-Val-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ile-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Boc-
Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF) and, according
to the
to sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium
hexafluorophosphate (HBTL)7 and diisopropylethylamine (DIEA). Removal of the
Fmoc
protecting group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2 to 7 The steps were performed in the same manner as Example 24.
Example 28
Cys-Phe-Gly-Arg-Lys-lle-Asp-Arg-Ile-Ser-Ser-
Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 28
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
2 o protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH,
Fmoc-Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pb~-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Boc-Cys(Acm)-OH,. They were dissolved in N,N-
dimethylformamide
(DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,
N, N', N'-
tetramethyl-uronium hexafluorophosphate (HBTU) and ~ diisopropylethylamine
(DIEA).
Removal of the Fmoc protecting group was achieved using a solution of 20%
(V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
s o Step 2-5 The steps were performed in the same manner as Example 21.
Example 29
MPA-AEEA-C ~s-Phe-Gly-Arg-Lys-Ile-Asp-Arg-Ile-Ser-Ser-Ser-Ser-
Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg Arg-His-CONH2
SEQ ID NO : 29
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)-
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
- 43 -
OH, Frnoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved
in N,N-dimethylformamide (DMF) and, according to the sequence, activated using
O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTLJ~
and
diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved using a
solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
i o Step 2-5 The steps were performed in the same manner as Example 21.
Example 30
Cyl -Phe-Gly-Arg-Lys-Ile-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-
Cys-Lys-Val-Leu-Arg-Arg-His-Lys(AEEA-MPA)-CONHZ
SEQ ID NO : 30
Step 1: Solid phase peptide synthesis was carned out on a 100 pmole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
His(Trt)-OH, Fmoc-Arg(Pb~-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ile-
a o OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH,
Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Cys(Acm)-OH. They were dissolved in
N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTLT)
and
diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved using a
2 s solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
Step 2 to 7 The steps were performed in the same manner as Example 24.
Example 31
Cyl -Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-
Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
3 o SEQ ID NO : 31
Step 1: Solid phase peptide synthesis was carried out on a 100 ,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pb~-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
35 Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
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-44
OH, Fmoc-Phe-OH, Boc-Cys(Acm)-OH. They were dissolved in N,N-dimethylformamide
(DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,
N, N', N'-
tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine
(DIEA).
Removal of the Fmoc protecting group was achieved using a solution of 20%
(V/V)
s piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 32
MPA-AEEA-Cyl -Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-
Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
io SEQ ID NO : 32
Step 1: Solid phase peptide synthesis was carried out on a 100 .mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
i5 Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved
in N,N-dimethylformamide (DMF) and, according to the sequence, activated using
O-
benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTLT)
and
a o diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved using a
solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 33
Cyl -Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-
z5 Cys-Lys-Val-Leu-Arg-Arg-His-Lys(AEEA-MPA)-CONH2
SEQ ID NO : 33
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
3 o Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ile-
OH, Fmoc-Arg(Pbfj-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Arg(Pbf]-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Cys(Acm)-OH. They were dissolved in
N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-
35 benzotriazol-1-yl-N, N, N', N'-tetrarnethyl-uronium hexafluorophosphate
(HBTU) and
SUBSTITUTE SHEET (RULE 26)
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- 45 -
diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved using a
solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
Step 2-7: The steps were performed in the same manner as Example 24.
s Example 34
Cy~-(NaMe-Phe)-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-
Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 34
Step 1: Solid phase peptide synthesis was carried out on a 100 .mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)-
Z o OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Na-Methyl-Phe-OH, Boc-Cys(Acm)-OH. They were dissolved in N,N-
15 dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(D1EA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 35
MPA-AEEA-Cys-(NaMe-Phe)-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-
Gly-Leu-Gly-C Is-Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 35
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
2 s protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH,
Fmoc-Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
3 o OH, Fmoc-N°'-Methyl-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-AEEA-OH, MPA-OH.
They
were dissolved in N,N-dimethylformarnide (DMF) and, according to the sequence,
activated
using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU)
and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved
using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
35 Step 2-5 The steps were performed in the same manner as Example 21.
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
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-46-
Example 36
Cy~ -(NaMe-Phe)-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-
Cys-Lys-Val-Leu-Arg-Arg-His-Lys(AEEA-MPA)-CONH2
SEQ ID NO : 36
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ile-
so OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-N"-Methlyl-Phe-OH, Boc-Cys(Acm)-OH. They were
dissolved in N,N-dimethylformamide (DMF) and, according to the sequence,
activated using
O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU) and
diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved using a
solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
Step 2-7: The steps were performed in the same manner as Example 24.
Example 37
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-(NaMe-Phe)-Gly-Arg-Lys-Met-
Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
a o SEQ ID NO : 37
Step 1: Solid phase peptide synthesis was carried out on a 100 pmole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pb~-OH, Fmoc-Gly-
OH, Fmoc-N"-Methlyl-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Pro-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide
(DMF)
3o and, according to the sequence, activated using O-benzotriazol-1-yl-N, N,
N', N'-tetramethyl-
uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal
of the
Fmoc protecting group was achieved using a solution of 20% (V/V) piperidine in
N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
Example 38
-47-
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-(NaMe-Phe)-Gly-Arg-Lys-Met-Asp-Arg-
Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-L~ys-Val-Leu-Arg-Arg-His-Lys(AEEA-MPA)-
CONH2
SEQ ID NO : 38
Step 1: Solid phase peptide synthesis was carried out on a 100 ,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Frnoc-
His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ile-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-
lo Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-N"-Methlyl-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-
Gly-
OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH. Fmoc-AEEA-OH, MPA-OH. They
were dissolved in N,N-dirnethylformamide (DMF) and, according to the sequence,
activated
using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate
(HBTU)
is and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was
achieved
using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20
minutes.
Step 2 to 7: The steps were performed in the same manner as Example 24.
Example 39
a o CYs-Phe-Gly-Arg-Lys-Ile-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-CONHZ
SEQ ID NO : 39
Step 1: Solid phase peptide synthesis was carried out on a 100 .mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Cys(Acm)-OHa,
Fmoc-Gly-
OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
25 Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-
OH,
Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH,
Boc-Cys(Acm)-OH,. They were dissolved in N,N-dimethylformamide (DMF) and,
according
to the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the
Fmoc
3 o protecting group was achieved using a solution of 20% (V/V) piperidine in
N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 40
MPA-AEEA-C Is-Phe-Gly-Arg-Lys-Ile-Asp-Arg-Ile-Ser-Ser-Ser-Ser-
3 5 Gly-Leu-Gly-Cys-CONH2
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
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- 48 -
SEQ H) NO : 40
Step 1: Solid phase peptide synthesis was carried out on a 100 .mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Cys(Acm)-OH, Fmoc-
Gly-OH,
Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-
OH,
s Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-
OH,
Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-
OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-dimethylformamide (DMF)
and, according to the sequence, activated using O-benzotriazol-1-yl-N, N, N',
N'-tetramethyl-
uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal
of the
io Fmoc protecting group was achieved using a solution of 20% (V/V) piperidine
in N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same mamier as Example 21.
Example 41
Cyl -Phe-Gly-Arg-Lys-Ile-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-
15 CYs-Lys(AEEA-MPA)-CONH2
SEQ ID NO : 41
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
2 o Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-
Arg(Pbf)-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Boc-Cys(Acm)-OH. They were dissolved in N,N-dimethylformamide
(DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,
N, N', N'-
tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine
(DIEA).
2 s Removal of the Fmoc protecting group was achieved using a solution of 20%
(V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2 to 7 The steps were performed in the same manner as Example 24.
Example 42
3 o CYs-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-CONH2
SEQ ID NO : 42
Step 1: Solid phase peptide synthesis was carried out on a 100 pmole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Cys(Acm)-OH, Fmoc-
Gly-OH,
Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-
OH,
35 Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-
OH,
Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Cys(Acm)-
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
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- 49 -
OH. They were dissolved in N,N-dimethylformamide (DMF) and, according to the
sequence,
activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-uronium
hexafluorophosphate
(HBTLT) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group
was
achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide
(DMF) for 20
minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 43
MPA-AEEA-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-
Gly-Leu-Gly-C ~ s-CONH2
i o SEQ H) NO : 43
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Cys(Acm)-OH, Fmoc-
Gly-OH,
Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-
OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-
OH,
i5 Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)
OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-dimethylformamide (DMF)
and, according to the sequence, activated using O-benzotriazol-1-yl-N, N, N',
N'-tetramethyl
uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal
of the
Fmoc protecting group was achieved using a solution of 20% (V/V) piperidine in
N,N
2 o dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 44
C Is-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-
Cys-Lys(AEEA-MPA)-CONH2
25 SEQ ID NO : 44
Step 1: Solid phase peptide synthesis was carried out on a 100 ,mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
3 o Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-
Gly-
OH, Fmoc-Phe-OH, Boc-Cys(Acm)-OH. They were dissolved in N,N-dimethylformamide
(DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,
N, N', N'-
tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine
(DIEA).
Removal of the Fmoc protecting group was achieved using a solution of 20%
(VlV)
3 5 piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
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-50-
Step 2 to 7: The steps were performed in the same manner as Example 24.
Example 45
Ser-Pro-Lys-Ile-Val-Gln-Gly-Ser-Gly- ~ys-Phe-Gly-Arg-Lys-Ile-Asp-
Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 45
Step 1: Solid phase peptide synthesis was carried out on a 100 pmole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
s o Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-
Arg(Pbf)-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Frnoc-Ser(tBu)-OH, Fmoc-Gly-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,
Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF) and,
according to
the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the
Frnoc
protecting group was achieved using a solution of 20% (V/U) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 46
MPA-AEEA-Ser-Pro-Lys-Ile-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-
Ile-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-~Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 46
Step 1: Solid phase peptide synthesis was carned out on a 100 pmole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly
3 o OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,
Fmoc-Ser(tBu)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTLT) and
diisopropylethylamine
3 5 (DIEA). Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
SUBSTITUTE SHEET (RULE 26)
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Step 2-5 The steps were performed in the same manner as Example 21.
Example 47
S er-Pro-Lys-lle-Val-Gln-Gly-S er-~s-Phe-Gly-Arg-Lys-lle-Asp-Arg-Ile-S er-
Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-Lys(AEEA-MPA)-CONHZ
s SEQ ID NO : 47
Step 1: Solid phase peptide synthesis was carried out on a 100 p.mole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
io Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Ile-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Pro-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-
15 dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2-7 The steps were performed in the same manner as Example 24.
Example 48
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-C~s-Phe-Gly-Arg-Arg-Met-Asp-
Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Arg-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 48
Step 1: Solid phase peptide synthesis was carned out on a 100 ,mole scale. The
following
2 s protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH,
Fmoc-Arg(Pbf)
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc
Cys(Acm)-OH, Fmoc-Gly-OH; Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Frnoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Arg(Pbf)-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Gly
s o OH, Frnoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Gly-
OH, Fmoc-Gln(Trt)-OH, Frnoc-Val-OH, Fmoc-Met-OH, Frnoc-Lys(Boc)-OH, Fmoc-Pro-
OH,
Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF) and,
according to
the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium
hexafluorophosphate (HBTU) and diisopropylethylarnine (DIEA). Removal of the
Fmoc
35 protecting group was achieved using a solution of 20% (V/V) piperidine in
N,N-
dimethylformamide (DMF) for 20 minutes.
SUBSTITUTE SHEET (RULE 26)
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Step 2-5 The steps were performed in the same maimer as Example 21.
Example 49
MPA-AEEA-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Arg-
Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-~Arg-Val-Leu-Arg Arg-His-CONH2
SEQ H) NO : 49
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf7-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
lo Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Arg(Pbt~-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,
Fmoc-Ser(tBu)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/VJ
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 50
Ser-Pro-Lys-Met-Val-Gln-Gly-Se~ -Phe-Gly-Arg-Arg-Met-Asp-Arg-Ile-Ser-
Ser-Ser-Ser-Gly-Leu-Gly-Cys~-Arg-Val-Leu-Arg-Arg-His-Lys(AEEA-MPA)-CONH2
SEQ ID NO : 50
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ile-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Arg(Pbf)-OH , Fmoc-
3 o Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acrn)-OH, Fmoc-Gly-OH,
Fmoc-
Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Pro-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
SUBSTITUTE SHEET (RULE 26)
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Step 2-7 The steps were performed in the same manner as Example 24.
Example 51
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-C s-Phe-Gly-Asp-Lys-Met-Asp-Arg-Ile-Ser-
S er-S er-S er-Gly-Leu-Gly-Cys-Lys-V al-Leu-Asp-Asp-His-C ONHZ
SEQ ID NO : 51
Step 1: Solid phase peptide synthesis was carried out on a 100 pmole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Asp(tBu)-
OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
io Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,
Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF) and,
according to
the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the
Fmoc
protecting group was achieved using a solution of 20% (V/V) piperidine in N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-5 The steps were performed in the same manner as Example 21.
Example 52
MPA-AEEA-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Asp-
Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cyls-Lys-Val-Leu-Asp-Asp-His-
CONH2
SEQ ID NO : 52
Step 1: Solid phase peptide synthesis was tamed out on a 100 mole scale. The
following
2s protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH,
Fmoc-Asp(tBu)
OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Frnoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly
s o OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,
Fmoc-Ser(tBu)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetrarnethyl-uronium hexafluorophosphate (HBTIJ) and
diisopropylethylarnine
3 5 (DIEA). Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
SUBSTITUTE SHEET (RULE 26)
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Step 2-5 The steps were performed in the same manner as Example 21.
Example 53
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-~Gl-y-~Cys-Phe-Gly-Asp-Lys-Met-Asp-Arg-Ile-Ser-
Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Asp-Asp-His-Lys(AEEA-MPA)-CONH2
s SEQ ID NO : 53
Step 1: Solid phase peptide synthesis was carried out on a 100 pmole scale.
The following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH Fmoc-
His(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
io Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Ile-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Pro-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-
15 dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
Step 2 to 7 The steps were performed in the same manner as Example 24.
Example 54
MPA-AEEA-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-~s-Phe-Gly-Arg-Lys-Met-
Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 54
Step 1: Solid phase peptide synthesis was carried out on a 100 pmole scale.
The following
2 s protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH,
Fmoc-Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
s o OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,
Fmoc-Ser(tBu)-OH, Fmoc-AEEA-OH, MPA-OH. They were dissolved in N,N-
dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
35 (DIEA). Removal of the Fmoc protecting group was achieved using a solution
of 20% (V/V)
piperidine in N,N-dimethylformamide (DMF) for 20 minutes.
SUBSTITUTE SHEET (RULE 26)
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Step 2-5 The steps were performed in the same manner as Example 21.
Example 55
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-
Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-Lys(AEEA-MPA)-CONH2
s SEQ ID NO : 55
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH,
Fmoc-
His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
so Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Ile-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Pro-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-
15 dimethylformamide (DMF) and, according to the sequence, activated using O-
benzotriazol-1-
yl-N, N, N', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and
diisopropylethylamine
(DIEA). Removal of the Fmoc protecting group was achieved using a solution of
20% (V/V)
piperidine in N,N-dimethylformarnide (DMF) for 20 minutes.
Step 2-7 The steps were performed in the same manner as Example 24.
Example 56
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-~ ys-Phe-Gly-Arg-Lys-Lys(AEEA-MPA)-Asp-
Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
SEQ ID NO : 56
Step 1: Solid phase peptide synthesis was carried out on a 100 p,mole scale.
The following
2 s protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH,
Fmoc-Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-
OH,
Fmoc-Asp(tBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
a o Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Frnoc-Lys(Boc)-OH,
Fmoc-Pro-OH, Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide
(DMF)
and, according to the sequence, activated using O-benzotriazol-1-yl-N, N, N',
N'-tetramethyl-
uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal
of the
3 5 Fmoc protecting group was achieved using a solution of 20% (V/V'
piperidine in N,N-
dimethylformamide (DMF) for 20 minutes.
SUBSTITUTE SHEET (RULE 26)
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Step 2-7 The steps were performed in the same manner as Example 24.
Example 57
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys\he-Gly-Arg-Lys-Met-Asp-Arg-Ile-
Lys(AEEA-MPA)-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His-CONH2
s SEQ ID NO : 57
Step 1: Solid phase peptide synthesis was carried out on a 100 mole scale. The
following
protected amino acids were sequentially added to resin: Fmoc-His(Trt)-OH, Fmoc-
Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-
Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-
lo Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Ile-OH, Fmoc-
Arg(Pbf)-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-
OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-
OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,
Boc-Ser(tBu)-OH. They were dissolved in N,N-dimethylformamide (DMF) and,
according to
i5 the sequence, activated using O-benzotriazol-1-yl-N, N, N', N'-tetramethyl-
uronium
hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the
Fmoc
protecting group was achieved using a solution of 20% (V/V' piperidine in N,N-
dimethylformamide (DMF) for 20 minutes.
Step 2-7 The steps were performed in the same manner as Example 24.
Purification procedure of the synthetised derivative
Each compound was purified by preparative reversed phase HPLC, using a Varian
z s (Dynamax) preparative binary HPLC system. The purification was performed
using a
Phenomenex Luna 10 ~ phenyl-hexyl, 50 mm x 250 mm column (particles lOp,)
equilibrated
with a water/TFA mixture (0.1 % TFA in H20 (solvent A) and acetonitrile/TFA
(0.1 % TFA in
CH3CN (solvent B). Fractions containing peptide were detected by UV absorbance
(Varian
Dynamax UVD In at 214 nm. Table 2 shows the retention time of compounds that
are NP
3 o peptides and derivatives according to the present invention.
SUBSTITUTE SHEET (RULE 26)
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TABLE 2
Compound Retention TimeCompound Retention Time
Example 1 27.0 A Example 16 29.8 A
Example 2 13.0 B Example 17 28.3
Exam le 3 28.1 A Example 18 31.0 A
Exam le 4 27.9 Example 19 27.2
Exam le 5 26.8 Exam le 20 28.8 A
Exam le 6 26.8 Example 21 23.3 A
Exam le 7 23.6 Example 54 24.9
Example 8 9.0 Exam le 55 24.1 A
Example 13 28.5 Example 56 23.5 A
Example 14 31.0 Example 57 24.2
Example 15 27.2
The retention times annotated with A, B and C have been obtained with gradient
of
elution shown in Tables 3, 4 and 5 respectively.
TABLE 3
Time (min) Solvent A (%) Solvent B (%) Flow (ml/min)
0 95.0 5.0 0.500
60 25.0 75.0 0.500
65 10.0 90.0 0.500
75 10.0 90.0 0.500
80 95.0 5.0 0.500
90 95.0 5.0 0.500
TABLE 4
Time (min) Solvent A (%) Solvent B (%) Flow (ml/min)
0 80.0 20.0 0.500
20 30.0 70.0 0.500
21 10.0 90.0 0.500
26 10.0 90.0 0.500
27 80.0 20.0 ~ 0.500
32 80.0 20.0 0.500
to
TABLE 5
Time (min) Solvent A (%) Solvent B (%) Flow (ml/min)
0 95.0 5.0 0.500
85.0 75.0 0.500
18 65.0 90.0 0.500
19 10.0 90.0 0.500
24 10.0 5.0 0.500
25 95.0 5.0 0.500
105 95.0 5.0 0.500
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Table 6 shows the predicted molecular weight (Predicted) and measured
molecular
weight (Measured) of compounds that are NP peptides and derivatives according
to the present
invention. All the molecular weights are expressed in g/mol. Molecular weight
has been
measured by Quadrupole Electro Spray mass spectroscopy. The predicted
molecular weight has
s been established by addition of the theoretical mass of each atom. The
differences between the
predicted molecular weight and the measured molecular weight are negligible
and indicate that
the compounds synthesized are the desired compounds.
TABLE 6
Compound PredictedMeasured Compound Predicted Measured
Exam le 3077.5 3078.5 Example 2565.1 2566.0
1 15
Exam le 3374.6 3377.0 Example 2861.2 2862.2
2 16
Exam le 3373.6 3377.1 Example 2391.1 2392.1
3 17
Example 3228.5 3230.3 Exam le 2687.2 2688.2
4 18
Example 3501.7 3504.0 Example 3091.5 3092.8
5 19
Example 3430.6 3432.5 Exam le 3387.6 3388.9
6 20
Example 3370.6 3372.3 Example 3460.8 3462.7
7 21
Example 3502.7 3504.5 Example 3756.9 3758.9
8 54
Example 2373.1 2374.0 Example 3885.0 3887.3
13 55
Example 2669.2 2670.1 Example 3753.9 3755.9
14 56
Determination of the efficiency of cyclisation of the peptide
Cyclisation is obtained by reduction of the thiol group of both cysteine
residues of the
is peptide so as to form an intramolecular disulphide bridge and details of
the process are in the
specification and are exemplified in Step 4 of Example 1 and in Step 4 of
Example 3. In order
to determine that the peptide has been successfully cyclised, an Ellman test
was performed on
the final cyclised material as taught in G.L. Ellman, Arch. Biochem. Biophys.,
82 (70) 1959 and
G.L. Ellinan, Biochem. Phannacol., 7 (68) 1961. The Ellman test allows
determination of thiol
a o groups that would not form disulphide bridges. The absence of free thiol
groups indicates that
the cyclisation was successful.
Also, analysis by LC/MS allows comparison of the intermediate of synthesis
obtained before the step of cyclisation and the final product obtained after
the cyclisation step.
z 5 Figure 1 shows in superposition the LC/MS spectrums of the intermediates
of synthesis of the
compound of Example 1 before cyclisation illustrated in dotted line (---) and
the corresponding
final products after cyclisation illustrated in continuous line (-), wherein
the cyclisation was
performed with iodine as exemplified in Step 4 of Example 1. It can be seen
that the
intermediates have a molecular ion fragment of 771.2 (M+4) that corresponds to
a mass of
3 0 3080.8 and the final products have a molecular ion fragment of 770.5 (M+4)
that corresponds to
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a mass of 3078Ø The reduction of the mass of 2.8 results from the loss of
two hydrogens
during the formation of the disulphide bridge. The sharpness of the peaks of
the linear
intermediates and the cyclic final products indicate that all the
intermediates were cyclised.
Moreover, no significant peak was seen at about 1232 (M+5) and/or 880.4 (M+7)
(not shown), which means that no dimer was synthesized; in other words no
intermolecular
disulphide bridge was generated.
to Ih vitvo conjugation
Preparation of ex vivo conjugates is used for ifz vita°o tests of the
derivative and for the
purposes of subsequent ift. vivo adminstration of the conjugate. Therefore,
the derivative is
conjugated to a blood component. Preferably, the blood component is human
serum albumin
(HSA). In examples 22-23-24, HSA is provided by Cortex-BiochemTM, San Leandro,
CA, USA.
In vitro conjugation examples
Example 58 : Preparation of 1 mM of the compound of Example 3:HSA conjugates.
In a 1500
NL Eppendorf''M tube, 450 ~I, of HSA 25% (g/100m1) is dispensed, and using a
variable speed
vortex machine, the HSA solution is vortexed. While vortexing, SO ~L of the
compound of
a o Example 3, at a concentration of l OmM in nanopure water, is added. The
resulting solution is
incubated at 37°C for 4 hours, and stored at 20°C.
Example 59 : Preparation of 1 mM of the compound of Example 4:HSA conjugates.
Conjugation to HSA is performed in the same manner as Example 58.
Example 60 : Preparation of 1 mM of the compound of Example S:HSA conjugates.
Conjugation to HSA is performed in the same manner as Example 58.
Example 61 : Preparation of 1 mM of the compound of Example 6:HSA conjugates.
3 o Conjugation to HSA is performed in the same manner as Example 58.
Example 62 : Preparation of 1 mM of the compound of Example 7:HSA conjugates.
Conjugation to HSA is performed in the same manner as Example 58.
Example 63 : Preparation of 1 mM of the compound of Example 14:HSA conjugates.
Conjugation to HSA is performed in the same manner as Example 58.
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Example 64 : Preparation of 1 mM of the compound of Example 18:HSA conjugates.
Conjugation to HSA is performed in the same manner as Example 58.
Example 65 : Prepare 1 mM of the compound of Example 54:HSA conjugates.
Conjugation
to HSA is performed in the same manner as Example 58.
Example 66 : Preparation of 1 mM of the compound of Example SS:HSA conjugates.
Conjugation to HSA is performed in the same manner as Example 58.
1o Example 67 : Preparation of 1 mM of the compound of Example 56:HSA
conjugates.
Conjugation to HSA is performed in the same manner as Example 58.
Example 68 : Preparation of 1 mM of the compound of Example 57:HSA conjugates.
Conjugation to HSA is performed in the same manner as Example 58.
Conjugate purity analysis
For analyzing the purity of the prepared conjugates, two tests are performed
by liquid
chromatography/mass spectrometry (LC/MS) (Electro Spray Ionization, Agilent HP
1100
Series): 1) quantifying the residual free derivatives with comparison to 1%
derivative reference
2 o and 2) detecting the conjugates with comparison to HSA.
Conjugate
purity results
The residual
free derivative
remaining
in solution
is:
Example 58: Conjugates of compound of Example 2.2%
3 with HSA:
Example Conjugates of compound of Example 4.4%
59: 4 with HSA:
Example 60: Conjugates of compound of Example 3.6%
5 with HSA:
Example 61: Conjugates of compound of Example <
6 with HSA: 1
Example 62: Conjugates of compound of Example <
7 with HSA: 1%
Example 63: Conjugates of compound of Example 1.2%
14 with HSA:
3 o Example Conjugates of compound of Example 1.3%
64: 18 with HSA:
Example 65: Conjugates of compound of Example 1.4%
54 with HSA:
Example 66: Conjugates of compound of Example 2.4%
55 with HSA:
Example 67: Conjugates of compound of Example 0.8%
56 with HSA:
Example 68: Conjugates of compound of Example 2.1%
57 with HSA:
Conjugate weight
Table 7 shows the predicted molecular weight (Predicted) and measured
molecular
weight (Measured) of conjugates of NP derivatives according to the present
invention. All the
molecular weights are expressed in glmol. Molecular weight has been measured
by Quadrupole
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Electro Spray mass spectroscopy. The predicted molecular weight has been
established by
addition of the theoretical mass of each atom. The differences between the
predicted molecular
weight and the measured molecular weight are negligible and indicate that the
compounds
synthesized are the desired compounds.
TABLE 7
Conjugate Predicted Measured
Example 58 69854 69853
Example 59 69709 69708
Exam le 60 69949 69943
Example 61 69878 69874
Example 62 69818 69814
Example 63 69118 69108
Exam le 64 69136 69128
Example 65 70204 70202
Exam le 66 70332 70329
Example 67 70201 70199
Example 68 70245 70243
i o Ih vitfo binding and activity assays
The potency of NP derivatives is evaluated as their ability to bind NPR
receptors in
guinea pig adrenal glands and to elevate cGMP levels in a rat primary lung
fibroblasts assay.
Others cell lines can be used to perform these iTa vitf°o assays such
as aortic smooth muscle
cells, glomeruli mesangial cells and adrenal cells. Human, rat, and ginea pig
cell lines or other
species cell lines can be used with a preference for human cell lines.
Isz vitro binding assays examples
Membranes for binding studies are prepared as follow. Adrenal glands were
collected from anesthetized normal Duncan Hartley Guinea Pig and homogenized
using a
a o polytron in 50 mM Tris-HCl buffer containing 150 mM NaCI, SmM MgCl2, SmM
MnCl2; pH
7.4 at 25°C. The homogenate was centrifuged for 10 minutes at 39,000 x
g (4°C). The pellet
was resuspended and washed. Finally, the membranes were resuspended in the
same buffer
supplemented with 1 mM Na2EDTA+0.2% BSA. Protein concentration is measured
using the
BCA protein assay kit (Pierce). The binding assay is done by incubation of
membranes with
2 s 0.016 nM laSI-rANF and increasing concentrations of either NP peptides or
NP derivatives
(10-5-10-11 M) for 60 minutes at 4°C. All assays were done in
duplicate. Separation of bound
and free radioactive rANF was achieved by rapid filtration through
polyethylenimine-treated
Whatman GF/C filters soaked in assay buffer. Filters were washed, dried and
counted for
radioactivity in a gamma-counter.
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Binding assays results of the NP derivatives comprising NP peptides of formula
I
are presented on Figure 2 and the binding assays results of the NP derivatives
comprising NP
peptides based on formula II are presented on Figure 3.
s In Figure 2, "Native ANP" is the peptide having the human ANP sequence that
has
been synthesized in our laboratories (see Example 1) and "hANP" is the
commercial peptide
provided by Phoenix Pharmaceuticals Inc., Belmont, CA, USA, and catalogue
number 005-
06. As it can be seen on Figure 2, native ANP and commercial hANP both
inhibited the
binding of lzsl-ANF to the receptor in a concentration-dependent manner with
apparent
to inhibition constants (Ki values) of 3.4 x 10-1°M and 6.0 x 10-
1°M, respectively. Conjugates of
NP derivatives of Examples 3 and 5 also inhibited the binding of lzsl-ANF to
the receptor of
adrenal glands in a concentration-dependent manner with apparent Ki values of
2.4 x 10-9M
and 2.9 x 10-9M respectively. Conjugates of NP derivatives of Examples 6 and 7
had a lower
binding affinity and avidity for the NPR receptors. The derivatives of
Examples 6 and 7 are
s5 modified in the loop in comparison with the derivatives of Examples 3 and
5, which are
modified at the N-terminus and C-terminus respectively.
Table 8 shows the concentrations at 50% of inhibition (EC50) and the
inhibition
constants (KI) that were calculated with the data from which originates the
graph in Figure 2.
TABLE 8
NP Peptides EC50 (M) KI
and
Conjugates
HANP 6.7230 e-0106.0340 e-010
Native ANP 3.8260 e-0103.4330 e-010
Example 3:HSA 2.7060 e-0092.4290 e-009
Exam le S:HSA 3.2330 e-0092.9020 e-009
Example 6:HSA 6.5110 e-0075.8440 e-007
Example 7:HSA 5.4730 e-0064.9110 e-006
2 s In Figure 3, "Native BNP" is the peptide having the human BNP sequence
that has
been synthesized in our laboratories (see Example 21). As it can be seen on
Figure 3, native
BNP inhibited the binding of lzsl-ANF to the receptor in a concentration-
dependent manner
with an apparent inhibition constant (Ki value) of 4.8 x 10-9M. Conjugates of
NP derivatives
of Examples 54 and 55 also inhibited the binding of lzsl-ANF to the receptor
of adrenal
3 o glands in a concentration-dependent manner with apparent Ki values of 1.5
x 10-$M and 5.5 x
10-$M respectively. Conjugates of NP derivatives of Examples 56 and 57 had a
lower binding
affinity and avidity for the NPR receptors. The derivatives of Examples 56 and
57 are
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modified in the loop in comparison with the derivatives of Examples 54 and 55,
which are
modified at the N-terminus and C-terminus respectively.
Table 9 shows the concentrations at 50% of inhibition (EC50) and the
inhibition
constants (I~ that were calculated with the data from which originates the
graph in Figure 3.
TABLE 9
NP Peptides and EC50 (M) KI
Conjugates
HBNP 5.4120 e-0094.8570 e-009
Exam le 54:HSA 1.7080 e-0081.5330 e-008
Exam le SS:HSA 6.0760 e-0085.4530 e-008
Example 56:HSA 3.1200 e-0072.8000 e-007
Example 57:HSA 2.8040 e-0072.5160 e-007
Ih vitro activity assays examples
For iyz vitYO activity studies, a human cervix epithelial adenocarcinoma cell
line was
used. Hela cells express high levels of natriuretic peptide receptors with
guanylate cyclase
activity.
One day prior cGMP experiments, cells are seeded in 48-wells plate (5X104
cells
per well) and incubated overnight. The day of the experiment cells are washed
twice in
serum-free media and then incubated with or without NP derivatives or native
ANP or BNP
for one hour, in presence of 3-isobutyl-1-methylxanthine to prevent eGMP
degradation.
a o Incubation is terminated by removing the assay medium and by adding HCl to
the cells for 10
minutes. The supernatants were then collected, centrifuged and cGMP levels are
assessed
using the direct cGMP EIA kit from Sigma.
All NP derivatives and conjugates were able to elevate cGMP in human Hela
cells
at concentration ranging from 10-6M to 10-9M, except for the conjugates of the
derivatives of
Examples 14, 18 and 56 as illustrated in Figures 4, 5 and 6. The EC50
(Effective
Concentration of a drug that causes 50% of the maximum response) have been
calculated for
each NP derivative and conjugate and are listed in Table 10. As it can be seen
from Table 10,
the increase in cGMP is comparable to that obtained from native ANP and no
significant (p<
3 0 ° 0.05) differences are observed between them, with exception for
the conjugates
Example 14:HSA, Example 18:HSA and Example 56:HSA. Assays were performed in
duplicata and each compound was tested three times.
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TABLE 10
NP Peptides and EC50 (M)
Conjugates
Native ANP 2.43 x 10'
Example 3:HSA 1.73 x 10'
Example 4:HSA 4.21 x 10-
Example S:HSA 3.67 x 10'
Exam le 13 2.72 x 10-
Exam le 14:HSA > 10'
Example 17 2.21 x 10'
Example 18:HSA > 10'
Native BNP 1.99 x 10'
Example 54:HSA 1.75 x 10'
Example 55:HSA 1.43 x 10'
Exam le 56:HSA > 10'
Example 57:HSA 3.36 x 10'
Analysis of the Stability in Human Plasma
Stability of conjugates of NP peptides is tested in human plasma in comparison
to the
corresponding free NP peptides so as to show protection of the conjugated NP
peptides against
1 o enzymatic degradation occurring in human plasma or to select the more
stable NP derivatives.
In the examples given below, the corresponding free NP peptide is human ANP,
called "hANP"
herein below, which was provided by Phoenix Pharmaceuticals Inc., Belmont, CA,
USA.
Conditions for the analysis of the stability in human plasma are as follow.
750 ~,L of
i5 human plasma (Biochemed Inc., Winchester, VA, USA) is poured in a 1500 gL
Eppendorf
Tube and 250 p,L of NP conjugates or hANP 1 mM is added to the plasma in order
to obtain a
final concentration of 0.25 mM of conjugates or hANP. The solutions are mixed
by vortexing
and the timer is started. The solutions are incubated at 37°C for 48
hours. An aliquot of 100 ~,L
is removed at time zero, 2 hrs, 4 hrs, 8 hrs, 12 hrs, 24 hrs, and 48 hrs. Each
aliquot is placed in a
2 o HPLC vial, snap freeze immediately on dry ice and stored at -80°C
until the LC/MS analysis.
'The LC/MS elution gradient of the peptides and the conjugates are
respectively
shown in Table 11 and 12; where solvent A is water/TFA mixture (0.1 % TFA in
H20) and
solvent B is acetonitrile/TFA (0.1% TFA in CH3CN).
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TABLE 11
Time (min) Solvent A (%) Solvent B (%) Flow (ml/min)
0 80.0 20.0 0.500
20 40.0 60.0 0.500
25 10.0 90.0 0.500
30 10.0 90.0 0.500
35 80.0 20.0 0.500
TABLE 12
Time (min) Solvent A (%) Solvent B (%) Flow (ml/min)
0 66.0 34.0 0.250
5 66.0 34.0 0.250
50.0 50.0 0.250
5.0 95.0 0.350
21 S.0 95.0 0.350
26 66.0 34.0 0.350
For each time point, results are reported as the percentage of peptide or
conjugate
peak height with respect to the total peak height of the sample. Figure 7
shows the results for
i o hANP (1), conjugates of Example 58 (~) and conjugates of Example 60 (1).
It can be seen from Figure 7, all the hANP is degraded after 24 hours of
incubation in
human plasma whereas more than 75% of the ANP conjugated with HSA is not
degraded after
48 hours. The resulting half life of hANP is about 4 hrs. The conjugates of
Example 58 (~)
i5 comprise an ANP sequence modified at the N-terminal (Example 23) and the
conjugates of
Example 60 (1) comprise an ANP sequence modified at the C-terminal (Example
25). Both
conjugates show similar results of stability in human plasma.
Analysis of the Stability towards NEP Enzyme
2 o Stability of conjugates of NP peptides is also tested in a NEP enzyme
solution in
comparison to the corresponding free NP peptides so as to show protection of
the conjugated
NP peptides against enzymatic degradation by NEP enzyme specifically. In the
examples given
below, the corresponding free NP peptide is human ANP, called "hANP" herein
below, which
was provided by Phoenix Pharmaceuticals Inc., Belmont, CA, USA.
Conditions for the analysis of the stability towards NEP enzyme degradation
are as
follow. The lyophilised enzymes contained in a vial of NEP enzyme (provided by
Calbiochem/Novabiochem Corporation, San Diego, CA, USA, product # 324762) are
solubilized with 100 ~L of 0.1 M Tris-HCl buffer pH 8Ø It was vortexed and
sonnicated to
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ensure a complete dissolution of the enzymes. One vial contains between 800
and 950 U of
enzymes. A solution of conjugates is prepared at 250 ~M with 0.1 M Tris-HCl
buffer pH 8Ø
Ten parts of the solution of conjugates or hANP (250 wM) are added to 1 part
of the NEP
enzyme solution (as above prepared). The resulting solution is vortexed and
incubated at 37°C
s under mixing conditions for 48 hours. An aliquot of 50 ~.L is removed at
time zero, 30 min,
1 hr, 2 hrs, 4 hrs, 8 hrs, 12 hrs, 24 hrs, and 48 hrs. Each aliquot is placed
in a vial, snap freeze
immediately on dry ice and stored at -80°C until analysis.
The site of hydrolysis of NEP on the sequence of ANP is the Cys-Phe peptidic
i o bond at the beginning of the loop, as illustrated in Figure 8. The BNP
sequence is also
cleaved by NEP at the same site, i.e. at the Cys-Phe peptidic bond at the
beginning of the
loop.
For detection of the non-hydrolysed NP peptide, radioimmunoassay (RIA) is
l5 performed using a commercial polyclonal antibody raised against human
native ANP (Product #
RGG-8798, Peninsula Laboratories Inc. Division of Bachem, San Carlos, CA,
USA).
For the radioimmunoassay, 50 ~L, of either NP conjugate calibration standards,
quality control samples, or diluted test samples in assay buffer (O.OSM
phosphate buffer, pH 7.5,
a o 0.08% sodium azide, 0.025M EDTA, and 0.1 % gelatin) is added to the
appropriately labeled 12
x 75 mm borosilicate glass test tubes. 50 ~T, of assay buffer is added to the
NSB (Non Specific
Binding) and zero-standard (Reference) tubes. Then, 300 p,I, of assay buffer
is added to each
NSB tube and 200 p,L of this same buffer is added to each of the other 12 x 75
mm borosilicate
glass test tubes. A volume of 100 ~.L, of rabbit anti-ANP IgG working
solution, at a
a 5 concentration of 2 pg/mL in assay buffer, is then added to all tubes
except TC (Total Counts)
and NSB tubes. Tube contents are mixed and incubated overnight (16 - 24 hours)
at
approximately 4°C. On the second day, 100 pL, of lzsl-hANP
(approximately 20,000
cpm/100p,L) is added to all tubes. Tube contents are mixed and incubated
overnight (16 - 24
hours) at approximately 4°C. On the third day, 1000 ~L of 0.6% charcoal
in O.OSM phosphate
3 o buffer is added to all tubes except TC tubes. Tubes are mixed and
incubated at approximately
4°C for approximately 30 minutes. After incubation, all tubes except TC
tubes are then
centrifuged at 4000 rpm for approximately 30 minutes at approximately
4°C. Free antigen is
separated from the bound antigen by decanting the supernatant. The
supernatants (bound
fractions) are then counted on a gamma counter (Packard Cobra II Auto-Gamma)
for at least 2
3 s minutes. The amount of [lzsI]-labeled antigen bound to the antibody is
inversely proportional to
the concentration of antigen in the tubes.
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For each time point of the incubation with NEP enzyme, results are reported as
the
percentage of peptide or conjugate with respect to the total amount of the
sample. Figure 9
shows the results for hANP (1), conjugates of Example 58 (~) and capped HSA
(~). "Capped
HSA" is albumin with a cysteine residue bonded to it.
It can be seen from Figure 9, most of the hANP is hydrolysed within 12 hours
whereas the conjugated ANP (conjugates of Example 58) take about 48 hrs to be
hydrolysed
completely by NEP enzyme in the test conditions. In order to prove that the
hydrolysis caused
by NEP enzyme occurs iii the ANP sequences and not in HSA, a control with
capped HSA is
i o used and shows that albumin is not (or almost not) subject to NEP
hydrolysis.
Pharmacokinetic studies
Pharmacokinetic studies of the derivatives are carried out in male Sprague-
Dawley
rats by subcutaneous (250 nmol/kg) or intravenous (50 nmol/kg) injection.
Serial blood
samples were taken at pre-dose and 5 min, 30 min, 1 hr, 2 hrs, 4 hrs, 8 hrs,
24 hrs, 48 hrs, 72 hrs
and 96 hrs post-agent administration. Blood samples were collected into tubes
containing K2-
EDTA and aprotinin, then centrifuged to obtain plasma and kept frozen until
analysis by
radioimmunoassay (RIA). A commercial polyclonal antibody raised against human
native ANP
(Product # RGG-8798, Peninsula Laboratories Inc. Division of Bachem, San
Carlos, CA, USA)
2 o is used to detect the compounds. The assay sensitivity is 300 to 10 000
pM. Specific
monoclonal antibodies need to be prepared and used for detecting each NP
derivative that
contains a NP peptide significatively different from the ANP and BNP. For
derivatives of ANP
and BNP, commercial antibodies are available. For the derivatives of NP
peptide having a high
homology with ANP or BNP, the commercially available antibodies may
successflly be used in
2 5 the RIA.
In Figure 10, the bioavailability of free NP peptides is compared with the
bioavailability of conjugated NP peptides. It can be seen that the conjugated
ANP (conjugates of
NP peptide of Example 3) administrered by intravenous injection (~) or by
subcutaneous
s o injection (~) are still bioavailable after 96 hrs whereas free ANP (NP
peptide of Example 3)
administered by intravenous injection (o) or by subcutaneous injection (O) are
not present in the
blood stream within 5 min.
In these rat studies, the half life of the conjugated ANP (conjugates of
Example 58)
35 administrered by intravenous injection (~) or by subcutaneous injection (~)
is 17,5 ~ 1,5 hours
and 14,8 ~ 0,6 hours respectively. The half life of free ANP (NP peptide of
Example 3)
administered by subcutaneous injection (o) is 0,2 ~ 0,06 hour and the one for
ANP
administered by intravenous injection (o) could not be calculated since it was
too short.
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Animal models of congestive heart failure are used to assess the optimal dose
response, the duration of action and the most effective NP derivatives and NP
conjugates. The
two following animal models can be used to do so: the spontaneous hypertensive
rats (SHR
rats) and the pacing model in dogs (Muders and Elsner, Pharm Res, 2000). Since
native BNP
is known to have no activity in rats, the derivatives of NP peptides having a
high homology
with BNP are not tested in the SHR rats; therefore dogs' models or other
models would be
used.
1 o SHR rats are genetically hypertensive rats, which develop significantly
elevated
systolic blood pressure (BP) by 4 weeks of age. As a consequence of sustained
elevated blood
pressure throughout their lifetimes, these rats develop congestive heart
failure by around 1
year of age. In addition to high blood pressure, this model is also
characterized by left
ventricular hypertrophy and left ventricular fibrosis. SHR rats have been used
previously in
i5 studies of the izz vivo effects of atrial natriuretic peptide. Single doses
of ANP analogues
produced a temporary drop in BP, while continuous infusions were required to
sustain a
decrease in systolic BP (DeMay et. al. J Pharm Exper Therap, 1987).
The pacing model in dogs, involves the implantation of programmable cardiac
a o pacemakers. After a surgical recovery period, the heart rate is increased
incrementally from
180 to 240 beats/min over a 31 to 38 day period. This model allows for the
study of different
stages of heart failure, evolving from the normal heart, to asymptomatic left
ventricular
dysfunction, to overt congestive heart failure (Luclmer et. al. Eur J Heart
Failure, 2000).
Characteristics of this model include increases of heart rate, increased
cardiac filling pressure,
25 low cardiac output, edema formation and activation of the sympathetic
nervous system and
other vasoconstrictor hormones (Arnolda et al, Austr. NZ J Med., 1999). The
pacing model
has been used previously in studies of the effects of both ANP and BNP on
heart failure
(Luchner et al, 2000; and Yamamoto et al, Am J Physiol, 1997).
3 o Isa vivo results
Tables 13 and 14 show izz vivo results in SHR rats of 7 week old and in
Winstar-
Kyoto rats of 7 week old respectively. The increase of urine secretion and the
increase of
cGMP expression have been measured 24 and 48 hours after injection of compound
of
Example 3. Concentrations of 1, 2 and 4 mg of compounds per kg of rats have
been tested in
s5 comparison with saline solution. Control values have been taken before
injection (pre-dose).
The urine secretion ( Vol.) is expressed in mL/day of urine exceeding the
value at pre-dose.
The cGMP expression (cGMP) is reported in rlmol/day and was measured by RIA
method.
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
-69-
TABLE 13
Saline 1 mg/k 2 mg/kg 4 mg/k
solution
Time Vol. cGMP Vol. cGMP Vol. cGMP Vol. cGMP
Point
Pre-Dose0.00.27.81.5 0.00.27.81.50.00.8 7.81.5 0.00.87.81.5
24 h 0.60.2192 -0.30.3292 2.20.7 353 2.30.8405
48 h 2.60.410.00.43.10.7191 4.21.2 194 2.00.7243
TABLE 14
Saline 1 mg/kg 2 mg/kg 4 m
solution /kg
Time Vol. cGMP Vol. cGMP Vol. cGMP Vol. cGMP
Point
Pre-Dose0.00.9 194 0.00.4194 0.00.4194 0.00.4194
24 h 1.00.9 305 1.50.7383 8.91.0533 2.30.8715
48 h 8.42.2 243 6.21.5243 8.50.6222 9.61.0365
1 o While the invention has been described in connection with specific
embodiments
thereof, it will be understood that it is capable of fixrther modifications,
and this application is
intended to cover any variations, uses or adaptations of the invention
following, in general, the
principles of the invention, and including such departures from the present
description as come
within known or customary practice within the art to which the invention
pertains, and as may
15 be applied to the essential features hereinbefore set forth, and as follows
in the scope of the
appended claims.
t
SUBSTITUTE SHEET (RULE 26)
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
i
SEQUENCE LISTING
<110> ConjuChem, Inc.
<120> Long lasting natriuretic peptide derivatives
<130> 2710
<140> unknown
<141> 2003-07-24
<150> US 60/400,199
<151> 2002-07-31
<150> US 60/400,413
<151> 2002-07-31
<160> 58
<210> l
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> AMIDATION
<222> 28
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Artificial Sequence: synthetic peptide
<400> 1
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25
<210> 2
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> SITE
<222> 28
<223> Xaa represents Tyr-COOH
<220>
<223> Description of Sequence: synthetic peptide
1/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<400> 2
Xaa Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25
<210> 3
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> AMIDATION
<222> 28
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 3
Xaa Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25
<210> 4
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-Ser
<220>
<22l> AMIDATION
<222> 28
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 4
Xaa Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25
<210> 5
2/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<211> 29
<212> PRT
<2l3> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> SITE
<222> 29
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 5
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr Xaa
20 25
<210> 6
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> SITE
<222> 17
<223> Xaa represents Lys(AEEA-MPA)
<220>
<221> AMTDATION
<222> 28
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 6
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly
1 5 10 15
Xaa Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25
<210> 7
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> SITE
<222> 12
<223> Xaa represents Lys(AEEA-MPA)
3/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<220>
<221> AMIDATION
<222> 28
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 7
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Xaa Asp Arg Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25
<210> 8
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 11 to 27
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 8
Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met
1 5 10 15
Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25 30
<210> 9
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 11 to 27
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Thr
<220>
<221> SITE
<222> 32
<223> Xaa represents Tyr-COOH
<220>
<223> Description of Sequence: synthetic peptide
<400> 9
Xaa Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met
1 5 10 15
Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25 30
4/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<210> 10
<2l1> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 11 to 27
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Thr
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 10
Xaa Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met
1 5 10 15
Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25 30
<210> 11
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> AMIDATION
<222> 28
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 11
Xaa Leu Asp Asp Ser Ser Cys Phe Gly Gly Asp Met Asp Asp Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Asp Xaa
20 25
<210> 12
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
5/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<221> DISULFID
<222> From 7 to 23
<220>
<221> AMIDATION
<222> 28
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 12
Ser Leu Asp Asp Ser Ser Cys Phe Gly Gly Asp Met Asp Arg Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Asp Xaa
20 25
<210> 13
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> AMIDATION
<222> 22
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 13
Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly
1 5 10 15
Cys Asn Ser Phe Arg Xaa
<210> 14
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Cys
<220>
<221> AMIDATION
<222> 22
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 14
6/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
Xaa Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly
1 5 10 15
Cys Asn Ser Phe Arg Xaa
<210> 15
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 3 to 19
<220>
<221> AMIDATION
<222> 24
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 15
Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly
1 5 10 15
Leu Gly Cys Asn Ser Phe Arg Xaa
<210> 16
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 3 to 19
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> AMIDATION
<222> 24
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 16
Xaa Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly
1 5 10 15
Leu Gly Cys Asn Ser Phe Arg Xaa
<210> 17
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
7/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<222> From 1 to 17
<220>
<221> AMIDATION
<222> 22
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 17
Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly
1 5 10 15
Cys Asn Ser Phe Arg Xaa
<210> 18
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Cys
<220>
<221> AMIDATION
<222> 22
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 18
Xaa Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly
1 5 10 15
Cys Asn Ser Phe Arg Xaa
<210> 19
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> SITE
<222> 8
<223> Xaa represents N-Methyl-Phe
<220>
<221> AMIDATION
<222> 28
<223> Xaa represents Tyr-CONH2
<220>
8/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<223> Description of Sequence: synthetic peptide
<400> 19
Ser Leu Arg Arg Ser Ser Cys Xaa Gly Gly Arg Met Asp Arg Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25
<210> 20
<211> 28
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 7 to 23
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> SITE
<222> 8
<223> Xaa represents N-Methyl-Phe
<220>
<221> AMIDATION
<222> 28
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 20
Xaa Leu Arg Arg Ser Ser Cys Xaa Gly Gly Arg Met Asp Arg Ile Gly
1 5 10 15
Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Xaa
20 25
<210> 21
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 21
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25 30
9/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<210> 22
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 3 to 19
<220>
<221> AMIDATION
<222> 25
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 22
Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly
1 5 10 15
Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25
<210> 23
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> AMIDATION
<222> 22
<223> Xaa represents Tyr-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 23
Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly
1 5 10 15
Cys Asn Ser Phe Arg Xaa
<210> 24
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 3 to 19
<220>
<221> SITE
<222> 26
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
10/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<400> 24
Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly
1 5 10 15
Leu Gly Cys Lys Val Leu Arg Arg His
20 25
<210> 25
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 3 to 19
<220>
<221> AMIDATION
<222> 25
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 25
Ser Gly Cys Phe Gly Arg Lys Ile Asp Arg Ile Ser Ser Ser Ser Gly
1 5 10 15
Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25
<210> 26
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 3 to 19
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> AMIDATION
<222> 25
<223> Xaa represents His-CONH2
<220>
<223> Desoription of Sequence: synthetic peptide
<400> 26
Xaa Gly Cys Phe Gly Arg Lys Ile Asp Arg Ile Ser Ser Ser Ser Gly
1 5 10 15
Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25
<210> 27
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
11/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<221> DISULFID
<222> From 3 to 19
<220>
<221> SITE
<222> 26
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 27
Ser Gly Cys Phe Gly Arg Lys Ile Asp Arg Ile Ser Ser Ser Ser Gly
1 5 10 15
Leu Gly Cys Lys Val Leu Arg Arg His Xaa
20 25
<210> 28
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> AMIDATION
<222> 23
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 28
Cys Phe Gly Arg Lys Ile Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Cys Lys Val Leu Arg Arg Xaa
<210> 29
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Cys
<220>
<221> AMIDATION
<222> 23
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 29
Xaa Phe Gly Arg Lys Ile Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
12/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
1 5 10 15
Cys Lys Val Leu Arg Arg Xaa
<210> 30
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> SITE
<222> 24
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 30
Cys Phe Gly Arg Lys Ile Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Cys Lys Val Leu Arg Arg His Xaa
<210> 31
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> AMIDATION
<222> 23
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 31
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Cys Lys Val Leu Arg Arg Xaa
<210> 32
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
13/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Cys
<220>
<221> AMIDATION
<222> 23
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 32
Xaa Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Cys Lys Val Leu Arg Arg Xaa
<210> 33
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 24
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 33
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Cys Lys Val Leu Arg Arg His Xaa
<210> 34
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 2
<223> Xaa represents N-alpha-methyl-Phe
<220>
<221> AMIDATION
<222> 23
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
14/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<400> 34
Cys Xaa Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Cys Lys Val Leu Arg Arg Xaa
<210> 35
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 2
<223> Xaa represents N-alpha-methyl-Phe
<220>
<22l> AMIDATION
<222> 23
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 35
Cys Xaa Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Cys Lys Val Leu Arg Arg Xaa
<210> 36
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 2
<223> Xaa represents N-alpha-methyl-Phe
<220>
<221> SITE
<222> 24
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 36
Cys Xaa Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Cys Lys Val Leu Arg Arg His Xaa
15/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<210> 37
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 11
<223> Xaa represents N-alpha-methyl-Phe
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 37
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Xaa Gly Arg Lys Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25 30
<210> 38
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 11
<223> Xaa represents N-alpha-methyl-Phe
<220>
<221> SITE
<222> 33
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 38
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Xaa Gly Arg Lys Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His
20 25 30
Xaa
<210> 39
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
16/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<221> DISULFID
<222> From 1 to 17
<220>
<221> AMIDATION
<222> l7
<223> Xaa represents Cys-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 39
Cys Phe Gly Arg Lys Ile Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Xaa
<210> 40
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Cys
<220>
<221> AMIDATION
<222> 17
<223> Xaa represents Cys-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 40
Xaa Phe Gly Arg Lys Ile Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Xaa
<210> 41
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 1
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<221> AMIDATION
<222> 18
<223> Xaa represents His-CONH2
17/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<220>
<223> Description of Sequence: synthetic peptide
<400> 41
Cys Phe Gly Arg Lys Ile Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
l 5 10 15
Cys Xaa
<210> 42
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> AMIDATION
<222> 17
<223> Xaa represents Cys-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 42
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Xaa
<210> 43
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Cys
<220>
<221> AMIDATION
<222> 17
<223> Xaa represents Cys-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 43
Xaa Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Xaa
<210> 44
<211> 18
<212> PRT
18/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 1 to 17
<220>
<221> SITE
<222> 18
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 44
Cys Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly
1 5 10 15
Cys Xaa
<210> 45
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 45
Ser Pro Lys Ile Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Ile Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25 30
<210> 46
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
19/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<400> 46
Xaa Pro Lys Ile Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Ile Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25 30
<210> 47
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 33
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 47
Ser Pro Lys Ile Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Ile Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His
20 25 30
Xaa
<210> 48
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 48
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Arg Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Arg Val Leu Arg Arg Xaa
20 25 30
<210> 49
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
20/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 49
Xaa Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Arg Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Arg Val Leu Arg Arg Xaa
20 25 30
<210> 50
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 33
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 50
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Arg Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Arg Val Leu Arg Arg His
20 25 30
Xaa
<210> 51
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 51
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Asp Lys Met Asp
21/26
CA 02488348 2004-12-02
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1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Asp Asp Xaa
20 25 30
<210> 52
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 52
Xaa Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Asp Lys Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Asp Asp Xaa
20 25 30
<210> 53
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 33
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 53
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Asp Lys Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Asp Asp His
20 25 30
Xaa
<210> 54
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
22/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 1
<223> Xaa represents MPA-AEEA-Ser
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 54
Xaa Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25 30
<210> 55
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 33
<223> Xaa represents Lys(AEEA-MPA)-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 55
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His
20 25 30
Xaa
<210> 56
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 15
<223> Xaa represents Lys(AEEA-MPA)
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
23/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<220>
<223> Description of Sequence: synthetic peptide
<400> 56
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Xaa Asp
1 5 10 15
Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25 30
<210> 57
<211> 32
<212> PRT
<213> Artificial Sequence
<220>
<221> DISULFID
<222> From 10 to 26
<220>
<221> SITE
<222> 19
<223> Xaa represents Lys(AEEA-MPA)
<220>
<221> AMIDATION
<222> 32
<223> Xaa represents His-CONH2
<220>
<223> Description of Sequence: synthetic peptide
<400> 57
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp
1 5 10 15
Arg Ile Xaa Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg Xaa
20 25 30
<210> 58
<211> 33
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Sequence: synthetic peptide
<220>
<221> VARIANT
<222> 1
<223> Xaa = R1-X1 where X1 is Thr or absent, and R1 is NH2 or a
N-terminal blocking group.
<220>
<221> VARIANT
<222> 2
<223> Xaa = Ser, Thr, Ala or absent.
<220>
<221> VARIANT
<222> 3
<223> Xaa = Pro, Hpr, Val, or absent.
<220>
<221> VARIANT
24/26
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WO 2004/011498 PCT/CA2003/001097
<222> 4
<223> Xaa = Lys, D-Lys, Arg, D-Arg, Asn, Gln or absent.
<220>
<221> VARIANT
<222> 5
<223> Xaa = Met, Leu, Ile, an oxidatively stable Met-replacement
amino acid, Ser, Thr or absent.
<220>
<221> VARIANT
<222> 6
<223> Xaa = Val, Ile, Leu, Met, Phe, Ala, D-Ala, Nle or absent.
<220>
<221> VARIANT
<222> 7
<223> Xaa = Gln, Asn, Arg, D-Arg, Asp, Lys, D-Lys or absent.
<220>
<221> VARIANT
<222> 8
<223> Xaa = Gly, Pro, Ala, D-Ala, Arg, D-Arg, Asp, Lys, D-Lys, Gln,
Asn or absent.
<220>
<221> VARIANT
<222> 9
<223> Xaa = Ser, Thr or absent.
<220>
<221> VARIANT
<222> 10
<223> Xaa = Gly, Pro, Ala, D-Ala, Ser, Thr or absent.
<220>
<221> VARIANT
<222> 12
<223> Xaa = Phe, Tyr, Leu, Val, Ile, Ala, D-Ala, Phe with an isosteric
replacement of its amide bond selected from N-alpha-methyl,
methyl amino, hydroxyl ethyl, hydrazino, ethylene, sulfonamide and
N-alkyl-b-aminopropionic acid, or a Phe-replacement amino acid
conferring NEP enzyme resistance.
<220>
<221> VARIANT
<222> 13
<223> Xaa = Gly, Ala, D-Ala or Pro.
<220>
<221> VARIANT
<222> 14
<223> Xaa = Arg, Lys, D-Lys, Asp, Gly, Ala, D-Ala or Pro.
<220>
<221> VARIANT
<222> 15
<223> Xaa = Lys, D-Lys, Arg, D-Arg, Asn, Gln or Asp.
<220>
<221> VARIANT
<222> 16
<223> Xaa = Met, Leu, Ile or an oxidatively stable Met-replacement
amino acid.
<220>
25/26
CA 02488348 2004-12-02
WO 2004/011498 PCT/CA2003/001097
<221> VARIANT
<222> 20
<223> Xaa = Ser, Gly, Ala, D-Ala or Pro.
<220>
<221> VARIANT
<222> 21
<223> Xaa = Ser, Gly, Ala, D-Ala, Pro, Val, Leu, or Ile.
<220>
<22l> VARIANT
<222> 22
<223> Xaa = Ser, Gly, Ala, D-Ala, Pro, Gln or Asn.
<220>
<221> VARIANT
<222> 24
<223> Xaa = Gly, Ala, D-Ala or Pro.
<220>
<221> VARIANT
<222> 26
<223> Xaa = Gly, Ala, D-Ala or Pro.
<220>
<221> VARIANT
<222> 28
<223> Xaa = Lys, D-Lys, Arg, D-Arg, Asn, Gln, His or absent.
<220>
<221> VARIANT
<222> 29
<223> Xaa =Val, Ile, Leu, Met, Phe, Ala, D-Ala, Nle, Ser, Thr or absent.
<220>
<221> VARIANT
<222> 30
<223> Xaa = Leu, Nle, Ile, Val, Met, Ala, D-Ala, Phe, Tyr or absent.
<220>
<221> VARIANT
<222> 31
<223> Xaa = Arg, D-Arg, Asp, Lys, D-Lys or absent.
<220>
<221> VARIANT
<222> 32
<223> Xaa = Arg, D-Arg, Asp, Lys, D-Lys, Tyr, Phe, Trp, Thr, Ser
or absent.
<220>
<221> VARIANT
<222> 33
<223> Xaa = X33-R2 where X33 is His, Asn, Gln, Lys, D-Lys, Arg, D-Arg
or absent, and R2 is COOH, CONH2 or a C-terminal blocking group.
<220>
<221> DISULFID
<222> From 11 to 27
<400> 58
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Asp Arg Ile Xaa Xaa Xaa Ser Xaa Leu Xaa Cys Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa '
26/26
