Note: Descriptions are shown in the official language in which they were submitted.
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LONG LASTING ANTI-ANGIOGENIC PEPTIDES
FIELD OF THE INVENTION
This invention relates to modified anti-angiogenic peptides. In
particular, this invention relates to modified kringle 5 peptides with long
duration of action for the treatment of diseases related to angiogenesis.
BACKGROUND OF THE INVENTION
Angiogenesis, the development of new blood vessels, is a highly
regulated and essential process of endothelial cell growth. Although
angiogenesis is a highly regulated process under normal conditions,
many diseases (characterized as "angiogenic diseases") are driven by
persistent unregulated angiogenesis. Unregulated, angiogenesis may
either cause a particular disease directly or exascerbate an existing
pathological condition. For example, ocular neovacularization has been
implicated as the most common cause of blindness and dominates
approximately 20 eye diseases. In certain existing conditions such as
arthritis, newly formed capillary blood vessels invade the joints and
destroy cartilage. In diabetes, new capillaries formed in the retina invade
the vitreous, bleed, and cause blindness. Growth and metastasis of
solid tumors are also angiogenesis-dependent (Folkman, J., Cancer
Research, 46: 467-473 (1986), Folkman, J., Journal of the National
Cancer Institute, 82: 4-6 (1989)).
Much research has been performed to identify anti-antiogenic
molecules. One angiogenic molecule of particular interest is
plasminogen. Of particular intererest is the kringle 5 region of
plasminogen and various peptides within the kringle 5 region. Both
plasminogen and the kringle 5 region of plasminogen have been shown
to interfere with the angiogenic process are thus known as anti-
angiogenic peptides.
While useful, kringle 5 peptides, like other peptides, suffer from
rapid kidney excretion, liver metabolism, and decomposition from
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2
endogeneous peptidases leading to very short plasma half-lives thereby
reducing their usefulness as anti-angiogenic agents. As a result of their
short half lives, peptides such as kringle 5 require constant infusion to
reach
adequate plasma levels sufficient for efficient therapy.
As a result, there is a need for long lasting anti-angiogenic peptides
such as kringle 5. Such long lasting peptides would be useful in treating
angiogenesis related diseases in mammals.
SUMMARY OF THE INVENTION
In order to meet these needs, the present invention is directed to
modified anti-angiogenic peptides. A preferred embodiment of this invention
is directed to modified kringle 5 peptides. The invention relates to novel
chemically reactive derivatives of anti-angiogenic peptides that can react
with
available functionalities on mobile blood proteins to form covalent linkages.
Specifically, the invention relates to novel chemically reactive derivatives
of
anti-angiogenic peptides such as kringle 5 peptides that can react with
available functionalities on mobile blood proteins to form covalent linkages.
The chemically reactive derivatives of the anti-angiogenic peptides are
capable of forming a peptidase stabilized anti-angiogenic peptide.
The invention is directed to a derivative of an anti-angiogenic peptide
that comprises an anti-angiogenic peptide to which is coupled a maleimide
group or succinimidyl group which reacts with an amino group, a hydroxyl
group or a thiol group on a blood component to form a stable covalent bond
therewith.
The present invention relates to modified anti-angiogenic peptides and
derivatives thereof and their use for providing an anti-angiogenic effect in a
patient and for the treatment of angiogenesis in a human. The modified
antiangiogenic peptide includes a reactive group capable of forming a
covalent bond with serum albumin.
In particular, the present invention relates to the following modified
kringle 5 peptides: NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Lys-NH2;
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NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-N H2 ; Nac-Tyr-Thr-Thr-Asn-Pro-Arg-
Lys-Leu-Tyr-Asp-Tyr-Lys-NH2; NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-
GIy-Pro-Trp-Aia-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH2 ;
NAc-Arg-Asn-Pro-As p-G ly-As p-Va I-G Iy-G Iy-P ro-Trp-Lys-N H2;
NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-(Ns-M PA)-N H2;
(M PA-AEEA)-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-N H2;
(M PA)-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-N H2;
NAc-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-( Ns-MPA)-NH2;
(M PA-AEEA)-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH2;
M PA)-Tyr-Th r-Th r-Asn-P ro-Arg-Lys-Le u-Tyr-Asp-Tyr-N H2;
NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-GIy-GIy-Pro-Trp-Ala-Tyr-Thr-Thr-
Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-( NE-M PA)-N H2;
(MPA-AEEA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-Ala-Tyr-
Th r-Th r-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-N H2;
(MPA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-
Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-N HZ;
NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-GIy-GIy-Pro-Trp-Lys-( NE-MPA)-NH2;
M PA-AEEA)-Arg-Asn-Pro-Asp-G Iy-Asp-Va I-GIy-GIy-Pro-Trp-N H2;
(M PA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-GIy-GIy-Pro-Trp-N H2;
NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-( Ns-MPA)-NH2;
(M PA-AEEA)-Arg-Lys-Leu-Tyr-Asp-Tyr-N Hz;
(M PA)-Arg-Lys-Leu-Tyr-Asp-Tyr-N H2;
NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Lys-( Ns-M PA)-N Hz;
(MPA-AEEA)-Pro-Arg-Lys-Leu-Tyr-Asp-NH2;
(MPA)-Pro-Arg-Lys-Leu-Tyr-Asp-NH2;
NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-( NE-AEEA-M PA)-N H2;
NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-( NE-AEEAn-MPA)-NH2; and
other modified kringle 5 peptides.
The modified anti-angiogenic peptides find use in the treatment of
angiogenesis in humans.
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DETAILED DESCRIPTION OF THE INVENTION
To ensure a complete understanding of the invention the
following definitions are provided:
Reactive Groups: Reactive groups are chemical groups capable
of forming a covalent bond. Such reactive groups are coupled or
bonded to an anti-angiogenic, or, more specifically, a kringle 5 peptide
of interest. Reactive groups will generally be stable in an aqueous
environment and will usually be carboxy, phosphoryl, or convenient acyl
group, either as an ester or a mixed anhydride, or an imidate, thereby
capable of forming a covalent bond with functionalities such as an amino
group, a hydroxy or a thiol at the target site on mobile blood
components. For the most part, the esters will involve phenolic
compounds, or be thiol esters, alkyl esters, phosphate esters, or the like.
Reactive groups include succimidyl and maleimido groups.
Functionalities: Functionalities are groups on blood
components with which reactive groups react to form covalent bonds.
Functionalities include hydroxyl groups for bonding to ester reactive
groups; thiol groups for bonding to imidates and thioester groups; amino
groups for bonding to carboxy, phosphoryl or acyl groups on reactive
groups and carboxyl groups for bonding to amino groups.
Blood Components: Blood components may be either fixed or
mobile. Fixed blood components are non-mobile blood components and
include tissues, membrane receptors, interstitial proteins, fibrin proteins,
collagens, platelets, endothelial cells, epithelial cells and their
associated membrane and membraneous receptors, somatic body cells,
skeletal and smooth muscle cells, neuronal components, osteocytes and
osteoclasts and all body tissues especially those associated with the
circulatory and lymphatic systems. Mobile blood components are blood
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components that do not have a fpced situs for any extended period of
time, generally not exceeding 5, more specifically one minute. These
blood components are not membrane-associated and are present in the
blood for extended periods of time and are present in a minimum
concentration of at least 0.1 pg/ml. Mobile blood components include
serum albumin, transferrin, ferritin and immunoglobulins such as tgM
and IgG. The half-life of mobile blood components is at least about 12
hours.
Protective Groups: Protectrve groups are chemical moieties
utilized to protect peptide derivatives from reacting with themselves.
Various protective groups are disclosed herein and in U.S. 5,493,007.
Such protective groups include acetyl, fluorenylmethyloxycarbonyl
(FMOC); t-butyloxycarbonyl (BOC), berizyloxycarbonyl (CBZ), and the
like. The specific protected amino acids are depicted in Table 1.
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TABLE 1
NATURAL AMINO ACIDS AND THEIR ABBREVIATIONS
3-Letter 1-Letter Protected Amino
Name Abbreviation Abbreviation Acids
Alanine Ala A Fmoc-Ala-OH
Arginine Arg R Fmoc-Arg(Pbf)-OH
Asparagine Asn N Fmoc-Asn(Trt)-OH
Aspartic acid Asp D Asp(tBu)-OH
Cysteine Cys C Fmoc-Cys(Trt)
Glutamic acid Glu E Fmoc-Glu(tBu)-OH
Glutamine Gin Q Fmoc-Gln(Trt)-OH
Glycine Gly G Fmoc-Gly-OH
Histidine His H Fmoc-His(Trt)-OH
Isoleucine lie I Fmoc-ile-OH
Leucine Leu L Fmoc-Leu-OH
Lysine Lys K Fmoc-Lys(Mtt)-OH
Methionine Met M Fmoc-Met-OH
Phenylalanine Phe F Fmoc-Phe-OH
Proline Pro P Fmoc-Pro-OH
Serine Ser S Fmoc-Ser(tBu)-OH
Threonine Thr T Fmoc-Thr(tBu)-OH
Tryptophan Trp W Fmoc-Trp(Boc)-OH
Tyrosine Tyr Y Boc-Tyr(tBu)-OH
Valine Val V Fmoc-Val-OH
Sensitive Functional Groups - A sensitive functional group is a
group of atoms that represents a potential reaction site on an anti-
angiogenic peptide. If present, a sensitive functional group may be
chosen as the attachment point for the linker-reactive entity modification.
Sensitive functional groups include but are not limited to carboxyl,
amino, thiol, and hydroxyl groups.
Modified Peptides - A modified anti-angiogenic peptide is a
peptide that has been modified by attaching a reactive group, and is
capable of forming a peptidase stabilized peptide through conjugation to
blood components. The reactive group may be attached to the anti-
angiogenic peptide either via a linking group, or optionally without using
a linking group. It is also contemplated that one or more additional
amino acids may be added to the anti-angiogenic peptide to facilitate
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the attachment of the reactive group. Modified peptides may be
administered in vivo such that conjugation with blood components
occurs in vivo, or they may be first conjugated to blood components in
vitro and the resulting peptidase stabilized peptide (as defined below)
administered in vivo. The terms "modified anti-angiogenic peptide" and
"modified peptide" may be used interchangeably in this application.
Peptidase Stabilized Anti-Angiogenic Peptides - A peptidase
stabilized anti-angiogenic peptide is a modified peptide that has been
conjugated to a blood component via a covalent bond formed
between the reactive group of the modified peptide and the
functionalities of the blood component, with or without a linking
group. Peptidase stabilized peptides are more stable in the presence
of peptidases in vivo than a non-stabilized peptide. A peptidase
stabilized anti-angiogenic peptide generally has an increased half life
of at least 10-50% as compared to a non-stabilized peptide of
identical sequence. Peptidase stability is determined by comparing
the half life of the unmodified anti-angiogenic peptide in serum or
blood to the half life of a modified counterpart anti-angiogenic
peptide in serum or blood. Half life is determined by sampling the
serum or blood after administration of the modified and non-modified
peptides and determining the activity of the peptide. In addition to
determining the activity, the length of the anti-angiogenic peptide
may also be measured by HPLC and Mass Spectrometry.
Linking Groups: Linking groups are chemical moieties that link
or connect reactive groups to anti-angiogenic peptides. Linking
groups may comprise one or more alkyl groups such as methyl,
ethyl, propyl, butyl, etc. groups, alkoxy groups, alkenyl groups,
alkynyl groups or amino group substituted by alkyl groups, cycloalkyl
groups, polycyclic groups, aryl groups, polyaryl groups, substituted
aryl groups, heterocyclic groups, and substituted heterocyclic
groups. Linking groups may also comprise poly ethoxy aminoacids
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such as AEA ((2-amino) ethoxy acetic acid) or a preferred linking
group AEEA ([2-(2-amino)ethoxy)]ethoxy acetic acid).
DETAILED DESCRIPTION OF THE INVENTION
Taking into account these definitions, the focus of this invention is
to modify anti-angiogenic peptides and particularly kringle 5 peptides to
improve bio-availability, extend half-life and distribution of the peptide in
vivo through conjugation of the peptide onto a protein carrier without
modifying its anti-angiogenesis properties. The carrier of choice (but not
limited to) for this invention would be albumin conjugated through its free
thiol by a kringle 5 peptide derivatized with a maleimide moiety.
1. Kringle 5 Peptides
As used herein, the term "kringle 5" refers to the region of
mammalian plasminogen having three disulfide bonds which contribute
to the specific three-dimensional confirmation defined by the fifth kringle
region of the mammalian plasminogen molecule. One such disulfide
bond links the cysteine residues located at amino acid positions 462 and
541, a second links the cysteine residues located at amino acid
positions 483 and 524 and a third links the cysteine residues located at
amino acid positions 512 and 536. The amino acid sequence of a
complete mammalian plasminogen molecule (the human plasminogen
molecule), including its kringle 5 region, is presented in (SEQ ID NO: 1).
The term "kringle 5 peptide fragments" refers to peptides with
anti-angiogenic activity of between 4 and 104 amino acids (inclusive)
with a substantial sequence homology to the corresponding peptide
fragment of mammalian plasminogen, an a-N-terminus at about amino
acid position 443 of intact mammalian plasminogen and an a-C-
terminus at about position 546 of SEQ ID NO:1; an a-N-terminus at
about amino acid position 513 of intact mammalian plasminogen and an
a-C-terminus at about position 523 of SEQ ID NO:1; an a-N-terminus at
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about amino acid position 525 of intact mammalian plasminogen and an
a-C-terminus at about position 535 of SEQ ID N0:1; an a-N-terminus at
about amino acid position 529 of intact mammalian plasminogen and an
a-C-terminus at about position 535 of SEQ ID NO:1; an a-N-terminus at
about amino acid position 529 of intact mammalian plasminogen and an
a-C-terminus at about position 534 of SEQ ID NO:1 and an a-N-
terminus at about amino acid position 150 of intact mammalian
plasminogen and an a-C-terminus at about position 156 of SEQ ID
NO:1.
In a preferred format, the kringle 5 peptide of the invention has
one or more of the following sequences:
Pro-Arg-Lys-Leu-Tyr-Asp-Lys-NH2 (SEQ ID N0:2);
Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH2 (SEQ ID N0:3);
Arg-Asn-Pro-Asp-G Iy-Asp-Va I-G Iy-G Iy-Pro-Trp-Ala-Tyr-Th r-Th r-Asn-Pro-
Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH2 (SEQ ID N0:4);
Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-Lys-NH2 (SEQ ID N0:5);
Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NHZ (SEQ ID N0:6);
Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NHz (SEQ ID NO:7);
Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH2 (SEQ ID N0:8);
Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-
Arg-Lys-Leu-Tyr-Asp-Tyr-NH2 (SEQ ID N0:9);
Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH2 (SEQ ID NO:10);
Arg-Lys-Leu-Tyr-Asp-Tyr-NH2 (SEQ ID NO:11);
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Pro-Arg-Lys-Leu-Tyr-Asp-Lys-NH2 (SEQ ID NO:12);
Pro-Arg-Lys-Leu-Tyr-Asp-NH2 (SEQ ID NO:13);
Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH2 (SEQ ID NO:14);
Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH2 (SEQ ID NO:15) and
Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp (SEQ ID N0:16).
Thus, it is to be understood that the present invention is
contemplated to encompass any derivatives or modifications of kringle 5
peptide fragments which have anti-angiogenic activity and includes the
entire class of kringle 5 peptide fragments described herein and
derivatives and modifications of those kringle 5 peptide fragments.
2. Modified Kringle 5 Peptides
This invention relates to modified anti-angiogenic peptides and, in
particular, modified kringle 5 peptides. The modified kringle 5 peptides
of the invention can react with available reactive functionalities on blood
components via covalent linkages. The invention also relates to such
modifications, such combinations with blood components and methods
for their use. These methods include extending the effective therapeutic
in vivo half life of the modified kringle 5 peptides.
To form covalent bonds with the functional group on a protein,
one may use as a chemically reactive group (reactive entity) a wide
variety of active carboxyl groups, particularly esters, where the hydroxyl
moiety is physiologically acceptable at the levels required to modify the
kringle 5 peptide. While a number of different hydroxyl groups may be
employed in these linking agents, the most convenient would be N-
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hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS),
maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxy
succinimide ester (GMBS) and maleimidopropionic acid (MPA).
Primary amines are the principal targets for NHS esters as
diagramed in the schematic below. Accessible a-amine groups present
on the N-termini of proteins react with NHS esters. However, a-amino
groups on a protein may not be desirable or available for the NHS
coupling. While five amino acids have nitrogen in their side chains, only
the s-amine of lysine reacts significantly with NHS esters. An amide
bond is formed when the NHS ester conjugation reaction reacts with
primary amines releasing N-hydroxysuccinimide as demonstrated in the
schematic below. These succinimide containing reactive groups are
herein referred to as succinimidyl groups.
o' o
O O H
R-CM-O-N + R-NHZ H-7-9 -= R~R'+ HO-N
NHS-Ester Reaction Scheme
In the preferred embodiments of this invention, the functional
group on the protein will be a thiol group and the chemically reactive
group will be a maleimido-containing group such as gamma-maleimide
butyrlamide (GMBA) or MPA. Such maleimide containing reactive
groups are herein referred to as "maleimido groups." The maleimido
group is 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
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with amines. A stable thioether linkage between the maleimido group
and the sulfhydryl is formed which cannot be cleaved under
physiological conditions as demonstrated in the following schematic.
o
H 0
~-SH+NR -~ P N-R
O O
O
I N-R H? ~
H HCr-C~R
0
Maleimide Reaction Scheme
The kringle 5 peptides and peptide derivatives of the invention
may be modified for specific labeling and non-specific labeling of blood
components.
A. Specific Labeling
Preferably, the modified angiogenic peptides of this invention are
designed to specifically react with thiol groups on mobile blood proteins.
Such reaction is preferably established by covalent bonding of a anti-
angiogenic peptide modified with a maleimide link (e.g. prepared from
GMBS, MPA or other maleimides) to a thiol group on a mobile blood
protein such as serum albumin or IgG.
Under certain circumstances, specific labeling with maleimides
(maleimido groups) offers several advantages over non-specific labeling
of mobile proteins with groups such as NHS and sulfo-NHS. Thiol
groups are less abundant in vivo than amino groups. Therefore, the
maleimide derivatives of this invention will covalently bond to fewer
proteins. For example, in albumin (the most abundant blood protein)
there is only a single thiol group. Thus, peptide-maleimide-albumin
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conjugates will tend to comprise approximately a 1:1 molar ratio of
peptide to albumin. In addition to aibumin, IgG molecules (class li) also
have free thiols. Since IgG molecules and serum albumin make up the
majority of the soluble protein in blood they also make up the majority of
the free thiol groups in blood that are available to covalently bond to
maleimide-modified peptides.
Further, even among free thiol-containing blood proteins, specific
labeling with maleimides leads to the preferential formation of peptide-
maleimide-albumin conjugates, due to the unique characteristics of
albumin itself. The single free thiol group of albumin, highly conserved
among species, is located at amino acid residue 34 (Cys34). It has been
demonstrated recently that the Cys34 of albumin has increased reactivity
relative to free thiols on other free thiol-containing proteins. This is due
in part to the very low pK value of 5.5 for the Cys' of albumin. This is
much lower than typical pK values for cysteines residues in general,
which are typically about 8. Due to this low pK, under normal
physiological conditions Cys34 of albumin is predominantly in the ionized
form, which dramatically increases its reactivity. In addition to the low
pK value of Cys', another factor which enhances the reactivity of Cys'
is its location, which is in a crevice close to the surface of one loop of
region V of albumin. This location makes Cys34 very available to ligands
of all kinds, and is an important factor in Cys34is biological role as free
radical trap and free thiol scavenger. These properties make Cys34
highly reactive with maleimide peptides, and the reaction rate
acceleration can be as much as 1000-fold relative to rates of reaction of
maleimide peptides with other free-thiol containing proteins.
Another advantage of peptide-maleimide-albumin conjugates is
the reproducibility associated with the 1:1 loading of peptide to albumin
specifically at Cys34. Other techniques, such as glutaraidehyde, DCC,
EDC and other chemical activations of, for example, free amines lack
this selectivity. For example, albumin contains 52 lysine residues, 25-
30 of which are located on the surface of albumin and accessible for
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conjugation. Activating these lysine residues, or altematively modifying
peptides to couple through these lysine residues, results in a
heterogenous population of conjugates. Even if 1:1 molar ratios of
peptide to albumin are employed, the yield will consist of multiple
conjugation products, some containing 0, 1, 2 or more peptides per
albumin, and each having peptides randomly coupled at any one of the
25-30 available lysine sites. Given the numerous combinations
possible, characterization of the exact composition and nature of each
batch becomes difficult, and batch-to-batch reproducibility is all but
impossible, making such conjugates less desirable as anti-angiogenic
peptides. Additionally, while it would seem that conjugation through
lysine residues of albumin would at least have the advantage of
delivering more anti-angiogenic agent per albumin molecule, studies
have shown that a 1:1 ratio of anti-angiogenic agent to albumin is
preferred. In an artiide by Stehle, et al., "The Loading Rate Determines
Tumor Targeting Properties of Methotrexate-Albumin Conjugates in
Rats," Anti-Cancer Drugs, Vol. 8, pp. 677-685 (1997), the authors
report 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 in vivo. It is postulated that at these higher
ratios, conformational changes to albumin diminish its effectiveness as
a therapeutic carrier.
Through controlled administration of maleimide-peptides in vivo,
one can control the specific labeling of albumin and !gG in vivo. In
typical administrations, 80-90% of the administered maleimide-peptides
will label albumin and less than 5% will label IgG. Trace labeling of free
thiols such as glutathione will also occur_ Such specific labeling is
preferred for in vivo use as it permits an accurate calculation of the
estimated half-life of the administered agent.
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In addition to providing controlled specific in vivo labeling,
maleimide-peptides can provide specific labeling of serum albumin and
IgG ex vivo. Such ex vivo labeling involves the addition of maleimide-
peptides to blood, serum or saline solution containing serum albumin
and/or IgG. Once modified ex vivo with maleimide-peptides, the blood,
serum or saline solution can be readministered to the blood for in vivo
treatment.
In contrast to NHS-peptides, maleimide-peptides are generally
quite stable in the presence of aqueous solutions and in the presence of
free amines. Since maleimide-peptides will only react with free thiols,
protective groups are generally not necessary to prevent the maleimide-
peptides from reacting with itself. In addition, the increased stability of
the peptide permits the use of further purification steps such as HPLC to
prepare highly purified products suitable for in vivo use. Lastly, the
increased chemical stability provides a product with a longer shelf life.
B. Non-Specific Labeling.
The kringle 5 peptides of the invention may also be modified for
non-specific labeling of blood components. Bonds to amino groups will
also be employed, particularly with the formation of amide bonds for
non-specific labeling. To form such bonds, one may use as a
chemically reactive group coupled to the kringle 5 peptide a wide variety
of active carboxyl groups, particularly esters, where the hydroxyl moiety
is physiologically acceptable at the levels required. While a number of
different hydroxyl groups may be employed in these linking agents, the
most convenient would be N-hydroxysuccinimide (NHS) and N-hydroxy-
sulfosuccinimide (sulfo-NHS), which form succinimidyl groups.
Other linking agents which may be utilized are described in U.S.
Patent 5,612,034.
The various sites with which the chemically reactive group of the
subject non-specific kringle 5 peptide derivatives may react in vivo
include cells, particularly red blood cells (erythrocytes) and platelets,
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and proteins, such as immunoglobulins, including IgG and IgM, serum
albumin, ferritin, steroid binding proteins, transferrin, thyroxin binding
protein, a-2-macroglobulin, and the like. Those receptors with which the
derivatized kringle 5 peptides react, which are not long-lived, will
generally be eliminated from the human host within about three days.
The proteins indicated above (including the proteins of the cells) will
remain at least three days, and may remain five days or more (usually
not exceeding 60 days, more usually not exceeding 30 days) particularly
as to the half life, based on the concentration in the blood.
For the most part, reaction will be with mobile components in the
blood, particularly blood proteins and cells, more particularly blood
proteins and erythrocytes. By "mobile" is intended that the component
does not have a fixed situs for any extended period of time, generally
not exceeding 5, more usually one minute, although some of the blood
component may be relatively stationary for extended periods of time.
Initially, there will be a relatively heterogeneous population of
functionalized proteins and cells. However, for the most part, the
population within a few days will vary substantially from the initial
population, depending upon the half-life of the functionalized proteins in
the blood stream. Therefore, usually within about three days or more,
IgG will become the predominant functionalized protein in the blood
stream.
Usually, by day 5 post-administration, IgG, serum albumin and
erythrocytes will be at least about 60 mole %, usually at least about 75
mole %, of the conjugated components in blood, with IgG, IgM (to a
substantially lesser extent) and serum albumin being at least about 50
mole %, usually at least about 75 mole %, more usually at least about
80 mole %, of the non-cellular conjugated components.
Preferably, the kringle 5 peptide derivative is conjugated to
albumin.
The desired conjugates of non-specific kringle 5 peptides to blood
components may be prepared in vivo by administration of the kringle 5
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peptide derivatives to the patient, which may be a human or other
mammal. The administration may be done in the form of a bolus or
introduced slowly over time by infusion using metered flow or the like.
If desired, the subject conjugates may also be prepared ex vivo
by combining blood with derivatized kringle 5 peptides of the present
invention, allowing covalent bonding of the derivatized kringle 5 peptides
to reactive functionalities on blood components and then returning or
administering the conjugated blood to the host. Moreover, the above
may also be accomplished by first purifying an individual blood
component or limited number of components, such as red blood cells,
immunoglobulins, serum albumin, or the like, and combining the
component or components ex vivo with the chemically reactive kringle 5
peptide derivatives. The functionalized blood or blood component may
then be returned to the host to provide in vivo the subject therapeutically
effective conjugates. The blood also may be treated to prevent
coagulation during handling ex vivo.
3. Synthesis of Modified Kringle 5 Peptides
A. Kringle 5 Peptide Synthesis
Kringle 5 peptide fragments may be synthesized by standard
methods of solid phase peptide chemistry known to those of ordinary
skill in the art. For example, kringle 5 peptide fragments may be
synthesized by solid phase chemistry techniques following the
procedures described by Steward and Young (Steward, J. M. and
Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical
Company, Rockford, III., (1984) using an Applied Biosystem synthesizer.
Similarly, multiple fragments may be synthesized then linked together to
form larger fragments. These synthetic peptide fragments can also be
made with amino acid substitutions at specific locations.
For solid phase peptide synthesis, a summary of the many
techniques may be found in J. M. Stewart and J. D. Young, Solid Phase
Peptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J.
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Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic
Press (New York), 1973. For classical solution synthesis see G.
Schroder and K. Lupke, The Peptides, Vol. 1, Acacemic Press (New
York). In general, these methods comprise the sequential addition of
one or more amino acids or suitably protected amino acids to a growing
peptide chain. Normally, either the amino or carboxyl group of the first
amino acid is protected by a suitable protecting group. The protected 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 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
removed sequentially or concurrently to afford the final polypeptide. By
simple modification of this general procedure, it is possible to add 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.
A particularly preferred method of preparing compounds of the
present invention involves solid phase peptide synthesis wherein 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 contained therein. Suitable protecting groups are 9-
fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),
benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl , t-
amyloxycarbonyl, isobornyloxycarbonyl, a, a-dimethyl-3,5-
dimethoxybenzyloxycarbonyl, o-nitrophenyisulfenyl, 2-cyano-t-
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butyloxycarbonyl, and the like. The 9-fluorenyl-methyloxycarbonyl
(Fmoc) protecting group is particularly preferred for the synthesis of
kringle 5 peptide fragments. Other preferred side chain protecting
groups are, for side chain amino groups like lysine and arginine,
2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl,
4-methoxybenzene-sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; for
tyrosine, benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl,
isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for
serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl,
Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for
asparticacid and glutamic acid, benzyl and t-butyl and for cysteine,
triphenylmethyl (trityl).
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 which 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 4-hydroxymethylphenoxymethyl-copoly(styrene-1%
divinylbenzene). The preferred solid support for a-C-terminal amide
peptides is the 4-(2',4'-dimethoxyphenyl-Fmoc-
aminomethyl)phenoxyacetamidoethyl resin available from Applied
Biosystems (Foster City, Calif.). The a-C-terminal amino acid is coupled
to the resin by means of N,N'-dicyclohexylcarbodiimide (DCC), N,N'-
diisopropylcarbodiimide (DIC) or O-benzotriazol-1-yl-N,N,N',N'-
tetramethyluronium-hexafluorophosphate (HBTU), with or without 4-
dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT),
benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-
hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl)phosphine
chloride (BOPCI), mediated coupling for from about 1 to about 24 hours
at a temperature of between 10 and 50 C. in a solvent such as
dichloromethane or DMF.
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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 (HBTU, 1 equiv.)
and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. The coupling of
successive protected amino acids can be carried out in an automatic
polypeptide synthesizer as is well known in the art. In a preferred
embodiment, the a-N-terminal amino acids of the growing peptide chain
are protected with Fmoc. The removal of the Fmoc protecting group
from the a-N-terminal side of the growing peptide is accomplished by
treatment with a secondary amine, preferably piperidine. Each
protected amino acid is then introduced in about 3-fold molar excess,
and the coupling is preferably carried out in DMF. The coupling agent is
normally O-benzotriazol-1-yl-N,N,N',N'-
tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1-
hydroxybenzotriazole (HOBT, 1 equiv.).
At the end of the solid phase synthesis, the polypeptide is
removed from the resin and deprotected, either in successively or in a
single operation. Removal of the polypeptide and deprotection can be
accomplished in a single operation by treating the resin-bound
polypeptide with a cleavage reagent comprising thianisole, water,
ethanedithiol and trifluoroacetic acid. In cases wherein the a-C-terminal
of the 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 using the cleavage cocktail described
above. The fully deprotected peptide is purified by a sequence of
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chromatographic steps employing any or all of the following types: ion
exchange on a weakly basic resin (acetate form); hydrophobic
adsorption chromatography on underivitized potystyrene-divinylbenzene
(for example, Amberlite XAD); silica gel adsorption chromatography; ion
exchange chromatography on carboxymethylcellulose; partition
chromatography, e.g. on Sephadex G-25, LH-20 or countercurrent
distribution; high performance liquid chromatography (HPLC), especially
reverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phase
column packing.
Molecular weights of these kringle 5 peptides are determined
using Fast Atom Bombardment (FAB) Mass Spectroscopy.
The kringle 5 peptides of the invention may be synthesized with
N- and C-terminal protecting groups.
1. N-Terminal Protective Groups.
The term "N-protecting group" refers to those groups intended to
protect the a-N-terminal of an amino acid or peptide or to otherwise
protect the amino group of an amino acid or peptide against undesirable
reactions during synthetic procedures. Commonly used N-protecting
groups are disclosed in Greene, "Protective Groups In Organic
Synthesis," (John Wiley & Sons, New York (1981)). Additionally,
protecting groups can be used as pro-drugs which are readily cleaved
in vivo, for example, by enzymatic hydrolysis, to release the biologically
active parent. a-N-protecting groups comprise loweralkanoyl groups
such as formyl, acetyl ("Ac"), propionyl, pivaloyl, t-butylacetyl and the
like; other acyl groups include 2-chloroacetyl, 2-bromoacetyl,
trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,
-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-
nitrobenzoyl and the like; sulfonyl groups such as benzenesulfonyl, p-
toluenesulfonyl and the like; carbamate forming groups such as
benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-
methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-
*Trade-Mark
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nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-
dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-
dimethoxybenzyioxycarbonyl, 4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-
dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-
biphenylyl)-1-methylethoxycarbonyl, a, a-dimethyt3,5-
dimethoxybenzyloxycarbonyl, benzhydrytoxycarbonyl, t
butyioxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,
ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-
trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantytoxycarbonyl, cyclohexyloxycarbonyl, phenytthiocarbonyl and
the like; arylalkyl groups such as benzyl, triphenylmethyl,
benzyloxymethyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like and
silyl groups such as trimethylsilyl and the like.
Preferred N-protecting groups are formyl, acetyl, benzoyl,
pivaloyl, t-butylacetyl, phenytsulfonyl, benzyl, t-butyloxycarbonyl (Boc)
and benzyloxycarbonyl (Cbz). For example, lysine may be protected at
the a-N-terminal by an acid labile group (e.g. Boc) and protected at the -
N-terminal by a base labile group (e.g. Fmoc) then deprotected
selectively during synthesis.
2. Carboxyl Protective GrouDs.
The term "carboxyl protecting group" refers to a carboxylic acid
protecting ester or amide group employed to block or protect the
carboxylic acid functionality while the reactions involving other functional
sites of the compound are performed. Carboxy protecting groups are
disclosed in Greene, "Protective Groups in Organic Synthesis" pp. 152-
186 (1981). Additionally, a carboxy protecting group can be used as a
pro-drug whereby the carboxy protecting group can be readily cleaved
in vivo, for example by enzymatic hydrolysis, to release the biologically
active parent. Such carboxy protecting groups are well know to those
skilled in the art.
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having been extensively used in the protection of carboxyl groups in the
penicillin and cephalosporin fields as described in U.S. Pat. Nos.
3,840,556 and 3,719,667. Representative carboxy protecting
groups are C, -Ca kmeralkyl (e.g., methyl, ethyl or t-butyl and the like);
arylalkyl such as phenethyl or benzyl and substituted derivatives thereof
such as alkoxybenzyl or nitrobenzyl groups and the like; arylaikenyl
such as phenylethenyl and the like; aryl and substituted derivatives
thereofsuch as 5-indanyl and the like; dialkylaminoalkyl such as
dimethylaminoethyl and the like); alkanoyloxyalkyl groups such as
acetoxymethyl, butyryloxymethyl, valeryloxymethyl, isobutyryloxymethyl,
isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl, 1-(pivaloyloxyl)-1-ethyl, 1-
methyl-1-(propionyloxy)-1-ethyl, pivaloyloxymethyl, propionyloxymethyl
and the like; cycloalkanoyloxyalkyl groups such as
cycloProPYlcarbonyloxymethyl, cyclobutylcarbonyloxymethyl,
cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl and the
like; aroyloxyalkyl such as benzoyloxymethyl, benzoyloxyethyl and the
like; arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl, 2-
benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl or
cycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl,
cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-l-ethyl and the like;
alkoxycarbonyloxyalkyl or cydoalkyloxycarbonyloxyalkyl such as
methoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl, 1-
ethoxycarbonyloxy-1 -ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl and the
like; aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl, 2-(5-
indanyloxycarbonyloxy)ethyl and the like; alkoxyalkylcarbonyloxyalkyl
such as 2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like;
arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl and
the like; arylaikenyloxycarbonyloxyalkyl such as 2-(3-phenylpropen-2-
yloxycarbonyloxy)ethyl and the like; alkoxycarbonylaminoalkyl such as t-
butyloxycarbonylaminomethyl and the like;
alkylaminocarbonylaminoalkyl such as
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methylaminocarbonylaminomethyl and the like; alkanoylaminoalkyl such
as acetylaminomethyl and the like; heterocycliccarbonyloxyalkyl such as
4-methylpiperazinylcarbonyloxymethyl and the like;
dialkylaminocarbonylalkyl such as dimethylaminocarbonylmethyl,
diethylaminocarbonylmethyl and the like; (5-(Ioweralkyl)-2-oxo-1,3-
dioxolen-4-yl)aikyl such as (5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and
the like; and (5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as (5-phenyl-2-
oxo-1,3-dioxolen-4-yl)methyl and the like.
Representative amide carboxy protecting groups are
aminocarbonyl and loweralkylaminocarbonyl groups.
Preferred carboxy-protected compounds of the invention are
compounds wherein the protected carboxy group is a 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 alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroyloxyalkyl or an
arylalkylcarbonyloxyalkyl ester. Preferred amide carboxy protecting
groups are loweralkylaminocarbonyl groups. For example, aspartic acid
may be protected at the a-C-terminal by an acid labile group (e.g. t-
butyl) and protected at the (i-C-terminal by a hydrogenation labile group
(e.g. benzyl) then deprotected selectively during synthesis.
B. Modification of Kringle 5 Peptides
The manner of producing the modified kringle 5 peptides of the
present invention will vary widely, depending upon the nature of the
various elements comprising the molecule. The synthetic procedures
will be selected so as to be simple, provide for high yields, and allow for
a highly purified product. Normally, the chemically reactive group will be
created at the last stage, for example, with a carboxyl group,
esterification to form an active ester will be the last step of the synthesis.
Specific methods for the production of derivatized kringle 5 peptides of
the present invention are described in examples below.
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Each kringle 5 peptide selected to undergo the derivatization with
a linker and a reactive agent will be modified according to the following
criteria: if a carboxylic group, not critical for the retention of
pharmacological activity is available on the original molecuie and no
other reactive functionality is present on the molecule, then the
carboxylic acid will be chosen as attachment point for the linker-reactive
group modification. If no carboxylic acids are available, then any other
functionalities not critical for the retention of pharmacological activity
will
be selected as attachment point for the linker-reactive group
modification. If several functionalities are available on kringle 5 peptide,
a combination of protecting groups will be used in such a way that after
addition of the linker/reactive group and deprotection of all the protected
functional groups, retention of pharmacological activity is still obtained.
If no reactive functionalities are available on the therapeutic agent,
synthetic efforts will allow for a modification of the original parent drug in
such a way that retention of biological activity and retention of receptor
or target specificity is obtained.
The chemically reactive group is at a site, so that when the
peptide is bonded to the blood component, the peptide retains a
substantial proportion of the parent compound's inhibitor activity.
Even more specifically, each kringle 5 peptide selected to
undergo the derivatization with a linker and a reactive group will be
modified according to the following criteria: if a terminal carboxylic group
is available on the kringle 5 peptide and is not critical for the retention of
pharmacological activity, and no other sensitive functional group is
present on the kringle 5 peptide, then the carboxylic acid will be chosen
as attachment point for the linker-reactive group modification. If the
terminal carboxylic group is involved in pharmacological activity, or if no
carboxylic acids are available, then any other sensitive functional group
not critical for the retention of pharmacological activity will be selected
as the attachment point for the linker-reactive group modification. If
several sensitive functional groups are available on a kringle 5 peptide,
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a combination of protecting groups will be used in such a way that after
addition of the linker/reactive entity and deprotection of all the protected
sensitive functional groups, retention of pharmacological activity is still
obtained. If no sensitive functional groups are available on the
therapeutic peptide, [or if a simpler modification route is desired],
synthetic efforts will allow for a modification of the original kringle 5
peptide in such a way that retention of biological activity and retention of
receptor or target specificity is obtained. In this case the modification
will occur at the opposite end of the peptide.
An NHS derivative may be synthesized from a carboxylic acid in
absence of other sensitive functional groups in the kringle 5 peptide.
Specifically, such a kringle 5 peptide is reacted with N-
hydroxysuccinimide in anhydrous CH2 C12 and EDC, and the product is
purified by chromatography or recrystallized from the appropriate
solvent system to give the NHS derivative.
Alternatively, an NHS derivative may be synthesized from a
kringle 5 peptide that contains an amino and/or thiol group and a
carboxylic acid. When a free amino or thiol group is present in the
molecule, it is preferable to protect these sensitive functional groups
prior to perform the addition of the NHS derivative. For instance, if the
molecule contains a free amino group, a transformation of the amine
into a Fmoc or preferably into a tBoc protected amine is necessary prior
to perform the chemistry described above. The amine functionality will
not be deprotected after preparation of the NHS derivative. Therefore
this method applies only to a peptide whose amine group is not required
to be freed to induce a pharmacological desired effect.
In addition, an NHS derivative may be synthesized from a kringle
5 peptide containing an amino or a thiol group and no carboxylic acid.
When the selected molecule contains no carboxylic acid, an array of
bifunctional linkers can be used to convert the molecule into a reactive
NHS derivative. For instance, ethylene glycol-
bis(succinimydylsuccinate) (EGS) and triethylamine dissolved in DMF
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and added to the free amino containing molecule (with a ratio of 10:1 in
favor of EGS) will produce the mono NHS derivative. To produce an
NHS derivative from a thiol derivatized molecule, one can use N-[1,-
maleimidobutyryloxy]succinimide ester (GMBS) and triethylamine in
DMF. The maleimido group will react with the free thiol and the NHS
derivative will be purified from the reaction mixture by chromatography
on silica or by HPLC.
An NHS derivative may also be synthesized from a kringle 5
peptide containing multiple sensitive functional groups. Each case is
have to be analyzed and solved in a different manner. However, thanks
to the large array of protecting groups and bifunctional linkers that are
commercially available, as described above, this invention is applicable
to any peptide with preferably one chemical step only to derivatize the
peptide or two steps by first protecting a sensitive group or three steps
(protection, activation and deprotection). Under exceptional
circumstances only, would one require to use multiple steps (beyond
three steps) synthesis to transform a kringle 5 peptide into an active
NHS or maleimide derivative.
A maleimide derivative may also be synthesized from a kringle 5
peptide containing a free amino group and a free carboxylic acid. To
produce a maleimide derivative from a amino derivatized molecule, one
can use N-[y-maleimidobutyryloxy]succinimide ester (GMBS) and
triethylamine in DMF. The succinimide ester group will react with the
free amino and the maleimide derivative will be purified from the
reaction mixture by crystallization or by chromatography on silica or by
HPLC.
Finally, a maleimide derivative may be synthesized from a kringle
5 peptide containing multiple other sensitive functional groups and no
free carboxylic acids. When the selected molecule contains no
carboxylic acid, an array of bifunctional crosslinking reagents can be
used to convert the molecule into a reactive NHS derivative. For
instance maleimidopropionic acid (MPA) can be coupled to the free
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amine to produce a maleimide derivative through reaction of the free
amine with the carboxylic group of MPA using HBTU/HOBt/DIEA
activation in DMF.
A large number of bifunctional compounds are available for
linking to entities. Illustrative entities include: azidobenzoyl hydrazide, N-
[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio)propionamide), bis-
sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate,
N-y-maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-
4-azidobenzoate, N-succinimidyl [4-azidophenyl]-1,3'-dithiopropionate,
N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, and
succinimidyl 4-[N-maleimidomethyl]cyclohexane-l-carboxylate.
4. Uses of the Modified Kringle 5 Peptides
As described earlier, angiogenesis includes a variety of
processes involving neovascularization of a tissue including "sprouting",
vasculogenesis, or vessel enlargement. With the exception of traumatic
wound healing, corpus leuteum formation and embryogenesis, it is
believed that the majority of angiogenesis processes are associated with
disease processes and therefore the use of the present therapeutic
methods are selective for the disease and do not have deleterious side
effects.
There are a variety of diseases in which angiogenesis is believed
to be important, which may be treatable with the modified peptides of
the invention. These diseases include, but not limited to, inflammatory
disorders such as immune and non-immune inflammation, chronic
articular rheumatism and psoriasis, disorders associated with
inappropriate or inopportune invasion of vessels such as diabetic
retinopathy, neovascular glaucoma, restenosis, capillary proliferation in
atherosclerotic plaques and osteoporosis, and cancer associated
disorders, such as solid tumors, solid tumor metastases, angiofibromas,
retrolental fibroplasia, hemangiomas, Kaposi sarcoma and the like
cancers which require neovascularization to support tumor growth.
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The modified kringle 5 peptides of the invention find use in
methods which inhibit angiogenesis in a diseased tissue ameliorates
symptoms of the disease and, depending upon the disease, can
contribute to cure of the disease. The modified peptides of the invention
are more stable in vivo and, as such, smaller amounts of the modified
peptide can be administered for effective treatment In one embodiment,
the invention contemplates inhibition of angiogenesis, per se, in a
tissue. The extent of angiogenesis in a tissue, and therefore the extent
of inhibition achieved by the present methods, can be evaluated by a
variety of method, for detecting a5B3 immunopositive immature and
nascent vessel structures by immunohistochemistry.
As described herein, any of a variety of tissues, or organs
comprised of organized tissues, can support angiogenesis in disease
conditions including skin, muscle, gut, connective tissue, joints, bones
and the like tissue in which blood vessels can invade upon angiogenic
stimuli.
In one related embodiment, a tissue to be treated with the
modified kringle 5 peptides of the invention is an inflamed tissue and the
angiogenesis to be inhibited is inflamed tissue angiogenesis where there
is neovascularization of inflamed tissue. In this class the method
contemplates inhibition of angiogenesis in arthritic tissues, such as in a
patient with chronic articular rheumatism, in immune or non-immune
inflamed tissues, in psoriatic tissue and the like.
The patient treated in the present invention in its many
embodiments is desirably a human patient, although it is to be
understood that the principles of the invention indicate that the invention
is effective with respect to all mammals, which are intended to be
included in the term "patient." In this context, a mammal is understood
to include any mammalian species in which treatment of diseases
associated with angiogenesis is desirable, particularly agricultural and
domestic mammalian species.
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In another related embodiment, a tissue to be treated with the
modified kringle 5 peptides of the invention is a retinal tissue of a patient
with diabetic retinopathy, macular degeneration or neovascular
glaucoma and the angiogenesis to be inhibited is retinal tissue
angiogenesis where there is neovascularization of retinal tissue.
In an additional related embodiment, a tissue to be treated with
the modified kringle 5 peptides of the invention is a tumor tissue of a
patient with a solid tumor, a metastases, a skin cancer, a breast cancer,
a hemangioma or angiofibroma and the like cancer, and the
angiogenesis to be inhibited is tumor tissue angiogenesis where there is
neovascularization of a tumor tissue. Typical solid tumor tissues
treatable by the present methods include lung, pancreas, breast, colon,
laryngeal, ovarian, and the like tissues.
Inhibition of tumor tissue angiogenesis is a particularly preferred
embodiment because of the important role neovascularization plays in
tumor growth. In the absence of neovascularization of tumor tissue, the
tumor tissue does not obtain the required nutrients, slows in growth,
ceases additional growth, regresses and ultimately becomes necrotic
resulting in killing of the tumor.
The present invention thus provides for a method of inhibiting
tumor neovascularization by inhibiting tumor angiogenesis according to
the present methods using the modified kringle 5 peptides of the
invention. Similarly, the invention provides a method of inhibiting tumor
growth by practicing the angiogenesis-inhibiting methods. The methods
are also particularly effective against the formation of metastases
because (1) their formation requires vascularization of a primary tumor
so that the metastatic cancer cells can exit the primary tumor and (2)
their establishment in a secondary site requires neovascularization to
support growth of the metastases.
In a related embodiment, the invention contemplates the practice
of the method in conjunction with other therapies such as conventional
chemotherapy directed against solid tumors and for control of
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establishment of metastases. The administration of the modified kringle
peptides of the invention is typically conducted during or after
chemotherapy, although it is preferably to inhibit angiogenesis after a
regimen of chemotherapy at times where the tumor tissue will be
5 responding to the toxic assault by inducing angiogenesis to recover by
the provision of a blood supply and nutrients to the tumor tissue. In
addition, it is preferred to administer the modified kringle 5 peptides after
surgery where solid tumors have been removed as a prophylaxis
against metastases. Insofar as the present methods apply to inhibition
of tumor neovascularization, the methods can also apply to inhibition of
tumor tissue growth, to inhibition of tumor metastases formation, and to
regression of established tumors using the modified kringle 5 peptides of
the invention.
Restenosis is a process of smooth muscle cell (SMC) migration
and proliferation at the site of percutaneous transiuminal coronary
angioplasty which hampers the success of angioplasty. The migration
and proliferation of SMC's during restenosis can be considered a
process of angiogenesis which is inhibited by the modified kringle 5
peptides of the present invention. Therefore, the invention also
contemplates inhibition of restenosis by inhibiting angiogenesis in a
patient following angioplasty procedures. For inhibition of restenosis, the
modified kringle 5 peptide is typically administered after the angioplasty
procedure for from about 2 to about 28 days, and more typically for
about the first 14 days following the procedure.
The present method for inhibiting angiogenesis in a tissue
comprises contacting a tissue in which angiogenesis is occurring, or is
at risk for occurring, with a composition comprising a therapeutically
effective amount of a modified kringle 5 peptide. The dosage ranges for
the administration of the modified kringle 5 peptide depend upon the
form of the peptide, and its potency, as described further herein, and are
amounts large enough to produce the desired effect in which
angiogenesis and the disease symptoms mediated by angiogenesis are
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ameliorated. The dosage should not be so large as to cause adverse
side effects, such as hyperviscosity syndromes, pulmonary edema,
congestive heart failure, and the like. Generally, the dosage will vary
with the age, condition, sex and extent of the disease in the patient and
can be determined by one of skill in the art. The dosage can also be
adjusted by the individual physician in the event of any complication.
As angiogenesis inhibitors, such modified kringle 5 peptides are
useful in the treatment of both primary and metastatic solid tumors and
carcinomas of the breast; colon; rectum; lung; oropharynx; hypopharynx;
esophagus; stomach; pancreas; liver; gallbladder; bile ducts; small
intestine; urinary tract including kidney, bladder and urothelium; female
genital tract including cervix, uterus, ovaries, choriocarcinoma and
gestational trophoblastic disease; male genital tract including prostate,
seminal vesicles, testes and germ cell tumors; endocrine glands
including thyroid, adrenal, and pituitary; skin including hemangiomas,
melanomas, sarcomas arising from bone or soft tissues and Kaposi's
sarcoma; tumors of the brain, nerves, eyes, and meninges including
astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,
neuroblastomas, Schwannomas and meningiomas; solid tumors arising
from hematopoietic malignancies such as leukemias and including
chloromas, plasmacytomas, plaques and tumors of mycosis fungoides
and cutaneous T-cell lymphoma/leukemia; lymphomas including both
Hodgkin's and non-Hodgkin's lymphomas; prophylaxis of autoimmune
diseases including rheumatoid, immune and degenerative arthritis;
ocular diseases including diabetic retinopathy, retinopathy of
prematurity, corneal graft rejection, retrolental fibroplasia, neovascular
glaucoma, rubeosis, retinal neovascularization due to macular
degeneration and hypoxia; abnormal neovascularization conditions of
the eye; skin diseases including psoriasis; blood vessel diseases
including hemagiomas and capillary proliferation within atherosclerotic
plaques; Osler-Webber Syndrome; myocardial angiogenesis; plaque
neovascularization; telangiectasia; hemophiliac joints; angiofibroma;
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wound granulation; diseases characterized by excessive or abnormal
stimulation of endothelial cells including intestinal adhesions, Crohn's
disease, atherosclerosis, scieroderma and hypertrophic scars (i.e.
keloids) and diseases which have angiogenesis as a pathologic
consequence including cat scratch disease (Rochele minalia quintosa)
and ulcers (Helicobacter pylori). Another use is as a birth control agent
which inhibits ovulation and establishment of the placenta.
The modified kringle 5 peptides of the present invention may also
be useful for the prevention of metastases from the tumors described
above either when used alone or in combination with radiotherapy
and/or other chemotherapeutic treatments conventionally administered
to patients for treating angiogenic diseases. For example, when used in
the treatment of solid tumors, the modified kringle 5 peptides of the
present invention may be administered with chemotherapeutic agents
such as alpha inteferon, COMP (cyclophosphamide, vincristine,
methotrexate and prednisone), etoposide, mBACOD (methortrexate,
bleomycin, doxorubicin, cyclophosphamide, vincristine and
dexamethasone), PRO-MACE/MOPP (prednisone, methotrexate
(w/leucovin rescue), doxorubicin, cyclophosphamide, taxol,
etoposide/mechlorethamine, vincristine, prednisone and procarbazine),
vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate,
platelet factor 4, angiostatin, LM-609, SU-1 01, CM-101, Techgalan,
thalidomide, SP-PG and the like. Other chemotherapeutic agents
include alkylating agents such as nitrogen mustards including
mechloethamine, melphan, chlorambucil, cyclophosphamide and
ifosfamide; nitrosoureas including carmustine, lomustine, semustine and
streptozocin; alkyl sulfonates including busulfan; triazines including
dacarbazine; ethyenimines including thiotepa and hexamethylmelamine;
folic acid analogs including methotrexate; pyrimidine analogues
including 5-fluorouracil, cytosine arabinoside; purine analogs including
6-mercaptopurine and 6-thioguanine; antitumor antibiotics including
actinomycin D; the anthracyclines including doxorubicin, bleomycin,
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mitomycin C and methramycin; hormones and hormone antagonists
including tamoxifen and cortiosteroids and miscellaneous agents
including cisplatin and brequinar. For example, a tumor may be treated
conventionally with surgery, radiation or chemotherapy and kringle 5
administration with subsequent kringle 5 administration to extend the
dormancy of micrometastases and to stabilize and inhibit the growth of
any residual primary tumor.
5. Administration of the Modified Kringle 5 Peptides
The modified kringle 5 peptides will be administered in a
physiologically 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 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 subject modified kringle 5 peptides will for the most part be
administered orally, parenterally, such as intravascularly (IV),
intraarterially (IA), intramuscularly (IM), subcutaneously (SC), or the like.
Administration may in appropriate situations be by transfusion. In some
instances, where reaction of the functional group is relatively slow,
administration may be oral, nasal, rectal, transdermal or aerosol, where
the nature of the conjugate allows for transfer to the vascular system.
Usually a single injection will be employed although more than one
injection may be used, if desired. The modified kringle 5 peptides 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
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or continuous administration, or the like. Preferably, the administration
will be intravascularly, where the site of introduction is not critical to
this
invention, preferably at a site where there is rapid blood flow, e.g.,
intravenously, peripheral or central vein. Other routes may find use
where the administration is coupled with slow release techniques or a
protective matrix. The intent is that the kringle 5 peptide, analog or
derivative be effectively distributed in the blood, so as to be able to react
with the blood components. The concentration of the conjugate will vary
widely, generally ranging from about 1 pg/mi to 50 mg/mI. The total
administered intravascularly will generally be in the range of about 0.1
mg/mI to about 10 mg/mI, more usually about 1 mg/mI to about 5 mg/mI.
By bonding to long-lived components of the blood, such as
immunoglobulin, serum albumin, red blood cells and platelets, a number
of advantages ensue. The activity of the modified kringle 5 peptide
compound is extended for days to weeks. Only one administration need
be given during this period of time. Greater specificity can be achieved,
since the active compound will be primarily bound to large molecules,
where it is less likely to be taken up intracellularly to interfere with other
physiological processes.
The formation of the covalent bond between the blood
component may occur in vivo or ex vivo. For ex vivo covalent bond
formation the modified kringle 5 peptide is added to blood, serum or
saline solution containing human serum albumin or IgG to permit
covalent bond formation between the modified kringle 5 peptide and the
blood component. In a preferred format, the kringle 5 peptide is
modified with maleimide and it is reacted with human serum albumin in
saline solution. Once the modified kringle 5 peptide has reacted with
the blood component, to form a kringle 5 peptide-protein conjugate, the
conjugate may be administered to the patient.
Alternatively, the modified kringle 5 peptide may be administered
to the patient directly so that the covalent bond forms between the
modified kringle 5 peptide and the blood component in vivo.
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6. Monitoring the Presence of Modified Kringle 5 Peptide
The blood of the mammalian host may be monitored for the
presence of the modified kringle 5 peptide compounds one or more
times. By taking a portion or sample of the blood of the host, one may
determine whether the kringle 5 peptide has become bound to the long-
lived blood components in sufficient amount to be therapeutically active
and, thereafter, the level of kringle 5 peptide compound in the blood. If
desired, one may also determine to which of the blood components the
kringle 5 peptide derivative molecule is bound. This is particularly
important when using non-specific kringle 5 peptides. For specific
maleimide-kringle 5 peptides, it is much simpler to calculate the half life
of serum albumin and IgG.
The modified kringle 5 peptides may be monitored using HPLC-
MS or antibodies directed to kringle 5 peptides.
A. HPLC-MS
HPLC coupled with mass spectrometry (MS) can be utilized to
assay for the presence of peptides and modified peptides as is well
known to the skilled artisan. Typically two mobile phases are utilized:
0.1% TFA/water and 0.1 % TFA/acetonitrile. Column temperatures can
be varied as well as gradient conditions. Particular details are outlined
in the Examples section below.
B. Antibodies
Another aspect of this invention relates to methods for
determining the concentration of the kringle 5 peptides and/or analogs,
or their derivatives and conjugates in biological samples (such as blood)
using antibodies specific to the kringle 5 peptides or peptide analogs or
their derivatives and conjugates, and to the use of such antibodies as a
treatment for toxicity potentially associated with such kringle 5 peptides
and/or their derivatives or conjugates. This is advantageous because
the increased stability and life of the kringle 5 peptides in vivo in the
patient might lead to novel problems during treatment, including
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increased possibility for toxicity. The use of anti-therapeutic agent
antibodies, either monoclonal or polyclonal, having specificity for a
particular kringle 5 peptides, can assist in mediating any such problem.
The antibody may be generated or derived from a host immunized with
the particular modified kringle 5 peptide, or with an immunogenic
fragment of the agent, or a synthesized immunogen corresponding to an
antigenic determinant of the agent. Preferred antibodies will have high
specificity and affinity for native, derivatized and conjugated forms of the
modified kringle 5 peptide. Such antibodies can also be labeled with
enzymes, fluorochromes, or radiolables.
Antibodies specific for modified kringle 5 peptides may be
produced by using purified kringle 5 peptides for the induction of
derivatized kringle 5 peptide-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 be used to monitore the presence of kringle 5
petides in the blood stream. Blood and/or surum samples may be
analyzed by SDS-PAGE and western blotting. Such techniques permit
the analysis of the blood or serum to determine the bonding of the
modified kringle 5 peptides to blood components.
The anti-therapeutic agent antibodies may also be used to treat
toxicity induced by administration of the modified kringle 5 peptide, and
may be used ex vivo or in vivo. Ex vivo methods would include immuno-
dialysis treatment for toxicity employing anti-therapeutic agent
antibodies fixed to solid supports. In vivo methods include
administration of anti-therapeutic agent antibodies in amounts effective
to induce clearance of antibody-agent complexes.
The antibodies may be used to remove the modified kringle 5
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peptides 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 modified kringle 5 peptides will bind to
the antibodies and the blood containing a low concentration of the
kringle 5 peptide, then may be returned to the patient's circulatory
system. The amount of modified kringle 5 peptide removed can be
controlled by adjusting the pressure and flow rate. Preferential removal
of the modified kringle 5 peptides from the plasma component of a
patient's blood can be effected, for example, by the use of a
semipermeable membrane, or by otherwise first separating the plasma
component from the cellular component by ways known in the art prior
to passing the plasma component over a matrix containing the anti-
therapeutic antibodies. Alternatively the preferential removal of kringle 5
peptide-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-therapeutic antibodies to
the exclusion of the serum component of the patient's blood.
The anti-therapeutic antibodies can be administered in vivo,
parenterally, to a patient that has received the modified kringle 5 peptide
or conjugates for treatment. The antibodies will bind the kringle 5
peptide compounds and conjugates. Once bound the kringle 5 peptide
activity will be hindered if not completely blocked thereby reducing the
biologically effective concentration of kringle 5 peptide compound in the
patient's bloodstream and minimizing harmful side effects. In addition,
the bound antibody-kringle 5 peptide complex will facilitate clearance of
the kringle 5 peptide compounds and conjugates from the patient's
blood stream.
The invention having been fully described is now exemplified by
the following non-limiting examples.
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EXAMPLES
General
Solid phase peptide synthesis of the Kringle-5 analogs on a 100
pmole scale was performed using manual solid-phase synthesis and a
Symphony Peptide Synthesizer using Fmoc protected Rink Amide
MBHA resin, Fmoc protected amino acids, O-benzotriazol-1 -y1-N, N, M,
M-tetramethyl-uronium hexafluorophosphate (HBTU) in N,1V
dimethylformamide (DMF) solution and activation with N-methyl
morpholine (NMM), and piperidine deprotection of Fmoc groups (Step
1). When required, the selective deprotection of the Lys(Aloc) 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 CHCI3:NMM:HOAc
(18:1:0.5) for 2 h (Step 2). The resin was then washed with CHC13 (6 x 5
mL), 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5
mL). In some instances, the synthesis was then re-automated for the
addition of one AEEA (aminoethoxyethoxyacetic acid) group, the
addition of acetic acid or the addition of a 3-maleimidopropionic acid
(MPA) (Step 3). Resin cleavage and product isolation was performed
using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by
precipitation by dry-ice cold Etz0 (Step 4). The products were purified
*
by preparative reverse phase HPLC using a Varian (Rainin) preparative
binary HPLC system:. gradient elution of 30-55% B (0.045% TFA in H20
(A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5 mUmin using a
*
Phenomenex Luna 10 p phenyl-hexyl, 21 mm x 25 cm column and UV
detector (Varian Dynamax*UVD ll) at ;L 214 and 254 nm. Purity was
determined 95% by RP-HPLC mass spectrometry using a Hewlett
Packard LCMS-1 100 series spectrometer equipped with a diode array
detector and using electro-spray ionization.
''Trade-Mark
1
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Example I
Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Lys-NH2.3TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-
Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH.
Deblocking of the Fmoc group the the N-terminal of the resin-bound
amino acid was performed with 20% piperidine in DMF for about 15-20
minutes. Coupling of the acetic acid was performed under conditions
similar to amino acid coupling. Final cleavage from the resin was
performed using cleavage mixture as described above. The product
was isolated by precipitation and purified by preparative HPLC to afford
the desired product as a white solid upon lyophilization
Example 2
Preparation of NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH2.3TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH.
Deblocking of the Fmoc group the the N-terminal of the resin-bound
amino acid was performed with 20% piperidine in DMF for about 15-20
minutes. Coupling of the acetic acid was performed under conditions
similar to amino acid coupling. Final cleavage from the resin was
performed using cleavage mixture as described above. The product
was isolated by precipitation and purified by preparative HPLC to afford
the desired product as a white solid upon lyophilization.
Example 3
Preparation of Nac-Ty r-Th r-Th r-As n -P ro-A rg -Lys -Leu -Ty r-As p-Tyr-
Lys-NH2.3TFA
Using automated peptide synthesis, the following protected
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amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-
Lys(Boc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-
OH, Fmoc-Tyr(tBu)OH. Deblocking of the Fmoc group the the N-
terminal of the resin-bound amino acid was performed with 20%
piperidine in DMF for about 15-20 minutes. Coupling of the acetic acid
was performed under conditions similar to amino acid coupling. Final
cleavage from the resin was performed using cleavage mixture as
described above. The product was isolated by precipitation and purified
by preparative HPLC to afford the desired product as a white solid upon
lyophilization.
Example 4
Preparation of NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-
A I a-Ty r-Th r-Th r-As n-P ro-A rg -Lys-Le u-Ty r-As p-Ty r-Lys-N H 2.4TFA
Using automated peptide synthesis, the following protected -
amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-
Lys(Boc)-OH, Fmoc-Tyr(tBu)OH. Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-
OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH,
Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH,
Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Arg(Pbf)-OH. Deblocking of the Fmoc group the the N-terminal
of the resin-bound amino acid was performed with 20% piperidine in
DMF for about 15-20 minutes. Coupling of the acetic acid was
performed under conditions similar to amino acid coupling. Final
cleavage from the resin was performed using cleavage mixture as
described above. The product was isolated by precipitation and purified
by preparative HPLC to afford the desired product as a white solid upon
lyophilization.
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Example 5
Preparation of NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-
Lys-NH2.2TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-
Lys(Boc)-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-
OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-
Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH.
Deblocking of the Fmoc group the the N-terminal of the resin-bound
amino acid was performed with 20% piperidine in DMF for about 15-20
minutes. Coupling of the acetic acid was performed under conditions
similar to amino acid coupling. Final cleavage from the resin was
performed using cleavage mixture as described above. The product
was isolated by precipitation and purified by preparative HPLC to afford
the desired product as a white solid upon lyophilization
Example 6
Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-(NE-MPA)-
NH2.2TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH. Deblocking of the Fmoc group the the N-terminal of the
resin-bound amino acid was performed with 20% piperidine in DMF for
about 15-20 minutes. Coupling of the acetic acid was performed under
conditions similar to amino acid coupling. Final cleavage from the resin
was performed using cleavage mixture as described above. The
product was isolated by precipitation and purified by preparative HPLC
to afford the desired product as a white solid upon lyophilization.
The selective deprotection of the Lys(Aloc) group was performed
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manually and accomplished by treating the resin with a solution of 3 eq
of Pd(PPh3)4 dissolved in 5 mL of CHCI3:NMM:HOAc (18:1:0.5) for 2 h
(Step 2). The resin was then washed with CHCI3 (6 x 5 mL), 20% HOAc
in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The
synthesis was then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Resin cleavage and product isolation
was performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et20 (Step 4). The product was
purified by preparative reversed phase HPLC using a Varian (Rainin)
preparative binary HPLC system: gradient elution of 30-55% B (0.045%
TFA in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5
mUmin using a Phenomenex Luna 10 p phenyl-hexyl, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at X214 and 254 nm.
Example 7
Preparation of (MPA-AEEA)-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NHZ.2TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1).
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH. The deprotection of the terminal Fmoc group is
accomplished using 20% piperidine (Step 2) followed by the coupling of
Fmoc-AEEA. Deprotection of the resulting Fmoc-AEEA-peptide with
piperidine 20% in DMF allow for the subsequent addition of the 3-MPA
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(Step 3). Resin cleavage and product isolation was performed using
86% TFA/5% TIS/5% H20/2% thioanisole and 2% phenol, followed by
precipitation by dry-ice cold Et20 (Step 4). The product was purified by
preparative reverse phase HPLC using a Varian (Rainin) preparative
binary HPLC system using a Dynamax C18, 60A, 8 pm, 21 mm x 25 cm
column equipped with a Dynamax C,a, 60A, 8 pm guard module, 21 mm
x 25 cm column and UV detector (Varian Dynamax UVD II) at k 214 and
254 nm. The product had >95% purity as determined by RP-HPLC
mass spectrometry using a Hewlett Packard LCMS-1 100 series
spectrometer equipped with a diode array detector and using electro-
spray ionization.
Example 8
Preparation of (MPA)-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH2.2TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1). Using automated peptide
synthesis, the following protected amino acids were sequentially added
to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH. The deprotection of
the terminal Fmoc group is accomplished using 20% piperidine (Step 2)
foilowed by the coupling of 3-MPA (Step 3). Resin cleavage and
product isolation was performed using 86% TFA/5% TIS/5% H20/2%
thioanisole and 2% phenol, followed by precipitation by dry-ice cold Et20
(Step 4). The product was purified by preparative reverse phase HPLC
using a Varian (Rainin) preparative binary HPLC system using a
Dynamax C18, 60A, 8 pm, 21 mm x 25 cm column equipped with a
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Dynamax C,a, 60A, 8 pm guard module, 21 mm x 25 cm column and UV
detector (Varian Dynamax UVD II) at k 214 and 254 nm. The product
had >95% purity as determined by RP-HPLC mass spectrometry using a
Hewlett Packard LCMS-1 100 series spectrometer equipped with a diode
array detector and using electro-spray ionization.
Example 9
Preparation of NAc-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-
Lys-( NE-MPA)-NH2.2TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-
Lys(Aloc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-
OH, Fmoc-Tyr(tBu)OH. Deblocking of the Fmoc group the the N-
terminal of the resin-bound amino acid was performed with 20%
piperidine in DMF for about 15-20 minutes. Final cleavage from the resin
was performed using cleavage mixture as described above. The
product was isolated by precipitation and purified by preparative HPLC
to afford the desired product as a white solid upon lyophilization
The selective deprotection of the Lys(Aloc) 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 CHCI3:NMM:HOAc (18:1:0.5) for 2 h
(Step 2). The resin was then washed with CHCI3 (6 x 5 mL), 20% HOAc
in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The
synthesis was then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Resin cleavage and product isolation
was performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et20 (Step 4). The product was
purified by preparative reverse phase HPLC using a Varian (Rainin)
preparative binary HPLC system: gradient elution of 30-55% B (0.045%
TFA in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5
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mUmin using a Phenomenex Luna 10 p phenyl-hexyl, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at X214 and 254 nm.
Example 10
Preparation of (MPA-AEEA)-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-
Asp-Tyr-NHZ.2TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yi-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1).
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-
Lys(Boc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-
OH, Fmoc-Tyr(tBu)OH. The deprotection of the terminal Fmoc group is
accomplished using 20% piperidine (Step 2) followed by the coupling of
Fmoc-AEEA. Deprotection of the resulting Fmoc-AEEA-peptide with
piperidine 20% in DMF allow for the subsequent addition of the 3-MPA
(Step 3). Resin cleavage and product isolation was performed using
86% TFA/5% TIS/5% H20/2% thioanisole and 2% phenol, followed by
precipitation by dry-ice cold EtzO (Step 4). The product was purified by
preparative reverse phase HPLC using a Varian (Rainin) preparative
binary HPLC system using a Dynamax C18, 60A, 8 pm, 21 mm x 25 cm
column equipped with a Dynamax C18, 60A, 8 pm guard module, 21 mm
x 25 cm column and UV detector (Varian Dynamax UVD II) at k 214 and
254 nm. The product had >95% purity as determined by RP-HPLC
mass spectrometry using a Hewlett Packard LCMS-1 100 series
spectrometer equipped with a diode array detector and using electro-
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spray ionization.
Example 11
Preparation of (MPA)-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-
Tyr-NH2.2TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 Nmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1 Using automated peptide
synthesis, the following protected amino acids were sequentially added
to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH. The
deprotection of the terminal Fmoc group is accomplished using 20%
piperidine (Step 2) followed by ttie coupling of 3-MPA (Step 3). Resin
cleavage and product isolation was performed using 86% TFA/5%
TIS/5% H20/2% thioanisole and 2% phenol, followed by precipitation by
dry-ice cold Et20 (Step 4). The product was purified by preparative
reverse phase HPLC using a Varian (Rainin) preparative binary HPLC
system using a Dynamax C,a, 60A, 8 pm, 21 mm x 25 cm column
equipped with a Dynamax C1B, 60A, 8 pm guard module, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at k 214 and 254 nm.
The product had >95% purity as determined by RP-HPLC mass
spectrometry using a Hewlett Packard LCMS-1 100 series spectrometer
equipped with a diode array detector and using electro-spray ionization.
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Example 12
Preparation of NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-
Ala-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-( Ns-MPA)-
NH2.3TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-
Lys(Aloc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-
OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH,
Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Arg(Pbf)-OH. Deblocking of the Fmoc group the the N-terminal
of the resin-bound amino acid was performed with 20% piperidine in
DMF for about 15-20 minutes. Coupling of the acetic acid was
performed under conditions similar to amino acid coupling. Final
cleavage from the resin was performed using cleavage mixture as
described above. The product was isolated by precipitation and purified
by preparative HPLC to afford the desired product as a white solid upon
lyophilization.
The selective deprotection of the Lys(Aloc) 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 CHCI3:NMM:HOAc (18:1:0.5) for 2 h
(Step 2). The resin was then washed with CHC13 (6 x 5 mL), 20% HOAc
in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The
synthesis was then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Resin cleavage and product isolation
was performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et20 (Step 4). The product was
purified by preparative reverse phase HPLC using a Varian (Rainin)
preparative binary HPLC system: gradient elution of 30-55% B (0.045%
TFA in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5
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mUmin using a Phenomenex Luna 10 p phenyl-hexyl, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at X 214 and 254 nm.
Example 13
Preparation of (MPA-AEEA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-
P ro-T rp-A l a-Ty r-Th r-T h r-As n-P ro-A rg -Lys-Le u-Ty r-As p-Ty r-
NHZ.3TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1).
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-
Lys(Boc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-
OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH,
Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Arg(Pbf)-OH. The deprotection of the terminal Fmoc group is
accomplished using 20% piperidine (Step 2) followed by the coupling of
Fmoc-AEEA. Deprotection of the resulting Fmoc-AEEA-peptide with
piperidine 20% in DMF allow for the subsequent addition of the 3-MPA
(Step 3). Resin cleavage and product isolation was performed using
86% TFA/5% TIS/5% H20/2% thioanisole and 2% phenol, followed by
precipitation by dry-ice cold Et2O (Step 4). The product was purified by
preparative reverse phase HPLC using a Varian (Rainin) preparative
binary HPLC system using a Dynamax C1e, 60A, 8 pm, 21 mm x 25 cm
column equipped with a Dynamax C18, 60A, 8 pm guard module, 21 mm
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x 25 cm column and UV detector (Varian Dynamax UVD II) at k 214 and
254 nm. The product had >95% purity as determined by RP-HPLC
mass spectrometry using a Hewlett Packard LCMS-1 100 series
spectrometer equipped with a diode array detector and using electro-
spray ionization.
Example 14
Preparation of (MPA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-
Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH2.3TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1). Using automated peptide
synthesis, the following protected amino acids were sequentially added
to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH Fmoc-Lys(Boc)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-
Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH,
Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-
Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH.
The deprotection of the terminal Fmoc group is accomplished
using 20% piperidine (Step 2) followed by the coupling of 3-MPA (Step
3). Resin cleavage and product isolation was performed using 86%
TFA/5% TIS/5% H2O/2% thioanisole and 2% phenol, followed by
precipitation by dry-ice cold EtzO (Step 4). The product was purified by
preparative reverse phase HPLC using a Varian (Rainin) preparative
binary HPLC system using a Dynamax C18, 60A, 8 pm, 21 mm x 25 cm
column equipped with a Dynamax C18, 60A, 8 pm guard module, 21 mm
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x 25 cm column and UV detector (Varian Dynamax UVD II) at k 214 and
254 nm. The product had >95% purity as determined by RP-HPLC mass
spectrometry using a Hewlett Packard LCMS-1 100 series spectrometer
equipped with a diode array detector and using electro-spray ionization.
Example 15
Preparation of NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-
Lys-( NE-MPA)-NH2.TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 Nmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1).
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-
Lys(Aloc)-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-
OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-
Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH.
The selective deprotection of the Lys(Aloc) 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 CHCI3:NMM:HOAc (18:1:0.5) for 2 h
(Step 2). The resin was then washed with CHCI3 (6 x 5 mL), 20% HOAc
in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The
synthesis was then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Resin cleavage and product isolation
was performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et2O (Step 4). The product was
purified by preparative reverse phase HPLC using a Varian (Rainin)
preparative binary HPLC system: gradient elution of 30-55% B (0.045%
TFA in HZO (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5
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mL/min using a Phenomenex Luna 10 p phenyl-hexyl, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at X214 and 254 nm.
Example 16
Preparation of (MPA-AEEA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-
Pro-Trp-NH2.TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1).
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-
Lys(Boc)-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-
OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-
Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH.
The deprotection of the terminal Fmoc group is accomplished using 20%
piperidine (Step 2) followed by the coupling of Fmoc-AEEA.
Deprotection of the resulting Fmoc-AEEA-peptide with piperidine 20% in
DMF allow for the subsequent addition of the 3-MPA (Step 3). Resin
cleavage and product isolation was performed using 86% TFA/5%
TIS/5% H20/2% thioanisole and 2% phenol, followed by precipitation by
dry-ice cold Et20 (Step 4). The product was purified by preparative
reverse phase HPLC using a Varian (Rainin) preparative binary HPLC
system using a Dynamax C18, 60A, 8 pm, 21 mm x 25 cm column
equipped with a Dynamax C18, 60A, 8 pm guard module, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at k 214 and 254 nm.
The product had >95% purity as determined by RP-HPLC mass
spectrometry using a Hewlett Packard LCMS-1 100 series spectrometer
equipped with a diode array detector and using electro-spray ionization.
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Example 17
Preparation of (MPA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-
Trp-NHZ.TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1). Using automated peptide
synthesis, the following protected amino acids were sequentially added
to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Trp-OH, Fmoc-
Pro-OH, Frnoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-
OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-
OH, Fmoc-Arg(Pbf)-OH. The deprotection of the terminal Fmoc group is
accomplished using 20% piperidine (Step 2) followed by the coupling of
3-MPA (Step 3). Resin cleavage and product isolation was performed
using 86% TFA/5% TIS/5% H20/2% thioanisole and 2% phenol,
followed by precipitation by dry-ice cold Et2O (Step 4). The product was
purified by preparative reverse phase HPLC using a Varian (Rainin)
preparative binary HPLC system using a Dynamax C1B, 60A, 8 pm, 21
mm x 25 cm column equipped with a Dynamax C18, 60A, 8 pm guard
module, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD
II) at ). 214 and 254 nm. The product had >95% purity as determined by
RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1 100
series spectrometer equipped with a diode array detector and using
electro-spray ionization.
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Example 18
Preparation of NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-( Ns-MPA)-
NH2.2TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH.
Deblocking of the Fmoc group the the N-terminal of the resin-bound
amino acid was performed with 20% piperidine in DMF for about 15-20
minutes. Coupling of the acetic acid was performed under conditions
similar to amino acid coupling. Final cleavage from the resin was
performed using cleavage mixture as described above. The product
was isolated by precipitation and purified by preparative HPLC to afford
the desired product as a white solid upon lyophilization.
The selective deprotection of the Lys(Aloc) 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 CHCI3:NMM:HOAc (18:1:0.5) for 2 h
(Step 2). The resin was then washed with CHCI3 (6 x 5 mL), 20% HOAc
in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The
synthesis was then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Resin cleavage and product isolation
was performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et20 (Step 4). The product was
purified by preparative reverse phase HPLC using a Varian (Rainin)
preparative binary HPLC system: gradient elution of 30-55% B (0.045%
TFA in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5
mUmin using a Phenomenex Luna 10 p phenyl-hexyl, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at a, 214 and 254 nm.
Example 19
Preparation of (MPA-AEEA)-Arg-Lys-Leu-Tyr-Asp-Tyr-NH2.2TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
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on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, N'-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1).
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH.
The deprotection of the terminal Fmoc group is accomplished using 20%
piperidine (Step 2) followed by the coupling of Fmoc-AEEA.
Deprotection of the resulting Fmoc-AEEA-peptide with piperidine 20% in
DMF allow for the subsequent addition of the 3-MPA (Step 3). Resin
cleavage and product isolation was performed using 86% TFA/5%
TIS/5% H20/2% thioanisole and 2% phenol, followed by precipitation by
dry-ice cold Et20 (Step 4). The product was purified by preparative
reverse phase HPLC using a Varian (Rainin) preparative binary HPLC
system using a Dynamax C,a, 60A, 8 pm, 21 mm x 25 cm column
equipped with a Dynamax C1e, 60A, 8 pm guard module, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at X 214 and 254 nm.
The product had >95% purity as determined by RP-HPLC mass
spectrometry using a Hewlett Packard LCMS-1 100 series spectrometer
equipped with a diode array detector and using electro-spray ionization
Example 20
Preparation of (MPA)-Arg-Lys-Leu-Tyr-Asp-Tyr-NH2.2TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, N', M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
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solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1). Using automated peptide
synthesis, the following protected amino acids were sequentially added
to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-
Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH. The deprotection of the terminal
Fmoc group is accomplished using 20% piperidine (Step 2) followed by
the coupling of 3-MPA (Step 3). Resin cleavage and product isolation
was performed using 86% TFA/5% TIS/5% H20/2% thioanisole and 2%
phenol, followed by precipitation by dry-ice cold Et20 (Step 4). The
product was purified by preparative reverse phase HPLC using a Varian
(Rainin) preparative binary HPLC system using a Dynamax C18, 60A, 8
pm, 21 mm x 25 cm column equipped with a Dynamax C,a, 60A, 8 pm
guard module, 21 mm x 25 cm column and UV detector (Varian
Dynamax UVD II) at k 214 and 254 nm. The product had >95% purity
as determined by RP-HPLC mass spectrometry using a Hewlett Packard
LCMS-1 100 series spectrometer equipped with a diode array detector
and using electro-spray ionization.
Example 21
Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Lys-(Ns-MPA)-
NHZ.2TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-
Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH.
Deblocking of the Fmoc group the the N-terminal of the resin-bound
amino acid was performed with 20% piperidine in DMF for about 15-20
minutes. Coupling of the acetic acid was performed under conditions
similar to amino acid coupling. Final cleavage from the resin was
performed using cleavage mixture as described above. The product
was isolated by precipitation and purified by preparative HPLC to afford
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the desired product as a white solid upon lyophilization.
The selective deprotection of the Lys(Aloc) 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 CHCI3:NMM:HOAc (18:1:0.5) for 2 h
(Step 2). The resin was then washed with CHCI3 (6 x 5 mL), 20% HOAc
in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The
synthesis was then re-automated for the addition of the 3-
maleimidopropionic acid (Step 3). Resin cleavage and product isolation
was performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et20 (Step 4). The product was
purified by preparative reverse phase HPLC using a Varian (Rainin)
preparative binary HPLC system: gradient elution of 30-55% B (0.045%
TFA in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5
mUmin using a Phenomenex Luna 10 p phenyl-hexyl, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at X 214 and 254 nm.
Example 22
Preparation of (MPA-AEEA)-Pro-Arg-Lys-Leu-Tyr-Asp-NH2.2TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, M, M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1).
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-
Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (Step
1). The deprotection of the terminal Fmoc group is accomplished using
20% piperidine (Step 2) followed by the coupling of Fmoc-AEEA.
Deprotection of the resulting Fmoc-AEEA-peptide with piperidine 20% in
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DMF allow for the subsequent addition of the 3-MPA (Step 3). Resin
cleavage and product isolation was performed using 86% TFA/5%
TIS/5% H20/2% thioanisole and 2% phenol, followed by precipitation by
dry-ice cold Et20 (Step 4). The product was purified by preparative
reverse phase HPLC using a Varian (Rainin) preparative binary HPLC
system using a Dynamax C1e, 60A, 8 pm, 21 mm x 25 cm column
equipped with a Dynamax C18, 60A, 8 pm guard module, 21 mm x 25 cm
column an+d UV detector (Varian Dynamax UVD II) at a, 214 and 254
nm. The product had >95% purity as determined by RP-HPLC mass
spectrometry using a Hewlett Packard LCMS-1 100 series spectrometer
equipped with a diode array detector and using electro-spray ionization.
Example 23
Preparation of (MPA)-Pro-Arg-Lys-Leu-Tyr-Asp-NH2.2TFA
Solid phase peptide synthesis of the modified Kringle 5 peptide
on a 100 pmole scale was performed on a Symphony Peptide
Synthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc
protected amino acids, O-benzotriazol-1-yl-N, N, N', M-tetramethyl-
uronium hexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF)
solution and activation with N-methyl morpholine (NMM), and piperidine
deprotection of Fmoc groups (Step 1). Using automated peptide
synthesis, the following protected amino acids were sequentially added
to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-
OH, Fmoc-Pro-OH. The deprotection of the terminal Fmoc group is
accomplished using 20% piperidine (Step 2) followed by the coupling of
3-MPA (Step 3). Resin cleavage and product isolation was performed
using 86% TFA/5% TIS/5% H20/2% thioanisole and 2% phenol,
followed by precipitation by dry-ice cold Et20 (Step 4). The product was
purified by preparative reverse phase HPLC using a Varian (Rainin)
preparative binary HPLC system using a Dynamax C1B, 60A, 8 pm, 21
mm x 25 cm column equipped with a Dynamax C18, 60A, 8 pm guard
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module, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD
II) at k 214 and 254 nm. The product had >95% purity as determined by
RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1 100
series spectrometer equipped with a diode array detector and using
electro-spray ionization.
Example 24
Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-(Ns--AEEA-
MPA)-NH2.2TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH (Step 1). Deblocking of the Fmoc group at the N-
terminal of the resin-bound amino acid was performed with 20%
piperidine in DMF for about 15-20 minutes. Coupling of the acetic acid
was performed under conditions similar to amino acid coupling.
The selective deprotection of the Lys(Aloc) 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 CHCI3:NMM:HOAc (18:1:0.5) for 2 h
(Step 2). The resin was then washed with CHCI3 (6 x 5 mL), 20% HOAc
in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The
synthesis was then re-automated for the addition of the AEEA
(aminoethoxyethoxyacetic acid) group and of the 3-maleimidopropionic
acid (MPA) (Step 3). Resin cleavage and product isolation was
performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol,
followed by precipitation by dry-ice cold Et20 (Step 4). The product was
purified by preparative reverse phase HPLC using a Varian (Rainin)
preparative binary HPLC system: gradient elution of 30-55% B (0.045%
TFA in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5
mUmin using a Phenomenex Luna 10 p phenyl-hexyl, 21 mm x 25 cm
column and UV detector (Varian Dynamax UVD II) at X 214 and 254 nm.
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Example 25
Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-( NE-AEEAõ
MPA)-NH2.2TFA
Using automated peptide synthesis, the following protected
amino acids were sequentially added to Rink Amide MBHA resin:
Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Pro-OH (Step 1). Deblocking of the Fmoc group at the N-
terminal of the resin-bound amino acid was performed with 20%
piperidine in DMF for about 15-20 minutes. Coupling of the acetic acid
was performed under conditions similar to amino acid coupling.
The selective deprotection of the Lys(Aloc) 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 CHCI3:NMM:HOAc (18:1:0.5) for 2 h
(Step 2). The resin was then washed with CHCI3 (6 x 5 mL), 20% HOAc
in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The
synthesis was then re-automated for the addition The synthesis was
then re-automated for the addition of n AEEA (aminoethoxyethoxyacetic
acid) groups and of the 3-maleimidopropionic acid (MPA) (Step 3).
Resin cleavage and product isolation was performed using 85% TFA/5%
TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice
cold Et20 (Step 4). The product was purified by preparative reverse
phase HPLC using a Varian (Rainin) preparative binary HPLC system:
gradient elution of 30-55% B (0.045% TFA in H20 (A) and 0.045% TFA
in CH3CN (B)) over 180 min at 9.5 mUmin using a Phenomenex Luna
10 p phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian
Dynamax UVD II) at a, 214 and 254 nm.
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Example 26
Peptide Stability Assay
A peptide stability assay was performed. (MPA)-Pro-Arg-Lys-
Leu-Tyr-Asp-Lys-NH2. 2TFA was synthesized as described above and
was identified MPA-K5. The non-modified counterpart peptide Pro-Arg-
Lys-Leu-Tyr-Asp-Lys-NH2 was also synthesized as described above
without the addition of 3-MPA and identified as K5.
K5 (MW = 1260.18, 918.12 freebase) was prepared as a 100 mM
stock solution in water. MPA-K5 (MW = 1411.17, 1069.11 freebase)
was prepared as a 100 mM stock solution in water. Human Serum
Albumin (HSA) was obtained as a 25% solution (ca 250 mg/mI, 3.75
mM) as Albutein available from Alpha Therapeutic. Human plasma
was obtained from Golden West Biologicals.
a. Stability of K5 in human plasma
K5 was prepared as a INM solution and dissolved in 25%
human serum albumin. The mixture was then incubated at 37 C in the
presence of human plasma to final concentration of 160 mM K5.
Aliquots of 100 NI were withdrawn from the plasma at 0, 4 hours and 24
hours. The 100 NI aliquots were mixed with 100 NI of blocking solution
(5 vol. 5%ZnSO4/3 vol. Acetonitrile/2 vol. Methanol) in order to
precipitate ali proteins. The sampie was centrifuged for 5 min at 10,000
g and the supernatant containing the peptide was recovered and filtered
through a 0.22 pm filter. The presence of free intact K5 peptide was
assayed by the HPLC/MS. The HPLC parameters for detection of K5
peptide in serum were as follows.
The HPLC method was as follows: A Vydac C18 250 X 4.6 mm,
5 p particle size column was utilized. The column temperature was
C with a flow rate of 0.5 mUmin. Mobile Phase A was 0.1%
30 TFA/water. Mobile Phase B was 0.1% TFA/acetonitrite. The injection
volume was 10N1.
The gradient was as follows:
*Trade-Mark
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Time(Minutes) %A %B
0 95 5
20 70 30
25 10 90
30 10 90
35 95 5
45 95 5
The proteins were detected at 214, 254 and 334 nm. For mass
spectral analysis, the ionization mode was API-electrospray (positive
mode) at an M/Z range of 300 to 2000. The gain was 3.0, fragmentor
120v, threshold 20, stepsize 0.1. The gas temp was 350 C and the
drying gas volume was 10.0 I/min. The Neb pressure was 24 psi and
the Vcap was 3500V. The HPLC method was as follows: A Vydac
C18 250 X 4.6 mm, 5 p particle size column was utilized. The column
temperature was 30 C with a flow rate of 0.5 mI/min. Mobile Phase A
was 0.1% TFA/water. Mobile Phase B was 0.1% TFA/acetonitrite. The
injection volume was 10pI.
The gradient was as follows:
Time(Minutes) %A %B
0 95 5
20 70 30
25 10 90
10 90
95 5
95 5
30 The proteins were detected at 214, 254 and 334 nm. For mass
spectral analysis, the ionization mode was API-electrospray (positive
mode) at an M/Z range of 300 to 2000. The gain was 3.0, fragmentor
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120v, threshold 20, stepsize 0.1. The gas temp was 350 C and the
drying gas volume was 10.0 I/min. The Neb pressure was 24 psi and
the Vcap was 3500V.
Time %K5 peptide in plasma
0 hrs. 100%
4hrs 9%
24 hrs 0%
After only 4 hours incubation in plasma only 9% of the
original K5 peptide remained. The results demonstrate that unmodified
K5 peptide is unstable in serum likely as a result of protease activity.
b. Stability of MPA-K5-HSA Conjugate in Plasma
MPA-K5 (modified K5 peptide) was incubated with 25% HSA for
2 hours at room temperature. The MPA-K5-HSA conjugate was then
incubated at 37 in the presence of human plasma at a final
concentration of 160 pm. After the specific incubation period (0, 4 and
24 hours) an aliquot of 100 NI was withdrawn and filtered through a 0.22
pm filter. The presence of intact conjugate was assayed by HPLC-MS.
The column was an Aquapore RP-300, 250 x 4.6 mm, 7p particle
size. The column temperature was 50 C. The mobile phase A was
0.1 % TFA/water. The mobile phase B was 0.1 % TFA/acetonitrile. The
injection volume was 1 NI. The gradient was as follows:
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Time (minutes) %A %B Flow(mI/min)
0 66 34 0.700
1 66 34 0.700
25 58.8 41.2 0.700
30 50 50 0.70
35 5 95 1.00
41 5 95 1.00
45 66 34 1.00
46 66 34 0.70
The peptide was detected at 214 mm for quantification. For mass
spectral analysis of the peptide, the ionization mode was API-
electrospray at 1280 to 1500 m/z range, gain 1.0, fragmentor 125V,
threshold 100, stepsize 0.40. The gas temperature was 350 C the
drying gas was 13.0 I/min. The pressure was 60psi and the Vcap was
6000V. The results are presented below.
Approximately 33% of circulating albumin in the bloodstream is
mercaptalbumin (SH-albumin) which is not blocked by endogenous
sulfhydryl compounds such as cysteine or glutathione and is therefore
available for reaction with maleimido groups. The remaining 66% of the
circulating albumin is capped or blocked by sulfhydryl compounds. The
HPLC MS assay permits the identification of capped-HSA, SH-albumin
and K5-MPA-albumin. The MPA covalently bonds to the free thiol on
the albumin. The stability of the three forms of albumin in plasma is
presented below.
Time %capped HSA % SH-Albumin %K5-MPA-HSA
0 hrs. 61.3 16.6 22.1
4 hrs. 64.6 16.05 19.35
24 hrs. 63 16.8 20.2
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The percentage of the three forms of human serum albumin
remained relatively constant througout the 24 assay period. In
particular, the percentage of K5-MPA-HSA remained refatively constant
throughout the 24 hour plasma assay. These results are in dramatic
contrast to the results obtained with unmodified K5 which decreased to
9% of the original amount of K5 in only 4 hours in plasma. The results
demonstrate that, in contrast to K5 which is quite unstable in plasma,
K5-MPA-HSA is quite stable from peptidase activity in plasma.
EXAMPLE 27
Endothelial Cell Migration Assay
The activity of modified anti-angiogenic peptides may be
determined with an endothelial cell migration assay. The endothelial cell
migration assay may be performed as described by Polverini, P. J. et al.,
Methods Enzymol, 198: 440-450 (1991). Briefly, bovine capillary
(adrenal) endothelial cells (BCE, which may be obtained from Judah
Folkman, Harvard University Medical School) are starved overnight in
DMEM containing 0.1 % bovine serum albumin (BSA). Cells are then
harvested with trypsin and resuspended in DMEM with 0.1 % BSA at a
concentration of 1.5 X 106 cells/mL. Cells are added to the bottom of a
48-well modified Boyden chamber (for example from Nucleopore
Corporation, Cabin John, Md.). The chamber is assembled and
inverted, and cells are allowed to attach for 2 hours at 37 C to
polycarbonate chemotaxis membranes (5 pm pore size) that is soaked
in 0.1% gelatin overnight and dried. The chamber is then reinverted
and test substances are added to the wells of the upper chamber (to a
total volume of 50 Ni); the apparatus is then incubated for 4 hours at
37 C. Membranes are recovered, fixed and stained (DiffQuick*, Fisher
Scientific, Pittsburgh, Pa.) and the number of cells that have migrated to
the upper chamber per 10 high power fields are counted. Background
migration to DMEM+0.1 % BSA may be subtracted and the data
reported as the number of cells migrated per 10
*Trade-Mark
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high power fields (400 x) or when results from multiple experiments are
combined, as the percent inhibition of migration compared to a positive
control.
Example 28
Preparation of HSA-Kringle 5 Conjugates
Modified kringle 5 peptides are dissolved in distilled water to a
final concentration of 100 mM. For the conjugation reaction, one volume
of 100 mM modified kringle 5 peptide is added to 99 volumes of 25%
HSA (Albutein , 25% solution, Alpha Therapeutic inc.) to get 1 mM
modified K5 : 3.75 mM HSA conjugates. The mixture is allowed to
incubate at room temperature for 2 hours. The presence of the
conjugate and the absence of unreacted modified K5 peptide are
determined by HPLC coupled with mass spectrometry.
Example 29
Effect of Modified Kringle 5 peptides on Endothelial Cell
Proliferation In Vitro.
The biological activity of free and HSA-conjugated Kringle 5
peptides may be determined in vitro using an endothelial cell
proliferation assay. Bovine aortic endothelial cells are plated at a
density of 2500 cells per well in a 96-well plate in Dulbecco's Modified
Eagle medium (DMEM, Gibco) containing 10% heat inactivated calf
serum. The cells were allowed to adhere for 24 hours, at 37 C in a 5%
CO2 incubator. The medium is then replaced with fresh DMEM (without
serum) containing varying concentrations of inhibitor (free K5 peptide
and HSA-Kringle 5 peptides). After 30 minutes at 37 C, bFGF (basic
fibroblast growth factor) may then be added to a final concentration of
1 ng/mL to stimulate growth. After 72 hours, the cell number may be
measured using the colorimetric substrate WST-1 (Boehringer
Mannheim) to determine the effect of modified K5 peptides on
endothelial cell proliferation in vitro.
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While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is capable of
further modifications and this application is intended to cover any varia-
tions, uses, or adaptations of the invention following, in general, the
principles of the invention and including such departures from the
present disclosure as come within known or customary practice within
the art to which the invention pertains and as may be applied to the
essential features hereinbefore set forth, and as follows in the scope of
the appended claims.
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1
SEQUENCE LISTING
<110> ConjuChem, Inc.
Beliveau, Richard
Bridon, Dominique
Rasamoelisolo, Michele
Thibaudeau, Karen
Huang, Xicai
<120> Long Lasting Anti-Angiogenic Peptides
<130> 2200
<140>
<141>
<150> 60/134,406
<151> 1999-05-17
<160> 16
*
<170> Patentln Ver. 2.1
<210> 1
<211> 790
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 1
Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala.Ser Leu Phe Ser
1 5 10 is
Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala
20 25 30
Lys Cys Glu Glu Asp Glu Giu Phe Thr Cys Arg Ala Phe Gln Tyr His
35 40 45
Ser Lys Glu Gin Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser
50 55 60
Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Vai Tyr
65 70 75 80
*Trade-Mark
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2
Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Ser
85 90 95
Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro
100 105 110
His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu
115 120 125
Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys
130 135 140
Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu
145 150 155 160
Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys
165 170 175
Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln
180 185 190
Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn
195 200 205
Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp
210 215 220
Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro
225 230 235 240
Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gin Cys Leu
245 250 255
Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser
260 265 270
Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn
275 280 285
Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys
290 295 300
Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser
305 310 315 320
Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser Pro
325 330 335
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3
Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro
340 345 350
Val Val Gln Asp Cys Tyr His Gly Asp Gly Gin Ser Tyr Arg Gly Thr
355 360 365
Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met
370 375 380
Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly
385 390 395 400
Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp
405 410 415
Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys
420 425 430
Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val Val
435 440 445
Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly
450 455 460
Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr
465 470 475 480
Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe
485 490 495
Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg
500 505 510
Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro
515 520 525
Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser
530 535 540
Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg
545 550 555 560
Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val
565 570 575
Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile
580 585 590
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Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro
595 600 605
Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn
610 615 620
Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu
625 630 635 640
Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val
645 650 655
Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val
660 665 670
Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln
675 680 685
Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile
690 695 700
Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln
705 710 715 720
Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys
725 730 735
Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr
740 745 750
Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn
755 760 765
Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu
770 775 780
Gly Val Met Arg Asn Asn
785 790
<210> 2
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
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<400> 2
Pro Arg Lys Leu Tyr Asp Lys
1 5
<210> 3
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 3
Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Lys
1 5 10
<210> 4
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 4
Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Ala Tyr Thr Thr Asn
1 5 10 15
Pro Arg Lys Leu Tyr Asp Tyr Lys
<210> 5
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 5
Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Lys
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6
1 5 10
<210> 6
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 6
Pro Arg Lys Leu Tyr Asp Tyr Lys
1 5
<210> 7
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 7
Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Lys
1 5 10
<210> 8
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 8
Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr
1 5 10
<210> 9
<211> 23
<212> PRT
CA 02373252 2001-11-06
WO 00/70665 PCT/IB00/00763
7
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 9
Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Ala Tyr Thr Thr Asn
1 5 10 15
Pro Arg Lys Leu Tyr Asp Tyr
<210> 10
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 10
Arg Lys Leu Tyr Asp Tyr Lys
1 5
<210> 11
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 11
Arg Lys Leu Tyr Asp Tyr
1 5
<210> 12
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
CA 02373252 2001-11-06
WO 00/70665 PCT/IB00/00763
8
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 12
Pro Arg Lys Leu Tyr Asp Lys
1 5
<210> 13
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 13
Pro Arg Lys Leu Tyr Asp
1 5
<210> 14
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 14
Pro Arg Lys Leu Tyr Asp Tyr Lys
1 5
<210> 15
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 15
Pro Arg Lys Leu Tyr Asp Tyr
1 5
CA 02373252 2001-11-06
WO 00/70665 9 PCT/IB00/00763
<210> 16
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
Peptide
<400> 16
Arg Asn Pro Asp Gly Asp Val Gly Gly Asp Val Gly Gly Pro Trp
1 5 10 15
