U.S. patent application number 10/235552 was filed with the patent office on 2003-02-06 for anti-invasive and anti-angiogenic compositions.
Invention is credited to Jones, Terence R., Mazar, Andrew P..
Application Number | 20030027768 10/235552 |
Document ID | / |
Family ID | 25412335 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030027768 |
Kind Code |
A1 |
Mazar, Andrew P. ; et
al. |
February 6, 2003 |
Anti-invasive and anti-angiogenic compositions
Abstract
A peptide compound having the sequence
Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu [SEQ ID NO:2] or a substitution
variant, addition variant or other chemical derivative thereof
inhibits cell invasion, endothelial tube formation or angiogenesis
in vitro. A number of substitution variants and addition variants
of this peptide, preferably capped at the N- and C-termini, as well
as peptidomimetic derivatives, are useful for treating diseases and
conditions mediated by undesired and uncontrolled cell invasion
and/or angiogenesis. Pharmaceutical compositions comprising the
above peptides and derivatives are administered to subjects in need
of such treatment in a dosage sufficient to inhibit invasion and/or
angiogenesis. The disclosed compositions and methods are
particularly useful for suppressing the growth and metastasis of
tumors.
Inventors: |
Mazar, Andrew P.;
(Escondido, CA) ; Jones, Terence R.; (San Diego,
CA) |
Correspondence
Address: |
VENABLE, BAETJER, HOWARD AND CIVILETTI, LLP
P.O. BOX 34385
WASHINGTON
DC
20043-9998
US
|
Family ID: |
25412335 |
Appl. No.: |
10/235552 |
Filed: |
September 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10235552 |
Sep 6, 2002 |
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09437136 |
Nov 10, 1999 |
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09437136 |
Nov 10, 1999 |
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08900327 |
Jul 25, 1997 |
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5994309 |
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Current U.S.
Class: |
514/9.4 ;
514/12.2; 514/13.3; 514/14.9; 514/19.8; 514/21.7; 530/328;
530/329 |
Current CPC
Class: |
C12Y 304/21073 20130101;
A61P 9/10 20180101; A61K 38/00 20130101; A61P 29/00 20180101; A61P
35/00 20180101; A61P 17/06 20180101; C12N 9/6462 20130101; A61P
35/04 20180101 |
Class at
Publication: |
514/16 ; 530/328;
530/329 |
International
Class: |
A61K 038/08; C07K
007/08; C07K 007/06 |
Claims
What is claimed is:
1. A peptide compound having the sequence
Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu [SEQ ID NO:2]or a substitution
variant, addition variant or other chemical derivative thereof,
which peptide, variant or derivative is capped or uncapped, wherein
said peptide, variant or derivative has one or more of the
following activities: (a) has at least about 20% of the biological
activity of Ac-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Am in one or more of
the following in vitro bioassays: (i) invasion in a Matrigel.RTM.
assay; (ii) endothelial tube formation on Matrigel.RTM., or (iii)
endothelial tube formation on a fibrin matrix in the presence of
basic fibroblast growth factor and vascular endothelial growth
factor; or (b) competes with labeled
Ac-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Am for binding to a cell or
molecule which has a binding site for
Ac-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Am.
2. A peptide according to claim 1 capped with an N-terminal acetyl
group and a C terminal amide group.
3. A substitution or addition variant of a peptide according to
claim 1, or a chemical derivative of the variant, which variant has
a sequence selected from the group consisting of: (a) SEQ ID NO:2
wherein the Glu at position 7 or 8 or both is replaced by one or
any two of the substituent amino acids Gln, Asp or Asn; (b) SEQ ID
NO:2 wherein Ser at position 3 or 4 or both is replaced by one or
any two of the substituent amino acids Thr, Ala, Gly, hSer or
Val.beta.OH; (c) SEQ ID NO:2 wherein the Lys at position 1 is
replaced by His, Arg, Gln, Orn, Cit or Hci; (d) SEQ ID NO:2 wherein
the Pro at position 2, 5 or 6 is replaced by Hyp; (e) an addition
variant of SEQ ID NO:2, wherein Leu, Ile, Val, Nva, Nle, Met, Ala,
or Gly is added to the C-terminal Glu or to any of said
substituents for Glu at position 8; (f) an addition variant of SEQ
ID NO:2, wherein any of the following peptides are added to the
C-terminal Glu or to any of said substituents for Glu at position
8: Leu-(Gly).sub.n; Ile-(Gly).sub.n; Val-(Gly).sub.n;
Nva-(Gly).sub.n; or Nle-(Gly).sub.n, wherein n=1-10; (g) an
addition variant of SEQ ID NO:2 wherein one or more of the
following residues or peptides is added to the N-terminal Lys or to
any of said N-terminal substituents of Lys at position 1: Gly,
Lys-(Gly).sub.n; Tyr-(Gly).sub.n; or Gly-(Gly).sub.n, wherein
n=1-10; and (h) a combination of one or more of (a)-(g).
4. A compound according to claim 1 which is a peptidomimetic
agent.
5. A multimer of a peptide or variant according to claim 1 which,
when the peptide is not a variant, has the formula:
(Lys-Pro-Ser-Ser-Pro-Pro-Glu-G-
lu-X.sub.m).sub.n-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu wherein X is
selected from the group consisting of C.sub.1-C.sub.20 alkyl,
C.sub.1-C.sub.20 alkenyl, C.sub.1-C.sub.20 alkynyl,
C.sub.1-C.sub.20 polyether containing up to 9 oxygen atoms and
Gly.sub.z, and wherein m=0 or 1, n=1-100 and z=1-10.
6. A pharmaceutical composition useful for inhibiting (i) invasion
of tumor cells or (ii) angiogenesis, comprising (a) a peptide,
variant or derivative according to claim 1; and (b) a
pharmaceutically acceptable carrier or excipient.
7. A pharmaceutical composition useful for inhibiting (i) invasion
of tumor cells or (ii) angiogenesis, comprising (a) a peptide,
variant or derivative according to claim 2; and (b) a
pharmaceutically acceptable carrier or excipient.
8. A pharmaceutical composition useful for inhibiting (i) invasion
of tumor cells or (ii) angiogenesis, comprising (a) a peptide,
variant or derivative according to claim 3; and (b) a
pharmaceutically acceptable carrier or excipient.
9. A pharmaceutical composition useful for inhibiting (i) invasion
of tumor cells or (ii) angiogenesis, comprising (a) a
peptidomimetic according to claim 4; and (b) a pharmaceutically
acceptable carrier or excipient.
10. A pharmaceutical composition useful for inhibiting (i) invasion
of tumor cells or (ii) angiogenesis, comprising (a) a peptide
multimer according to claim 5; and (b) a pharmaceutically
acceptable carrier or excipient.
11. A method for inhibiting the invasiveness of tumor cells
comprising contacting said cells with an effective amount of a
peptide, variant or derivative according to claim 1.
12 A method for inhibiting cell migration, invasion,
migration-induced cell proliferation or angiogenesis in a subject
having a disease or condition associated with undesired cell
migration, invasion, migration-induced proliferation, or
angiogenesis, comprising administering to said subject an effective
amount of a pharmaceutical composition according to claim 6.
13. A method according to claim 12 wherein said disease or
condition is primary tumor growth, tumor invasion or metastasis,
atherosclerosis, post-balloon angioplasty vascular restenosis,
neointima formation following vascular trauma, vascular graft
restenosis, fibrosis associated with a chronic inflammatory
condition, lung fibrosis, chemotherapy-induced fibrosis, wound
healing with scarring and fibrosis, psoriasis, deep venous
thrombosis, or another disease or condition in which angiogenesis
is pathogenic.
14. A method according to claim 13 wherein said disease is tumor
growth, invasion or metastasis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/900,327, filed Jul. 25, 1997, from which
priority is claimed pursuant to 35 U.S.C. .sctn.120, and which
disclosure is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention in the fields of biochemistry, organic
chemistry and medicine relates to peptide compounds and methods of
their use to treat diseases and conditions associated with
movement, migration and adhesion of cells including diseases that
involve angiogenesis such as tumor invasion and metastasis.
[0004] 2. Description of the Background Art
[0005] Several disease processes have been demonstrated to require
the invasion or migration of cells as part of their pathology.
These include tumor invasion, tumor metastasis, pathological
angiogenesis, inflammation, and endometriosis (Liotta et al., 1991;
Fox et al., 1996; Osborn, 1990; Mareel et al., 1990; Aznavoorian et
al., 1993; Lennarz and Strittmatter, 1991; Fernandez-Shaw et al.,
1995).
[0006] In the case of tumor angiogenesis, quiescent endothelial
cells can become motile in response to a variety of angiogenic
growth factors as well as to changes in the basement membrane
induced by tumor cells and various accessory cells found within a
tumor (Blood and Zetter, 1990; Liotta et al., 1991; Odedra and
Weiss, 1991). Neovascularization of a tumor enables the metastatic
spread of aggressive tumor cells by (1) providing a route of escape
for the metastatic cells as well as (2) nurturing the tumor by
providing a growth-conducive environment (Cornelius et al., 1995;
Blood and Zetter, 1990; Weaver et al., 1997; Weinstat-Saslow and
Steeg, 1994; Leek et al., 1994).
[0007] The process of tumor metastasis may be viewed as
bi-directional, comprising the following steps
[0008] (1) endothelial cells migrate into a tumor in response to a
chemotactic gradient produced by the tumor cells or by accessory
cells (stromal cells, leukocytes); and
[0009] (2) aggressive tumor cells concomitantly invade toward the
developing neovasculature.
[0010] The process of invasion may further fuel angiogenesis by the
proteolytic release of growth/angiogenic factors bound to
extracellular matrix (ECM), including basic fibroblast growth
factor (bFGF), vascular endothelial growth factor (VEGF), and
hepatocyte growth factor (HGF) as well as other factors including
interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating
factor (GM-CSF). Also generated are proteolytic fragments of the
ECM which are themselves chemotactic for both tumor cells and
endothelial cells (Fox et al., 1996; Leek et al., 1994; Vlodavsky
et al., 1990; Sweeney et al., 1991; Taipale and Keski-Oja,
1997).
[0011] It has been suggested that only 1-2% of the total cells in a
tumor are capable of metastasis. As this statement is based on a
static view of the tumor phenotype, it is probably inaccurate. In
reality, metastasis appears to depend on disseminated tumor cells
becoming exposed to an environment which supports their spread and
survival (Weaver et al., 1997). In the majority of patients
presenting with a clinically detectable primary tumor, metastasis
has already occurred (Welch, 1997). Metastatic disease occurs when
the disseminated foci of tumor cells seed a tissue which supports
their growth and propagation, and this secondary spread of tumor
cells is responsible for the morbidity and mortality associated
with the majority of cancers. Clinical management of metastatic
disease is often unsuccessful with conventional cytotoxic
therapies. Metastasis differs substantially from the growth of the
primary tumor in that it involves the simultaneous outgrowth of
many foci which are phenotypically similar from the standpoint of
their aggressiveness This outgrowth is dependent on the ability of
cells that have metastasized to invade locally and to recruit
neovessels.
[0012] By preventing interaction of adhesion molecules, the
important process of cell migration/invasion and angiogenesis can
be diminished or halted, with a number of important consequences
for those diseases and conditions which are caused in part by
undesirable cell migration, invasion and angiogenesis. In addition
to vascular phenomena, such cell migration/invasion is important in
tumor metastasis, which can be suppressed by the compositions and
methods disclosed herein. Administration of effective amounts of
these compositions will also disrupt the molecular interactions
required for angiogenesis.
[0013] The art recognizes the need for novel treatments of subjects
with cancer, in particular patients with metastatic cancer who have
the poorest prognosis. Such treatment should be as devoid as
possible of undesired side effects such as those associated with
conventional chemotherapy and some of the experimental
biotherapies. The present invention is directed to this objective.
Inhibition of tumor cell invasion and endothelial cell migration
(an important component of the angiogenic process) provide a novel
approach to treating subjects with metastatic cancer. By inhibiting
the local spread of tumor cells and angiogenesis at metastatic
sites, metastatic foci should be induced to regress due to
deprivation of their blood supply thus encouraging the subsequent
expression of the cells' endogenous apoptotic program.
[0014] Furthermore, the inhibition of invasion of tissue by
leukocytes and the concomitant angiogenesis would be useful for
treating inflammation and other disease processes wherein cellular
invasiveness is part of the pathogenic process. Inflammation and
tumor invasion and metastasis and angiogenesis are known to involve
similar mechanisms and extracellular factors (Liotta et al., 1991;
Fox et al., 1996; Osborn, 1990; Mareel et al., 1990; Aznavoorian et
al., 1993; Lennarz and Strittmatter, 1993).
[0015] Blasi et al. (U.S. Pat. No. 5,416,006) discloses plasminogen
activators and their chemical modification, in particular
phosphorylated uPA and tPA as thrombolytic agents. These workers
examined phosphorylated uPA by generating tryptic phosphopeptides
therefrom and noted the existence of KPSSPPEELK [SEQ ID NO:1]
(corresponding to positions 136-145 of uPA). This decapeptide was
not tested for any function, nor ascribed any properties of
functional relevance. More importantly, as disclosed herein, this
peptide (unphosphorylated), capped or uncapped, is inactive in an
in vitro assay of cell invasion.
[0016] Citation of the above documents is not intended as an
admission that any of the foregoing is pertinent prior art. All
statements as to the date or representation as to the contents of
these documents is based on the information available to the
applicant and does not constitute any admission as to the
correctness of the dates or contents of these documents.
SUMMARY OF THE INVENTION
[0017] The present invention provides methods and compositions for
treating diseases and processes mediated by undesired and
uncontrolled cell invasion and/or angiogenesis by administering to
an animal a composition comprising an oligopeptide, chemical
derivative or peptidomimetic in a dosage sufficient to inhibit the
invasion and/or angiogenesis. The present invention is particularly
useful for treating or for suppressing the growth of tumors.
Administration of the composition to a human or subject with
prevascularized metastasized tumors will prevent the growth or
expansion of those tumors.
[0018] Thus, the present invention is directed to a peptide
compound having the sequence Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu (also
abbreviated in single letter amino acid code as KPSSPPEE) [SEQ ID
NO:2] or a substitution variant, addition variant or other chemical
derivative thereof. The preferred peptide, variant or derivative is
"capped" at the amino and carboxyl termini, wherein (a) acetyl
(abbreviated as "Ac") is boun*d to the N at the amino-terminus and
(b) an amido group (abbreviated as "Am") is bound to the C-terminal
carboxyl group. In general, this capped peptide will be written
"Ac-KPSSPPEE-Am" throughout this document using the single letter
amino acid code and indicating the blocking groups as Ac and Am.
This compound is also designated ".ANG.6" and will therefore be
referred to by this name as well.
[0019] The peptide, variant or derivative of this invention has one
or more of the following activities:
[0020] (a) at least about 20% of the biological activity of
Ac-KPSSPPEE-Am in one or more of the following in vitro bioassays:
(i) invasion in a Matrigel.RTM. assay;
[0021] (ii) endothelial tube formation on Matrigel.RTM., or (iii)
endothelial tube formation on a fibrin matrix in the presence of
basic fibroblast growth factor and vascular endothelial growth
factor; or
[0022] (b) binding activity such that it competes with labeled
Ac-KPSSPPEE-Am for binding to a cell or molecule which has a
binding site for Ac-KPSSPPEE-Am.
[0023] In a preferred embodiment, the peptide or peptide variant is
capped at both ends with an N-terminal acetyl group and a C
terminal amide group.
[0024] A preferred substitution or addition variant of the peptide,
or a chemical derivative of the variant, has an amino acid sequence
selected from the group consisting of:
[0025] (a) SEQ ID NO:2 wherein the Glu at position 7 or 8 or both
is replaced by one or any two of the substituent amino acids Gln,
Asp or Asn;
[0026] (b) SEQ ID NO:2 wherein Ser at position 3 or 4 or both is
replaced by one or any two of the substituent amino acids Thr, Ala,
Gly, hSer (homoserine) or Val.beta.OH (.beta.-hydroxyvaline);
[0027] (c) SEQ ID NO:2 wherein the Lys at position 1 is replaced by
His, Arg, Gln, Orn (ornithine), Cit (citrulline) or Hci
(homocitrulline);
[0028] (d) SEQ ID NO:2 wherein the Pro at position 2, 5 or 6 is
replaced by Hyp (hydroxyproline);
[0029] (e) an addition variant of SEQ ID NO:2, wherein Leu, Ile,
Val, Nva (norvaline), Nle (norleucine), Met, Ala, or Gly is added
to the C-terminal Glu or to any C-terminal substituent for Glu at
position 8 as disclosed above.
[0030] (f) an addition variant of SEQ ID NO:2, wherein any of the
following peptides are added to the C-terminal Glu or to the C
terminal substituent for Glu at position 8: Leu-(Gly).sub.n;
Ile-(Gly).sub.n; Val-(Gly).sub.n; Nva-(Gly).sub.n; or
Nle-(Gly).sub.n, wherein n=1-10.
[0031] (g) an addition variant of SEQ ID NO:2 wherein one or more
of the following residues or peptides is added to the N-terminal
Lys, or to any N-terminal substituent of Lys at position 1 as
disclosed: Gly, Lys-(Gly).sub.n; Tyr-(Gly).sub.n; or
Gly-(Gly).sub.n, wherein n=1-10; and
[0032] (h) a combination of one or more of (a)-(g).
[0033] In a preferred embodiment, the chemical derivative above is
a peptidomimetic agent.
[0034] Also provided is a multimer of the peptide or variant above,
which, when the peptide is not a variant, has the formula:
(KPSSPPEE-X.sub.m).sub.n,-KPSSPPEE wherein X is selected from the
group consisting of C.sub.1-C.sub.20 alkyl, C.sub.1-C.sub.20
alkenyl, C.sub.1-C.sub.20 alkynyl, C.sub.1-C.sub.20 polyether
containing up to 9 oxygen atoms and Gly.sub.m, and wherein m=0 or
1, n=1-100 and z=1-10.
[0035] The invention is further directed to a pharmaceutical
composition useful for inhibiting invasion of tumor cells or
angiogenesis, comprising (a) any of the above peptides, variants or
chemical derivatives including a peptidomimetic or a multimeric
peptide and (b) a pharmaceutically acceptable carrier or
excipient.
[0036] Also included is a method for inhibiting the invasiveness of
tumor cells comprising contacting the cells with an effective
amount of a peptide, variant or derivative as above.
[0037] In another embodiment, a method is provided for inhibiting
tumor invasion or metastasis in a subject comprising administering
to the subject any of the above pharmaceutical compositions.
[0038] Also provided is a method for inhibiting cell migration,
invasion, migration-induced cell proliferation or angiogenesis in a
subject having a disease or condition associated with undesired
cell migration, invasion, migration-induced proliferation, or
angiogenesis comprising administering to the subject an effective
amount of a pharmaceutical composition as described above.
[0039] In any of the foregoing methods, the disease or condition
being treated may be primary tumor growth, tumor invasion or
metastasis, atherosclerosis, post-balloon angioplasty vascular
restenosis, neointima formation following vascular trauma, vascular
graft restenosis, fibrosis associated with a chronic inflammatory
condition, lung fibrosis, chemotherapy-induced fibrosis, wound
healing with scarring and fibrosis, psoriasis, deep venous
thrombosis, or another disease or condition in which angiogenesis
is pathogenic. The treatment methods are most preferred for tumor
growth, invasion or metastasis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a graph showing the inhibitory effect of
Ac-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Am (5 .mu.M) on the in vitro
invasion of both human (PC-3) and rat (Mat BIII) tumor cell lines
in a Matrigel.RTM. system as described in the Examples. The left
bar of each pair is the control group and the right bar represents
cells responding in the presence of the peptide.
[0041] FIG. 2 is a graph showing the effect of various peptides on
the in vitro invasion of PC-3 cells in a Matrigel.RTM. system as
described in the Examples. All compounds were tested at a 5 .mu.M.
The following peptides were examined:
Ac-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Am (Ac-KPSSPPEE-Am) [SEQ ID
NO:2] and the following variants of SEQ ID NO:2:
Ac-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Am (Ac-KPSSPPE-Am),
Ac-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Am (Ac-PSSPPEE-Am) as well as SEQ ID
NO:1 (Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-Leu-Lys) either capped
(Ac-KPSSPPEELK-Am) or uncapped (KPSSPPEELK).
[0042] FIG. 3 shows that .ANG.6 inhibits the activation of
plasminogen in a clot lysis assay. Fibrin clots were formed by
supplementing plasma with [.sup.125I] fibrinogen. Clotting was
initiated by adding thrombin and calcium to the plasma mixture.
Clots were washed to remove any free radioactivity, plasminogen and
the activator to be tested were added to the clot and the release
of [.sup.125I] fibrin degradation products was monitored. The data
presented in this figure is at 30 minutes after initiation of clot
lysis. scuPA-suPAR: dose response with .ANG.6 on clot lysis by
scuPA-suPAR complex; scuPA: dose response on clot lysis by scuPA
alone; scuPA/suPAR+scrambled pep: dose response with a scrambled
version of .ANG.6 (Ac-PSESPEKP-Am).
[0043] FIGS. 4 and 5 show the results of HUVEC proliferation
assays. HUVEC were plated on gelatin and allowed to adhere for 4
hrs in the presence of 2% FBS. bFGF (1 ng/mL) and the test
compounds were added after 4 hours and the % proliferation (where
bFGF induced proliferation was 100% and proliferation in the
absence of bFGF was 0) was determined using MTS. FIG. 4: the .ANG.6
(50 .mu.g/mL) concentration was kept constant and the concentration
of cisplatin ("CDDP") was varied;
[0044] FIG. 5: CDDP was kept constant at a sub-optimal dose (l
1g/mL) and .ANG.6 was varied.
[0045] FIG. 6 shows the migration of HMVEC on Type I collagen.
Membranes contained in transwell chambers (8.0 .mu.m) were coated
with Type I Collagen. Microvessel endothelial cells (HMVECs,
2.times.10.sup.5/well) were added to the top chamber of each well
and bFGF (10 ng/mL) was added to each bottom chamber as a
chemoattractant. Inhibitor was added to both chambers and the
migration allowed to proceed for 6 hrs at 37.degree. C. The top of
each filter was scraped to remove cells that had not migrated and
the cells adhering to the bottom of the filter were fixed and
stained using DiffQuick and the cells were quantitated by counting.
The results are expressed as a percentage of the cells migrated in
response to bFGF alone.
[0046] FIG. 7 shows that .ANG.6 inhibits the invasion of Mat B-III
rat breast cancer cells and MDA-MB-231 human breast cancer cells
through Matrigel
[0047] FIG. 8 shows the inhibition of Mat B-III invasion by .ANG.6
and its variants. A: Control (no compound); B: .ANG.6; C: .ANG.6
lacking the C-terminal Glu; D: .ANG.6 lacking the N-terminal Lys;
E: .ANG.6 extended by LeuLys at the C-terminus (corresponds to
amino acids 144 and 145 in uPA); F: same as E except C-terminal is
not capped; G: .ANG.6, N and C termini uncapped; H: .ANG.6 extended
by Leu at C-terminus (capped).
[0048] FIG. 9 is a PAGE gel pattern of biotin-.ANG.6 crosslinked to
MDA-MB-231 cells. Cells were incubated in the presence of
biotin-.ANG.6 (200 .mu.M) for 2 hrs. DSS (1 mM) was added and
cross-linking was allowed to proceed for 15 minutes. The cells were
washed and extracted using PBS/1% Triton X-114. Cell particulate
was removed by centrifugation and the supernatants were heated to
37.degree. C. for 5 minutes to induce phase separation. Aqueous and
detergent (membrane) phases were separated by centrifugation and
each phase was resolved by SDS-PAGE and analyzed by western blot
using strepatavidin-HRPO.
[0049] A: Aqueous phase, 2.sup.nd wash; B: Detergent Phase; C:
Aqueous phase, 1.sup.st wash; D: MW markers;
[0050] E: Biotin-uPA.
[0051] FIG. 10 shows that .ANG.6 inhibits angiogenesis in a CAM
assay. Filter disks saturated with either compound or compound
+bFGF (0.3 ng) were placed on the CAM (7 day old) and vessel
formation was observed for 4 days. Major vessels were quantitated
at this time.
[0052] FIG. 11 shows the results of CAM assays. A: Control CAM; B:
CAM treated with 1.2 .mu.g/disk of .ANG.6.
[0053] FIGS. 12 and 13 show that .ANG.6 inhibits Mat B-III tumor
growth and metastasis. FIG. 12: The volume of the primary challenge
tumor was determined using caliper measurements. Inhibition of
tumor growth by .ANG.6 was most effective when the tumors were
small. FIG. 13: Macroscopic metastasis was also inhibited by .ANG.6
treatment. Control animals received vehicle (PBS) only.
[0054] FIG. 14 shows results of in vitro and in vivo studies using
.ANG.6 and TAM. TopPanel: Invasion of Mat B-III cells through
Matrigel A: Control; B: TAM (1 .mu.M); C: .ANG.6 (5 .mu.M); D:
.ANG.6 (50 .mu.M); E: .ANG.6 (50 .mu.M)+TAM (1 .mu.M). Bottom
Panel: Mat B-III tumor bearing animals were treated with TAM (3
mg/kg/day), .ANG.6 (75 mg/kg/day) or a combination of TAM +.ANG.6.
Tumor volumes were determined using caliper measurements. Control
received vehicle (PBS) only.
[0055] FIG. 15 shows that .ANG.6 inhibits the growth of primary
challenge tumors in an MDA-MB-231 xenograft model. Nude mice (n=5
per group) were challenged with 2.times.10.sup.5 tumor cells
co-injected into the mammary fat pad of the mice with Matrigel.
Treatment of the mice with .ANG.6 (IP, 1 mg bid) was initiated when
the tumor nodules had become palpable (beginning of week 1 on
graph). Control animals received vehicle (PBS) only.
[0056] FIG. 16 shows results of combination therapy of U87 tumors
inoculated s.c. in nude mice. U87 cells (1.times.10.sup.5) were
inoculated sc on the back of a nude mouse (n=5). Tumors were staged
to 50-100 mm.sup.3 (day 0) at which time treatment was started with
either .ANG.6 alone (75 mg/kg/day given IP bid), cisplatin (CDDP)
alone (3 mg/kg/day given every other day from day 4.times.6
administrations), or a combination of .ANG.6+CDDP. Tumor volumes
were determined using caliper measurements. Treatment was
discontinued at day 20 and the mice were euthanized one week
later.
[0057] FIG. 17 shows the number of tumor foci using Ki-67 staining
of U87 tumors. Formalin-fixed sections were stained with mouse
anti-Ki-67 followed by peroxidase detection. Positive foci were
quantitated by capturing digital images of the slides and
determining the number of pixels associated with the staining on
each slide.
[0058] FIG. 18 shows the effects of .ANG.6 on orthotopically
inoculated human U87 GBM cells. U87 cells were inoculated into the
ventricles of nude mice and treatment was initiated 72 hrs after
inoculation. Tumor volume was determined using caliper measurements
after euthanasia and necropsy.
[0059] FIG. 19 shows the dose dependent response of U87 tumor
growth to .ANG.6. Mice (n=4 per group) were treated as previously
described and treatment was discontinued after 21 days.
[0060] FIG. 20 shows the effect of .ANG.6 and CDDP on survival of
mice with orthotopically inoculated U87 GBM tumors. A: Control; B:
.ANG.6 only; C: CDDP only; D: .ANG.6+CDDP.
[0061] FIG. 21 shows the results of combination treatment of 3LL
tumors with .ANG.6+cyclophosphamide. 3LL cells (1.5.times.10.sup.5
cells) were injected into the tail vein of C57/B1 mice. Treatment
with .ANG.6 (75 mg/kg/day IP b.i.d) and cyclophosphamide (4 mg/kg
IP on day 4) was continued for 19 days. Animals were euthanized on
day 20 and the lungs removed and analyzed macroscopically and
histologically for the presence of tumor. The average number of
tumors in each group (n=7) is shown.
[0062] FIG. 22 shows the plasma concentration of .ANG.6 in mice
following a single intravenous injection of 37.5 mg/kg.
[0063] FIG. 23 shows the plasma concentration of .ANG.6 in
Cynomolgus monkeys following a single intravenous injection of 37.5
mg/kg.
[0064] FIG. 24 shows allometric scaling of plasma clearance in
animals.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0065] The present inventors have discovered a novel peptide and
related compounds which act as inhibitors of angiogenesis and
invasiveness and have devised various methods for using this
peptide for diagnosis, therapy and receptor identification. The
peptide is a potent and specific inhibitor of (a) cell invasion,
(b) angiogenesis at tumor sites including sites of metastasis, and
(c) inflammatory responses.
[0066] In addition, the peptide and its derivatives are designed to
be highly soluble in aqueous buffer and body fluids but not in
lipids. This property limits non-specific partitioning into
membranes. Non-specific partitioning of compounds into and across
membranes is a frequent cause of toxicity. The compounds of this
invention have minimal toxicity because, owing to their possessing
Coulombic charge, they are not expected to partition into cells.
The target(s) of the compositions are extracellular, and method(s)
of this invention are predicated on the compositions acting first
in the extracellular space. Therefore, it is desirable to maintain
the compounds in the extracellular space.
[0067] Additional pharmacological advantage is obtained due to the
high solubility limit of the compounds, allowing their delivery in
high concentrations in the absence of co-solvents or extraordinary
excipients.
[0068] Compounds of the invention have been shown by the present
inventors (see Example II) to block the invasion of both human and
rat tumor cells in vitro in the Matrigel.RTM. system.
[0069] In addition, they block endothelial cell tube formation in
response to bFGF and VEGF in either a fibrin matrix or when the
endothelial cells are plated on Matrigel.RTM..
[0070] The compounds of the invention also inhibit experimental
metastasis in a xenograft model in nu/nu mice using the human
prostatic carcinoma cell line, PC-3, transfected with the green
fluorescent protein (GFP) as a reporter. Finally, the compounds
also inhibit tumor progression, spontaneous metastasis and
angiogenesis in a syngeneic rat model of breast cancer.
[0071] The Peptide Compositions
[0072] The original inhibitory capped peptide discovered by the
present inventors has 8 amino acid residues with a molecular weight
of 911 Da. This preferred peptide is characterized by the
sequence:
[0073] CH.sub.3CO-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-NH.sub.2 [SEQ ID
NO:2]
[0074] The amino and carboxyl termini are preferably blocked or
"capped" with acetyl (CH.sub.3CO--, bound to the amino-terminal N;
also abbreviated as "Ac") and amido (--NH.sub.2 bound to the
C-terminal carboxyl group; also abbreviated as "Am"), respectively.
This peptide will also be referred to below in single letter code
indicating the blocking groups as Ac and Am groups:
Ac-KPSSPPEE-Am.
[0075] The N-terminal capping function is preferably in a linkage
to the terminal amino group and may be selected from the group
consisting of:
[0076] formyl;
[0077] alkanoyl, having from 1 to 10 carbon atoms, such as acetyl,
propionyl, butyryl;
[0078] alkenoyl, having from 1 to 10 carbon atoms, such as
hex-3-enoyl;
[0079] alkynoyl, having from 1 to 10 carbon atoms, such as
hex-5-ynoyl;
[0080] aroyl, such as benzoyl or 1-naphthoyl;
[0081] heteroaroyl, such as 3-pyrroyl or 4-quinoloyl;
[0082] alkylsulfonyl, such as methanesulfonyl;
[0083] arylsulfonyl, such as benzenesulfonyl or sulfanilyl;
[0084] heteroarylsulfonyl, such as pyridine-4-sulfonyl;
[0085] substituted alkanoyl, having from 1 to 10 carbon atoms, such
as 4-aminobutyryl;
[0086] substituted alkenoyl, having from 1 to 10 carbon atoms, such
as 6-hydroxy-hex-3-enoyl;
[0087] substituted alkynoyl, having from 1 to 10 carbon atoms, such
as 3-hydroxy-hex-5-ynoyl;
[0088] substituted aroyl, such as 4-chlorobenzoyl or
8-hydroxy-naphth-2-oyl;
[0089] substituted heteroaroyl, such as
2,4-dioxo-1,2,3,4-tetrahydro-3-met- hyl-quinazolin-6-oyl;
[0090] substituted alkylsulfonyl, such as
2-aminoethanesulfonyl;
[0091] substituted arylsulfonyl, such as
5-dimethylamino-1-naphthalenesulf- onyl;
[0092] substituted heteroarylsulfonyl, such as
1-methoxy-6-isoquinolinesul- fonyl;
[0093] carbamoyl or thiocarbamoyl;
[0094] substituted carbamoyl (R'--NH--CO) or substituted
thiocarbamoyl (R'--NH--CS) wherein R' is alkyl, alkenyl, alkynyl,
aryl, heteroaryl, substituted alkyl, substituted alkenyl,
substituted alkynyl, substituted aryl, or substituted
heteroaryl;
[0095] substituted carbamoyl (R'--NH--CO) and substituted
thiocarbamoyl (R'--NH--CS) wherein R' is alkanoyl, alkenoyl,
alkynoyl, aroyl, heteroaroyl, substituted alkanoyl, substituted
alkenoyl, substituted alkynoyl, substituted aroyl, or substituted
heteroaroyl, all as above defined;
[0096] Lys-(Gly).sub.n where n=1 4; or Tyr-(Gly).sub.n where n=1
4.
[0097] The C-terminal capping function can either be in an amide
bond with the terminal carboxyl or in an ester bond with the
terminal carboxyl. Capping functions that provide for an amide bond
are designated as NR.sup.1R.sup.2 wherein R.sup.1 and R.sup.2 may
be independently drawn from the following group:
[0098] hydrogen;
[0099] alkyl, preferably having from 1 to 10 carbon atoms, such as
methyl, ethyl, isopropyl;
[0100] alkenyl, preferably having from 1 to 10 carbon atoms, such
as prop-2-enyl;
[0101] alkynyl, preferably having from 1 to 10 carbon atoms, such
as prop-2-ynyl;
[0102] substituted alkyl having from 1 to 10 carbon atoms, such as
hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl,
halogenoalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl;
[0103] substituted alkenyl having from 1 to 10 carbon atoms, such
as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl,
alkylthioalkenyl, halogenoalkenyl, cyanoalkenyl, aminoalkenyl,
alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl,
carboxyalkenyl, carbamoylalkenyl;
[0104] substituted alkynyl having from 1 to 10 carbon atoms, such
as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl,
alkylthioalkynyl, halogenoalkynyl, cyanoalkynyl, aminoalkynyl,
alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl,
carboxyalkynyl, carbamoylalkynyl;
[0105] aroylalkyl having up to 10 carbon atoms, such as phenacyl or
2-benzoylethyl;
[0106] aryl, such as phenyl or 1-naphthyl;
[0107] heteroaryl, such as 4-quinolyl;
[0108] alkanoyl having from 1 to 10 carbon atoms, such as acetyl or
butyryl;
[0109] aroyl, such as benzoyl;
[0110] heteroaroyl, such as 3-quinoloyl;
[0111] OR' or NR'R" where R' and R" are independently hydrogen,
alkyl, aryl, heteroaryl, acyl, aroyl, sulfonyl, sulfinyl, or
SO.sub.2--R'" or SO--R'" where R'" is substituted or unsubstituted
alkyl, aryl, heteroaryl, alkenyl, or alkynyl.
[0112] Capping functions that provide for an ester bond are
designated as OR, wherein R may be: alkoxy; aryloxy; heteroaryloxy;
aralkyloxy; heteroaralkyloxy; substituted alkoxy; substituted
aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or
substituted heteroaralkyloxy.
[0113] Either the N-terminal or the C-terminal capping function, or
both, may be of such structure that the capped molecule functions
as a prodrug (a pharmacologically inactive derivative of the parent
drug molecule) that undergoes spontaneous or enzymatic
transformation within the body in order to release the active drug
and that has improved delivery properties over the parent drug
molecule (Bundgaard, 1985).
[0114] Judicious choice of capping groups allows the addition of
other activities on the peptide. For example, the presence of a
sulfhydryl group linked to the N- or C-terminal cap will permit
conjugation of the derivatized peptide to other molecules.
[0115] Capping of the peptide is intended primarily to to increase
plasma half life, as has been demonstrated for many peptides (e.g.,
Powell et al., Ann Repts Med. Chem. 28:285-294, 1993). Any capping
group which serves this function is intended. However, the uncapped
form is still useful as a template for peptidomimetic design (see
below) and may have acceptable activity in vitro.
[0116] Production of Peptides and Derivatives
[0117] General Chemical Synthetic Procedures
[0118] The peptides of the invention may be prepared using
recombinant DNA technology. However, given their length, they are
preferably prepared using solid-phase synthesis, such as that
generally described by Merrifield, J. Amer. Chem. Soc., 85:2149-54
(1963), although other equivalent chemical syntheses known in the
art are also useful. Solid-phase peptide synthesis may be initiated
from the C-terminus of the peptide by coupling a protected
.alpha.-amino acid to a suitable resin. Such a starting material
can be prepared by attaching an .alpha.-amino-protected amino acid
by an ester linkage to a chloromethylated resin or to a
hydroxymethyl resin, or by an amide bond to a BHA resin or MBHA
resin.
[0119] The preparation of the hydroxymethyl resin is described by
Bodansky et al., 1966. Chloromethylated resins are commercially
available from BioRad Laboratories, Richmond, Calif. and from Lab.
Systems, Inc. The preparation of such a resin is described by
Stewart et al., 1969. BHA and MBHA resin supports are commercially
available and are generally used only when the desired polypeptide
being synthesized has an unsubstituted amide at the C-terminus.
[0120] The amino acids can be coupled to the growing peptide chain
using techniques well known in the art for the formation of peptide
bonds. For example, one method involves converting the amino acid
to a derivative that will render the carboxyl group of the amino
acid more susceptible to reaction with the free N-terminal amino
group of the growing peptide chain. Specifically, the C-terminal of
the protected amino acid can be converted to a mixed anhydride by
the reaction of the C-terminal with ethyl chloroformate, phenyl
chloroformate, sec-butyl chloroformate, isobutyl chloroformate, or
pivaloyl chloride or the like acid chlorides. Alternatively, the
C-terminal of the amino acid can be converted to an active ester,
such as a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, a
pentafluorophenyl ester, a p-nitrophenyl ester, a
N-hydroxysuccinimide ester, or an ester formed from
1-hydroxybenzotriazole. Another coupling method involves the use of
a suitable coupling agent, such as N,N'-dicyclohexylcarbodiimide or
N,N'-diisopropylcarbodiimide. Other appropriate coupling agents,
apparent to those skilled in the art, are disclosed in Gross et al.
1979, which is hereby incorporated by reference.
[0121] The .alpha.-amino group of each amino acid employed in the
peptide synthesis must be protected during the coupling reaction to
prevent side reactions involving their active .alpha.-amino
function. Certain amino acids contain reactive side-chain
functional groups (e.g., sulfhydryl, amino, carboxyl, and hydroxyl)
and such functional groups must also be protected with suitable
protecting groups to prevent a chemical reaction from occurring at
either (1) the .alpha.-amino group site or (2) a reactive side
chain site during both the initial and subsequent coupling
steps.
[0122] In the selection of a particular protecting group to be used
in synthesizing the peptides, the following general rules are
typically followed. Specifically, an .alpha.-amino protecting group
(1) should render the .alpha.-amino function inert under the
conditions employed in the coupling reaction, (2) should be readily
removable after the coupling reaction under conditions that will
not remove side-chain protecting groups and will not alter the
structure of the peptide fragment, and (3) should substantially
reduce the possibility of racemization upon activation, immediately
prior to coupling.
[0123] On the other hand, a side-chain protecting group (1) should
render the side chain functional group inert under the conditions
employed in the coupling reaction, (2) should be stable under the
conditions employed in removing the .alpha.-amino protecting group,
and (3) should be readily removable from the desired
fully-assembled peptide under reaction conditions that will not
alter the structure of the peptide chain.
[0124] It will be apparent to those skilled in the art that the
protecting groups known to be useful for peptide synthesis vary in
reactivity with the agents employed for their removal. For example,
certain protecting groups, such as triphenylmethyl and
2-(p-biphenyl)isopropyloxycarbonyl, are very labile and can be
cleaved under mild acid conditions. Other protecting groups, such
as t-butyloxycarbonyl (BOC), t-amyloxycarbonyl,
adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, are less
labile and require moderately strong acids for their removal, such
as trifluoroacetic, hydrochloric, or boron trifluoride in acetic
acid. Still other protecting groups, such as benzyloxycarbonyl (CBZ
or Z), halobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl
cycloalkyloxycarbonyl, and isopropyloxycarbonyl, are even less
labile and require even stronger acids, such as hydrogen fluoride,
hydrogen bromide, or boron trifluoroacetate in trifluoroacetic
acid, for their removal. Suitable protecting groups, known in the
art are described in Gross et al. 1981.
[0125] Among the classes of amino acid protecting groups useful for
protecting the .alpha.-amino group or for protecting a side chain
group are included the following.
[0126] (1) For an .alpha.-amino group, three typical classes of
protecting groups are:
[0127] (a) aromatic urethane-type protecting groups, such as
fluorenylmethyloxycarbonyl (FMOC), CBZ, and substituted CBZ, such
as, p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
p-bromobenzyloxycarbonyl, and p-methoxybenzyloxycarbonyl,
o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,
2,6-dichlorobenzyloxycarbonyl, and the like;
[0128] (b) aliphatic urethane-type protecting groups, such as BOC,
t-amyloxycarbonyl, isopropyloxycarbonyl,
2-(p-biphenyl)isopropyloxycarbon- yl, allyloxycarbonyl and the
like; and
[0129] (c) cycloalkyl urethane-type protecting groups, such as
cyclopentyloxycarbonyl, adamantyloxycarbonyl, and
cyclohexyloxycarbonyl.
[0130] The preferred .alpha.-amino protecting groups are BOC and
FMOC.
[0131] (2) For the side chain amino group present in Lys,
protection may be by any of the groups mentioned above in (1) such
as BOC, 2-chlorobenzyloxycarbonyl and the like.
[0132] (3) For the guanidino group of Arg, protection may be
provided by nitro, tosyl, CBZ, adamantyloxycarbonyl,
2,2,5,7,8-pentamethylchroman-6-s- ulfonyl,
2,3,6-trimethyl-4-methoxyphenylsulfonyl, or BOC groups.
[0133] (4) For the hydroxyl group of Ser or Thr, protection may be,
for example, by t-butyl; benzyl (BZL); or substituted BZL, such as
p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and
2,6-dichlorobenzyl.
[0134] (5) For the carboxyl group of Asp or Glu, protection may be,
for example, by esterification using such groups as BZL, t-butyl,
cyclohexyl, cyclopentyl, and the like.
[0135] (6) For the imidazole nitrogen of His, the benzyloxymethyl
(BOM) or tosyl moiety is suitably employed as a protecting
group.
[0136] (7) For the phenolic hydroxyl group of Tyr, a protecting
group such as tetrahydropyranyl, tert-butyl, trityl, BZL,
chlorobenzyl, 4-bromobenzyl, and 2,6-dichlorobenzyl are suitably
employed. The preferred protecting group is
bromobenzyloxycarbonyl.
[0137] (8) For the side chain amino group of Asn or Gln, xanthyl
(Xan) is preferably employed.
[0138] (9) For Met, the amino acid is preferably left
unprotected.
[0139] (10) For the thio group of Cys, p-methoxybenzyl is typically
employed.
[0140] The first C-terminal amino acid of the growing peptide
chain, e.g., Glu, is typically protected at the .alpha.-amino
position by an appropriately selected protecting group such as BOC.
The BOC-Glu-(y-cyclohexyl)-OH can be first coupled to a
benzylhydrylamine resin using isopropylcarbodiimide at about
25.degree. C. for two hours with stirring or to a chloromethylated
resin according to the procedure set forth in Horiki et al., 1978.
Following the coupling of the BOC-protected amino acid to the resin
support, the .alpha.-amino protecting group is usually removed,
typically by using trifluoroacetic acid (TFA) in methylene chloride
or TFA alone. The .alpha.-amino group de-protection reaction can
occur over a wide range of temperatures, but is usually carried out
at a temperature between about 0.degree. C. and room
temperature.
[0141] Other standard .alpha.-amino group de-protecting reagents,
such as HCl in dioxane, and conditions for the removal of specific
.alpha.-amino protecting groups are within the skill of those
working in the art, such as those described in Lubke et al., 1975,
which is hereby incorporated by reference. Following the removal of
the .alpha.-amino protecting group, the unprotected .alpha.-amino
group, generally still side-chain protected, can be coupled in a
stepwise manner in the intended sequence.
[0142] An alternative to the stepwise approach is the fragment
condensation method in which pre-formed peptides of short length,
each representing part of the desired sequence, are coupled to a
growing chain of amino acids bound to a solid phase support. For
this stepwise approach, a particularly suitable coupling reagent is
N,N'-dicyclohexylcarbodiimide or diisopropylcarbodiimide. Also, for
the fragment approach, the selection of the coupling reagent, as
well as the choice of the fragmentation pattern needed to couple
fragments of the desired nature and size are important for success
and are known to those skilled in the art.
[0143] Each protected amino acid or amino acid sequence is usually
introduced into the solid-phase reactor in amounts in excess of
stoichiometric quantities, and the coupling is suitably carried out
in an organic solvent, such as dimethylformamide (DMF),
CH.sub.2Cl.sub.2 or mixtures thereof. If incomplete coupling
occurs, the coupling procedure is customarily repeated before
removal of the N-amino protecting group in preparation for coupling
to the next amino acid. Following the removal of the .alpha.-amino
protecting group, the remaining .alpha.-amino and
side-chain-protected amino acids can be coupled in a stepwise
manner in the intended sequence. The success of the coupling
reaction at each stage of the synthesis may be monitored. A
preferred method of monitoring the synthesis is by the ninhydrin
reaction, as described by Kaiser et al., 1970. The coupling
reactions can also be performed automatically using well-known
commercial methods and devices, for example, a Beckman 990 Peptide
Synthesizer.
[0144] Upon completion of the desired peptide sequence, the
protected peptide must be cleaved from the resin support, and all
protecting groups must be removed. The cleavage reaction and
removal of the protecting groups is suitably accomplished
concomitantly or consecutively with de-protection reactions. When
the bond anchoring the peptide to the resin is an ester bond, it
can be cleaved by any reagent that is capable of breaking an ester
linkage and of penetrating the resin matrix. One especially useful
method is by treatment with liquid anhydrous hydrogen fluoride.
This reagent will usually not only cleave the peptide from the
resin, but will also remove all acid-labile protecting groups and,
thus, will directly provide the fully de-protected peptide. When
additional protecting groups that are not acid-labile are present,
additional de-protection steps must be carried out. These steps can
be performed either before or after the hydrogen fluoride treatment
described above, according to specific needs and circumstances.
[0145] When a chloromethylated resin is used, the hydrogen fluoride
cleavage/de-protection treatment generally results in the formation
of the free peptide acids. When a benzhydrylamine resin is used,
the hydrogen fluoride treatment generally results in the free
peptide amides. Reaction with hydrogen fluoride in the presence of
anisole and dimethylsulfide at 0.degree. C. for one hour will
typically remove the side-chain protecting groups and,
concomitantly, release the peptide from the resin.
[0146] When it is desired to cleave the peptide without removing
protecting groups, the protected peptide-resin can be subjected to
methanolysis, thus yielding a protected peptide in which the
C-terminal carboxyl group is methylated. This methyl ester can be
subsequently hydrolyzed under mild alkaline conditions to give the
free C-terminal carboxyl group. The protecting groups on the
peptide chain can then be removed by treatment with a strong acid,
such as liquid hydrogen fluoride. A particularly useful technique
for methanolysis is that of Moore et al., 1977, in which the
protected peptide-resin is treated with methanol and potassium
cyanide in the presence of a crown ether.
[0147] Other methods for cleaving a protected peptide from the
resin when a chloromethylated resin is employed include (1)
ammoniolysis and (2) hydrazinolysis. If desired, the resulting
C-terminal amide or hydrazide can be hydrolyzed to the free
C-terminal carboxyl moiety, and the protecting groups can be
removed conventionally. The protecting group present on the
N-terminal .alpha.-amino group may be removed either before, or
after, the protected peptide is cleaved from the support.
Purification of the peptides of the invention is typically achieved
using chromatographic techniques, such as preparative HPLC
(including reverse phase HPLC), gel permeation, ion exchange,
partition chromatography, affinity chromatography (including
monoclonal antibody columns), and the like, or other conventional
techniques such as countercurrent distribution or the like.
[0148] Amino Acid Substitution and Addition Variants
[0149] Also included in this invention are peptides in which at
least one amino acid residue and preferably, only one, has been
removed and a different residue inserted in its place. For a
detailed description of protein chemistry and structure, see
Schulz, G. E. et al., Principles of Protein Structure,
Springer-Verlag, New York, 1979, and Creighton, T. E., Proteins:
Structure and Molecular Principles, W. H. Freeman & Co., San
Francisco, 1984, which are hereby incorporated by reference. The
types of substitutions which may be made in the peptide molecule of
the present invention are conservative substitutions and are
defined herein as exchanges within one of the following groups:
[0150] 1. Small aliphatic, nonpolar or slightly polar residues:
e.g., Ala, Ser, Thr, Gly;
[0151] 2. Polar, negatively charged residues and their amides:
e.g., Asp, Asn, Glu, Gln;
[0152] 3. Polar, positively charged residues: e.g., His, Arg,
Lys;
[0153] Pro, because of its unusual geometry, tightly constrains the
chain. Substantial changes in functional properties are made by
selecting substitutions that are less conservative, such as
between, rather than within, the above groups (or two other amino
acid groups not shown above), which will differ more significantly
in their effect on maintaining (a) the structure of the peptide
backbone in the area of the substitution (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. Most substitutions according to the present
invention are those which do not produce radical changes in the
characteristics of the peptide molecule. Even when it is difficult
to predict the exact effect of a substitution in advance of doing
so, one skilled in the art will appreciate that the effect can be
evaluated by routine screening assays, preferably the biological
assays described below. Modifications of peptide properties
including redox or thermal stability, hydrophobicity,
susceptibility to proteolytic degradation or the tendency to
aggregate with carriers or into multimers are assayed by methods
well known to the ordinarily skilled artisan.
[0154] One group of preferred substitution variants of KPSSPPEE
have the Glu at position 7 or 8 (or both) of SEQ ID NO:2 replaced
by one or any two of Gln, Asp or Asn.
[0155] Other derivatives may further include substitution of the
Ser at position 3 or 4 (or both) of SEQ ID NO:2 with one or any two
of the following: Thr, Ala, Gly, hSer or Val.beta.OH.
[0156] Furthermore, the Lys at position 1 of SEQ ID NO:2 may be
replaced by His, Arg, Gln, Orn, Cit or Hci.
[0157] Other derivatives have Pro at position 2, 5 or 6 replaced by
Hyp (hydroxyproline).
[0158] It is noteworthy that any and all combinations of the
foregoing substitutions are within the scope of this invention.
[0159] Also included in this invention are addition variants
wherein two or more residues are added to the C-terminus after Glu
(or after any of its above substituents) in SEQ ID NO:2. These
residues may be Leu-(Gly).sub.n, Ile-(Gly).sub.n, Val-(Gly).sub.n,
Nva-(Gly).sub.n, or Nle-(Gly).sub.n, wherein Nva is norvaline, Nle
is norleucine, and n=1-10.
[0160] Also included in this invention are addition variants
wherein one or more residues is/are added to the N-terminus before
Lys (or any of its above substituents) in SEQ ID NO:2. These
residues may be Gly, Lys-(Gly).sub.n, Tyr-(Gly).sub.n, or
Gly-(Gly).sub.n wherein n=1-10.
[0161] Another preferred derivative of this invention is a 9-mer
addition variant wherein any one of the following amino acids is
added to the C-terminus after Glu (or any of its above
substituents) in SEQ ID NO:2: Leu, Ile, Val, Nva, Nle, Met, Ala, or
Gly.
[0162] In general, preferred peptide addition variants may have up
to about 30 additional amino acids, more preferably about 20, most
preferably 11. The functional limitations placed on the peptide
variant, and the ease by which these activities can be tested using
conventional means, would permit one skilled in the art to
ascertain whether an addition (or any other type of) variant would
affect the peptide's activity. In view of the structural
description provided herein, it would be to determine whether a
peptide variant falls within the scope of this invention.
[0163] Uncapped peptides of any of the foregoing sequences having
free N- and C-termini, for example, uncapped NH.sub.2-KPSSPPEE-OH
[SEQ ID NO:2].
[0164] Chemical Derivatives
[0165] "Chemical derivatives" of KPSSPPEE [SEQ ID NO:2] contain
additional chemical moieties not normally a part of the peptide.
Covalent modifications of the peptide are included within the scope
of this invention. Such modifications may be introduced into the
molecule by reacting targeted amino acid residues of the peptide
with an organic derivatizing agent that is capable of reacting with
selected side chains or terminal residues.
[0166] The capped peptides discussed above are examples of
preferred chemical derivatives of the "natural" uncapped peptide.
Any of the above combination of substitution or addition variants
may be capped with any of the capping groups disclosed herein.
[0167] Other examples of chemical derivatives of the peptide
follow.
[0168] Lysinyl and amino terminal residues are derivatized with
succinic or other carboxylic acid anhydrides. Derivatization with a
cyclic carboxylic anhydride has the effect of reversing the charge
of the lysinyl residues. Other suitable reagents for derivatizing
.alpha.-amino-containing residues include imidoesters such as
methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;
2,4 pentanedione; and transaminase-catalyzed reaction with
glyoxylate.
[0169] Carboxyl side groups, aspartyl or glutamyl, may be
selectively modified by reaction with carbodiimides
(R--N.dbd.C.dbd.N--R') such as
1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues can be converted to asparaginyl and
glutaminyl residues by reaction with ammonia.
[0170] Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, methylation of the amino group of lysine (Creighton,
supra, pp. 79-86), acetylation of the N-terminal amine, and
amidation of the C-terminal carboxyl groups.
[0171] For every single peptide sequence disclosed herein, this
invention includes the corresponding retro-inverso sequence wherein
the direction of the peptide chain has been inverted and wherein
all the amino acids belong to the D-series. For example the
retro-inverso analogue of the natural L-series peptide KPSSPPEE is
EEPPSSPK which is composed of D-series amino acids and in which E
is the N-terminus and K is the C-terminus. For example the
retro-inverso analogue of the natural L-series capped peptide
Ac-KPSSPPEE-Am is Ac-EEPPSSPK-Am which is composed of D-series
amino acids and in which the N-terminal E is acetylated and the
C-terminal K is amidated. The complete range of N-terminal capping
groups and the complete range C-terminal capping groups specified
for the L-series peptides are also intended for the D-series
peptides.
[0172] Also included are peptides wherein one or more D-amino acids
has/have been substituted for one or more L-amino acids.
Additionally, modified amino acids or chemical derivatives of amino
acids may be provided such that the peptide contains additional
chemical moieties or modified amino acids not normally a part of a
natural protein. Such derivatized moieties may improve the
solubility, absorption, biological half life, and the like.
Moieties capable of mediating such effects are disclosed, for
example, in Remington's Pharmaceutical Sciences, 16th ed., Mack
Publishing Co., Easton, Pa. (1980).
[0173] Multimeric Peptides
[0174] The present invention also includes longer peptides in which
the basic peptidic sequence of about 7-9 amino acids is repeated
from about two to about 100 times, with or without intervening
spacers or linkers. A multimer of the peptide KPSSPPEE is shown by
the following formula (KPSSPPEE-X.sub.m).sub.n-KPSSPPEE wherein m=0
or 1, n=1-100. X is a spacer group, preferably C.sub.1-C.sub.20
alkyl, C.sub.1-C.sub.20 alkenyl, C.sub.1-C.sub.20 alkynyl,
C.sub.1-C.sub.20 polyether containing up to 9 oxygen atoms or
Gly.sub.z. (z=1-10).
[0175] It is understood that such multimers may be built from any
of the peptide variants described herein. Moreover, a peptide
multimer may comprise different combinations of peptide monomers,
both KPSSPPEE and the disclosed variants thereof. Such oligomeric
or multimeric peptides can be made by chemical synthesis or by
recombinant DNA techniques as discussed herein. When produced
chemically, the oligomers preferably have from 2-8 repeats of the
basic peptide sequence. When produced recombinantly, the multimers
may have as many repeats as the expression system permits, for
example from two to about 100 repeats.
[0176] Peptidomimetics
[0177] A preferred type of chemical derivative of the peptides
described herein is a peptidomimetic compound which mimics the
biological effect of KPSSPPEE, capped or uncapped. A peptidomimetic
agent may be an unnatural peptide or a non-peptide agent which has
the stereochemical properties of KPSSPPEE, capped or uncapped, such
that it has the binding activity or biological activity of
KPSSPPEE, capped or uncapped. Hence, this invention includes
compounds wherein a peptidomimetic compound is coupled to a
peptide, for example,
[0178] X -PPEE
[0179] wherein X is a peptidomimetic which mimics KPSS; the peptide
portion may include a normal or a retro-inverso sequence.
[0180] Peptidomimetic compounds, either agonists, substrates or
inhibitors, have been described for a number of bioactive peptides
such as opioid peptides, VIP, thrombin, HIV protease, etc. Methods
for designing and preparing peptidomimetic compounds are known in
the art (Kempf D J, Methods Enzymol 241:334-354 (1994); Hruby, V.
J., Biopolymers 33:1073-82 (1993); Wiley, R. A. et al., Med. Res.
Rev. 13:327-384 (1993); Claeson, G., Blood Coagul. Fibrinolysis
5:411-436 (1994), which references are incorporated by reference in
their entirety). These methods are used to prepare capped or
uncapped KPSSPPEE peptidomimetics which possess at least the
binding capacity and specificity of the peptide and preferably also
possess the biological activity. Knowledge of peptide chemistry and
general organic chemistry available to those skilled in the art are
sufficient for the design and testing of such compounds.
[0181] For example, such peptidomimetics may be identified by
inspection of the cystallographically-derived three-dimensional
structure of a peptide of the invention, for example KPSSPPEE,
capped or uncapped, either free or bound in complex with its
receptor(s). Alternatively, the structure of a peptide of the
invention bound to its receptor(s) can be gained by the techniques
of nuclear magnetic resonance spectroscopy. The better knowledge of
the stereochemistry of the interaction of, say, KPSSPPEE, capped or
uncapped, with its receptor will permit the rational design of such
peptidomimetic agents.
[0182] All the foregoing peptides, variants and chemical
derivatives including peptidomimetics and multimeric peptides must
have the biological activity and/or the binding activity of
KPSSPPEE as follows: at least about 20% of the activity of
Ac-KPSSPPE-Am in an in vitro assay of cell invasiveness or an in
vitro assay of endothelial tube formation and/or angiogenesis.
These activities are characterized in greater detail below.
Alternatively, or in addition, the peptide, variant or chemical
derivatives should compete with labeled Ac-KPSSPPEE-Am for binding
to a ligand or binding partner for Ac-KPSSPPEE-Am, whether this be
a cellular receptor (tested in a binding assay with whole cells or
fractions thereof), an isolated receptor or any other
Ac-KPSSPPEE-Am-binding molecule.
[0183] Moreover, the peptides, variants or derivatives of the
present invention do not have biological activities previously
associated with urokinase plasminogen activator (uPA). That is they
do no block the binding of uPA to the uPA receptor. These peptides
lack thrombolytic activity, a hallmark of uPA.
[0184] Additional Discussion of Peptides, Variants and
Peptidomimetics
[0185] Minor modifications of the amino acid sequence might affect
activity if those modifications are selected purely at random.
However, one skilled in the art of peptide and peptidomimetic
design would follow a well-established set of "rules" in creating
useful variants and derivatives. For example, it is expected that
the KPSSPPEE peptide modified by replacing Ser with either Thr,
Ala, or Gly possesses the level of activity disclosed above.
According to Bowie et al. (Science 247:1306-1310, 1990), if a
particular property of a side chain, such as charge or size, is
important at a given position, only side chains that have the
required property will be allowed. Conversely, if the chemical
identity of the side chain is unimportant, then many different
substitutions will be permitted. Studies based on these notions
revealed that proteins are surprisingly tolerant of amino acid
substitutions (Bowie et al., supra at page 1306). Thus the art
recognizes and accepts certain types of changes in proteins and in
peptides. Such acceptable modifications delineate a genus of
peptides wherein each species predictably has the requisite type
and/or level of activity.
[0186] Further, Bordo and Argos, J. Mol. Biol. 217:721-729 (1991),
reported a statistical analysis of protein sequences and provided
guidelines for "safe" amino acid substitutions in protein design,
and by analogy, peptide design. It is axiomatic that proteins with
similar functions are topographically similar at least in those
regions responsible for activity. Based on the fact that the
peptide KPSSPPEE contains 3 prolines out of 8 amino acids, the
present inventors predicted that this peptide would have a single
major conformer in solution, perhaps differing only in proline
isomerization. This was also predicted by molecular dynamics
simulations. Hence, KPSSPPEE would not be expected to assume
multiple conformers in solution. The PP region of this peptide
forms a specialized conformational motif known as a "proline turn"
(or "bend"). The stiffness of this peptide (and its preliminary 3D
structure) has been confirmed by 2D NMR.
[0187] In addition to applying this topographical criterion to the
design and production of peptides with sequence homology or
acceptable sequence substitutions, this criterion can be used as a
basis for generating chemical derivatives of KPSSPPEE, including
the peptidomimetics described above. This is fundamental to
structure-based drug design and modeling. Although the solution
structure of a free peptide may not exactly mimic its bound
conformation, the solution structure does provide a starting
scaffold for optimizing derivatives which mimic the peptide's
activity. In fact, such scaffolds could not be derived in the
absence of the basic topographical information about this peptide,
either free or bound. If a derivative is prepared with a
structure/topography similar to that of KPSSPPEE and the requisite
biological and binding activity as disclosed herein, then it is
within the scope of the present invention.
[0188] If a peptide or peptidomimetic is designed in accordance
with this invention based on either the sequence or the topography
(structure) of KPSSPPEE, and it has the bioactivity stated above,
then it must be similar in conformation to KPSSPPEE and therefore
falls within the scope of the invention. The assessment of activity
in bioassays or binding assays such as those described herein is
routine in the and is the logical way to determine whether a
compound is active. A useful substitution variant, addition variant
or other chemical derivative of KPSSPPEE is a compound that has
been designed based on the sequence or topographical structure of
KPSSPPEE.
[0189] Systematic approaches in the art that allow the optimization
of a peptide and development of peptidomimetics (Hruby et al.,
Biochem J. 268:249-262 (1990); and Hruby, 1993, supra) flow from a
single starting point: the identification of a peptide lead
compound. For example, the most preferred peptide lead compound of
this invention is KPSSPPEE. The peptide and peptidomimetic design
approaches disclosed herein and/or known in the art for generating
an optimized compound are not possible without first identifying an
active lead peptide, so that these designed peptides or
peptidomimetics constitute a genus of compounds "around" the
original peptide. Schemes for preparing active derivatives of the
parent peptide have been described (e.g., Moore et al., Adv.
Pharmacol. 33:91-141 (1995); Giannis and Rubsam, Adv. Drug Research
29:1-78 (1997)). Although each approach may require some
experimentation, it is neither random nor undue. By following
accepted schemes practiced by those skilled in the art, one can
generate families of similarly acting compounds.
[0190] Diagnostic and Prognostic Compositions
[0191] Further, the peptides can be labeled for detection and used,
for example, to detect a binding site for the peptide on the
surface or in the interior of a cell. Thus, the fate of the peptide
can be followed in vitro or in vivo by using the appropriate method
to detect the label. The labeled peptide may also be utilized in
vivo for diagnosis and prognosis, for example to image occult
metastatic foci or for other types of in situ evaluations.
[0192] Example of suitable detectable labels are radioactive,
fluorogenic, chromogenic, or other chemical labels. Useful
radiolabels, which are detected by a gamma counter or a
scintillation counter or by autoradiography include .sup.3H,
.sup.125I, .sup.131I, .sup.35S and .sup.14C. In addition, .sup.131I
is also useful as a therapeutic isotope (see below).
[0193] Common fluorescent labels include fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine.
[0194] The fluorophore, such as the dansyl group, must be excited
by light of a particular wavelength to fluoresce. See, for example,
Haugland, Handbook of Fluorescent Probes and Research Chemicals,
Sixth Edition, Molecular Probes, Eugene, Oreg., 1996). In general,
a fluorescent reagent is selected based on its ability to react
readily with an amino function. Examples of such fluorescent probes
include the Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)
fluorophores which span the visible spectrum (U.S. Pat. Nos.
4,774,339; 5,187,288; 5,248,782; 5,274,113; 5,433,896; 5,451,663).
A preferred member of this group is
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid.
[0195] Fluorescein, fluorescein derivatives and fluorescein-like
molecules such as Oregon Green.TM. and its derivatives, Rhodamine
Green.TM. and Rhodol Green.TM., are coupled to amine groups using
the isocyanate, succinimidyl ester or dichlorotriazinyl-reactive
groups. The long wavelength rhodamines, which are basically
Rhodamine Green.TM. derivatives with substituents on the nitrogens,
are among the most photostable fluorescent labeling reagents known.
Their spectra are not affected by changes in pH between 4 and 10,
an important advantage over the fluoresceins for many biological
applications. This group includes the tetramethylrhodamines,
X-rhodamines and Texas Red derivatives. Other preferred
fluorophores for derivatizing the peptide according to this
invention are those which are excited by ultraviolet light.
Examples include cascade blue, coumarin derivatives, naphthalenes
(of which dansyl chloride is a member), pyrenes and pyridyloxazole
derivatives.
[0196] In yet another approach, one or more amino groups is allowed
to react with reagents that yield fluorescent products, for
example, fluorescamine, dialdehydes such as o-phthaldialdehyde,
naphthalene-2,3-dicarboxylate and anthracene-2,3-dicarboxylate.
7-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives, both chloride and
fluoride, are useful to modify amines to yield fluorescent
products.
[0197] Those skilled in the art will recognize that known
fluorescent reagents modify groups other than amines, such as
thiols, alcohols, aldehydes, ketones, carboxylic acids and amides.
Hence, fluorescent substrates can readily be designed and
synthesized using these other reactive groups.
[0198] The peptide can also be labeled for detection using
fluorescence-emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the peptide
using such metal chelating groups as diethylenetriaminepentaacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The peptide
can be made detectable by coupling it to a chemiluminescent
compound. The presence of the chemiluminescent-tagged peptide is
then determined by detecting the presence of luminescence that
arises during the course of a chemical reaction. Examples of
particularly useful chemiluminescers are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate
ester. Likewise, a bioluminescent compound may be used to label the
peptide. Bioluminescence is a type of chemiluminescence found in
biological systems in which a catalytic protein increases the
efficiency of the chemiluminescent reaction. The presence of a
bioluminescent protein is determined by detecting the presence of
luminescence. Important bioluminescent compounds for purposes of
labeling are luciferin, luciferase and aequorn.
[0199] In yet another embodiment, colorimetric detection is used,
based on chromogenic compounds (chromophores) with high extinction
coefficients.
[0200] In situ detection of the labeled peptide may be accomplished
by removing a histological specimen from a subject and examining it
by microscopy under appropriate conditions to detect the label.
Those of ordinary skill will readily perceive that any of a wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection.
[0201] The term "diagnostically labeled" means that the peptide has
attached to it a diagnostically detectable label. There are many
different labels and methods of labeling known to those of ordinary
skill in the art. Examples of the types of labels which can be used
in the present invention include radioactive isotopes, paramagnetic
isotopes, and compounds which can be imaged by positron emission
tomography (PET). Those of ordinary skill in the art will know of
other suitable labels for binding to the peptides used in the
invention, or will be able to ascertain such, by routine
experimentation. Furthermore, the binding of these labels to the
peptide or derivative can be done using standard techniques known
to those of ordinary skill in the art.
[0202] For diagnostic in vivo radioimaging, the type of detection
instrument available is a major factor in selecting a given
radionuclide. The radionuclide chosen must have a type of decay
which is detectable by a given type of instrument. In general, any
conventional method for visualizing diagnostic imaging can be
utilized in accordance with this invention. Another factor in
selecting a radionuclide for in vivo diagnosis is that the
half-life of a radionuclide be long enough so that it is still
detectable at the time of maximum uptake by the target issue, but
short enough so that deleterious radiation of the host is
minimized. In one preferred embodiment, a radionuclide used for in
vivo imaging does not emit particles, but produces a large number
of photons in a 140-200 keV range, which may be readily detected by
conventional gamma cameras.
[0203] For in vivo diagnosis, radionuclides may be bound to peptide
either directly or indirectly by using an intermediary functional
group. Intermediary functional groups that are often used to bind
radioisotopes, which exist as metallic ions, to peptides are the
chelating agents, DTPA and EDTA. Examples of metallic ions which
can be bound to peptides are .sup.99Tc, .sup.123I, .sup.111In,
.sup.131I, .sup.97Ru, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.72As,
.sup.89Zr, and .sup.201Tl. Generally, the dosage of peptide labeled
for detection for diagnostic use will vary depending on
considerations such as age, condition, sex, and extent of disease
in the patient, counterindications, if any, and other variables, to
be adjusted by the individual physician. Dosage can vary from 0.01
mg/kg to 100 mg/kg.
[0204] In another embodiment, the peptides or derivatives of the
present invention are used as affinity ligands for binding the
peptide's receptor in assays, preparative affinity chromatography
or solid phase separation. Such compositions may also be used to
enrich, purify or isolate cells to which the peptide or derivative
binds, preferably through a specific receptor-ligand interaction.
The peptide or derivative is immobilized using common methods known
in the art, e.g. binding to CNBr-activated Sepharose.RTM. or
Agarose.RTM., NHS-Agarose.RTM. or Sepharose.RTM., epoxy-activated
Sepharose.RTM. or Agarose.RTM., EAH-Sepharose.RTM. or Agarose.RTM.,
streptavidin-Sepharose.RTM. or Agarose.RTM. in conjunction with
biotinylated peptide or derivatives. In general the peptides or
derivatives of the invention may be immobilized by any other method
which is capable of immobilizing these compounds to a solid phase
for the indicated purposes. See, for example Affinity
Chromatography: Principles and Methods (Pharmacia LKB
Biotechnology). Thus, one embodiment is a composition comprising
any of the peptides, derivatives or peptidomimetics described
herein, bound to a solid support or a resin. The compound may be
bound directly or via a spacer, preferably an aliphatic chain
having about 2-12 carbon atoms.
[0205] By "solid phase" or "solid support" or "carrier" is intended
any support or carrier capable of binding the peptide or
derivative. Well-known supports, or carriers, in addition to
Sepharose.RTM. or Agarose.RTM. described above are glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,
natural and modified celluloses such as nitrocellulose,
polyacrylamides, polyvinylidene difluoride, other agaroses, and
magnetite, including magnetic beads. The carrier can be totally
insoluble or partially soluble. The support material may have any
possible structural configuration so long as the coupled molecule
is capable of binding to receptor material. Thus, the support
configuration may be spherical, as in a bead, or cylindrical, as in
the inside surface of a test tube or microplate well, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, bottom surface of a microplate well,
etc.
[0206] Antibodies and Their Uses
[0207] The present invention also provides antibodies specific for
an epitope defined by the peptide sequence KPSSPPEE or specific for
a chemical derivative thereof or a peptidomimetic thereof. Such
antibodies may be polyclonal, monoclonal, bispecific, chimeric or
antiidiotypic, and include antigen-binding fragments thereof. Any
immunoassay known in the art may be used to detect the binding of
such an antibody to a peptide, chemical derivative thereof or
peptide oligomer according to this invention. Preferred assays are
enzyme immunoassays or radioimmunoassay. The following references
(incorporated by reference in their entirety) describe the
production, purification, testing and use of antibodies: Hartlow,
E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988; Campbell, A., In:
Laboratory Techniques in Biochemistry and Molecular Biology, Volume
13 (Burdon, R., et al., eds.), Elsevier, Amsterdam (1984)); Work,
T. S. et al., Laboratory Techniques and Biochemistry in Molecular
Biology, North Holland Publishing Company, NY, 1978; Weintraub, B.,
Principles of Radioimmunoassays, Seventh Training Course on
Radioligand Assay Techniques, The Endocrine Society, March, 1986;
Butler, J. E. (ed.), Immunochemistry of Solid-Phase Immunoassay,
CRC Press, Boca Raton, 1991; Butler, J. E., In: STRUCTURE OF
ANTIGENS, Vol. 1, Van Regenmortel, M., ed., CRC Press, Boca Raton
1992, pp. 209-259; Butler, J. E., In: van Oss, C. J. et al., (eds),
IMMUNOCHEMISTRY, Marcel Dekker, Inc., New York, 1994, pp. 759-803;
Voller, A. et al. (eds)., Immunoassays for the 1980's, University
Park Press, Baltimore, 1981.
[0208] Antibodies of this invention are used to detect the presence
of or measure the amount of the peptide epitope in a biological
material or other sample by direct or competitive immunoassay. The
antibodies can be coupled to a solid support and used in affinity
chromatography to isolate and purify material containing the
peptide epitope. Conversely, as described above, the peptide,
variant or chemical derivative of this invention, bound to a solid
support, is used to enrich or purify specific antibodies.
Antiidiotypic antibodies can be used to gain a knowledge of the
structure of a peptide, variant or chemical derivative of this
invention when bound to a receptor for it.
[0209] Biological Assay of Anti-Invasive Activity
[0210] The compositions of the invention are tested for their
anti-invasive capacity in a Matrigel.RTM. invasion assay system as
described in detail by Kleinman et al., 1986 and Parish et al.,
1992, which references are hereby incorporated by reference in
their entirety. The assay is performed with a cell line, more
preferably a tumor cell line, most preferably the rat breast cancer
(Mat BIII) line or the human prostate cancer (PC-3) line (Xing and
Rabbani, 1996; Hoosein et al., 1991).
[0211] Matrigel.RTM. is a reconstituted basement membrane
containing type IV collagen, laminin, heparan sulfate proteoglycans
such as perlecan, which bind to and localize bFGF, vitronectin as
well as transforming growth factor-.beta. (TGF.beta.),
urokinase-type plasminogen activator (uPA), tissue plasminogen
activator (tPA), and the serpin known as plasminogen activator
inhibitor type 1 (PAI-1) (Chambers et al., 1995).
[0212] It is accepted in the art that results obtained in this
assay for compounds which target extracellular receptors or enzymes
are predictive of the efficacy of these compounds in vivo (Rabbani
et al., 1995).
[0213] Biological Assay of Anti-Angiogenic Activity
[0214] The compounds of this invention are tested for their
anti-angiogenic activity in one of two different assay systems in
vitro.
[0215] Endothelial cells, for example, human umbilical vein
endothelial cells (HUVEC) or human microvascular endothelial cells
(HMVEC) which can be prepared or obtained commercially, are mixed
at a concentration of 2.times.10.sup.5 cells/mL with fibrinogen (5
mg/mL in phosphate buffered saline (PBS) in a 1:1 (v/v) ratio.
Thrombin is added (5 units/mL final concentration) and the mixture
is immediately transferred to a 24-well plate (0.5 mL per well).
The fibrin gel is allowed to form and then VEGF and bFGF are added
to the wells (each at 5 ng/mL final concentration) along with the
test compound. The cells are incubated at 37.degree. C. in 5%
CO.sub.2 for 4 days at which time the cells in each well are
counted and classified as either rounded, elongated with no
branches, elongated with one branch, or elongated with 2 or more
branches. Results are expressed as the average of 5 different wells
for each concentration of compound. Typically, in the presence of
angiogenic inhibitors, cells remain either rounded or form
undifferentiated tubes (e.g. 0 or 1 branch).
[0216] This assay is recognized in the art to be predictive of
angiogenic (or anti-angiogenic) efficacy in vivo (Min et al.,
1996).
[0217] In an alternate assay, endothelial cell tube formation is
observed when endothelial cells are cultured on Matrigel.RTM.
(Schnaper et al., 1995). Endothelial cells (1.times.10.sup.4
cells/well) are transferred onto Matrigel.RTM.-coated 24-well
plates, and tube formation is quantitated after 48 hrs. Inhibitors
are tested by adding them either at the same time as the
endothelial cells or at various time points thereafter.
[0218] This assay models angiogenesis by presenting to the
endothelial cells a particular type of basement membrane, namely
the layer of matrix which migrating and differentiating endothelial
cells might be expected to first encounter. In addition to bound
growth factors, the matrix components found in Matrigel.RTM. (and
in basement membranes in situ) or proteolytic products thereof may
also be stimulatory for endothelial cell tube formation which makes
this model complementary to the fibrin gel angiogenesis model
previously described (Blood and Zetter, 1990; Odedra and Weiss,
1991). The compounds of this invention inhibit endothelial cell
tube formation in both assays, which suggests that the compounds
will also have anti-angiogenic activity.
[0219] In vivo Testing of Compositions in Animal Models of Human
Tumors
[0220] The peptides, peptidomimetics and conjugates are tested for
therapeutic efficacy in several well established rodent models
which are considered to be highly representative of a broad
spectrum of human tumors. The approaches are described in detail in
Geran, R. I. et al., "Protocols for Screening Chemical Agents and
Natural Products Against Animal Tumors and Other Biological Systems
(Third Edition)", Canc. Chemother. Reports, Part 3, 3:1-112, which
is hereby incorporated by reference in its entirety. All general
test evaluation procedures, measurements and calculations are
performed in accordance with this reference, including mean
survival time, median survival time, calculation of approximate
tumor weight from measurement of tumor diameters with vernier
calipers; calculation of tumor diameters; calculation of mean tumor
weight from individual excised tumors; and ratios between treated
and control groups ratio for any measure (T/C ratios).
[0221] A. Rat Model of Tumor Progression
[0222] The effects of the compounds are tested on tumor progression
in a rat syngeneic model of breast cancer (Xing and Rabbani, 1996).
Mat Bill rat breast tumor cells (1.times.10.sup.6 cells in PBS, 0.1
mL per rat) are inoculated into the mammary fat pads of female
Fisher rats. The test compound is dissolved in PBS (200 mM stock),
sterile filtered and dispensed in vivo at a dose of up to about 100
mg/kg/day) using a 14-day Alza osmotic mini-pump implanted
intraperitoneally at the time of inoculation. Control animals
receive vehicle (PBS) alone. Animals are euthanized at day 14 and
examined for metastasis in the spleen, lungs, liver, kidney and
lymph nodes. In addition, the primary tumors are excised,
quantitated, and prepared for immunohistochemistry.
[0223] B. 3LL Lewis Lung Carcinoma: Primary Tumor Growth
[0224] This tumor line arose spontaneously in 1951 as carcinoma of
the lung in a C57BL/6 mouse (Cancer Res 15:39, 1955. See, also
Malave, I. et al., J. Nat'l. Canc. Inst. 62:83-88 (1979)). It is
propagated by passage in C57BL/6 mice by subcutaneous (sc)
inoculation and is tested in semiallogeneic C57BL/6.times.DBA/2
F.sub.1 mice or in allogeneic C3H mice. Typically six animals per
group for subcutaneously (sc) implant, or ten for intramuscular
(im) implant are used. Tumor may be implanted sc as a 2-4 mm
fragment, or im or sc as an inoculum of suspended cells of about
0.5-2.times.10.sup.6-cells. Treatment begins 24 hours after implant
or is delayed until a tumor of specified size (usually
approximately 400 mg) can be palpated. The test compound is
administered ip daily for 11 days
[0225] Animals are followed by weighing, palpation, and measurement
of tumor size. Typical tumor weight in untreated control recipients
on day 12 after im inoculation is 500-2500 mg. Typical median
survival time is 18-28 days. A positive control compound, for
example cyclophosphamide at 20 mg/kg/injection per day on days 1-11
is used. Results computed include mean animal weight, tumor size,
tumor weight, survival time For confirmed therapeutic activity, the
test composition should be tested in two multi-dose assays.
[0226] C. 3LL Lewis Lung Carcinoma: Primary Growth and Metastasis
Model
[0227] This model has been utilized by a number of investigators.
See, for example, Gorelik, E. et al., J. Nat'l. Canc. Inst.
65:1257-1264 (1980); Gorelik, E. et al., Rec. Results Canc. Res.
75:20-28 (1980); Isakov, N. et al., Invasion Metas. 2:12-32 (1982);
Talmadge J. E. et al., J. Nat'l. Canc. Inst. 69:975-980 (1982);
Hilgard, P. et al., Br. J. Cancer 35:78-86(1977)). Test mice are
male C57BL/6 mice, 2-3 months old. Following sc, im, or
intra-footpad implantation, this tumor produces metastases,
preferentially in the lungs. With some lines of the tumor, the
primary tumor exerts anti-metastatic effects and must first be
excised before study of the metastatic phase (see also U.S. Pat.
No. 5,639,725).
[0228] Single-cell suspensions are prepared from solid tumors by
treating minced tumor tissue with a solution of 0.3% trypsin. Cells
are washed 3 times with PBS (pH 7.4) and suspended in PBS.
Viability of the 3LL cells prepared in this way is generally about
95-99% (by trypan blue dye exclusion). Viable tumor cells
(3.times.10.sup.4-5.times.10.sup.6) suspended in 0.05 ml PBS are
injected subcutaneously, either in the dorsal region or into one
hind foot pad of C57BL/6 mice. Visible tumors appear after 3-4 days
after dorsal sc injection of 10.sup.6 cells. The day of tumor
appearance and the diameters of established tumors are measured by
caliper every two days.
[0229] The treatment is given as one or two doses of peptide or
derivative, per week. In another embodiment, the peptide is
delivered by osmotic minipump.
[0230] In experiments involving tumor excision of dorsal tumors,
when tumors reach about 1500 mm.sup.3 in size, mice are randomized
into two groups: (1) primary tumor is completely excised; or (2)
sham surgery is performed and the tumor is left intact. Although
tumors from 500-3000 mm.sup.3 inhibit growth of metastases, 1500
mm.sup.3 is the largest size primary tumor that can be safely
resected with high survival and without local regrowth. After 21
days, all mice are sacrificed and autopsied.
[0231] Lungs are removed and weighed. Lungs are fixed in Bouin's
solution and the number of visible metastases is recorded. The
diameters of the metastases are also measured using a binocular
stereoscope equipped with a micrometer-containing ocular under
8.times. magnification. On the basis of the recorded diameters, it
is possible to calculate the volume of each metastasis. To
determine the total volume of metastases per lung, the mean number
of visible metastases is multiplied by the mean volume of
metastases. To further determine metastatic growth, it is possible
to measure incorporation of .sup.125IdUrd into lung cells (Thakur,
M. L. et al., J. Lab. Clin. Med. 89:217-228 (1977). Ten days
following tumor amputation, 25 .mu.g of fluorodeoxyuridine is
inoculated into the peritoneums of tumor-bearing (and, if used,
tumor-resected mice). After 30 min, mice are given 1 .mu.Ci of
.sup.125IdUrd (iododeoxyuridine). One day later, lungs and spleens
are removed and weighed, and a degree of .sup.125IdUrd
incorporation is measured using a gamma counter.
[0232] In mice with footpad tumors, when tumors reach about 8-10 mm
in diameter, mice are randomized into two groups: (1) legs with
tumors are amputated after ligation above the knee joints; or (2)
mice are left intact as nonamputated tumor-bearing controls.
(Amputation of a tumor-free leg in a tumor-bearing mouse has no
known effect on subsequent metastasis, ruling out possible effects
of anesthesia, stress or surgery). Mice are killed 10-14 days after
amputation. Metastases are evaluated as described above.
[0233] Statistics: Values representing the incidence of metastases
and their growth in the lungs of tumor-bearing mice are not
normally distributed. Therefore, non-parametric statistics such as
the Mann-Whitney U-Test may be used for analysis.
[0234] Study of this model by Gorelik et al. (1980, supra) showed
that the size of the tumor cell inoculum determined the extent of
metastatic growth. The rate of metastasis in the lungs of operated
mice was different from primary tumor-bearing mice. Thus in the
lungs of mice in which the primary tumor had been induced by
inoculation of larger doses of 3LL cells (1-5.times.10.sup.6)
followed by surgical removal, the number of metastases was lower
than that in nonoperated tumor-bearing mice, though the volume of
metastases was higher than in the nonoperated controls. Using
.sup.125IdUrd incorporation as a measure of lung metastasis, no
significant differences were found between the lungs of
tumor-excised mice and tumor-bearing mice originally inoculated
with 1.times.10.sup.6 3LL cells. Amputation of tumors produced
following inoculation of 1.times.10.sup.5 tumor cells dramatically
accelerated metastatic growth. These results were in accord with
the survival of mice after excision of local tumors. The phenomenon
of acceleration of metastatic growth following excision of local
tumors had been repeatedly observed (for example, see U.S. Pat. No.
5,639,725). These observations have implications for the prognosis
of patients who undergo cancer surgery.
[0235] D. Experimental Metastasis Models
[0236] The compounds of this invention are also tested for
inhibition of late metastasis using an experimental metastasis
model (Crowley et al., 1993). Late metastasis involves the steps of
attachment and extravasation of tumor cells, local invasion,
seeding, proliferation and angiogenesis.
[0237] Human prostatic carcinoma cells (PC-3) transfected with a
reporter gene, preferably the green fluorescent protein (GFP) gene,
but as an alternative with a gene encoding the enzymes
chloramphenicol acetyl-transferase (CAT), luciferase or LacZ. This
permits utilization of either of these markers (fluorescence
detection of GFP or histochemical colorimetric detection of
enzymatic activity) for following the fate of these cells. Cells
are injected, preferably iv, and metastases identified after about
14 days, particularly in the lungs but also in regional lymph
nodes, femurs and brain. This mimics the organ tropism of naturally
occurring metastases of prostate cancer. For example,
GFP-expressing PC-3 cells (1.times.10.sup.6 cells per mouse) are
injected iv into the tail veins of nude (nu/nu) mice. Animals are
also implanted with mini-pumps (sub-dermally on the back)
dispensing either the test compound (at least about 100 mg/kg/day)
or vehicle. The animals are euthanized after 14 days and their
organs prepared for histological examination. Single metastatic
cells and foci are visualized and quantitated by fluorescence
microscopy or light microscopic histochemistry or by grinding the
tissue and quantitative colorimetric assay of the detectable
label.
[0238] For a compound to be useful in accordance with this
invention, it should demonstrate anti-tumor activity in the above
models, for example, blocking tumor progression, angiogenesis
and/or metastasis.
[0239] Angiogenesis
[0240] Angiogenesis is measured by determining microvessel density
using immunostaining for CD31 (also known as platelet-endothelial
cell adhesion molecule or PECAM). Results are reported as the
average microvessel density of 5 fields each from 5 different
sections (Penfold et al., 1996). Typically, the whole tumor is
excised, sectioned and the sections examined histologically for
microvessel density using appropriate stains or labels for other
markers.
[0241] Pharmaceutical and Therapeutic Compositions and Their
Administration
[0242] The compounds that may be employed in the pharmaceutical
compositions of the invention include all of those compounds
described above, as well as the pharmaceutically acceptable salts
of these compounds. Pharmaceutically acceptable acid addition salts
of the compounds of the invention containing a basic group are
formed where appropriate with strong or moderately strong,
non-toxic, organic or inorganic acids in the presence of a basic
amine by methods known to the art. Exemplary of the acid addition
salts that are included in this invention are maleate, fumarate,
lactate, oxalate, methanesulfonate, ethanesulfonate,
benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide,
sulfate, phosphate and nitrate salts.
[0243] Pharmaceutically acceptable base addition salts of compounds
of the invention containing an acidic group are prepared by known
methods from organic and inorganic bases and include, for example,
nontoxic alkali metal and alkaline earth bases, such as calcium,
sodium, potassium and ammonium hydroxide; and nontoxic organic
bases such as triethylamine, butylamine, piperazine, and
tri(hydroxymethyl)methylamine.
[0244] As stated above, the compounds of the invention possess the
ability to inhibit invasiveness or angiogenesis, properties that
are exploited in the treatment of cancer, in particular metastatic
cancer. A composition of this invention may be active per se, or
may act as a "pro-drug" that is converted in vivo to the active
form.
[0245] The compounds of the invention, as well as the
pharmaceutically acceptable salts thereof, may be incorporated into
convenient dosage forms, such as capsules, impregnated wafers,
tablets or injectable preparations. Solid or liquid
pharmaceutically acceptable carriers may be employed.
[0246] Preferably, the compounds of the invention are administered
systemically, e.g., by injection. When used, injection may be by
any known route, preferably intravenous, subcutaneous,
intramuscular, intracranial or intraperitoneal. Injectables can be
prepared in conventional forms, either as solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions.
[0247] Solid carriers include starch, lactose, calcium sulfate
dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin,
acacia, magnesium stearate and stearic acid. Liquid carriers
include syrup, peanut oil, olive oil, saline, water, dextrose,
glycerol and the like. Similarly, the carrier or diluent may
include any prolonged release material, such as glyceryl
monostearate or glyceryl distearate, alone or with a wax. When a
liquid carrier is used, the preparation may be in the form of a
syrup, elixir, emulsion, soft gelatin capsule, sterile injectable
liquid (e.g., a solution), such as an ampoule, or an aqueous or
nonaqueous liquid suspension. A summary of such pharmaceutical
compositions may be found, for example, in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton Pa.
(Gennaro 18th ed. 1990).
[0248] The pharmaceutical preparations are made following
conventional techniques of pharmaceutical chemistry involving such
steps as mixing, granulating and compressing, when necessary for
tablet forms, or mixing, filling and dissolving the ingredients, as
appropriate, to give the desired products for oral, parenteral,
topical, transdermal, intravaginal, intranasal, intrabronchial,
intracranial, intraocular, intraaural and rectal administration.
The pharmaceutical compositions may also contain minor amounts of
nontoxic auxiliary substances such as wetting or emulsifying
agents, pH buffering agents and so forth.
[0249] Though the preferred routes of administration are systemic
the pharmaceutical composition may be administered topically or
transdermally, e.g., as an ointment, cream or gel; orally;
rectally; e.g., as a suppository, parenterally, by injection or
continuously by infusion; intravaginally; intranasally;
intrabronchially; intracranially intra-aurally; or
intraocularly.
[0250] For topical application, the compound may be incorporated
into topically applied vehicles such as a salve or ointment. The
carrier for the active ingredient may be either in sprayable or
nonsprayable form. Non-sprayable forms can be semi-solid or solid
forms comprising a carrier indigenous to topical application and
having a dynamic viscosity preferably greater than that of water.
Suitable formulations include, but are not limited to, solution,
suspensions, emulsions, creams, ointments, powders, liniments,
salves, and the like. If desired, these may be sterilized or mixed
with auxiliary agents, e.g., preservatives, stabilizers, wetting
agents, buffers, or salts for influencing osmotic pressure and the
like. Preferred vehicles for non-sprayable topical preparations
include ointment bases, e.g., polyethylene glycol-1000 (PEG-1000);
conventional creams such as HEB cream; gels; as well as petroleum
jelly and the like.
[0251] Also suitable for topic application are sprayable aerosol
preparations wherein the compound, preferably in combination with a
solid or liquid inert carrier material, is packaged in a squeeze
bottle or in admixture with a pressurized volatile, normally
gaseous propellant. The aerosol preparations can contain solvents,
buffers, surfactants, perfumes, and/or antioxidants in addition to
the compounds of the invention.
[0252] For the preferred topical applications, especially for
humans, it is preferred to administer an effective amount of the
compound to an infected area, e.g., skin surface, mucous membrane,
eyes, etc. This amount will generally range from about 0.001 mg to
about 1 g per application, depending upon the area to be treated,
the severity of the symptoms, and the nature of the topical vehicle
employed.
[0253] The compositions of the invention may further comprise one
or more additional compounds that are anti-tumor agents, such as
mitotic inhibitors, e.g., vinblastine; alkylating agents, e.g.,
cyclophosphamide; folate inhibitors, e.g., methotrexate, piritrexim
or trimetrexate; antimetabolites, e.g., 5-fluorouracil and cytosine
arabinoside; intercalating antibiotics, e.g., adriamycin and
bleomycin; enzymes or enzyme inhibitors, e.g., asparaginase;
topoisomerase inhibitors, e.g., etoposide; or biological response
modifiers, e.g., interferon. In fact, pharmaceutical compositions
comprising any known cancer therapeutic in combination with the
peptides disclosed herein are within the scope of this
invention.
[0254] The composition of the invention may also comprise one or
more other medicaments, preferably anti-infectives such as
antibacterial, anti-fungal, anti-parasitic, anti-viral, and
anti-coccidial agents. Exemplary antibacterial agents include, for
example, sulfonamides such as sulfamethoxazole, sulfadiazine or
sulfadoxine; DHFR inhibitors such as trimethoprim, bromodiaprim or
trimetrexate; penicillins; cephalosporins; aminoglycosides;
bacteriostatic inhibitors of protein synthesis; the
quinolonecarboxylic acids and their fused isothiazole analogs; and
the like.
[0255] Other Therapeutic Compositions
[0256] In another embodiment, the compounds of this invention are
"therapeutically conjugated" and used to deliver a therapeutic
agent to the site of where the compounds home and bind, such as
sites of tumor metastasis or foci of infection/inflammation. The
term "therapeutically conjugated" means that the compound,
preferably a peptide, peptide derivative, or peptidomimetic, is
conjugated to a therapeutic agent. The therapeutic agents used in
this manner act are directed either to the underlying cause or the
components of the processes of tumor invasion, angiogenesis or
inflammation. Examples of agents used to treat inflammation are the
steroidal and non-steroidal anti-inflammatory drugs, many of which
inhibit prostaglandin synthesis.
[0257] Other therapeutic agents which can be coupled to the
compounds according to the method of the invention are drugs,
radioisotopes, lectins and other toxins. The therapeutic dosage
administered is an amount which is therapeutically effective, and
will be known to one of skill in the art. The dose is also
dependent upon the age, health, and weight of the recipient, kind
of concurrent treatment, if any, frequency of treatment, and the
nature of the effect desired, such as, for example,
anti-inflammatory effects or anti-bacterial effect.
[0258] Lectins are proteins, commonly derived from plants, that
bind to carbohydrates. Among other activities, some lectins are
toxic. Some of the most cytotoxic substances known are protein
toxins of bacterial and plant origin (Frankel, A. E. et al., Ann.
Rev. Med. 37:125-142 (1986)). These molecules binding the cell
surface and inhibition cellular protein synthesis. The most
commonly used plant toxins are ricin and abrin; the most commonly
used bacterial toxins are diphtheria toxin and Pseudomonas exotoxin
A. In ricin and abrin, the binding and toxic functions are
contained in two separate protein subunits, the A and B chains. The
ricin B chain binds to the cell surface carbohydrates and promotes
the uptake of the A chain into the cell. Once inside the cell, the
ricin A chain inhibits protein synthesis by inactivating the 60S
subunit of the eukaryotic ribosome Endo, Y. et al., J. Biol. Chem.
262: 5908-5912 (1987)). Other plant derived toxins, which are
single chain ribosomal inhibitory proteins, include pokeweed
antiviral protein, wheat germ protein, gelonin, dianthins,
momorcharins, trichosanthin, and many others (Strip, F. et al.,
FEBS Lett. 195:1-8 (1986)). Diphtheria toxin and Pseudomonas
exotoxin A are also single chain proteins, and their binding and
toxicity functions reside in separate domains of the same protein
chain with full toxin activity requiring proteolytic cleavage
between the two domains. Pseudomonas exotoxin A has the same
catalytic activity as diphtheria toxin. Ricin has been used
therapeutically by binding its toxic .alpha.-chain, to targeting
molecules such as antibodies to enable site-specific delivery of
the toxic effect. Bacterial toxins have also been used as
anti-tumor conjugates. As intended herein, a toxic peptide chain or
domain is bound to a compound of this invention and delivered in a
site-specific manner to a target site where the toxic activity is
desired, such as a metastatic focus. Conjugation of toxins to
protein such as antibodies or other ligands are known in the art
(Olsnes, S. et al., Immunol. Today 10:291-295 (1989); Vitetta, E.
S. et al., Ann. Rev. Immunol. 3:197-212 (1985)).
[0259] Examples of therapeutic radioisotopes which can be bound to
the compound for use in accordance with according the methods of
the invention, are .sup.125I, .sup.131I, .sup.90Y, .sup.67Cu,
.sup.217Bi, .sup.211At, .sup.212Pb, .sup.47Sc, and .sup.109Pd.
[0260] Cytotoxic drugs that interfere with critical cellular
processes including DNA, RNA, and protein synthesis, have been
conjugated to antibodies and subsequently used for in vivo therapy.
Such drugs, including but are not limited to daunorubicin,
doxorubicin, methotrexate, and Mitomycin C are also coupled to the
compounds of this invention and use therapeutically in this
form.
[0261] Therapeutic Methods
[0262] This invention includes methods for inhibiting cellular
invasion, chiefly by tumor cells, or angiogenesis, primarily
induced by tumor cells in a subject. By inhibiting invasion by
cells or angiogenesis, the method results in inhibition of tumor
metastasis. In this method, a vertebrate subject, preferably a
mammal, more preferably a human, is administered an amount of the
compound effective to inhibit invasion or angiogenesis. The
compound or pharmaceutically acceptable salt thereof is preferably
administered in the form of a pharmaceutical composition as
described above.
[0263] Doses of the compounds preferably include pharmaceutical
dosage units comprising an effective amount of the peptide. By an
effective amount is meant an amount sufficient to achieve a steady
state concentration in vivo which results in a measurable reduction
in any relevant parameter of disease and may include growth of
primary or metastatic tumor, any accepted index of inflammatory
reactivity, or a measurable prolongation of disease-free interval
or of survival. For example, a reduction in tumor growth in 20% of
patients is considered efficacious (Frei III, E., The Cancer
Journal 3:127-136 (1997)). However, an effect of this magnitude is
not considered to be a minimal requirement for the dose to be
effective in accordance with this invention.
[0264] In one embodiment, an effective dose is at least equal to,
preferably 10-fold and more preferably 100-fold higher than the 50%
inhibitory concentration (IC.sub.50) of the compound in an in vivo
assay as described herein.
[0265] The amount of active compound to be administered depends on
the precise peptide or derivative selected, the disease or
condition, the route of administration, the health and weight of
the recipient, the existence of other concurrent treatment, if any,
the frequency of treatment, the nature of the effect desired, for
example, inhibition of tumor metastasis, and the judgment of the
skilled practitioner.
[0266] A preferred dose for treating a subject, preferably
mammalian, more preferably human, with a tumor is an amount of up
to about 100 milligrams of active compound per kilogram of body
weight.
[0267] Typical single dosages of the peptide are between about 1
.mu.g and about 100 mg/kg body weight. For topical administration,
dosages in the range of about 0.01-20% concentration of the
compound, preferably 1-5%, are suggested. A total daily dosage in
the range of about 10 milligrams to about 7 grams is preferred for
oral administration. The foregoing ranges are, however, suggestive,
as the number of variables in regard to an individual treatment
regime is large, and considerable excursions from these recommended
values are expected.
[0268] An effective amount or dose of the peptide for inhibiting
invasion in vitro is in the range of about 1 picogram to about 0.5
nanograms per cell. Effective doses and optimal dose ranges may be
determined in vitro using the methods described herein.
[0269] The compounds of the invention may be further characterized
as producing an inhibitory effect on cell migration and invasion,
on angiogenesis, on tumor metastasis or on inflammatory reactions.
The compounds are especially useful in producing an anti-tumor
effect in a mammalian host, preferably human, harboring a
tumor.
[0270] The foregoing compositions and treatment methods are useful
for inhibiting cell migration and invasion or migration-induced
cell proliferation in a subject having a disease or condition
associated with undesired cell invasion, migration-induced
proliferation, angiogenesis or metastasis. Such diseases or
conditions may include primary growth or solid tumors or leukemias
and lymphomas, metastasis, invasion and/or growth of tumor
metastases, atherosclerosis, myocardial angiogenesis, post-balloon
angioplasty vascular restenosis, neointima formation following
vascular trauma, vascular graft restenosis, coronary collateral
formation, deep venous thrombosis, ischemic limb angiogenesis,
telangiectasia, pyogenic granuloma, corneal diseases, rubeosis,
neovascular glaucoma, diabetic and other retinopathy, retrolental
fibroplasia, diabetic neovascularization, macular degeneration,
endometriosis, arthritis, fibrosis associated with chronic
inflammatory conditions including psoriasis scleroderma, lung
fibrosis, chemotherapy-induced fibrosis, wound healing with
scarring and fibrosis; peptic ulcers, fractures, keloids, and
disorders of vasculogenesis, hematopoiesis, ovulation,
menstruation, pregnancy and placentation, or any other disease or
condition in which invasion or angiogenesis is pathogenic.
[0271] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE I
[0272] Synthesis of
Acetyl-Lys-Pro-Ser-Ser-Pro-Pro-Glu-Glu-NH.sub.2
[0273] The starting material was p-methyl-benzhydrylamine resin
substituted at a level of 0.70 mEq per gram of resin. Each of the
L-amino acids, starting with glutamic acid, was added in sequence
in a synthesis cycle consisting of the three steps of TFA
deprotection, coupling and capping. The completed peptide was
subjected to HF cleavage and then purified.
[0274] 1. TFA De-Protection
[0275] The starting resin was conditioned before adding the first
glutamic acid, or, in the case of subsequent cycles, the BOC
protecting group was removed from the .alpha.-amino nitrogen of the
starting material by treating the resin with 50% trifluoroacetic
acid (TFA) in dichloromethane (DCM) (two to three volumes per resin
volume). The mixture was stirred at room temperature for 30 minutes
and then drained. The resin was then washed once with an equal
volume of isopropanol for one minute and washed twice with an equal
volume of methanol, each wash taking one minute.
[0276] 2. Coupling
[0277] The de-protected resin was washed twice with an equal volume
of 10% triethylamine in DCM, each wash taking one minute, and
washed twice with an equal volume of methanol, each wash taking one
minute, and washed twice with an equal volume of DCM, each wash
taking one minute. A BOC-protected amino acid (three equivalents,
dissolved in DCM or in a mixture of DCM and N,N'-dimethylformamide
(DMF)) and 1-hydroxybenzotriazole (1 M solution in DMF, three
equivalents) was added to the resin, and the mixture was stirred
for a few seconds. Dicyclohexylcarbodiimide (DCC) (1M solution in
DCM, three equivalents) was then added, and the whole mixture was
stirred for 60-120 minutes. The resin was washed twice with an
equal volume of methanol and then washed twice with an equal volume
of DCM. A small sample was taken for a ninhydrin test to assess the
completeness of coupling. Generally, if incomplete, the coupling
step 2 is repeated. If complete, the synthesis is continued with
the capping step 3.
[0278] All amino acids were used as .alpha.-BOC derivatives. Side
chain protecting groups were as follows:
1 Amino acid Protecting group Histidine Benzyloxymethyl Asparagine
Xanthyl Glutamine Xanthyl Serine O-benzyl Threonine O-benzyl
Tyrosine 2-Brompo-Z Lysine 2-Chloro-Z Glutamic acid Cyclohexyl
Aspartic acid Cyclohexyl
[0279] 3. Capping
[0280] The resin was stirred with an equal volume of acetic
anhydride (20% solution in DCM) for 5 minutes at room temperature.
The resin was washed twice with an equal volume of methanol and
then washed twice with an equal volume of DCM.
[0281] 4. HF Cleavage
[0282] The resin bearing the desired amino acid sequence (1.0 gram)
was placed in a Teflon reaction vessel, and anhydrous anisole (1
mL) was added. The vessel was cooled with liquid N.sub.2, and
anhydrous HF (10 mL) was distilled into it. The temperature was
raised with ice water to 0.degree. C. The mixture was stirred at
this temperature for one hour, and then the HF was distilled off at
0.degree. C. The residue was washed with anhydrous ether, and the
peptide was extracted with a 1:1 mixture of
CH.sub.3CN:H.sub.2O.
[0283] 5. Purification
[0284] The lyophilized powder was dissolved in 0.1% TFA buffer and
loaded onto a Waters C18 preparative column (2 inches diameter,
15-20 .mu.m particle size, 300 .ANG. pore size). The loaded column
was eluted with a two-component eluent applied as a linear
gradient, starting with 0% of solution A in solution B and
finishing with 40% of solution A in solution B. Solution A was 0.1%
TFA in H.sub.2O, and solution B was 0.1% TFA in CH.sub.3CN.
Fractions exhibiting purity equal to or better than that desired
were pooled and lyophilized to render the purified final product as
the trifluoroacetate salt.
EXAMPLE II
Anti-Invasive and Anti-Proliferative Activity of Capped KPSSPPEE
(.ANG.6) and Related Peptides
[0285] Several peptides were tested for anti-invasive capacity in a
Matrigel.RTM. invasion assay system as indicated above (Kleinman et
al., supra; Parish et al., supra). Several invasive human tumor
lines (PC-3, MDA-MB-231) and non-human tumors (3LL, Mat B-III) were
examined. The rat breast cancer line Mat B III and the human
prostate cancer line PC-3 were initially used.
[0286] Tumor cells (5.times.10.sup.5/mL, in a volume of 200 .mu.L)
in serum-free RPMI 1640 medium were added to a disposable transwell
invasion chamber coated with Matrigel.RTM. (Becton Dickinson,
Lincoln Park, N.J.). The invasion chambers were placed in 24-well
tissue culture plates filled with serum-free RPMI-1640 and the
plates were placed in an atmosphere of 5% CO.sub.2 in humidified
air at 37.degree. C. for 48-72 hours. The chambers were then
removed, inverted, and the cells which had invaded (and now
appeared on the bottom face of the invasion chamber) were fixed and
stained using Diff-Quick.RTM. (Scientific Products). Cells were
counted in 10 different fields on each filter and an average
obtained. Typically, 3-5 replicates were performed at each
concentration of compound tested.
[0287] The invasion of all the cell lines tested thus far has been
inhibited by .ANG.6. FIG. 1 shows that Ac-KPSSPPEE-Am inhibited the
invasion of both rat and human prostate cancer line PC-3 and rat
breast cancer line Mat B III. Typical results with both MDA-MB-231
and Mat B-III cells are presented in FIG. 7.
[0288] This peptide was not cytotoxic to the cells nor did it
inhibit cell proliferation. Thus the observed effect was not a side
effect of cytotoxicity and could be ascribed to a mechanism of
action distinct from that of cytotoxic or cytostatic agents.
[0289] Tests were also conducted on shorter related, capped
peptides having the sequence Ac-PSSPPEE-Am (a deletion variant of
SEQ ID NO:2 which lacks the N-terminal Lys) and Ac-KPSSPPE-Am (a
deletion variant of SEQ ID NO:2 lacking one of the C-terminal Glu
residues). Also tested was a similar longer peptide, KPSSPPEELK
[SEQ ID NO:1] (Blasi et al., U.S. Pat. No. 5,416,006) and its
capped counterpart, Ac-KPSSPPEELK-Am).
[0290] To identify the minimal sequence required for activity as
well as to assess the role of the capping group for activity,
.ANG.6 and variants of .ANG.6 were tested for their ability to
inhibit Mat B-III invasion through Matrigel (FIG. 8). .ANG.6 was
found to be optimal.
[0291] It is noteworthy that all the peptides other than
Ac-KPSSPPEE-Am showed little or no activity in this assay,
indicating that SEQ ID NO:2 was the minimal required size for
activity (FIG. 2). The results also indicated that addition of Leu
and Lys at the C terminus of KPSSPPEE abrogated its biological
activity, regardless of whether the termini were capped or
uncapped.
[0292] The anti-invasive and anti-migratory properties of prompted
the inventors to test whether .ANG.6 inhibited the matrix
metalloproteinases MMP2 and MMP9. HT1080 cells were grown in the
presence of dexamethasone for 24 hrs and the supernatant collected
and treated with phenylmercuric acetate to activate proMMPs. MMP
activity was measured using the EnzCheck gelatinase assay
(Molecular Probes). .ANG.6 did not inhibit gelatinase activity at
concentrations up to 100 .mu.M.
[0293] .ANG.6 was also tested for its ability to inhibit the
proliferation of cells other than endothelial cells in vitro. No
anti-proliferative effects were observed when .ANG.6 was tested
against U87, MDA-MB-231, Mat B-III, HeLa, CHO, HepG2 or SMC (aortic
smooth muscle cells). Further, .ANG.6 did not potentiate the
anti-proliferative activity of CDDP against U87 cells.
EXAMPLE III
Inhibition of Plasminogen Activation
[0294] Single chain uPA (scuPA) complexed with a soluble form
(suPAR) of the uPA receptor (uPAR) is able to activate plasminogen
as efficiently as uPA, in the absence of activation by plasmin
(Higazi A. A. R. et al., (1995) J Biol Chem 270: 17375-17380).
scuPA remains as a single chain molecule, yet complex formation
with suPAR induces a conformational change in scuPA, such that an
active site capable of activating plasminogen was formed. This
scuPA-suPAR complex mimics the scuPA-uPAR complex formed on the
cell surface and activation of plasminogen by scuPA bound to
cell-surface uPAR has indeed been demonstrated (Manchanda N. et
al., (1991) J Biol Chem. 266: 12752-12758). In addition, the
scuPA-suPAR complex has been demonstrated to mediate clot lysis
(fibrin turnover) in vitro and was more efficient in this assay
than uPA alone. The scuPA-uPAR (or scuPA-suPAR) complex is very
resistant to inhibition by endogenous uPA inhibitors (PAIs)
(Higazi, A R, Mazar A et al. (1996) Blood 87:3545-3549), in
contrast to uPA, which is rapidly quenched in the presence of
PAIs.
[0295] Since .ANG.6 was originally derived from uPA, its ability to
inhibit various activities of uPA were tested, including binding to
uPAR and the activation of plasminogen. .ANG.6 had no effect in any
of these assays except that it inhibited scuPA-suPAR mediated clot
lysis (FIG. 3).
[0296] .ANG.6 did not affect the activation of plasminogen by
scuPA-suPAR when small, chromogenic substrates were used, and it
did not affect the activity of either uPA or plasmin directly. The
requirement of protein co-factors for the activation of plasminogen
has been demonstrated by (Higazi et al., (1998) Blood 92,
2075-2083). .ANG.6 may affect formation of a tertiary or ternary
complex required for the activation of plasminogen in the clot
lysis assay.
EXAMPLE IV
Actions of .ANG.6 on Endothelial Cells
[0297] .ANG.6 was tested for its ability to inhibit HUVEC
proliferation. No inhibition was observed at concentrations as high
as 312 .mu.g/mL (340 .mu.M) when HUVEC were grown on gelatin in 2%
FBS (FIG. 4). .ANG.6 did not potentiate the anti-proliferative
activity of cisplatin (CDDP) against endothelial cells (FIG.
5).
[0298] The ability of .ANG.6 to inhibit endothelial cell migration
was evaluated using HUVEC and lung and dermal microvessel
endothelial cells (HMVEC) with essentially identical results.
Typical results are presented in FIG. 6.
EXAMPLE V
Receptor Studies
[0299] The present inventors prepared .ANG.6-biotin (conjugated to
the N-terminus) and used this as a probe for receptor binding and
identification. This conjugate was cross-linked to whole cells and
cell extracts in an attempt to identify candidate receptors for
.ANG.6. After cross-linking, the extracts were resolved by SDS-PAGE
and transferred to PVDF membranes. Cross-linked products were
detected using streptavidin-HRPO. After analyzing the cross-linked
products from several cell line extracts, the only candidate
product thus far identified corresponds to a molecular weight of
about 30 kDa (FIG. 9). Further, this product seems to be present
only in the detergent phase of the extract, suggesting that it is
membrane bound. The more intensely staining high-molecular weight
products likely bind biotin directly, as they could not be competed
with unbiotinylated .ANG.6.
EXAMPLE VI
Additional in vivo Actions of .ANG.6 and Related Peptides
[0300] A. Angiogenesis in Chick Chorioallantoic Membrane (CAM)
[0301] .ANG.6 was tested for its effect on bFGF-induced
angiogenesis in a 7 day old chick CAM assay. FIG. 10 shows results
that are the average of 10 eggs for each condition tested. .ANG.6
inhibited angiogenesis in this system. In addition to inhibiting
major vessel formation, .ANG.6 also inhibited branching
morphogenesis; this effect is qualitatively evident in the
stereomicroscopic images of the CAMs (FIG. 11).
[0302] The CAM assay serves as a useful system for measuring the
effects of .ANG.6 and related peptides or derivatives on events
associated with the angiogenic "switch" as these events are
observable in near-real time.
[0303] B. Tests for Inhibition of Tumor Angiogenesis
[0304] Angiogenesis induced by tumor growth and metastasis in vivo
is examined in the models systems described above. Mice injected
with 3LL cells are treated either with peptide or with vehicle and
are sacrificed at various time points. Angiogenesis is assessed by
determining microvessel density (MVD) using an antibody specific
for microvascular endothelium or other markers of growing blood
vessels, such as PECAM (CD31). Such an antibody is employed in
conventional immunohistological methods to immunostain tissue
sections as described by Penfold et al., supra. A large number of
such antibodies is commercially available, for example the JC70
mAb. The MVD are correlated with other measures of tumor behavior
including lymph node status and primary tumor size and rate of
growth. In humans as reported by Penfold et al., supra, tumor MVD
correlates with lymph node metastasis and is independent of tumor
size, growth rate or type of histological differentiation. Only MVD
showed a significant association with lymph node metastasis.
[0305] The compounds are given i.v., i.p., or by osmotic minipump.
Typical dosages are 100-250 mg/kg/day. At various time points, two
animals are sacrificed, and the tumor tissue and surrounding tissue
is prepared for histological examination. Results are reported as
the average microvessel density of 5 fields each from 5 different
sections. The following seven compounds are tested: Ac-KPSSPPEE-Am
(SEQ ID NO:2), Ac-KPTTPPEE-Am (disubstitution variant at positions
3 and 4), Ac-KPSSPPDD-Am (disubstitution variant at positions 7 and
8), Ac-RPSSPPEE-Am (substitution variant at position 1),
Ac-PSSPPEE-Am (deletion variant, position 1 of SEQ ID NO:2
deleted), Ac-KPSSPPE-Am (deletion variant, position 8 of SEQ ID
NO:2 deleted), and Ac-KPPSSPPEELK-Am (SEQ ID NO:1).
[0306] The following results are obtained. In the rats treated with
Ac-KPSSPPEE-Am, Ac-KPTTPPEE-Am, Ac-KPSSPPDD-Am and Ac-RPSSPPEE-Am,
there is a significant reduction in the number of microvessels in
the region of the primary tumor at the subcutaneous inoculation
site as compared to controls. Peptides Ac-PSSPPEE-Am, Ac-KPSSPPE-Am
and Ac-KPPSSPPEELK-Am had no significant effect on angiogenesis.
Therefore, the four indicated compounds have anti-angiogenic
activity which is responsible at least in part for their
effectiveness as antitumor agents.
[0307] C. Growth and Metastasis of Rat Mat B-III Breast Cancer
[0308] The rat syngeneic breast cancer system (Xing and Rabbani,
1996) employs Mat BIII rat breast cancer cells. When Mat B-III
cells (1.times.10.sup.6 cells) are inoculated into the mammary fat
pad of female Fisher rats, they form large tumors which metastasize
to regional lymph nodes (LNs) and other distal sites within 14-20
days.
[0309] .ANG.6 was initially delivered prophylactically (infusion
starting on the day of tumor inoculation) using an Alzet mini-pump
that delivered 75 mg/kg/day. This treatment had substantial
anti-tumor activity including inhibition of tumor growth and LN
metastasis. Intraperitoneal delivery of .ANG.6 (75 mg/kg/day given
IP b.i.d.) produced similar results on tumor growth (FIG. 12) and
metastasis (FIG. 13). Metastasis was quantitated by counting the
number of macroscopic foci without regard for their size.
[0310] .ANG.6 was clearly more effective when the tumors were
smaller. However, despite the increase in growth rate of the
primary challenge tumors, the formation of macroscopic metastatic
foci continued to be suppressed during the course of this treatment
schedule. Representative LNs were excised from both control and
.ANG.6-treated animals, fixed in formalin and embedded in paraffin
for histological examination. Tumor cells were found in LNs from
.ANG.6-treated rats, despite the lack of macroscopic
metastases.
[0311] Histological analysis of the primary tumor also revealed
extensive necrosis at the tumor periphery. Typically, in the
primary tumors, the central core is spontaneously necrotic.
However, in .ANG.6-treated rats, the periphery was 50-75% necrosed
in all tumors evaluated. The only remaining viable tumor cells
formed perivascular cuffs around what appeared to be pre-existing
blood vessels. Each of these cuffs was approximately 5 cells in
width, consistent with Folkman's observations that a tumor could
expand no farther than 5 cells away from its blood supply without
initiating the formation of neovessels (Folkman J (1992) Semin
Cancer Biol 3:65-7112).
[0312] Many of the cells in the .ANG.6-treated groups stained
positive in the TUNEL assay for apoptosis, indicating that
apoptosis of tumor cells was occurring. Because TUNEL detects
fragmented DNA, other mechanisms of cell death (including necrosis)
might also have contributed.
[0313] Factor VIII staining of tumor sections in .ANG.6-treated
animals also revealed a 40-60% decrease in Factor VIII-positive
foci (data not shown).
[0314] In a comparative analysis of related peptides, the following
results will be obtained. In the rats treated with Ac-KPSSPPEE-Am
(.ANG.6), Ac-KPTTPPEE-Am, Ac-KPSSPPDD-Am and Ac-RPSSPPEE-Am, there
is a significant reduction in the size of the primary tumor and in
the number of metastases in the spleen, lungs, liver, kidney and
lymph nodes (enumerated as discrete foci). Upon histological and
immunohistochemical analysis, it is seen that in treated animals,
there is increased necrosis and signs of apoptosis. Large necrotic
areas are seen in tumor regions lacking in neovascularization. In
contrast, treatment with peptides Ac-PSSPPEE-Am, Ac-KPSSPPE-Am and
Ac-KPPSSPPEELK-Am will fail to cause a significant change in tumor
size or metastasis.
[0315] D. Treatment of Mat B-III Tumors with .ANG.6+Tamoxifen
("TAM")
[0316] The present inventors evaluated the ability of .ANG.6 to
potentiate the activity of TAM, an anti-estrogen used in the
treatment of human estrogen receptor-positive breast cancer. One
promise of anti-angiogenic therapy is the potential for preventing
or inhibiting tumorigenesis, a prophylactic outcome, in patient
populations at risk for a particular type of cancer. TAM may be
beneficial as a prophylactic treatment for some patients at risk of
developing breast cancer (although this is controversial because it
TAM could accelerate the formation of certain subtypes of breast
cancer. TAM is part of the accepted treatment regimen in early
stage breast cancer. Thus, the combination of an anti-angiogenic
agent such as the compounds of this invention, will have
prophylactic and therapeutic effects in on early stage breast
cancer.
[0317] Estrogen receptor-positive Mat B-III tumors were used to
test combination treatment with .ANG.6 (75 mg/kg/day) and TAM (3
mg/kg/day). In contrast to previous studies, the Mat B-III tumors
(inoculated in the mammary fat pad) were staged to 40-50 mm prior
to the initiation of treatment. Treatment was continued for 8 days
during which time primary tumor growth was measured using calipers
(FIG. 14). The combination was a more potent antitumor therapeutic
than TAM or .ANG.6 alone.
[0318] E. Treatment of Xenografted Human MDA-MB-231 Tumors with
.ANG.6
[0319] The results obtained with the Mat B-III model were extended
to a human tumor xenograft model of MDA-MB-231 human breast cancer
cells. These cells were first transfected with green fluorescent
protein (GFP) to simplify the detection and visualization of
metastases. The growth curves of the GFP-transfected MDA-MB-231
cells (MDA-MB-231 GFP) were indistinguishable from the parental
cells. The in vitro invasive activity of both cell lines was also
identical indicating that GFP transfection did not alter cellular
behavior.
[0320] The tumor cells (5.times.10.sup.5) were suspended in 0.1 mL
of Matrigel and injected into the mammary fat pad of female BALB/c
nu/nu mice. Treatment (75 mg/kg/day) was initiated when the tumors
were palpable (10 mm.sup.3, approximately 4 weeks after inoculation
of tumor cells) and continued for 5 weeks. Tumor volumes were
determined twice per week using caliper measurements. The animals
were euthanized at the end of the 5.sup.th week of treatment,
necropsied and examined for macroscopic metastases. Sections
prepared from LNs, lung, liver, spleen and kidney were analyzed for
microscopic dissemination of tumor cells using fluorescence
microscopy to detect GFP-positive foci. As shown in FIG. 15, .ANG.6
treatment inhibited tumor growth by >80%.
[0321] .ANG.6 was ineffective in this model if the tumors were
staged to 50-100 mm.sup.3 prior to initiating treatment. The
bi-phasic nature of the growth curve appears to represent the
growth rate of the tumor before (slow growth or dormancy) and after
(fast growth) the angiogenic switch (which occurs at the inflection
point of the curve). Thus, if .ANG.6 is found to inhibit events
associated with the angiogenic switch, to be effective it should be
administered before the switch. Folkman's group (Bergers et al.,
Science 284: 808-812) recently demonstrated the stage-specific
nature of angiogenesis inhibitors. Most angiogenesis inhibitors do
not cause regression of established tumors when used
alone--anti-angiogenic therapy appears to be most efficacious in
animal models when it is targeted to a specific stage of tumor
progression. The activity of .ANG.6 is consistent with this
notion.
[0322] TUNEL and Factor VIII staining of tumor sections revealed
results similar to those observed in the rat studies reported
above. Tumors from .ANG.6-treated animals demonstrated a
significant increase in TUNEL-positive foci as well as a decrease
in Factor VIII-positive hot spots.
[0323] Macroscopic metastases were enumerated and their size
measured with calipers (Table I, below). Microscopic dissemination
of tumor cells was quantitated by counting GFP-positive foci in
representative sections from lung, liver, kidney and spleen.
Disseminated tumor cells were not evident in kidney sections.
Because disseminated cells may exist in a dormant state for many
years or, in some cases, never progress, one cannot posit an
absolute correlation between the presence of disseminated cells and
metastasis. Nevertheless, the foci in liver and lungs did appear to
be true metastases as the cells were not single foci but seem to
have formed larger colonies in the control animals. Sections from
.ANG.6-treated animals appeared to have a greater number of single
foci (vs. larger colonies), indicating lack of metastatic
progression.
[0324] Macrometastases were determined by excising involved LNs and
determining the tumor volume by caliper measurement. Microscopic
tumor foci were quantitated by sectioning and fixing target organs,
then visualizing GFP-labeled cells using fluorescence microscopy
(at 200.times. enlargement). The number of disseminated tumor foci
represents the average of 5 fields per section from 3 different
sections per organ.
2TABLE I (a) Macroscopic Metastases Lymph Nodes Lungs Group Number
Size (mm.sup.3) Number Size Control 4.5 .+-. 1.2 27 .+-. 3 4.2 .+-.
1.8 N.D. .ANG.6-treated 1.2 .+-. 0.2 7 .+-. 3 1 .+-. 0 N.D. (b)
Disseminated GFP-positive Tumor Foci Lung Liver Spleen Control 4.23
.+-. 0.18 18.83 .+-. 0.36 35.67 .+-. .82 .ANG.6-treated 2.09 .+-.
0.30 3.11 .+-. 0.14 28.30 .+-. 2.80
[0325] F. Treatment of U87 Human Glioblastoma U87 Xenografts
[0326] 1. Growth of Primary Tumors after Subcutaneous
Implantation
[0327] Tumors were established by subcutaneous injection of human
U87 glioblastoma ("GBM") cells sc into nude mice. U87 tumors were
staged to 50-100 mm.sup.3 prior to initiating treatment. .ANG.6,
cisplatin (CDDP) and the combination of .ANG.6+CDDP were tested
(FIG. 16). CDDP was tested at a dose of 3 mg/kg/day given every
other day from day 4.times.6 administrations, which converts to
approximately 6 mg/m.sup.2 per administration. This is a
substantially lower dose than that typically given to human
patients (20-40 mg/m.sup.2 for most tumors although doses as high
as 200 mg/m.sup.2 have been reported in neuroblastomas; the dose
depends on the regimen used) since dose limiting toxicity (as
exemplified by weight loss) occurs in mice at doses greater than 6
mg/m.sup.2. Thus, the full benefit of CDDP+.ANG.6 as a combination
therapy may exceed that which was observed here.
[0328] As the combination treatment of .ANG.6+CDDP was highly
effective in inhibiting tumor growth in this model, the present
inventors assessed the proliferative (mitotic) index in these
tumors. Typically, human GBM is not characterized by rapid
proliferation and only 15% of the tumor cells are typically
proliferating (CLINICAL ONCOLOGY (1995) Abeloff, M. D. et al., eds.
Churchill-Livingstone, New York). For this reason, anti-metabolites
are not efficacious in treating this type of tumor. Though
alkylating agents (such as CDDP and BCNU) have been the most
successful in treating GBM in the clinic (as they induce apoptosis
by damaging tumor cell DNA), they are not selective for rapidly
dividing cell populations and are quite toxic.
[0329] Anti-angiogenic therapy is expected to produce both
anti-proliferative and pro-apoptotic effects that would "prime" the
tumor chemotherapy with these alkylating agents. The proliferative
index in the U87 tumors was evaluated using Ki-67 staining followed
by digitization of the staining intensity and quantitation. Three
sections from each animal were evaluated for Ki-67 positive
staining (FIG. 18). .ANG.6 treatment inhibited proliferation by
50%, which was not enhanced by combination treatment with CDDP, as
predicted by the inventors. .ANG.6 did not inhibit U87 cell
proliferation directly nor did it potentiate the pro-apoptotic
activity of CDDP in vitro. Similar results were obtained against
other human GBM cell lines, including those designated 308 and
U251.
[0330] Dose Response Studies
[0331] The effect of different doses of .ANG.6 on U87 tumor growth
sc was evaluated (FIG. 19). Tumor growth was almost completely
suppressed in animals treated with the combination of the .ANG.6
(150 mg/kg/day) and CDDP as long as treatment was continued. Tumors
grew when treatment was discontinued. Tumor regression (defined as
a tumor that was no longer palpable) was observed in 1 or 4 mice in
this group, a response that was durable throughout the course of
the experiment. This individual mouse is being analyzed for the
presence of microscopic tumor. Once information with an even higher
dose of .ANG.6 (300 mg/kg/day) been obtained, the results will be
extrapolated to the orthotopic model where the effects of higher
doses of .ANG.6 on angiogenesis and survival will be evaluated.
[0332] 2. U87 Implanted Orthotopically
[0333] U87 tumors were surgically inoculated into the cerebral
ventricles of mice. The animals were allowed to recover for 72
hours at which time treatment with .ANG.6 (75 mg/kg/day IP bid),
CDDP (3 mg/kg/day given every other day from day 4.times.6
administrations), or a combination of .ANG.6+CDDP was
initiated.
[0334] Animals were treated for 21 days at which time they were
euthanized and their brains evaluated for the presence of tumor.
Transverse sections were stained with hematoxylin and eosin
(H&E). The combination of .ANG.6 and CDDP was significantly
more effective in inhibiting U87 tumor growth than either agent
alone.
[0335] Tumor sections were also evaluated for microvessel density
using antibodies specific for mouse-CD31 in immmunostaining. Very
few CD31+foci were evident in animals treated with the combination
of .ANG.6+CDDP. In fact, CDDP and .ANG.6 alone both inhibited
angiogenesis to some extent. Qualitatively, .ANG.6 appeared not
only to inhibit the number of vessels but also the differentiation
of the vessels as fewer branching vessels were observed in the
.ANG.6-treated tumors.
[0336] 3. Survival of Mice Implanted Orthotopically with U87
[0337] Tumors were established as in the previous section, and the
same therapeutic regimens were employed. Treatment was discontinued
at day 21, and survival was measured. Control animals appeared
moribund around day 25 and all of the animals in this group were
dead by day 30. In contrast, the combination treatment group showed
a significant increase in survival when compared to the control
group or to the groups receiving either .ANG.6 or CDDP alone (FIG.
20).
[0338] G. Treatment of Murine Lewis Lung Carcinoma (3LL)
Experimental Metastasis/Lung Colonization
[0339] Lewis Lung Carcinoma cells (3LL, 1.5.times.10.sup.5 cells
per mouse) were injected i.v. into C57BL/6 mice. Mice were treated
with cyclophosphamide (CY: 300 mg/kg once on day 4) alone, .ANG.6
alone (treated from day 0-day 19 at 75 mg/kg/day) or a combination
of CY+.ANG.6. The animals were euthanized on day 19 and the lungs
harvested for analysis. Macroscopic metastases were counted and the
lungs analyzed histologically. Combination treatment reduced the
total number of lung colonies when compared to control or CY only
groups (FIG. 21).
[0340] Histological evaluation of tumors from the combination
treatment group revealed that the lungs were mostly free of tumor.
In lungs having small amounts of residual tumor, hemorrhagic
necrosis was observed in some of the foci.
[0341] Additional Studies of Metastasis
[0342] In addition to .ANG.6, other related peptide compounds
described above are tested for efficacy in vivo in the 3LL model
(as provided above) as well as the PC-3 model. PC-3 cells
transfected with the gene encoding the enzyme chloramphenicol
acetyl-transferase (CAT) are inoculated into mice i.v. at doses of
1.times.10.sup.6 cells per mouse. These mice are implanted with a
minipump, as above, which dispenses 100 mg/kg/day of the peptide or
vehicle over a period of 14 or 21 days. At termination of
treatment, the animals are euthanized and the tumor marker probe is
assayed in regional lymph nodes, femurs, lungs, and brain.
[0343] The following results are obtained in both systems. In mice
treated with Ac-KPSSPPEE-Am (.ANG.6), Ac-KPTTPPEE-Am,
Ac-KPSSPPDD-Am and Ac-RPSSPPEE-Am, metastasis is markedly
inhibited. These results indicate that these compounds interfere
with the metastatic process. In contrast, mice treated with
peptides Ac-PSSPPEE-Am, Ac-KPSSPPE-Am and Ac-KPPSSPPEELK-Am have no
reduction in metastases.
[0344] Other uPAR-binding peptides with utility as tumor-targeted
imaging agents that have been discovered by the present inventors
are the subject of an issued patent (U.S. Pat. No. 5,942,492) and
co-pending, commonly assigned patent applications U.S. Ser. No.
09/285,783 filed Apr. 5, 1999; U.S. Ser. No. 09/1891,816 filed Oct.
29, 1999; and provisional application No. 60/157,012, filed Oct. 1,
1999 (all of which documents are incorporated by reference in their
entirety). Some of these agents are useful as radiodiagnostics for
evaluating tumor size and dissemination. These can be used in
monitoring patients during therapy. Some of these reagents have
been designed to carry a Tc.sup.99m .gamma.-emitting nuclide to the
surface of solid tumors and tumor vessels. These same constructs
are also useful in localizing a Tc.sup.94 isotope for PET
imaging.
EXAMPLE VII
Pharmacological and Pharmacodynamic Evaluation of .ANG.6
[0345] 1. Pharmacodynamic Markers
[0346] Evaluation has begun of several markers that are expected to
be associated with a therapeutic response of tumors to .ANG.6.
Molecules associated with angiogenesis will be evaluated for their
utility as pharmacodynamic markers of .ANG.6 activity. Certain
markers (bFGF, MMPs) will be detected in urine
[0347] 2. Pharmacology
[0348] Liquid Chromatography/Mass Spectroscopy (LC/MS) Assay
Development
[0349] An LC/MS assay developed for the detection of .ANG.6 in
plasma has been validated for mouse and monkey plasma. The
sensitivity is 10 ng in 0.1 mL of plasma. This test can be
similarly validated for human plasma under GLP conditions to be
used for human pharmacology. Protein binding studies are presently
underway. Because the peptide can be recovered from plasma after
simple precipitation of total protein (.ANG.6 is in the
supernatant), the undesired effect caused by extensive binding of
the peptide to other proteins is not expected. .ANG.6 is extremely
soluble and can be formulated in physiological buffers and
excipients to high concentration (>100 mg/mL).
[0350] Plasma Stability of .ANG.6
[0351] .ANG.6 was stable in plasma at room temperature for 24 hrs
(tested at 10 .mu.g/mL). .ANG.6 is stable as a lyophilized powder
for at least 2 months (degradation is <1% by HPLC) and in PBS or
water at 4.degree. C. for at least 2 weeks.
[0352] Pharmacokinetics in Mice
[0353] Pharmacokinetic analysis was carried out by Gilbert Lam,
MicroConstants. The plasma concentration profile of .ANG.6 is
depicted in FIG. 22. The pharmacokinetics of .ANG.6 is
characterized by a mono-exponential decline following a single
bolus dose. The terminal half-life is 0.2 hour. The systemic
clearance "CL" is 2.0 L/h/kg, which is moderate when compared to
the liver blood flow of the mouse. .ANG.6 has a small volume of
distribution at steady-state, (V.sub.SS=0.4064 L/kg). Low V.sub.SS
results in high plasma concentrations of the peptide.
[0354] Pharmacokinetics in Monkeys
[0355] The plasma concentration profile of .ANG.6 is depicted in
FIG. 23. .ANG.6 pharmacokinetics is characterized by a
mono-exponential decline following a single bolus dose. The
terminal half-life is 0.4 hour. The systemic clearance, CL, is
0.042 L/h/kg which is low when compared to either the kidney blood
flow or the liver blood flow of the monkey. .ANG.6 has a small
volume of distribution at steady-state, V.sub.SS=0.0252 L/kg. Low
V.sub.SS results in high plasma concentrations of the peptide.
[0356] Mouse Plasma Therapeutic Levels
[0357] The therapeutic plasma levels in mice receiving .ANG.6 are
being evaluated. Plasma concentration of .ANG.6 will be measured in
blood samples obtained from the dose-response studies in the
glioblastoma model (above). This information is combined with
allometric scaling data (see below) to predict therapeutic doses in
man. This information will then be combined with toxicology data to
establish starting doses for a Phase I trial.
[0358] Allometric Scaling
[0359] .ANG.6 is a particularly good candidate for allometric
scaling analysis. The distribution of this compound is simple (i.e.
after iv administration, the compound is not absorbed by, or
distributed in, tissue but rather is restricted to the plasma) and
is proportional to the plasma volume and therefore body weight.
This makes the prediction of clearance in man fairly
straightforward. Based on allometric scaling, it is expected that
the CL in man is about 0.0061 L/h/kg (0.43 L/h for a 70 kg person)
(FIG. 24). Extrapolations can also be made for other parameters
such as t.sub.1/2.
[0360] 3. Toxicology
[0361] Acute toxicity was evaluated at three different doses of
.ANG.6 (1500 mg/kg, 500 mg/kg and 250 mg/kg). Mice (n=6) were
infused (i.v.) with a bolus of .ANG.6 over 5-10 minutes and
observed for 3 days. No evidence for overt toxicity was observed,
and all the animals survived and apparently tolerated the .ANG.6
well.
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[0396] The references cited above are all incorporated by reference
herein, whether specifically incorporated or not.
[0397] Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations, and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
[0398] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present 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 as follows in the scope of the appended
claims.
[0399] While this invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present 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 as follows in the scope of the appended
claims.
Sequence CWU 1
1
9 1 10 PRT Artificial Sequence Description of Artificial Sequence
corresponds to positions 136-145 of the human protein urokinase
plasminogen activator (uPA) 1 Lys Pro Ser Ser Pro Pro Glu Glu Leu
Lys 1 5 10 2 8 PRT Artificial Sequence Description of Artificial
Sequence corresponds to positions 136-143 of uPA 2 Lys Pro Ser Ser
Pro Pro Glu Glu 1 5 3 4 PRT Artificial Sequence Description of
Artificial Sequence substitution, deletion or addition variants of
SEQ ID NO2 3 Pro Pro Glu Glu 1 4 7 PRT Artificial Sequence
Description of Artificial Sequence substitution, deletion or
addition variants of SEQ ID NO2 4 Pro Ser Ser Pro Pro Glu Glu 1 5 5
7 PRT Artificial Sequence Description of Artificial Sequence
substitution, deletion or addition variants of SEQ ID NO2 5 Lys Pro
Ser Ser Pro Pro Glu 1 5 6 8 PRT Artificial Sequence Description of
Artificial Sequence substitution, deletion or addition variants of
SEQ ID NO2 6 Lys Pro Thr Thr Pro Pro Glu Glu 1 5 7 8 PRT Artificial
Sequence Description of Artificial Sequence substitution, deletion
or addition variants of SEQ ID NO2 7 Lys Pro Ser Ser Pro Pro Asp
Asp 1 5 8 8 PRT Artificial Sequence Description of Artificial
Sequence substitution, deletion or addition variants of SEQ ID NO2
8 Arg Pro Ser Ser Pro Pro Glu Glu 1 5 9 4 PRT Artificial Sequence
Description of Artificial Sequence substitution, deletion or
addition variants of SEQ ID NO2 9 Lys Pro Ser Ser 1
* * * * *