Note: Descriptions are shown in the official language in which they were submitted.
CA 02352477 2001-05-23
WO 00131244 PCT/US99/2?243
COMPOSITIONS COMPRISING ANGIOSTATIN AND KRINGLE 5 REGION OF
PLASMINOGEN AND METHODS OF USE THEREOF
Field of the Invention
The present invention relates to compositions useful to inhibit proliferation
and/or migration of endothelial cells. The compositions comprise a combination
of
angiostatin protein plus the kringle-5 region of plasnainogen. The
compositions of the
present invention are capable of inhibiting angiogenesis related diseases and
modulating angiogenic processes.
to Background of the Invention
As used herein, the term "angiogenesis" means the generation of new blood
vessels into tissue or organ. Under nonrnal physiological conditions, humans
or
animals undergo angiogenesis only in very restricted situations. For example,
angiogenesis is normally observed in wound healing, fetal and embryonic
15 development and formation of the corpus luteum, endometrium and placenta.
The
team "endothelium" means a thin layer of flat endothelial cells that lines
serous
cavities, lymph vessels, and blood vessels.
Endothelial cells and pericytes, surrounded by a basement membrane, form
capillary blood vessels. Angiogenesis begins with the erosion of the basement
2o membrane by enzymes released by endothelial cells and leukocytes. The
endothelial
cells, which line the lumen of blood vessels, then protrude through the
basement
membrane. Angiogenic stimulants induce the endol:helial cells to migrate
through the
eroded basement membrane. The migrating cells form a "sprout" off the parent
blood
vessel, where the endothelial cells undergo mitosis and proliferate. The
endothelial
25 sprouts merge with each other to form capillary loops, creating new blood
vessels.
Pathological angiogenesis occurs in a number of disease states, for example
tumor metastasis and abnormal growth by endothelial cells, and suppos-ts the
pathological damage seen in these conditions. The diverse pathological disease
states
in which abnormal angiogenesis is present have been grouped together as
"angiogenic
3o dependent" or "angiogenic associated" disorders. F'or a review of
angiogenesis and
its relation to tumor growth, see PCT publication W'U 95/29242 and references
cited
therein, hereby incorporated by reference in its entirety.
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Angiogenesis is tightly regulated by both positive and negative signals.
Angiogenic stimulators, such as fibroblast growth factor (FGF), and vascular
endothelial growth factor (VEGF), are potent mitogc.ns for endothelial cell
proliferation and strong chemoattractants for endothelial cell migration.
These
positive regulators can promote neovascularization l:o sustain the expansion
of both
primary and metastatic tumors (Cwoss, J. L. et. al., ('.I 993) J. Natl. Cancer
Iycst.
8S(2):121-i31; Kim, K.J. et. al., (1993) Nature 362(6243):841-844). Among the
negative regulators described to date, Angiostatin ranks as one of the most
effective
endogenous inhibitors of angiogenesis (O'Reilly, Mf. S. et. al., (1994}
Cel179:315-
to 328; O'Reilly, M: S. et. al., (1996) Nat.. Meal. 2:689-692; Wu, Z. et. al.,
(1997)
Biochem. Biophys. Res. Commun. 236:651-654). Angiostatin comprises an internal
fragment of plasrninogen and consists of four tx-iple--looped kringle domains
constrained by three disulfide bonds. Angiostatin was shown to inhibit
endothelial cell
proliferation in vitro and to suppress growth factor-:induced angiogenesis in
vivo
is (O'Reilly (1994), supra). Inhibition of angiogenesis by treatment with
angiostatin
results in significant suppression of tumor growth in both murine and human
tumor
models (O'Reilly {1994); O'Reilly (1996); Wu (1997), supra).
Angiostatin has been described as a potent angiogenesis inhibitor that could
markedly suppress the growth of a variety of tumors, including carcinomas of
lung,
20 prostate, colon, and breast (Cao, Y. et. al., {1998) J. Clin. Invest.
101(S): IOSS-1063;
O'Reilly, et. al ., ( 1996) supra; O'Rei Ily, et. aL, ( 1994) supra).
The individual kringle domains of angiostatin have distinct anti-proliferative
and anti-migratory activities toward endothelial cells (Cao, Y. et. al., (
1996) J. Biol.
Chem. 271:29461-29467; Ji, W.R. et. al., ( 1998) FASEB Jrnl {in press)). It
has been
2S documented that the first three kringles of angiostatin exhibit potent
inhibitory
activities on endothelial cell proliferation whereas lcringle 4 has a marginal
effect. It
was also shown that the intact kringle structure is essential for the anti-
proliferative
activities of angiostatin.
The kringle S of human plasminogen displays high structural similarity and
3o about S0% sequence identity to the four kringles of angiostatin. Kringle S
was
reported to inhibit bFGF-elicited endothelial cell growth in a dose-dependent
manner
(Ji, W.R. et. al., (1998) Biochem Biophys Res Commun. 247(2):414-4.19; Cao, Y.
et.
al., (1997) J. Biol. Chem. 272:22924-22928). These data suggest that lmingle
S, like
angiostatin, may have potent anti-angiogenic activities.
2
CA 02352477 2001-05-23
wo 00/3124a PCTNS99/272a3
Summary of the Invention
In accordance with the present invention, compositions are provided
comprising an Angiostatin molecule plus a kringie 5 molecule. Applicants
herein
provide evidence that a combination of Angiostatin plus a kringle 5 molecule
exhibit
a synergistic effect, and shows superior results over Angiostatin, the kningle
S region
alone, or kringles 1-5 of plasminogen.
The present invention provides methods and compositions for treating diseases
and processes mediated by undesired arid uncontrolled angiogenesis by
administering
1 o to a human or animal a composition comprising Anl;iostatin and the kringle
5 region
of plasminogen. The present invention is particularly useful for treating, or
for
repressing the growth of, tumors. Administration of compositions of the
present
invention to a human or animal with prevascularized metastasized tumors will
prevent
the growth or expansion of those tumors.
is The methods of the present invention encompass the use of a single
composition comprising Angiostatin and the kringle: 5 region of plasminogen.
Additionally, the methods of the present invention encompass the use of a
composition comprising Angiostatin and a composition comprising the kringle 5
region ~of plasminogen, said compositions administered simultaneously or
2o sequentially.
All references cited herein, whether supra or it f~a, are hweby incorporated
by
reference in their entirety.
Brief Description of the Figures
25 Figure 1 is a graph showing the additive anti-endothelial cell migratory
activities of kringle 5 of human plasminogen in combination with Angiostatin.
Figure
lA shows additive inhibition of HCTVEC cell migration by kringle 5 of human
plasminogen in combination with Angiostatin; Figure 1B shows additive
inhibition of
BCE cell migration by kringle 5 of human plasminagen in combination with
3o Angiastatin. (Abbreviations: KS = kringle 5; AST == Angiostatin; bFGF =
basic
fibroblast growth factor).
CA 02352477 2001-05-23
WO 00131244 PCT/US99127243
Detailed Description of the lmvention
Angiostatin has been described as a potent angiogenesis inhibitor that can
markedly suppress the growth of a variety of tumors, including carcinomas of
lung,
prostate, colon, and breast. Angiostatin and the sequences of mouse, human,
rhesus
monkey, porcine and bovine plasminogen are given iin U.S. Patent No.
5,639,725, the
disclosure of which is incorporated herein by reference in its entirety.
The kringle 5 of human plasminogen displays high structural similarity and
about 50% sequence identity to the four k~ingles of angiostatin. Kringle 5 was
reported to inhibit bFGF-elicited endothelial cell growth in a dose-dependent
manner.
to These data suggest that kringle 5, like angiostatin, may have potent anti-
angiogenic
activities.
The present invention for the first time presents evidence that combination
therapy, involving the administration of Angiostatin and a kringle 5 fi~agment
of
plasminogen, exhibits superior angiogenesis inhibition over Angiostatin alone,
kringle
15 5 alone, and a kringle 1-5 region of plasminogen.
Compositions are provided comprising a kringle 5 region of plasminogen and
Angiostatin. The source plasminogen (i.e., the plasminogen from which the
kringle 5
region andlor the Angiostatin is derived) may be from the same species (e.g.,
human)
or from different species (e.g., a kringle 5 region from murine plasminogen
and
2o Angiostatin comprising kringles I-4 from human pl~usminogen). A kringle 5
region
from plasminogen, or biologically active portion thereof, and said
Angiostatin, may
be deuived, for example, fi~om murine plasminogen, lhuman plasrninogen, Rhesus
plasminogen, porcine plasrninogen, canine plasminogen, or bovine plasminogen.
Preferably, the kringle 5 region and the Angiostatin we derived from the same
25 species.
Additionally, the kringle 5 fragment and the Angiostatin may be administered
in the same composition, or they may be administered in separate compositions.
If
separate compositions are used, the compositions may be administered
simultaneously
or sequentially.
so Also encompassed within the scope of the present invention are variations
of
the kringle 5 fi~agrnent, including biologically active fragments, and
biologically
active analogs involving amino acid deletions, additions and/or substitutions.
"Biologically active fragment" includes fi-agments of the kringle 5 region
that
maintain the same biological activity of the kringle :S region. "Biologically
active
CA 02352477 2001-05-23
WO 00/31244 PCT/US99/27243
analogs" includes variations of the kringle 5 region that do not materially
alter the
biological activity (i.e., anti-angiogenic activity) of the I~;ringle S
fragment. Included
within the scope of the invention are changes made to txie kringle 5 fragment
that
increase anti-angiogenic activity.
Preferred analogs include a kr~ingle 5 fragment and Angiostatin molecule
whose sequences differ from the wild-type sequence by one or more conservative
amino acid substitutions or by one or more non-conservative amino acid
substitutions,
deletions or insertions which do not abolish the biological activity of the
molecules.
Conservative substitutions typically include the substitu~.tion of one amino
acid for
to another with similes characteristics, e.g., substitutions within the
following groups:
valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid,
glutamic
acid; asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine,
tyrosine. Other canservative amino acid substitutions c;an be taken from the
table
below.
Table 1
Conservative amino acid replacements
For Amino Acid Code Replace with any of
Alanine A D-Ala, Gly, beta-Ala, lL-Cys, D-Cys
Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg,
Met, Ile, D-
M et, D-I1 e, Orn, D-Orn
Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln
Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln
Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr
Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln
Glycine G Ala, D-Ala, Pro, D-Pro, f3-Ala, Acp
Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met
Leucine L D-Leu, Val, D-Val, Met, D-Met
Lysine K D-Lys, Arg, D-Arg, horno-Arg, D-homo-Arg,
Met, D-Met,
11 e, D-I1 e, Osn, D-Orn
Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu,
Val, D-Val
Phenylalanine F D-Phe, Tyr, D-Thr, L-lDopa, His, D-His,
Tip, D-Trp, Trans-
3,4, or S-phenylproline, cis-3,4, or 5-phenylproline
Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid,
D- or L-1-
oxazolidine-4-cwboxylic acid
Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met,
Met(O), D-
Met(O), L-Cys, D-Cys;
Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,
Met(O), D-
Met(O), Val, D-Val
Tyrosine Y D-Tyr, Phe, D-Phe, L-:Dopa, His, D-His
Valine ~ V ~ D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met
5
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Other analogs within the invention are those with modifications which
increase protein or peptide stability; such analogs m;~y contain, for example,
one or
more non-peptide bonds (which replace the peptide bonds) in the protein or
peptide
sequence. Also included are analogs that include re:>idues other than
naturally
occurring L-amino acids, e.g., D-amino acids or non-naturally occuuring or
synthetic
amino acids, e.g., fi or y amino acids.
The kringle 5 regions and Angiostatin useful in the present invention may be
obtained by synthetic means, i.e., chemical synthesis of the polypeptide from
its
1 o component amino acids, by methods known to those; of ordinary skill in the
aat. For
example, the solid phase procedure described in Houghton et al., Proc.
Natl.'Acad.
Sci. 82, 5131-5135 (1985) rnay be employed. The polypeptides may be obtained
by
production in prokaryotic or euka~yotic host cells expressing a DNA sequence
coding
for all or pant of the desired fragment, or by in vitro translation of the
mRNA encoded
15 by a DNA sequence coding for the desired peptide. Techniques for the
production of
a kringle 5 fragment and Angiostatin by these means ai-e known in the an, and
are
described herein.
The kringle S region and Angiostatin produced in this manner may then be
isolated and purified to some degree using vaa-ious protein purification
techniques.
2o For example, chromatographic procedures such as ion exchange
chromatography, gel
filtration chromatography and immunoaffinity chromatography may be employed.
If the polypeptides of the invention (i.e., a kringle 5 fragment and
Angiostatin)
aa~e made through recombinant techniques, the DNA, sequences useful to prepare
the
polypeptides of the invention can be obtained using various methods well known
to
25 those of ordinary skill in the art. The expression vectors can be partly or
wholly
synthesized chemically and/or paz-tly or wholly prepared through genetic
engineering
techniques. Fragments can be sequentially ligated (via appropriate temninal
restz~iction sites or complementary terminal sequences) so as to form the
col~~ect linear
sequence of nucleotides.
3o Expression vehicles of the invention for production of the anti-angiogenic
polypeptides of the invention include plasrnids or other vectors. In general,
such
vectors contain control sequences that allow expression in various types of
hosts.
Suitable expression vectors containing the desired coding and contl-ol
sequences may
6
CA 02352477 2001-05-23
WO OOI31244 PCT/US99/2'7243
be constructed using standard recombinant DNA techniques known in the as~t,
many
of which are described in Sambrook et al. (1989), Molecular Cloning: A
Laboratory
Manual, 2nd edition, Cold Spuing Harbor Laboratory, Cald Spring Harbor, NY.
An expression vector as contemplated by the; present invention is capable of
directing the replication of the vector in bacteria, ye~~st, insect, andlor
mammalian
cells. One class of vectors utilizes yeast DNA elements that provide
autonomously
replicating origins such as the yeast 2~. element or A,RS 1 sequence which
yield
extrachromosornal plasmids. A second class of vectors relies upon the
integration of
the desired gene sequences into the host cell chromosome. The vectors may also
to incorporate a bacterial origin of replication. Suitable bacterial origins
of replication
include, for example, the ColEl, pSC101 and MI3 origins of replication.
Expression vectors useful of the present invc,ntion typically contain a
promoter
located 5' to (i.e., upstream of) the DNA sequence to be expressed, and a
transcription
termination sequence located 3' to (i.e., downstream ofj the sequence to be
expressed.
15 Suitable promoters include, for example, the yeast ADHI promoter. The
promoter
sequence may also be inducible, to allow modulation of expression {e.g., by
the
presence or absence of nutrients or other inducers in the growth medium).
Examples
include the yeast GALI, CUPl, and MET35 promoters. Suitable termination
sequences include, for example, the yeast CYCI teumination and polyadenylation
2o sequences.
The expression vectors may also include other regulatory sequences for
optimal expression of the desired product. Such sequences include secretoiy
leader
sequences, which provide for secretion of the expressed product or direct
membrane
localization, and restriction enzyme recognition sequences, which provide
sites for
25 cleavage by restriction endonucleases. All of these materials are known in
the ay-t and
most a~-e commercially available.
A suitable expression vector may also include marking sequences, which
allow phenotypic detection and/or selection of tt-ansformed yeast or bacterial
cells.
Such a marker may provide prototrophy to an auxooophic host (e.g., amino acid
30 biosynthetic genes), biocide resistance or supersens~itivity (e.g.,
antiobiotic resistance)
or a phenotypically detectable signal (e.g,, fluorescence). The selectable
marker gene
can be either directly linked to the DNA gene sequences to be expressed, or
introduced into the same cell by transformation. Examples of yeast selectable
markers include Basidium pullularrs AURI-C gene, the S. cerevisiae URA3 or
LEU2
7
CA 02352477 2001-05-23
WD 00/31244 PCT/US99/27243
genes and the like. Examples of bacterial selectable markers include the
ampicillin
resistance gene. A preferred vector is pYESII, containing the ColEl and 2p.
origins of
replication, the yeast URA3 and bacterial ampR genes, and the yeast GALI
promoter
sequence (Inviti~ogen).
s In a further alternative, the constructs may be introduced into a cell by
transformation in conjunction with a gene allowing for selection where the
construct
will become integrated into the host genome or persist episomally. Usually,
the
constZ-uct will be part of a vector having homologous sequences for
integration or a
replication system recognized by the host cell.
1o The compositions of the present invention, comprising a kringle 5 fragment
of
plasminogen (or a biologically active fragment or analog thereof) and
Angiostatin, are
useful to treat angiogenic associated disorders. The present invention
includes the
method of tz-eating an angiogenic-associated disorder with an effective amount
of a
composition comprising a kringle 5 fragment and Angiostatin. As described
above, a
1s single composition comprising a lcringle 5 fragment and Angiostatin may be
used, or
sepwate compositions (a first comprising a kringle 5 fiagment and a second
comprising Angiostatin) may be administered simultaneously or sequentially.
The angiogenesis mediated diseases include, but are not limited to, solid
tumors, blood bom tumors such as leukemias; tumor metastasis; benign tumors,
for
2o example, hemangiomas, acoustic acuromas, neurofibrornas, trachomas, and
pyogenic
granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for
example,
diabetic retinopathy, retinopathy of prematurity, macular degenet-ation,
corneal graft
rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-
Webber
Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia;
25 hemophiliac joints; angiofibroma; and wound granulation. The compositions
of the
present invention are useful in treatment of disease of excessive or abnormal
stimulation of endothelial cells. These disorders include, but are not limited
to,
intestinal adhesions, atherosclerosis, scleroderma, and hypeW-ophic scars,
i.e., keloids.
The compositions can also be used as birth control agents by preventing
30 vascularization required for embryo implantation.
The compositions and methods of the present invention may be used in
combination with other compositions and procedures for the treatment of
angiogenic
associated disorders. For example, a tumor may be treated conventionally with
surgery, radiation or chemotherapy, and then compositions comprising a kiingle
S
CA 02352477 2001-05-23
WO 00!31244 PCTNS99/2'7243
region and Angiostatin may be subsequently administered to the patient to
extend the
dormancy of micrometastases and to stabilize any residual primary tumor.
The present invention also provides pharmaceutical (i.e., therapeutic)
compositions comprising a kringle 5 region of plasrr~inogen (or a biologically
active
fragment or analog thereof) and Angiostatin, optionally in combination with at
least
one additional active compound, and any pharmaceutically acceptable carrier,
adjuvant or vehicle. "Additional active compounds''' encompasses, but is not
limited
to, an agent or agents selected from the group consisting of an
irnmunosuppressant, an
anti-cancer agent, an anti-viral agent, an anti-inflammato;y agent, an anti-
fungal
1o agent, an antibiotic, or an anti-vascular hypeiproliferation compound.
The term "pharmaceutically acceptable cawier, adjuvant or vehicle" refers to a
cazTier, adjuvant or vehicle that may be administered to a subject,
incorporated into a
composition of the present invention, and which does not destroy the
pharmacological
activity thereof. Pharmaceutically acceptable carriers, adjuvants and vehicles
that
15 may be used in the pharmaceutical compositions of the present invention
include, but
are not limited to, the following: ion exchangers, alumina, aluminum stearate,
lecithin,
self emulsifying drug delivery systems ("SEDDS") such as d(-tocopherol
polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage
forms
such as Tweens or other similar polymeric delivery ;matrices, serum proteins
such as
2o human serum albumin, buffer substances such as phosphates, giycine, sorbic
acid,
potassium sorbate, partial glyceride mixtures of saturated vegetable fatty
acids, water,
salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate,
potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyroHdone, cellulose-based substances,
25 polyethylene glycol, sodium carboxyrnethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool
fat.
Cyclodextrins such as a-, (3- and y-cyclodextrin, or chemically modified
derivatives
such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-(3-
cyclodextrins, or other solubilized derivatives may also be used to enhance
delivery of
3o the compositions of the present invention.
The compositions of the present invention rr~ay contain other therapeutic
agents as descuibed below, and may be formulated, :For example, by employing
conventional solid or liquid vehicles or diluents, as well as pharmaceutical
additives
of a type appropriate to the mode of desired administration (for example,
excipients,
9
CA 02352477 2001-05-23
WO 00/31244 PCT/US99/27243
binders, preseZVatives, stabilizers, flavors, etc.) according to techniques
such as those
well known in the aart of phaz~naceutical foimularion.
The compositions of the present invention rr~ay be administered by any
suitable means, for example, orally, such as in the fcn~rn of tablets,
capsules, granules
or powders; sublingually; buccally; parenterally, such as by subcutaneous,
intravenous, inta~amuscular, or intrastemal injection or infusion techniques
(e.g., as
sterile injectable aqueous or non-aqueous solutions or suspensions}; nasally
such as
by inhalation spray; topically, such as in the form of a cream or ointment; or
rectally
such as in the form of suppositories; in dosage unit fornnulations containing
non-toxic,
l0 pharmaceutically acceptable vehicles or diluents. T'he present compositions
may, for
example, be administered in a form suitable for immediate release or extended
release. Immediate release or extended release may be achieved by the use of
suitable
pharmaceutical compositions comprising a ki-ingle 5 fragment and Angiostatin,
or,
particularly in the case of extended release, by the use of devices such as
subcutaneous implants or osmotic pumps. The present compositions may also be
administered liposomally.
Exemplary compositions for oral administration include suspensions which
may contain, for example, microciystalline cellulose for imparting bulk,
alginic acid
or sodium alginate as a suspending agent, methylcellulose as a viscosity
enhances, arid
2o sweeteners or flavoring agents such as those known in the aa~t; and
immediate release
tablets which may contain, for example, microczyst~alline cellulose, dicalcium
phosphate, starch, magnesium stearate andlor lactose and/or other excipients,
binders,
extenders, disintegrants, diluents and lubricants such as those known in the
art. The
present compounds may also be delivered through t:he oral cavity by sublingual
andlor
2s buccal administr ation. Molded tablets, compressed tablets or freeze-dried
tablets are
exemplary forms which may be used. Exemplary compositions include those
formulating the present compositions with fast dissolving diluents such as
rnannitol,
lactose, sucrose andlor cyclodextrins. Also included in such fomnulations may
be
high molecular weight excipients such as celluloses (avicel) or polyethylene
glycols
30 {PEG}. Such foumulations may also include an excipient to aid mucosal
adhesion
such as hydroxy propyl cellulose {HPC), hydroxy propyl methyl cellulose
(HPMC),
sodium carboxy methyl cellulose (SCMC), malefic ;anhydride copolymer {e.g.,
Gantrez), and agents to control release such as polyaciylic copolymer (e.g.,
Carbopol
CA 02352477 2001-05-23
WO 00/31244 PCTIUS99/27243
934). Lubricants, glidants, flavors, coloring agents .and stabilizers may also
be added
for ease of fabrication and use.
Exemplazy compositions for nasal aerosol or inhalation administration include
solutions in saline which may contain, for example, benzyl alcohol or other
suitable
preservatives, absorption promoters to enhance bioavailability, andlor other
solubilizing or dispersing agents such as those knovvn in the art.
Exemplary compositions for parenteral administration include injectable
solutions or suspensions which may contain, for example, suitable non-toxic;
parenterally acceptable diluents or solvents, such as mannitol, 1,3-
butanediol, water,
io Ringer's solution, an isotonic sodium chloride solution, or other suitable
dispersing or
wetting and suspending agents, including synthetic mono- or diglycerides, and
fatty
acids, including oleic acid. The team "parenteral" as used herein includes
subcutaneous, inti~acutaneous, intravenous, inti-amuscular, inti~aanicular,
intt~aaz-terial,
intrasynovial, inh-asternal, int<-athecal, intt-alesional and inti-acranial
injection or
15 infusion techniques.
Exemplary compositions for rectal administration include suppositories which
may contain, for example, a suitable non-irritating c;xcipient, such as cocoa
butter,
synthetic glyceride esters or polyethylene glycols, which are solid at
ordinary
temperatures, but liquify and/or dissolve in the rectal cavity to release the
active
2o compounds (i.e., the kringle 5 fragment and Angiostatin).
Exemplary compositions for topical administration include a topical carrier
such as Plastibase (mineral oil gelled with polyethylene).
The effective amount of a compound of the present invention may be
determined by one of ordinary skill in the art, and includes exemplary dosage
amounts
25 for an adult human of from about 0.1 to 500 mg/kg of body weight of active
compound per day, which may be administered in a single dose or in the form of
individual divided doses, such as from 1 to 5 times per day. It will be
understood that
the specific dose level and fi~equency of dosage for any particular subject
may be
varied and will depend upon a variety of factors including the activity of the
specific
3o compound employed, the metabolic stability and length of action of that
compound,
the species, age, body weight, general health, sex and diet of the subject,
the mode
and time of administration, rate of excretion and clearance, drug combination,
and
severity of the particular condition. Preferred subjects for treatment include
animals,
11
CA 02352477 2001-05-23
WO 00131244 PCT/US99/27243
most preferably mammalian species such as humans, and domestic animals such as
dogs, cats and the like, subject to angiogenic associated disorders.
The compositions of the present invention may be employed alone or in
combination with other suitable therapeutic agents useful in the treatment of
s angiogenic associated disorders, such as angiogenesis inhibitors other than
those of
the present invention, antiinflammatories, antiproliferatives,
chemotherapeutic agents,
immunosuppressants, and antimicrobials.
Other therapeutic agents, when employed in combination with the
compositions of the present invention,-may be used, for example, in those
amounts
to indicated in the Physicians' Desk Reference {PDR) or as otherwise
determined by one
of ordinary skill in the art.
The following examples ar-e further illustrative of the present invention.
These examples are not intended to limit the scope of the present invention,,
and
provide further understanding of the invention.
Example l: Gene construction, expression, and purification of recombinant
human
angiostatin
The human angiostatin cDNA was amplified from a human plasminogen
cDNA template {American Type Culture Collection, Rockville, MD) by standard
2o polymerase chain reaction (PCR) with the foilowinl; two primers:
5'-GCGGATCCATGAAAGTGTATCTCTCAGAGTGCAAG (forward
primer for residue 98-458); and
5'-GCGGATCCTCACTATTCTGTTCCTCiAGCATTTTTTCAG (reverse
primer for residue 98-458).
The amplified cDNA fragment was Iigated into the BamHI site of the
pMelBacA vector (InVitrogen, San Diego, CA). The angiostatin cDNA plasmid was
then co-transfected into S~ cells with viral BaculoCroldTM DNA (PharMingen,
San
Diego, CA). Briefly, lx 106 Sf~ cells were seeded in a T25 tissue culture
flask and
incubated at 27°C with 1 ml of the transfection solution containing 2
~g of transfer
3o vector DNA, 0.5 pg of BaculoGoldTM DNA, and 6 ~.l of Cellfectin (Gibco BRL,
Gaithersburg, MD). The transfection solution was removed 4 hours post-
transfection
and replaced with 3 m1 of Sf900II medium (Gibco BRL, Gaithersburg, MD). Four
days after incubation, the viral supernatant was harvested and individual
clones were
identified by limiting dilution. The clone with the lhighest protein
expression, as
12
CA 02352477 2001-05-23
WO 00/31244 PCT/US99/27243
determined by SDS-PAGE/Coomassie blue staining, was amplified in Sf9 cells for
protein production. High Five insect cells (InVitrogen, San Diego, CA} (1.5 x
10~
cells/rnl) were infected with approximately 1 x I 0' viral panicleslml of the
recombinant virus. After 48 houm, the culture supernatant was collected by
centrifugation at 5,000 x g for 30 minutes. The supernatant was then applied
to a
lysine-Sepharose column and angiostatin protein eluted with s-aminocaproic
acid as
previously described (Wu, Z., et. al., (1997) Biochem. Biophys. Res. Commun.
236:651-654).
Example 2: Production of recombinant knngle 5 of human plasminogen
The cDNA for K4-5 was amplified from HPg cDNA template by PCR as previously
described {Menhart, N., et. al., {1993) Biochemistry 32:8799-8806). The
amplified
cDNA was inserted between the Avr Il and the Not I sites of the pPIC9K vector
(InVitrogen, San Diego, CA). The transfer plasmid, pPIC9K[K4-KS of HPg], was
then linearized with restriction endonuclease Sac I and transformed into the
KM71
strain of Pichia pasto~is by electi~oporation. Isolation of high yield clones
and the
following high bio-mass fermentation were performed as previously described
(NiIsen, S. L., et. al., (1997) Biotech. ar~cl Applied Biochem. 25:63-74). The
K4-KS
protein of human plasminogen was purified from the fermentation medium by
lysine
2o affinity chromatography as described (Chang, Y., et. al., (1997)
Biochemistry
36(25):7652-7663). The purified products were extensively dialyzed against
water;
lyophilized, and digested with elastase at room temperature for 16 hours in
0.1 M
phosphate/15 mM s-aminocaproic acid (pH 7.8) at a protein: elastase ratio of
1:250
(w/w). The digest was then reapplied to a lysine-Sepharose chromatography
column
z5 and the kringle 5 fragment collected in the flow-through. The kringle S
protein was
extensively dialyzed against water and lyophilized.
Example 3: Anti-Endothelial Cell Proliferative/Mil;ratory Activity of Kringle
5 in
Combination With Angiostatin
3o Two primary endothelial cell types, human umbilical vein endothelial cell
(HLTVEC) and bovine capillary endothelial {BCE) cell, were chosen foa- the
Boyden-
chamber based migration assay. Briefly, HUVEC cells were obtained commercially
(Clonetics, San Diego, CA). Bovine capillary endothelial cells were harvested
fi-orn
bovine adrenal glands as previously described (Fol~kman, J. et. al., (1979}
Proc. Natl.
13
CA 02352477 2001-05-23
WO OOI31244 PCT/US99/27243
Acad. Sci. USA 76:5217-5221). BCE cells were maintained in DMEM in the
presence
of 10% bovine calf serum, 1% antibiotics, and 3 nglrnl of bFGF (PeproTech,
Rockhill, NJ).
To evaluate endothelial cell migration, a Boyden Chamber-based assay was
performed. First, polycarbanate membranes with 8 l.~m pore sizes (Neuro Probe
Inc.,
Cabin John, MD) were coated with 100 ~tg/ml of collagen type I following
manufacturer's instructions (Becton Dickinson, Bedford, MA). BCE cells between
passage 10 to 14, or I-IWEC cells between passage I to 3, were harvested with
0.05%
tiypsin solution, washed, resuspended to a density of 75,000 cells/ml in DMEM
to containing 10% bovine calf serum and 10 ng/ml of bFGF, and incubated at
37°C for
30 minutes. During cell incubation, various concentrations of kningle 5,
Angiostatin,
or a combination of kringle 5 and Angiostatin samples were loaded into the
lower
chambers. The collagen-coated membrane filter ways placed on top of the lower
chamber and the top chamber then attached. After the 30 minute incubation,
endothelial cells were loaded into the top chamber and incubated at 37"C for 4
hours.
The chemotaxis chamber was then dismantled and the filter membrane removed.
The
non-migrated cells were scraped off the upper surface of the membrane with
cotton
swabs three times. After rinsing with PBS, the membrane was fixed with 10%
buffered formalin for 45 minutes and then stained with Gill No. 2 hematoxyiin
overnight (J.B. Baker, Phillipsburg, NJ). The membrane was then rinsed with
PBS
and mounted with Cytoseal (Stephens Scientific, Riverdale, NJ). Each sample
was
tested in quach-uplicate and a representative field in each well was counted
at 100x
magnification to determine the number of migrated cells.
Table 2 below summarizes the inhibitory activities of kningle 5 ("KS"),
Angiostatin ("AST"), KS+AST combination, andkongle I-5 ("Kl-5") of
pl asminogen.
Table 2
Protein lCso (nM) ICso (nM)
(HUVEC),............................................._..._.....................
................~BCE)..............................................._..........
....
.................................... 489.1
.........._........................_..._.:........._.......................
AST 650.2
K5 100.0 89.4
K5 + AST 9.3 8.8
...~,."~,., K1-' w,~"~""~.."~,., 8~~ "~",.,N." 33.7-
~.~"w .,~.,~"..""~.,~.,.",.
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CA 02352477 2001-05-23
WO 00/31244 PCT/US99/27243
For HUVEC migration, the ICso's (protein concentrations at 50% of
inhibition) of angiostatin and kringle 5 are approximately 0.651ZM and 0.10
p.M,
respectively (Figure lA & Table 2). A combination of these two agents produced
a
significant increase of the anti-migratory activity wiith an ICso of about 9.3
nM. This
one-log increase in anti-endothelial cell activity implies that kringle S and
angiostatin
may inhibit angiogenesis via different yet coordinated pathways. For BCE cell
migration, the ICsc~'s of angiostatin and lcringle 5 were approximately 0.49
p.M and
0.09 ~.M, respectively {Figure 1B & Table 2). A combination of these two
proteins
resulted in a nearly one log increase in. the anti-migratory activity (ICso =
8.8 nM).
to This synergism suggests that kringle S and angiostatin may function in
cooperation to
block angiogenesis. As a reference, the recombinant kringle I-5 protein was
generated in the Bacculovirus expression system. It is shown to potently
inhibit
ITEJVEC and BCE cell migration in a dose-dependent manner. The anti-migratory
activities of lcringle I -5 are close to those of angiost,atin and kringle 5
combinations
for HUVEC (ICso of kringle 1-5 = 8 nM), but is approximately 4-fold less
potent for
BCE migration (ICso = 33.7 nM for BCE). This shows that the combination of a
kringle 5 fragment of plasminogen plus Angiostatin shows superior activity
even over
a ki-ingle I-5 fragment of plasminogen. Ki-ingle 5 of human pIasminogen may
thus be
used in combination with Angiostatin to potentiate ias anti-angiogenic
functions.
Although the present invention has been described in some detail by way of
illustration and example for purposes of clarity and understanding, it will be
apparent
that certain changes and modifications may be practiced within the scope of
the
appended claims.
15
