Note: Descriptions are shown in the official language in which they were submitted.
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HEPATOCYTE GROWTH FACTOR ANTAGONISTS
Field of the Invention
The field of the invention is treatment of chronic
renal disease.
Background of the Invention
Hepatocyte growth factor (HGF) is a heterodimeric
molecule derived from a preproprecursor protein of 728
amino acids, which is proteolytically processed by a
specific protease to form mature HGF (Miyazawa et al.,
1993, J. Biol. Chem. 268:10024-10028).
HGF binds to cells through the tyrosine kinase
receptor c-met. Binding of HGF to c-met has the
following effects: Growth of renal epithelial cells is
stimulated; the motility of cells is enhanced; and,
renal tubule formation is induced (Santos et al., I993,
Dev. Biol. 159:535-548; Cantley et al., 1994, Am. J.
Physiol. 267:F271-F280). Thus, HGF may be characterized
as a mitogen, a motogen and a morphogen, respectively,
and is believed to play a role in renal development.
In addition, HGF is involved in renal remodeling
following injury as reflected by increased levels of HGF
and its receptor c-met in the kidney following
nephrectomy or ischemia (Joannidis et al., 1994, Am. J.
Physiol. 267:F231-F236). Further, HGF has been observed
to effect an improvement in renal function following
mercuric chloride assault or ischemia (Kawaida et al.,
1994, Proc. Natl. Acad. Sci. USA 91:4357-4361; Miller et
al., 1994, Am. J. Physiol. 266:F129-F134). Glomerular
hypertrophy occurs during renal remodeling in many
chronic renal diseases. This event is associated with
glomerular basement membrane expansion, proliferation of
mesangial and epithelial cells, and the accumulation of
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collagen and fibronectin. The role of HGF in glomerular
extracellular matrix expansion is not known.
Chronic renal failure is the progressive loss of
functional renal mass, accompanied by compensatory
growth and remodeling. The molecular and cellular
events that take place during chronic renal failure
include release of growth factors, proliferation of
glomerular mesangial cells and expansion of
extracellular matrix (Klahr et al., 1988, New Engl. J.
Med. 3l8:1657-1666; Striker et al., 1989, In: Klahr,
(Ed.) Seminars in Nephrology, pp. 318, Philadelphia,
W.B. Saunders); Ebihara et al., 1993, J. Am. Soc.
Nephrol. 3:1387-1397). The components of the
extracellular matrix which change during chronic renal
failure include collagen, fibronectin, and laminin
(Ebihara et al., supra; Tarsio et al., 1988, Diabetes
37:532-539; Yoshioka et al.) 1989, Kidney Int. 35:1203-
1211). In the renal ablation model, fibronectin and
collagen a1 immunocytochemical staining is elevated in
areas of increased cellularity of glomeruli at 2 and 6
weeks after nephrectomy of 5/6 of the renal mass, when
chronic renal failure is established (Foelge et al.,
1992, Lab. Invest. 66:485-497).
Several protein factors have been implicated in
stimulating mitogenesis of mesangial cells or
extracellular matrix production during chronic renal
failure. These include thrombin (Xu et al., 1995, Am.
J. Pathol. 146:101-110; Sraer et al., 1993, Ren. Fail.
15:343-348; Albrightson et al., 1992, J. Pharmacol. Exp.
Ther. 263:404-412), epidermal growth factor (Byyny et
al., 1972, Endocrinology 90:1261-1266; Killion et al.,
2993, J. Urol. 150:1551-1556), insulin-like growth
factor-1 (Stiles et al., 1985, Endocrinology 117:2397-
2401; Fagin et al., I987, Endocrinology 120:718-724),
and transforming growth factor-b1 (Coimbra et al., 1991,
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Am. J. Pathol. l38:223-234; Okuda et al., 1990, J. Clip.
Invest. 86:453-462).
° There remains a need for compositions for treatment
of chronic renal disease which serve to diminish or
~ 5 ablate disease leading to renal failure and either death
or dependence upon dialysis.
Summary of the Invention
The invention relates to a method of treating a
human having chronic renal disease comprising
administering a hepatocyte growth factor antagonist to
the human.
Brief Description of the Drawings
Figure 1 is a series of graphs depicting
extracellular acidification rates of transformed mouse
mesangial cells (MMC-SV40 cells) (Panels A-C), or normal
human mesangial cells (Panel D) determined by
microphysiometry. Panel A: MMC-SV40 cells treated with
HGF (100 ng/ml) for 10 minutes in the presence or
absence of 0.1~ bovine serum albumin (BSA) or 0.5~ fetal
bovine serum (FBS). Panel B: MMC-SV40 cells treated
with HGF (100 ng/ml) for 2, 5, 10, and 15 minutes.
Panel C: MMC-SV40 cells treated with 4 concentrations
of HGF. Panel D: Normal human mesangial cells treated
with 100 ng/ml HGF; duplicate traces are shown.
Figure 2 is a graph depicting extracellular
acidification rates of MMC-SV40 cells treated with HGF
(50 ng/ml). Cells were perfused with control medium or
medium containing different concentrations of RO-32-
0432, a protein kinase inhibitor described by Birchall
et al., 1994, J. Pharmacol. Exp. Ther. 268:922-929.
Figure 3 is a graph depicting tritiated thymidine
incorporation of normal human mesangial cells (HMC},
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MMC-SV40 cells, or mouse mesangial cells transfected
with a luciferase reporter plasmid driven by the
collagen a1 (IV) promoter (MMC-COL cells) treated with
different concentrations of HGF for 24 hours.
Figure 4 is a graph depicting collagen a1 (IV)
promoter activity in MMC-COL cells in response to
different concentrations of HGF. Data are the means
SD of quadruplicates.
Figure 5 is a graph depicting creatinine clearance
in lean and obese (diabetic) mice assessed at 21 days
after either vehicle or HGF implants. HGF significantly
decreased creatinine levels in obese (diabetic) mice (*
< 0.05; n=5-6). LV - lean vehicle; LH - lean HGF; OV -
obese vehicle; OH - obese HGF treated mice.
Detailed Descrit~ton of the Invention
It has been discovered according to the present
invention, that long term administration of HGF results
in chronic renal disease. Thus, although HGF may be
useful for treatment of acute renal disease, when this
compound is administered to a mammal for prolonged
periods of time, renal function is decreased. It has
also been discovered that treatment of cells with HGF
results in increased fibronectin mRNA and transcription
in mesangial and epithelial cells. Thus, the present
invention is based on the discovery that HGF contributes
to renal disease in part by activating fibronectin and
collagen synthesis, thereby causing glomerulosclerosis.
The invention relates to a method of treating
chronic renal disease in a mammal comprising
administering an antagonist of HGF to the mammal.
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By "chronic renal disease" as used herein, is meant
progressive loss of renal function as measured by
glomerular filtration rate.
Antagonists of HGF include, but are not limited to,
small non-peptide molecules, peptides comprising
specific portions of HGF, peptidometics having anti-HGF
activity, antibodies directed against HGF, nucleic acids
having a sequence which is complementary to all or a
portion of the nucleic acid encoding HGF and small
chemical compounds which inhibit HGF-specific protease.
By "HGF antagonist activity" as used herein, is
meant a compound which inhibits the normal activity of
HGF, such as determined, for example, in any one or more
of the assays described herein. By way of example,
treatment of mouse mesangial cells with HGF results in
an increase in the acidification rate of these cells.
Thus, a compound having HGF antagonist activity is
defined as one which inhibits an HGF-induced increase in
acidification rate in mouse mesangial cells.
To identify an antagonist of HGF, a test compound
is assessed for HGF antagonist activity in one or more
of the assays described herein in the experimental
examples section, or in any other assay for measurement
of HGF function. Preferably, initially, an in vitro
test is used to identify a compound having HGF
antagonist activity. Such in vitro tests include, but
are not limited to, tests which assess the affect of the
test compound on HGF binding to cell membranes of cells
which are known to respond to HGF, on HGF-induced
acidification rate of mesangial cells and tests which
assess the affect of the test compound on HGF-induced
extracellular matrix gene expression.
To assess the effect of a test compound on HGF
binding, [125I~_labeled HGF is incubated with cell
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membranes from A498 cells attached to scintillation
beads plus or minus the-test compound. If the compound
inhibits HGF binding to the membrane, then the [1252_
labeled HGF will not be in the proximity of the
scintillation bead resulting in decreased signal. A
secondary assay used below would determine if this
compound is an agonist or antagonist.
To assess the effect of a test compound on cell
acidification rates, mesangial cells are incubated in
the presence or absence of HGF plus or minus the test
compound. The acidification rates of the cells are
measured by microphysiometry as described herein and the
effect of the test compound on HGF-induced cell
acidification is determined. A reduction in the
acidification rates of cells treated with HGF and the
test compound, compared with the acidification rates in
cells treated with HGF alone, is an indication that the
test compound has HGF antagonist activity.
To assess the effect of a test compound on
extracellular matrix gene expression, cells are
incubated in the presence or absence of HGF plus or
minus the test compound under the conditions described
herein in the experimental examples section. The
expression of mRNA associated with extracellular matrix
genes (e.g., collagen al(IV) and fibronectin~ is
measured and compared among the different sets of cells.
A reduction in extracellular matrix mRNA expression in
cells treated with HGF and the test compound, compared
with extracellular matrix mRNA expression in cells
treated with HGF alone, is another indication that the
test compound possesses HGF antagonist activity.
The test compound may also be tested for HGF
antagonist activity in an in vivo assay. As will be
apparent from the data provided in the experimental
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examples section, long term treatment of mice with HGF
induces renal failure. -Thus, another exemplary way to
assess the HGF antagonist activity of a test compound is
to administer HGF alone HGF plus test compound to mice
over at least a 21 day period. Additional controls
include mice which are administered a suitable placebo
compound. Creatine clearance is assessed in each set of
mice as a measure of renal function. An increase in
creatine clearance in mice administered HGF plus the
test compound, compared with creative clearance in mice
administered HGF alone, is another indication that the
test compound has HGF antagonist activity.
HGF antagonists which comprise peptides comprising
specific portions of HGF include, but are not limited
to, those which encompass the first krinkle domain near
the N-terminal of HGF. An example of such a peptide is
the truncated HGF peptide NK2 (Chan et al., 1991,
Science 254:1382-1385).
To obtain a substantially pure preparation of a
peptide comprising a portion of HGF, the peptide may be
produced by cloning and expressing HGF DNA encoding the
desired portion of HGF. HGF DNA and amino acid
sequences are provided in Miyazawa et al., 1991, Eur. J.
Biochem. 197:15-22 (SEQ ID NOs: 1 and 2). An isolated
DNA encoding the desired portion of HGF is cloned into
an expression vector and the protein is expressed
therefrom. Procedures for cloning and expression of
peptides are well known in the art and are described,
for example, in Sambrook et al. (1989, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, New
York). Peptide so expressed may be obtained using
ordinary peptide purification procedures well known in
the art.
As used herein, the term "substantially pure"
describes a compound, e.g., a protein or polypeptide
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which has been separated from components which naturally
accompany it. Typically, a compound is substantially
pure when at least 10~, more preferably at least 20~,
more preferably at least 50~, more preferably at least
60~, more preferably at least 75~, more preferably at
least 90~, and most preferably at least 98~ of the total
material (by volume, by wet or dry weight, or by mole
percent or mole fraction) in a sample is the compound of
interest. Purity can be measured by any appropriate
method, e.g., in the case of polypeptides by column
chromatography, gel electrophoresis or HPLC analysis. A
compound, e.g., a protein, is also substantially
purified when it is essentially free of naturally
associated components or when it is separated from the
native contaminants which accompany it in its natural
state.
The present invention also includes analogs of
peptides obtained according to the methods of the
invention. Analogs can differ from naturally occurring
peptides by conservative amino acid sequence differences
or by modifications which do not affect sequence, or by
both.
For example, conservative amino acid changes may be
made, which although they alter the primary sequence of
the peptide, do not normally alter its function.
Conservative amino acid substitutions typically include
substitutions within the following groups:
glycine, alanine;
valine, isoleucine, leucine;
aspartic acid, glutamic acid;
asparagine, glutamine;
serine, threonine;
lysine, arginine;
phenylalanine, tyrosine.
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Modifications (which do not normally alter primary
sequence) include in vivo, or in vitro chemical
derivatization of peptides, e.g., acetylation, or
carboxylation. Also included are modifications of
glycosylation, e.g., those made by modifying the
glycosylation patterns of a peptide during its synthesis
and processing or in further processing steps; e.g., by
exposing the peptide to enzymes which affect
glycosylation, e.g., mammalian glycosylating or
deglycosylating enzymes. Also embraced are sequences
which have phosphorylated amino acid residues, e.g.,
phosphotyrosine, phosphoserine, or phosphothreonine.
Also included are peptides which have been modified
using ordinary molecular biological techniques so as to
improve their resistance to proteolytic degradation or
to optimize solubility properties. Analogs of such
peptides include those containing residues other than
naturally occurring L-amino acids, e.g., D-amino acids
or non-naturally occurring synthetic amino acids. The
peptides of the invention are not limited to products of
any of the specific exemplary processes listed herein.
Thus, the present invention also relates to active
fragments of HGF having HGF antagonistic activity. A
specific polypeptide is considered to have HGF
antagonistic activity if it inhibits the action of HGF
as described herein.
As used herein, the term "fragment," as applied to
a HGF peptide, will ordinarily be at least about 10
contiguous amino acids, typically at least about 20
contiguous amino acids, more typically at least about 50
continuous amino acids and usually at least about 78
contiguous amino acids in length.
Nucleic acid sequence complementary to HGF may be
generated using the sequence of HGF provided in Nakamura
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et al. (supra). Administration of antisense
oligonucleotides to mammals is now common in the art and
may be accomplished by using any of the administration
techniques described herein. Nucleic acid complementary
to nucleotides 78-950 of HGF (SEQ ID NO: 1) or portions
thereof may be obtained by cloning HGF DNA or portions
thereof into an expression vector such that RNA is
expressed therefrom in the antisense orientation (i.e.,
complementary) to HGF mRNA.
An "isolated DNA", as used herein, refers to a DNA
sequence, segment, or fragment which has been purified
from the sequences which flank it in a naturally
occurring state, e.g., a DNA fragment which has been
removed from the sequences which are normally adjacent
to the fragment, e.g., the sequences adjacent to the
fragment in a genome in which it naturally occurs. The
term also applies to DNA which has been substantially
purified from other components which naturally accompany
the DNA, e.g.) RNA or DNA or proteins which naturally
accompany it in the cell.
"Complementary" as used herein, refers to the
subunit sequence complementarity between two nucleic
acids, e.g., two DNA or RNA molecules. When a
nucleotide position in both of the molecules is occupied
by nucleotides normally capable of base pairing with
each other, then the nucleic acids are considered to be
complementary to each other at this position. Thus, two
nucleic acids are complementary to each other when a
substantial number (at least 50~) of corresponding
positions in each of the molecules are occupied by
nucleotides which normally base pair with each other
(e. g., A:T and G:C nucleotide pairs).
The invention should also be construed to include
DNAs which are substantially homologous to HGF DNA or
portions thereof, which DNAs are useful for the
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production of HGF complementary nucleic acid, or for the
production of HGF peptides. Preferably, DNA which is
substantially homologous is about 50~ homologous, more
preferably about 70~ homologous, even more preferably
about 80~ homologous and most preferably about 90~
homologous to DNA obtained using the method of the
invention.
"Homologous" as used herein, refers to the subunit
sequence similarity between two polymeric molecules,
e.g., between two nucleic acid molecules, e.g., two DNA
molecules or two RNA molecules, or between two
polypeptide molecules. When a subunit position in both
of the two molecules is occupied by the same monomeric
subunit, e.g., if a position in each of two DNA
molecules is occupied by adenine, then they are
homologous at that position. The homology between two
sequences is a direct function of the number of matching
or homologous positions, e.g., if half (e. g., five
positions in a polymer ten subunits in length) of the
positions in two compound sequences are homologous then
the two sequences are 50~ homologous, if 90~ of the
positions, e.g., 9 of 10, are matched or homologous, the
two sequences share 90~ homology. By way of example,
the DNA sequences 3'ATTGCCS' and 3'TATGCG5' share 50~
homology.
Anti-HGF antibodies are easily generated by
immunization of a mammal with the HGF peptide identified
herein. Protocols for the generation of antibodies
(either monoclonal or polyclonal antibodies) to a known
peptide are described in Harlow et al. (1988, In:
Antibodies, A Laboratory Manual, Cold Spring Harbor,
NY), which protocols can be easily followed by the
skilled artisan. Polyclonal antibodies to HGF may be
raised in any suitable mammal, such as a mouse or a
rabbit. Monoclonal anti-HGF antibodies are generated by
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immunization of a mouse with HGF peptide followed by
production of hybridoma cells capable of secreting anti-
HGF antibody. Other means of producing monoclonal
antibodies, such as antibodies which are expressed by
bacteriophage, are now also well known in the art and
are described, for example, in Marks et al. t1991, J.
Mol. Biol. 222:581-597), Barbas (1995, Nature Medicine
1:837-839), de Kruif et a~. (1995, J. Mol. Biol. 248:97-
105) and Sternberg et al. (1995, Proc. Natl. Acad. Sci.
USA 92:1609-1613).
Peptidometics having HGF-antagonist-like activity
may also be designed and used according to the present
invention. Additional information describing
administration of peptidometics is provided in
PCT/US93/01201 and U.S. Patent No. 5,334,702, which are
hereby incorporated herein by reference. Any of the
techniques described in either of these two references
may be employed in the present invention for the
administration of peptidometics.
An HGF antagonist may also include smal:i molecules
having HGF antagonist activity as defined herein which
are not peptide or nucleic acid molecules.
Examples of HGF antagonists useful in the method of
the present invention include, but are not limited to,
those described in Faletto et al. (WO 94/06909) Roos et
al. (W094/06456), JP05208998 and Aaronson et al., (WO
92/05184).
Kidney diseases which are treatable using the
method of the invention include, but are not limited to,
polycystic disease, diabetic nephropathy, focal
segmental glomerulosclerosis, hypertension-induced
nephropathy, hypernephroma, and the like.
Protocols for treatment of mammals with a chronic
renal disease involving administration of an antagonist
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of HGF will be apparent to those skilled in the art and
will vary depending upon-the type of disease and the
type and age of the mammal. Treatment regimes which are
contemplated include a single dose or dosage which is
administered hourly, daily, weekly or monthly, or
yearly. Dosages may vary from 1-1000 mg/kg of body
weight of the antagonist, and will be in a form suitable
for delivery of the compound. The route of
administration may also vary depending upon the disorder
to be treated.
The invention contemplates administration of an HGF
antagonist to humans for the purpose of alleviating or
ablating chronic renal disease. The protocol which is
described below for administration of HGF to a human is
provided as an example of how to administer HGF to a
human. This protocol should not be construed as being
the only protocol which can be used, but rather, should
be construed merely as an example of the same. Other
protocols will become apparent to those skilled in the
art when in possession of the instant invention.
Essentially, for administration to humans, the HGF
antagonist is dissolved in about 1 ml of saline and
doses of 1-1000 mg per kg of body weight are
administered orally or intra-venously once per day to
several times per day. Renal function is monitored
throughout the administration period.
The antagonist of HGF is prepared for
administration by being suspended or dissolved in a
pharmaceutically acceptable carrier such as saline,
salts solution or other formulations apparent to those
skilled in such administration. The compositions of the
invention may be administered to a mammal in one of the
traditional modes (e. g., orally, parenterally,
transdermally or transmucosally), in a sustained release
formulation using a biodegradable biocompatible polymer,
or by on-site delivery using micelles, gels and
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liposomes, or rectally (e. g., by suppository or enema)
or nasally (e.g., by nasal spray). Thus) an HGF
antagonist may be administered to the mammal by any
route in order that it eventually reaches the target
area in the mammal, i.e., the kidney, wherein it exerts
its effects. The appropriate pharmaceutically
acceptable carrier will be evident to those skilled in
the art and will depend upon the route of
administration.
Compounds having HGF antagonist activity also
include compounds which are formulated so as to target
specific types of cells. For example, it is now known
in the art to encapsulate or otherwise formulate
compounds such that they are directed to specific
receptors on cells. Such formulations include antibody-
tagging formulations, receptor-ligand binding
formulations and the like.
The invention is further described in detail by
reference to the following experimental examples. These
examples are provided for purposes of illustration only,
and are not intended to be limiting unless otherwise
specified. Thus, the invention should in no way be
construed as being limited to the following examples,
but rather, should be construed to encompass any and all
variations which become evident as a result of the
teaching provided herein.
The experimental examples described herein provide
procedures and results which establish that antagonists
of HGF are useful for treatment of chronic renal disease
since, according to the data provided herein, treatment
of cells in vitro with HGF results in the synthesis in
the production of compounds associated with chronic
renal disease. Similarly, long term treatment of
animals in vivo with HGF results in a reduction of renal
function.
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Examflles
Cell culture
Mouse mesangial cell cultures were established from
glomeruli obtained from the kidneys of 8 to 10 week old
SJL/J (H-2B) mice (Wolf et al., 1992, Am. J. Pathol.
140:95-107y. Cells were grown in Dulbecco's modified
Eagle's medium (DMEM) containing 2 mM L-glutamine and
180 mg/dl glucose, supplemented with 10~ FBS and 100U/ml
penicillin/streptomycin at 37°C in 5~ C02. Cells were
subcultured by rinsing with phosphate buffered saline
(PBS) and incubation with 0.05 trypsin supplemented
with 20 mM EDTA.
Mouse mesangial cells transformed with noncapsid
forming SV40 virus to establish a permanent cell line
(Wolf et al., supra) are designated as MMC-SV40 cells.
These cells exhibit many features of differentiated
mesangial cells (Wolf et al., supra). A stable
transfection was performed on MMC-SV40 cells with a
reporter construct HB35, which expresses luciferase
driven by a "minigene" comprised of the 5' flanking and
first intron regions of the marine COL4A1 gene (Fumo et
al., 1994, Am. J. Physiol. 267:F632-F638). The stable
transformants are designated as MMC-COL cells. These
cells exhibited patterns of growth and protein synthesis
in response to elevated glucose concentration similar to
MMC-SV40 cells (Fumo et al., supra).
Cryopreserved human mesangial cells (passage 3)
were purchased from Clonetics Corp. (San Diego, CA) and
were grown in Clonetics Mesangial Cell Growth Medium
(MsGM) supplemented with 5~ FBS, 50 mg/ml Gentamicin and
50 ng/ml Amphotericin-B. Human mesangial cells were
grown according to the methods provided by Clonetics
Corp. and were used in these experiments bet:.~een passage
6-8.
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Microphysiometry
The cytosensor micr-ophysiometer is based on a pH
sensitive silicon sensor which is part of a microvolume
flow chamber in which cells are immobilized (McConnel et
al., 1992, Science 257:1906-1912). Mouse mesangial
cells were subcultured with trypsin from 150 cm2 flasks
into capsule cups (Molecular Devices, Inc., Sunnyvale,
CA) having a polycarbonate membrane of 3 mm pore size at
a density of 300,000 cells per cup. Cells were allowed
to attach for 24 hours in the medium specified for each
cell type. After spacer rings and insert cups were
fitted into the capsule cups, the assembled units were
transferred to the sensor chamber and perfused at 100
ml/min with bicarbonate-free RPMI 1640 medium (Molecular
Devices, Inc.) in the microphysiometer. Acidification
rate was measured as a change in pH overtime, which was
determined when the pumps were turned off for 30 seconds
in 2 minute intervals.
Mitogenesis assay
The mitogenic effect of HGF on mouse mesangial
cells was measured as the amount of [3H] thymidine
incorporated into newly synthesized DNA. Cells were
subcultured into 24 well dishes (2.5 x 10' cells per well
and Incubated in growth media for 72 hours.
Subconfluent cultures were made quiescent by placing
them for 48 hours in DMEM medium containing 2 mM L-
glutamine and 100 mg/dl glucose, the medium being
further supplemented with 3~ FBS and 100/m1 penicillin
and 100 mg/ml streptomycin. HGF was diluted in
unsupplemented DMEM medium and then added to wells in
triplicate for 24 hours. Cells were pulsed with [3H]
thymidine during the last 4 hours of the 24 hour
incubation period. Cells were washed with PBS, and then
5~ trichloroacetic acid was added to precipitate
~s
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proteins and nucleic acids and to remove unincorporated
[3H] thymidine. The precipitate was then dissolved by
adding 0.5 m1 of 0.5 N NaOH and 400 ml aliquots were
added to scintillation fluid and counted on a Taurus
liquid scintillation counter (ICN Biomedicals, Inc.,
Huntsville, AL).
Luciferase assay
MMC-COL cells were cultured in 24 well plates at
25,000 cells per well in growth medium for 48 hours.
Cells were then incubated in the same medium used to
make the cells quiescent in the proliferatio~:~ assay and
were incubated for an additional 48 hours before HGF was
added. At the times indicated, cells were lysed using
500 ml of a buffer containing 0.1 mM potassium phosphate
and 1 mM dithiothreitol, pH 7.2 with 1 ~ Triton X-100
(luciferase activity) or were trypsinized (cell number).
The lysed cells were centrifuged and 100 ml of the
supernatant was added in duplicate to wells of a 96 well
microliter plate. Light emission was measured directly
at room temperature using a Microlumat LB96P luminometer
(Wallac Inc., Gaithersburg, MD) and integrated over a
20-s period following the automated injection of 100 ml
of a luciferin reaction mixture. This mixture contained
a stock buffer of 0.1 mM potassium phosphate, 10 mM ATP,
20 mM MgCl2 and 1 mM dithiothreitol, pH 7.2, and freshly
added 0.8 mg/ml of D-luciferin (Boehringer Mannheim,
Indianapolis, IN). Luciferase activity was expressed as
relative light units (RLU) per number of cells as
determined from similarly treated cultures of cells in
wells which were trypsinized and counted (Coulter
Electronic LTD, Luton, Beds, England).
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Northern blot hybridization
MMC-SV40 cells were cultured in 150 cm2 flasks and
incubated for 3 to 4 days in the growth medium described
above. The medium was changed to DMEM medium containing
2 mM L-glutamine and 100 mg/dl glucose and supplemented
with 3~ FBS and 100U/ml penicillin and 100 mg/ml
streptomycin for 48 hours before the addition of HGF.
Cells were incubated for 2, 6, and 16 hours with HGF.
Total RNA was extracted from mouse cells by guanidinium
thiocyanate denaturation and acidified phenol-chloroform
extraction (Chomczynski et al., 1987, Anal. Biochem.
162:156-159). Total RNA (10 mg/lane) was fractionated
on 0.2 M formaldehyde-1~ agarose gels and transferred to
nylon membranes (Nylon-1; Bethesda Research
Laboratories, Bethesda, MD) in 4X SSC. Equivalent
loading and transfer were verified by methylene blue
staining. Random primed [32P]DNA probes were made for
fibronectin that recognize an mRNA of 7.6 kb. The
fibronectin clone was purchased from ATCC.
Hybridizations were performed with 106 cpm/ml of labeled
DNA in 50~ formamide, 225 mM NaCl, 20 mM NaH2P09, 1.5 mM
EDTA, 1~ sodium dodecyl sulfate, 0.5~ dry milk, 100
mg/ml yeast total RNA and 300 mg/ml salmon DNA, at 42°C
for 16 hours. Blots were washed with a final stringency
of 0.2 X SSC, 0.2~ sodium dodecyl sulfate at 65°C.
Membranes were exposed to phosphor imaging plate and
bands were quantified with ImageQuant software
(Molecular Dynamics, Inc.).
Animal experiments
Alzet mini-osmotic pumps {Alza, Palo Alto, CA) were
filled with either HGF at 14.6 ng/ml or vehicle (350 mM
NaCl, 10 mM phosphate pH 7.3, 625 mg/ml human serum
albumin. Lean and obese mice of the C57BL/Ks strain
with the recessive db mutation were purchased from the
18
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Jackson laboratory (Bar Harbor, ME). Mice received the
implants in the peritoneal cavity under Ketalar (60
mg/kg) and were killed 21 days later. Twenty-four hour
urine samples were collected in metabolic cages on the
day of sacrifice. At the time of sacrifice, blood was
also collected. Urine and serum creatinine levels were
determined by a Synchron Clinical System AS8 (Beckman;
Columbia, MD). Renal function was determined by
calculating creatinine clearance.
HGF mediated acidification rates
In order to evaluate the effect of FBS or BSA on
HGF mediated acidification rates, MMC-SV40 cells were
pretreated with 0.1~ BSA, 0.5~ FBS or both for 30
minutes. This experiment was performed because
recombinant HGF obtained from R&D Systems, Minneapolis,
MN occurs in the single chain precursor form which
requires enzymatic activation. Proteases in the serum
or proteases secreted by cells may activate HGF, or
proteases contained in FBS may be required. In
addition, HGF, like other growth factors, tends to
adhere to plastic tubing and thus, a carrier molecule
for delivery of HGF to the cells may be required.
Following the pre-incubation period, cells were exposed
to 100 mg/ml HGF for 10 minutes in the presence or
absence of 0.1 ~ BSA or 0.5~ FBS.
In the presence of FBS, a peak of HGF-induced
acidification was observed to be attenuated although the
baseline response was reset at 30~ higher than prior to
HGF administration. BSA caused a dramatic attenuation
of the peak response to HGF with or without FBS (Figure
1, Panel A). Therefore, in the remaining experiments
HGF was administered in the absence of FBS or BSA in the
running medium.
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The effect of the length of time of exposure of
cells to HGF on acidification rates was assessed by
treating MMC-SV40 cells with 100 ng/ml HGF for 2, 5, 10,
and 15 minutes. While incubation of the cells in the
presence of HGF for 5, 10, and 15 minutes each yielded
similar peaks of acidification rates, a 5 minute
exposure time was chosen for further experiments because
of the rapid recovery of the cells following this
treatment (Figure 1, Panel B).
The HGF dose response was assessed by treating MMC-
SV40 cells for 5 minutes with 3) 10, 30, or 100 ng/ml of
HGF. A concentration of 30 ng/ml HGF elicited the
maximum increase in acidification rate with no further
increase at 100 ng/ml HGF. (Figure 1, Panel C).
To determine if non-transformed mesangial cells
also respond to HGF, primary human mesangial cells were
treated at passage 7. HGF at a concentration of 100
ng/ml induced an increase in the acidification rate of
human mesangial cells (Figure 1, Panel D).
The involvement of PKC mediated second messenger
systems in mesangial responses to HGF was evaluated
using the PKC inhibitor RO-32-0432. Transformed mouse
mesangial cells were pre-treated with 1, 3, or 5 mM R0-
32-0432 for 30 minutes and HGF at a concentration of 50
ng/ml was added for 5 minutes in the presence of the
pretreatment medium. RO-32-0432 partially blocked the
HGF-induced acidification rate in a concentration
dependent fashion (Figure 2).
The effect of HGF oa mitogeaesis
Proliferation of normal human and immortalized
mouse mesangial cells was evaluated by tritiated
thymidine incorporation. Human glomerular mesangial
cells were serum starved far 48 hours. Mouse mesangial
cells were grown in 3~ serum. Each set of cells was
treated with increasing doses of HGF up to 100 ng/ml for
CA 02270867 1999-05-04
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24 hours. HGF did not affect tritiated thymidine
incorporation at any doses tested in bath normal human
and immortalized mouse mesangial cells (Figure 3).
The affect of HGF on extracellular matrix gene
expression
The effect of HGF on extracellular matrix gene
expression was evaluated by assessing collagen al(IV)
promoter activation of a luciferase reporter gene in
MMC-COL cells and by northern blot hybridization
analysis of collagen al(IV) and fibronectin mRNA in MMC-
SV40 cells.
HGF-induced activation of the collagen promoter in
mouse mesangial cells in a dose-dependent fashion.
Collagen promoter activity doubled in the presence of 30
ng/ml HGF compared with untreated cells (Figure 4).
MMC-SV40 cells were treated with 50 nglml HGF in
the presence or absence of 1 mM 5-amino-2-(4-
aminoanilino)benzenesulfonic acid (ICN Biomedicals,
Inc., Costa Mesa, CA), a protein kinase C inhibitor.
Northern blots indicated that collagen a1 (IV) mRNA
levels were increased in the mouse mesangial cells by
HGF and that the inhibitor blocked the HGF-induced
increase (data not shown).
MMC-SV40 cells were also treated with 50 ng/m1 HGF
for 2, 6, and 16 hours in the presence of 3o serum.
Northern blots indicated that HGF induced an increase in
fibronectin mRNA levels at 6 and 16 hours following HGF
administration (data not shown).
The effect of HGF on renal function
The effect of HGF on renal function was determined
by evaluating creatinine clearance following chronic HGF
administration for 21 days in normal and diabetic mice.
Creatinine clearance in normal and diabetic vehicle
implanted mice was 400 and 454 ml/minute/100g,
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CA 02270867 1999-OS-04
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respectively. HGF caused a decrease in creatinine
clearance in both groups to 283 and 287* ml/minute/100
g, respectively (*p < 0.05 vs vehicle group; n = 5-6
mice; Figure 5) .
The data presented herein establish that glomerular
mesangial cells respond to HGF exhibiting changes in
acidification rate and extracellular matrix gene
expression. The effect of HGF on mesangial cells is
mediated in part by protein kinase C second messenger
systems.
In contrast to the mitogenic activity of HGF on
epithelial cells, HGF has no effect on proliferation of
mouse or human mesangial cells. HGF has distinct
effects on glomerular mesangial cells during chronic
renal failure. Moreover, in addition to other proteins
known to affect renal malfunction, the present data
demonstrate that HGF also contributes to matrix
production in mesangial cells and therefore chronic
renal disease.
In summary, HGF stimulates the extracellular
acidification rate of mesangial cells and increases gene
expression of fibronectin and collagen al(IV) in
glomerular mesangial cells. Therefore, HGF contributes
to glomerulosclerosis by activating mesangial cells to
increase extracellular matrix deposition. Further,
treatment of animals for 21 days with HGF (long term
treatment) reduces creatinine clearance suggesting a
reduction in renal function. Thus, chronic elevation of
HGF observed in many chronic renal diseases likely
contributes to decreased renal function by causing
expansion of extracellular matrix in the glomerulus.
The disclosures of each and every patent, patent
application and publication cited herein are hereby
incorporated herein by reference in their er~~irety.
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While this invention has been disclosed with
reference to specific embodiments, it is apparent that
other embodiments and variations of this invention may
be devised by others skilled in the art without
departing from the true spirit and scope of the
invention. The appended claims are intended to be
construed to include all such embodiments and equivalent
variations.
23
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: Brooks, David
Laping, Nicholas
(ii) TITLE OF THE INVENTION: HEPATOCYTE GROWTH FACTOR ANTAGONISTS
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKline Beecham Corporation
(B) STREET: 709 Swedeland Road
(C) CITY: King of Prussia
(D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19406
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
{C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 1.5
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: TO BE ASSIGNED
(B) FILING DATE: HEREWITH
(C) CLASSIFICATION: UNKNOWN
{vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/030,342
(B) FILING DATE: 05-NOV-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Baumeister) Kirk
(B) REGISTRATION NUMBER: 33,833
{C) REFERENCE/DOCKET NUMBER: P50585P
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-270-5096
(B) TELEFAX: 6l0-270-5090
(C) TELEX:
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1201 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
{v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 78...947
(D) OTHER INFORMATION:
24
CA 02270867 1999-OS-04
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(xi) SEQUENCE DESCRIPTION: 5EQ NO:1;
ID
TAGGCACTGA TCCTCCAGAG GGATCCGCCA 60
CTCCGAACAG
GATTCTTTCA
CCCAGGCATC
GCCCGTCCAG TGG CCA CTG 110
CAGCACC GTG GCC CTG
ATG ACC
AAA
CTC
CTG
Met Pro Leu
Trp Ala Leu
Val
Thr
Lys
Leu
Leu
1 5 10
CTGCAG CATGTCCTC CTGCATCTC CTCCTGCTC CCCATC GCCATCCCC 158
LeuGln HisValLeu LeuHisLeu LeuLeuLeu ProIle AlaIlePro
15 20 25
TATGCA GAGGGACAA AGGAAAAGA AGAAATACA ATTCAT GAATTCAAA 206
TyrAla GluGlyGln ArgLysArg ArgAsnThr IleHis GluPheLys
30 35 40
AAATCA GCAAAGACT ACCCTAATC AAAATAGAT CCAGCA CTGAAGATA 254
LysSer AlaLysThr ThrLeuIle LysIleAsp ProAla LeuLysIle
45 50 55
AAAACC AAAAAAGTG AATACTGCA GACCAATGT GCTAAT AGATGTACT 302
LysThr LysLysVal AsnThrAla AspGlnCys AlaAsn ArgCysThr
6C 65 70 75
AGGAAT AAAGGACTT CCATTCACT TGCAAGGCT TTTGTT TTTGATAAA 350
ArgAsn LysGlyLeu ProPheThr CysLysAla PheVal PheAspLys
80 85 90
GCAAGA AAACAATGC CTCTGGTTC CCCTTCAAT AGCATG TCAAGTGGA 398
AlaArg LysGlnCys LeuTrpPhe ProPheAsn SerMet SerSerGly
95 l00 105
GTGAAA AAAGAATTT GGCCATGAA TTTGACCTC TATGAA AACAAAGAC 446
ValLys LysGluPhe GlyHisGlu PheAspLeu TyrGlu AsnLysAsp
110 1l5 120
TACATT AGAAACTGC ATCATTGGT AAAGGACGC AGCTAC AAGGGAACA 494
TyrIle ArgAsnCys IleIleGly LysGlyArg SerTyr LysGlyThr
125 130 135
GTATCT ATCACTAAG AGTGGCATC AAATGTCAG CCCTGG AGTTCCATG 542
ValSer IleThrLys SerGlyIle LysCysGln ProTrp SerSerMet
140 145 150 155
ATACCA CACGAACAC AGCTTTTTG CCTTCGAGC TATCGG GGTAAAGAC 590
IlePro HisGluHis SerPheLeu ProSerSer TyrArg GlyLysAsp
160 165 170
CTACAG GAAAACTAC TGTCGAAAT CCTCGAGGG GAAGAA GGGGGACCC 638
LeuGln GluAsnTyr CysArgAsn ProArgGly GluGlu GlyGlyPro
175 180 185
TGGTGT TTCACAAGC AATCCAGAG GTACGCTAC GAAGTC TGTGACATT 686
TrpCys PheThrSer AsnProGlu ValArgTyr GluVal CysAspIle
190 195 200
CCTCAG TGTTCAGAA GTTGAATGC ATGACCTGC AATGGG GAGAGTTAT 734
ProGln CysSerGlu ValGluCys MetThrCys AsnGly GluSerTyr
205 210 215
CGAGGT CTCATGGAT CATACAGAA TCAGGCAAG ATTTGT CAGCGCTGG 782
ArgGly LeuMetAsp HisThrGlu SerGlyLys IleCys GlnArgTrp
220 225 230 235
CA 02270867 1999-OS-04
WO 98I19696 PCT/US97/19891
GAT CAT CAG ACA CCA CAC CGG CAC AAA TTC TTG CCT GAA AGA TAT CCC 830
Asp His Gln Thr Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro
240 245 2S0
GAC AAG GGC TTT GAT GAT AAT TAT TGC CGC AAT CCC GAT GGC CAG CCG 878
Asp Lys Gly Phe Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro
255 260 265
AGG CCA TGG TGC TAT ACT CTT GAC CCT CAC ACC CGC TGG GAG TAC TGT 926
Arg Pro Trp Cys Tyr Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys
270 275 280
GCA ATT AAA ACA TGC GAG ACA TAACATGGGC TCTCAACTGA TGGTGAACTT CTTCT 982
Ala Ile Lys Thr Cys Glu Thr
285 290
GGTGAGTGAC AGAGGCTGCA GTGAAGAATA ATGAGTCTAA TAGAAGTTTA TCACAGATGT 1042
CTCTAATCTC TATAGCTGAT CCCTACCTCT CTCGCTGTCT TTGTACCCAG CCTGCATTCT 1102
GTTTCGATCT GTCTTTTAGC AGTCCATACA ATCATTTTTC TACATGCTGG CCCTTACCCA 1162
GCTTTTCTGA ATTTACAATA AAAACTATTT TTTAACGTG 1201
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
;A) LENGTH: 290 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
;v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln .'is Val Leu
1 5 10 15
Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln
20 25 30
Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr
35 40 45
Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
50 55 60
Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu
65 70 75 80
Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys
85 90 95
Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe
100 105 110
Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys
115 120 125
Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140
Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu His
145 150 155 160
Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr
165 170 175
Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser
180 185 190
Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu
195 200 205
26
CA 02270867 1999-OS-04
WO 98I19696 PCT/US97/19891
Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp
210 215 220
His Thr Glu Ser Gly Lys Ile-Cys Gln Arg Trp Asp His Gln Thr Pro
225 230 235 . 240
His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp
245 250 255
Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr
260 265 270
Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys
275 280 285
Glu Thr
290
27
