Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF MODULATING APOPTOSIS BY
ADMINISTRATION OF RELAXIN AGONISTS OR
ANTAGONISTS
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BACKGROUND OF THE INVENTION
The growth and development of normal tissues is achieved by
programmed cell proliferation, differentiation and cell death. Cell
proliferation and
differentiation are required for the formation of new cells and tissues.
Conversely,
programmed cell death, also referred to as apoptosis, is required to remove
existing
cells, including immature or damaged cells. Apoptosis naturally occurs in
virtually all
tissues of the body. Apoptosis plays a critical role in tissue homeostasis,
that is, it
ensures that the number of new cells produced are correspondingly offset by an
equal
number of cells that die. For example, the cells in the intestinal lining
divide so
rapidly that the body must eliminate cells after only three days in order to
prevent the
overgrowth of the intestinal lining.
The disruption of the genetic program, by either abnormally increasing
or decreasing rate of cell proliferation and/or apoptosis can result in
abnormal tissue
development. For example, decreases in cell proliferation below normal levels
can
lead to immature tissues and other tissue abnormalities. Increases in cell
proliferation
above normal levels are thought to be major events in the development of
neoplasia
and cancer, as well as other cell proliferative disorders. Abnormal increases
in
apoptosis can also lead to precancerous lesions. Precancerous lesions include
lesions
of the breast (that can develop into breast cancer), lesions of the prostate
(that can
develop into prostate cancer) or skin (that can develop into malignant
melanoma or
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basal cell carcinoma), colonic adenomatous polyps (that can develop into colon
cancer), and other such neoplasia. Such lesions exhibit a strong tendency to
develop
into malignant tumors or cancer.
Precancerous lesions can result from an accumulation of insults to
existing cells in various tissues of the body. Such insults can include
exposure to
sunlight, radiation, mutagens and carcinogens normally found in the diet,
chemicals
such as pesticides, herbicides, preservatives, and the like. These insults can
result in
the accumulation of mutations in the cells, which can lead to hyperplastic
conditions
(i.e., abnormal increases in cell number), such as, for example, hyperplasia
of liver,
kidney, spleen, thymus, intestine, lung or prostate tissues. The down-
regulation of
apoptosis can also lead to the accumulation of cells in these hyperplastic
conditions.
An abnormal increase in apoptosis can interfere with normal
development and/or differentiation of tissues. For example, apoptosis is
required
during pregnancy and for maturation of the male reproductive tract tissues. An
abnormal increase in apoptosis can also interfere with the formation of new
cells and
tissues, thereby preventing normal tissue maturation or development.
Thus, there is a need for methods of modulating apoptosis by
administering agonists or antagonists of apoptosis. In particular, there is a
need for
methods of treating conditions associated, directly or indirectly, with
abnormally high
or low rates of apoptosis. The present invention satisfies this need by
providing
methods for the administration of relaxin agonists or antagonists to treat
relaxin-
associated tissue abnormalities by modulating apoptosis in such tissues.
SUMMARY OF THE INVENTION
The present invention relates to the discovery that relaxin is associated
with the development of body tissues. Knockouts of the gene encoding relaxin
result
in various abnormalities in the development of various body tissues, including
smaller
size of, increased collagen deposition in, and immaturity of such tissues.
Conversely,
relaxin-responsive cell accumulation leads to abnormalities in body tissues.
The
present invention provides methods of modulating apoptosis in tissues by
administering a relaxin agonist or a relaxin antagonist.
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In one aspect, the present invention provides methods for modulating
apoptosis in a subject by administering to a subject in need thereof an
effective amount
of a relaxin agonist for a period of time sufficient to decrease apoptosis in
cells
expressing a relaxin receptor. Tissues which are typically affected by the
administration of the relaxin agonist useful in methods according to the
present
invention include, for example, liver, kidney, spleen, thymus, brain, heart,
intestine,
skin, lung, the male reproductive tract, and the female reproductive tract.
Male
reproductive tract tissues include, for example, prostatic, epididymal,
seminiferous
tissues, tissues of the testes, and the like. Female productive tract tissues
include, for
example, uterus, cervix, the interpubic ligament, connective tissues within
the pelvic
girdle, and the like.
In certain embodiments, the administration of the relaxin agonist
typically reduces the number of apoptotic cells. In other embodiments, the
relaxin
agonist stimulates maturation of the tissue. For example, a relaxin agonist
can
stimulate maturation of male reproductive tract tissue, such as prostatic
tissue,
epididymal tissue, seminiferous tissue, testicular tissue or sperm. In an
embodiment,
the maturation results in an increase in cell number in an under-developed
testes, such
as, for example, an increase in the number of mature testicular cells. In
another
embodiment, maturation of male reproductive tract tissue results in an
increase in the
number of viable sperm cells, as compared with tissue not contacted with the
relaxin
agonist. In yet another embodiment, fibrosis can be reduced by the relaxin
agonist,
and/or excessive collagen deposition can be reduced.
The subject in need of administration of the relaxin agonist can have a
relaxin-deficient condition, such as, for example, immature tissue, excessive
collagen
deposition and/or a low sperm count. For example, the immature tissue can be
immature male productive tissue (e.g., underdeveloped testes). Such immature
tissue
can be present in an otherwise mature or immature animal.
The relaxin agonist can be relaxin, a relaxin analog, a small molecule
relaxin effector or a relaxin nucleic acid. The relaxin is typically
vertebrate relaxin
and more typically is human relaxin.
Compositions comprising a relaxin agonist useful in the methods
according to the present invention can be formulated for administration, for
example,
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by infusion, injection, oral delivery, nasal delivery as well as
intrapulmonary, rectal,
transdermal, interstitial or subcutaneous delivery. Such compositions also can
be
formulated for delayed release of the relaxin agonists into the tissues and
circulation of
the subject. Particular embodiments comprise compositions formulated for
infusion or
injection, or by intrapulmonary, subcutaneous or transdermal delivery. In
certain
embodiments, nucleic acids encoding relaxin agonists can formulated for
administration to the subject in a vector encoding the relaxin agonist. For
example, the
vector can be an expression vector which expresses the relaxin agonist in the
cells.
Alternatively, relaxin or relaxin analog nucleic acids can be formulated for
delivery to
the subject. The subjects can be pre-pubescent or post-pubescent.
In another aspect, the present invention provides methods for
modulating apoptosis in a subject in need thereof by administering an
effective amount
of a relaxin antagonist for a period of time sufficient to increase apoptosis
in a cell
population expressing a relaxin receptor. In an embodiment, the relaxin
antagonist
inhibits binding of relaxin to relaxin receptor. In another embodiment, the
relaxin
antagonist reduces relaxin-associated tissue remodeling.
Tissues which are typically affected by the administration of the relaxin
antagonist useful in methods according to the present invention include, for
example,
liver, kidney, spleen, thymus, brain, heart, intestine, skin, lung, the male
reproductive
tract, the female reproductive tract, and the like. Male reproductive tract
tissues
include, for example, prostatic, epididymal, seminiferous tissues, tissues of
the testes,
and the like. In certain embodiments, the male reproductive tract tissue can
be
prostatic tissue, and can be mature or immature. Suitable target female
productive
tract tissues include, for example, uterus, cervix, the interpubic ligament,
connective
tissues within the pelvic girdle, and the like. Alternatively, the cell
population can
comprise cells expressing a relaxin receptor such as, for example,
fibroblasts,
osteoblasts, monocytes epithelial cells, endothelial cells, and the like.
The relaxin antagonist can be, for example, a relaxin binding agent, a
relaxin receptor binding agent, a relaxin antisense nucleic acid, and the
like. The
relaxin binding agent can be, for example, an anti-relaxin antibody, a soluble
relaxin
receptor, a small molecule relaxin antagonist, and the like. The relaxin
receptor
binding agent can be, for example, an anti-relaxin receptor antibody, a
relaxin analog,
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a small molecule relaxin receptor antagonist, and the like. Antibodies that
bind relaxin
or relaxin receptor can include, for example, polyclonal antibodies,
monoclonal
antibodies, an Fab, Fab', an F(ab')2, an Fv, a single heavy chain, a chimeric
antibody,
and the like.
Compositions comprising a relaxin antagonist useful in the methods of
the present invention can be forinulated for administration, for example, by
infusion,
injection, oral delivery, nasal delivery as well as intrapulmonary, rectal,
transdermal,
interstitial or subcutaneous delivery. Compositions can also be formulated for
delayed
release of the relaxin antagonist into the tissues and circulation of the
subject.
Particular embodiments comprise compositions formulated for infusion or
injection or
by intrapulmonary, subcutaneous or transdermal delivery.
In certain embodiments, nucleic acids encoding relaxin antagonists can
also be administered to the subject in a vector encoding the relaxin
antagonist. In one
embodiment, the vector is an expression vector which expresses the relaxin
antagonist
in the cell population. Alternatively, relaxin or relaxin receptor antisense
nucleic acids
can be delivered directly to the subject, according to any of the methods
described
above. In a typical embodiment, administration of the relaxin antagonist
increases
apoptosis to reduce unwanted cell accumulation. For example, the unwanted
cells can
be hyperplasia, hypertrophy, cancer or neoplasia.
DEFINITIONS
Prior to setting forth the invention in more detail, it may be helpful to a
further understanding of the invention to set forth definitions of certain
terms as used
hereinafter.
The term "nucleic acid" refers to a polymer composed of a multiplicity
of nucleotide units (ribonucleotide or deoxyribonucleotide or related
structural
variants) linked via phosphodiester bonds. A nucleic acid can be of
substantially any
length, typically from about six (6) nucleotides to about 109 nucleotides, or
larger.
Unless otherwise stated, the conventional notation used herein for nucleic
acids is as
follows: the left-hand end of single-stranded nucleic acid is the 5' end; the
left-hand
direction of double-stranded nucleic acid is referred to as the 5' direction.
The
direction of 5' to 3' addition of nascent RNA transcripts is referred to as
the
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transcription direction. Sequence regions on the DNA strand having the same
sequence as the RNA and which are 5' to the 5' end of the RNA transcript are
referred
to as "upstream sequences;" sequence regions on the DNA strand having the same
sequence as the RNA and which are 3' to the 3' end of the coding RNA
transcript are
referred to as "downstream sequences".
Nucleic acids include RNA, mRNA, cDNA, genomic DNA, synthetic
fonns, and mixed polymers, both sense and antisense strands, and can also be
chemically or biochemically modified or can contain non-natural or derivatized
nucleotide bases, as will be readily appreciated by those skilled in the art.
Such
modifications include, for example, labels, methylation, substitution of one
or more of
the naturally occurring nucleotides with an analog, intemucleotide
modifications such
as uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoamidates,
and carbamates), charged linkages (e.g., phosphorothioates and
phosphorodithioates),
pendent moieties (e.g., polypeptides), intercalators (e.g., acridine and
psoralen),
chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic
acids). Also
included are synthetic molecules that mimic nucleic acids in their ability to
bind to a
designated sequence via hydrogen bonding and other chemical interactions. Such
molecules are known in the art and include, for example, those in which
peptide
linkages substitute for phosphate linkages in the backbone of the molecule.
The terms "amino acid" or "amino acid residue", as used herein, refer
to L amino acids or to D amino acids as described further below. The commonly
used
one- and three-letter abbreviations for amino acids are used herein (see,
e.g., Alberts et
al., Molecular Biology of the Cell, Garland Publishing, Inc., New York (3d ed.
1994)).
The term "conservative substitution," when describing a polypeptide,
refers to a change in the amino acid composition of the polypeptide that does
not
substantially alter the polypeptide's activity. Thus, a "conservative
substitution" of a
particular amino acid sequence refers to substitution of those amino acids
that are not
critical for polypeptide activity or substitution of amino acids with other
amino acids
having similar properties (e.g., acidic, basic, positively or negatively
charged, polar or
non-polar, and the like) such that the substitution of even critical amino
acids does not
substantially alter activity. Conservative substitution tables providing
functionally
similar amino acids are well known in the art. For example, the following six
groups
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each contain amino acids that are conservative substitutions for one another:
1)
Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid
(E); 3)
Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine
(L), Methionine (M), Valine (V); and 6) Phenylala.nine (F), Tyrosine (Y),
Tryptophan
(W). (See also Creighton, Proteins,W W. H. Freeman and Company (1984)). In
addition,
individual substitutions, deletions or additions that alter, add or delete a
single amino acid
or a small percentage of amino acids in an encoded sequence are also
"conservative
substitutions".
The term "relaxin analog" refers to a modified relaxin polypeptide that
increases or decreases the functional activity of the molecule or its
interaction with a
relaxin receptor.
The term "soluble," in the context of a polypeptide, refers to the ability
of the polypeptide to be dissolved in (i.e., to be molecularly or ionically
dispersed in)
an aqueous solution, such as water, blood or plasma.
The term "small molecule effector," in the context of relaxin or a
relaxin receptor, refers to an agent that binds to a relaxin, or to a relaxin
receptor, and
stimulates the activity of relaxin or relaxin receptor.
The term "small molecule antagonist," in the context of a relaxin or a
relaxin receptor, refers to an agent that binds to a relaxin, or to a relaxin
receptor, and
reduces or inhibits the activity of relaxin or relaxin receptor.
The term "effective amount" means a dosage sufficient to provide
amelioration of a symptom or treatment for an abnormality, such as a disease,
disorder
or condition, being treated. The dosage will vary depending on the subject,
the
abnormality being treated, and the treatment being effected.
The terms "subnormal," "subnormally" and "underdeveloped," in the
context of body tissue and/or cells, refer to a smaller size, decreased cell
number,
and/or immature developmental state, as compared with the size, cell number
and/or
developmental state of a tissue and/or cells of a normal subject of the same
age and
species of the subject.
The term "tissue" refers to a population of cells, generally consisting of
cells of the same kind that perform the same, or a similar, function. A tissue
can be
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part of an organ or bone or it can be a loose association of cells, such as
cells of the
immune system.
The term "maturation," in the context of body tissue, refers to the
developmental progression and/or differentiation of the tissue towards its
full
developed state.
The Willi "mature," in the context of body tissue, refers to the
achievement of full development, differentiation and/or growth of the tissue.
The term "immature," in the context of body tissue, refers to a tissue
that has not fully developed and/or differentiated.
The term "pre-pubescent male" refers to a male that has not completed
the process of puberty.
The term "post-pubescent male" refers to a male that has completed the
process of puberty.
The term "puberty" refers to the sequence of events by which a child
becomes a young adult, characterized by the beginning of gametogenesis,
secretion of
gonadal hormones, development of secondary sexual characteristics, and
reproductive
function.
The term "accumulation," in the context of body tissue, refers to an
increase in the number of normal (i.e., non-mutant, non-malignant) cells in
the tissue.
The teinis "biologically active" and "functionally active" refer to the
ability of a molecule (e.g., a relaxin agonist or antagonist) to bind to a
relaxin or
relaxin receptor and to stimulate or inhibit apoptosis, cell accumulation/
and/or tissue
maturation, in a body tissue.
The term "relaxin-deficient condition" refers to a disease, disorder or
condition of a subject, in which relaxin levels, or relaxin-receptor levels,
in the
relevant tissue, cell(s), or in the subject, are below normal.
The term "relaxin-responsive" refers to an increase in cell number, or in
the state of maturity (i.e., tissue development and/or differentiation), in
response to the
binding of relaxin to a relaxin receptor.
The term "relaxin-associated" refers to a property, condition or
response of a cell or tissue by which the cell or tissue is affected, directly
or indirectly,
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by an increase or decrease in functionally active relaxin levels, or in
functionally
active relaxin receptor.
The terms "hyperplasia" or "hyperplastic tissue" refer to an increase in
the number of cells in a tissue.
The terms "hypertrophy" or "hypertrophic" refer to an increase in the
size of a tissue.
The terms "cancer" and "malignancy" generally refer to the various
types of malignant neoplasms, most of which invade surrounding tissues.
The terms "cancerous" and "malignant" relate to cells or tissue having
properties of cancer or a malignancy.
The term "tissue remodeling" refers to the formation of new cells or
tissues and the destruction of existing cells through the apoptotic pathway,
in response
to the signals mediated by relaxin or a relaxin-receptor.
The term "apoptosis" refers to a regulated network of biochemical
events which lead to a selective form of cell suicide, and is characterized by
readily
observable morphological and biochemical phenomena, such as the fragmentation
of
the deoxyribonucleic acid (DNA), condensation of the chromatin, which may or
may
not be associated with endonuclease activity, chromosome-migration,
margination in
cell nuclei, the formation of apoptotic bodies, mitochondrial swelling,
widening of the
mitochondria' cristae, opening of the mitochondrial permeability transition
pores
and/or dissipation of the mitochondria' proton gradient.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention provides methods for the administration of
relaxin agonists or antagonists for the modulation of apoptosis. The relaxin
agonists
and antagonists can be used to reduce, manage, treat or prevent relaxin-
associated
abnormalities. Relaxin-associated abnormalities include diseases, disorder or
conditions of a subject that are associated with increased or decreased levels
of
apoptosis, as compared with levels of apoptosis in a normal subject. Relaxin-
associated abnormalities include, for example, relaxin-deficient conditions
and relaxin-
responsive conditions.
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In one aspect, a relaxin agonist is administered to a subject to treat a
relaxin-associated abnormality having increased apoptosis, as compared with
comparable cells from a normal subject (i.e., not having the relaxin-
associated
abnormality). Such relaxin-associated abnormalities can include, for example,
immature body tissue, increased collagen deposition, fibrosis (i.e., the
presence of
abnoinial amounts of fibrous tissue, as compared with normal tissue), low
tissue
weight, increased apoptosis of cells in a cell population in a tissue, and the
like.
Relaxin-associated abnormalities are typically associated with decreased
levels of
relaxin, and/or relaxin receptor, as compared with normal cell or tissues.
In certain embodiments, the relaxin-associated abnormality is a relaxin-
deficient condition, such as, for example, immature tissue, the presence of
excessive
collagen, a low sperm count, and the like. The immature tissue can be, for
example,
immature male productive tract tissue (e.g., immature pro static, epididymal,
seminiferous or testicular tissue or sperm). Immature tissue can be
characterized, for
example, by increased collagen deposition, low weight, and/or decreased cell
number,
as compared with a comparable sample of normal tissue. Such tissue also can be
characterized by its lack of organization, by incomplete development and/or by
a lack
of, or incomplete, differentiation, as compared with a comparable sample of
normal
tissue. In certain other embodiments, fibrosis can be reduced, such as, for
example,
dermal fibrosis, lung fibrosis, kidney fibrosis, or age-related fibrosis of
these or other
organs or tissues.
The relaxin agonist is administered in an amount effective to reduce,
manage, treat or prevent the relaxin-associated abnormality. For example,
administration of a relaxin agonist can decrease apoptosis of target cells,
stimulate
maturation, development and/or differentiation of immature tissue, increase
tissue
weight, increase cell number in the tissue, decrease in collagen deposition,
and the
like.
In another aspect according to the present invention, a relaxin
antagonist is administered to a subject to reduce, manage, treat or prevent a
relaxin-
associated abnormality by increasing apoptosis in the target cells. Such
relaxin
associated abnormalities can be, for example, relaxin-associated cell
accumulation,
decreased apoptosis relative to normal cells or tissue, hyperplasia,
hypertrophy, cancer,
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neoplasia, and the like. In certain embodiments, the relaxin antagonist can
reduce
relaxin-associated tissue remodeling, reduce unwanted cell accumulation,
reduce or
prevent hyperplasia, hypertrophy, cancer, neoplasia, and the like.
Body tissues having relaxin-associated or relaxin-responsive tissue
abnormalities can include, for example, tissues of the brain, heart, liver,
kidney,
spleen, thymus, intestine, skin, lung, male reproductive tract (e.g.,
prostate,
epididymis, seminal vesicles or testes), or female reproductive tract (e.g.,
the uterus,
cervix, the interpubic ligament or connective tissues of the pelvic girdle).
Relaxin Agonists
In one aspect according to the present invention, a relaxin agonist is
administered to a subject to treat a relaxin-associated abnormality. The
relaxin agonist
can be, for example, a relaxin, a relaxin analog, a small molecule relaxin
effector, a
relaxin nucleic acid, and the like.
Relaxin and Relaxin Analogs
In one aspect of the invention, the relaxin agonist is a relaxin
polypeptide or a fragment or analog of a relaxin polypeptide. The term
"relaxin"
refers to vertebrate relaxin polypeptides, including full length relaxin
polypeptide or a
portion of the relaxin polypeptide that retains biological activity.
Relaxin has been well defined in its natural human form, animal form,
and in its synthetic form. In particular, relaxin has been extensively
described in U.S.
Patent Nos. 5,166,191 and 4,835,251. In this application, "relaxin" generally
refers to the
terms "relaxin", "human relaxin", "native relaxin", and "synthetic relaxin" as
defined in
U.S. Patent No. 5,166,191 and the terms "human relaxin" and "human relaxin
analogs" as
defined in U.S. Patent No. 4,835,251. In a typical embodiment, the relaxin is
human
relaxin, as described in, for example, U.S. Patent Nos. 5,179,195; 5,023,321;
and
4,758,516. "Relaxin" in this application will also refer to relaxin as
isolated in pigs, rats,
horses, or other mammalian or vertebrates, and relaxin produced by recombinant
techniques using cDNA clones for rat, porcine or other mammalian or vertebrate
relaxin(s).
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Methods of making relaxin and its analogs are known in the art. In
addition, methods for isolating and purifying relaxin are known in the art.
Several
sources for these methods are identified in U.S. Patent No. 5,166,191,
including the
following references: U.S. Patent No. 4,835,251, Barany et al., The Peptides
2:1
(1980), Treager et al., Biology of Relaxin and its Role in the Human, pp. 42-
55;
EP 0 251 615; EP 0 107 782; EP 0 107 045; and WO 90/13659.
Additional methods of making relaxin are described in U.S. Patent No.
5,464,756, and PCT/US94/06997. Relaxin can also be prepared by synthesis of
the A and
B chains, and purification and assembly thereof, as described in European
Patent
0 251 615 (published Jan. 7, 1988). For in vitro assembly of relaxin, a 4:1
molar ratio of
A to B chains is generally employed. The resulting product is then purified by
any means
known to one of ordinary skill in the art, including, for example, reverse-
phase HPLC, ion
exchange chromatography, gel filtration, dialysis, and the like, or any
combination of such
procedures. Unprocessed or partially processed forms of relaxin, such as
preprorelaxin or
prorelaxin, can also be used.
In specific embodiments, relaxin polypeptides include the H1 and H2
forms of human relaxin. It has been reported that the predominant species of
human
relaxin is the H2 relaxin form with a truncated B chain (i.e., relaxin H2(B29
A24)),
wherein the four C-terminal amino acids of the B-chain are absent so that the
B-chain
ends with a serine at position 29. Either this form (referred to as designated
"short
relaxin" or "long relaxin," which contains a B chain of 33 amino acids) can be
used.
Relaxin agonists further includes analogs, such as naturally-occurring
amino acid sequence variants of relaxin. Relaxin analogs also include those
altered by
substitution, addition or deletion of one or more amino acid residues that
provide for
functionally active relaxin polypeptides. Such relaxin analogs include, but
are not
limited to, those containing as a primary amino acid sequence all or part of
the amino
acid sequence of a relaxin polypeptide, including altered sequences in which
one or
more functionally equivalent amino acid residues are substituted for residues
within
the sequence, resulting in a silent functional change (e.g., a conservative
substitution).
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In another aspect, the relaxin agonist is a polypeptide consisting of or
comprising a fragment of a relaxin polypeptide having at least 10 contiguous
amino
acids of the relaxin polypeptide. Alternatively, the fragment contains at
least 20 or 25
contiguous amino acids of the relaxin polypeptide. In other embodiments, the
fragments are not larger than 20 or 30 amino acids.
The relaxin analog can be a polypeptide comprising regions that are
substantially similar to a relaxin polypeptide or fragments thereof (e.g., in
various
embodiments, at least 60%, 70%, 75%, 80%, 90%, or even 95% identity or
similarity
over an amino acid sequence of identical size), or when compared to an aligned
sequence in which the alignment is done by a computer sequence
comparison/alignment program known in the art, or which coding nucleic acid is
capable of hybridizing to a relaxin nucleic acid, under high stringency,
moderate
stringency, or low stringency conditions (infra). (See, e.g., Smith and
Waterman, Adv.
AppL Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Biol. 48:443 (1970);
Pearson and Lipman, Proc. NatL Acad. ScL USA 85:2444 (1988); GAP, BESTFIT,
FASTA, and TEASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, WI); Ausubel et al. (eds.), Current
Protocols in Molecular Biology, 4th ed., John Wiley and Sons, New York
(1999)).
Relaxin agonists further comprise functionally active relaxin polypeptides,
analogs or
fragments that bind to a relaxin receptor.
Relaxin agonists, such as relaxin polypeptides, analogs and fragments
can be produced by various methods known in the art. The manipulations which
result
in their production can occur at the gene or polypeptide level. For example,
cloned
relaxin nucleic acids can be modified by any of numerous strategies known in
the art
(see, e.g., Sambrook et al., Molecular Cloning: A Laboratoiy Manual, 3d Ed.,
Cold
Spring Harbor Laboratory Press, New York (2001); Ausubel et al., Current
Protocols
in Molecular Biology, 4th ed., John Wiley and Sons, New York (1999)), such as
making
conservative substitutions, deletions, insertions, and the like. The sequence
can be cleaved
at appropriate sites with restriction endonuclease(s), followed by further
enzymatic
modification if desired, isolated, and ligated in vitro. In the production of
the relaxin
nucleic acids
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encoding an analog or fragment, the modified nucleic acid typically remains in
the
proper translational reading frame, so that the reading frame is not
interrupted by
translational stop signals or other signals that interfere with the synthesis
of the relaxin
- analog or fragment. The relaxin nucleic acid can also be mutated in vitro
or in vivo to
create and/or destroy translation initiation and/or termination sequences. The
relaxin
nucleic acid can also be mutated to create variations in coding regions and/or
to form
new restriction endonuclease sites or destroy preexisting ones and to
facilitate further
in vitro modification. Any technique for mutagenesis known in the art can be
used,
including but not limited to, chemical mutagenesis, in vitro site-directed
mutagenesis
(see, e.g., Hutchison et al., J. Biol. Chem. 253:6551-60 (1978)), the use of
TAB
linkers (Pharmacia), and the like. (See generally Sambrook et al., supra;
Ausubel et
al., supra.)
In a specific embodiment, relaxin analogs are prepared from relaxin-
encoding nucleic acids that are altered to introduce aspartic acid codons at
specific
position(s) within at least a portion of the relaxin coding region. (See,
e.g., U.S. Patent
No. 5,945,402, the disclosure of which is incorporated by reference herein.)
The
resulting analogs can be treated with dilute acid to release a desired analog,
thereby
rendering the protein more readily isolated and purified. Other relaxin
analogs are
disclosed in U.S. Patent Nos. 4,656,249; 5,179,195; 5,945,402; 5,811,395; and
5,795,807.
Manipulations of the relaxin polypeptide sequence can also be made at
the polypeptide level. Included within the scope of the invention are relaxin
polypeptides, analogs or fragments that are differentially modified during or
after
synthesis (e.g., in vivo or by in vitro translation). Such modifications
include
conservative substitution, glycosylation, acetylation, phosphorylation,
amidation,
derivatization by known protecting/blocking groups, proteolytic cleavage,
linkage to
an antibody molecule, another polypeptide or other cellular ligand, and the
like. Any
of numerous chemical modifications can be carried out by known techniques,
including, but not limited to, specific chemical cleavage (e.g., by cyanogen
bromide),
enzymatic cleavage (e.g., by tr3psin; chymotrypsin, papain, V8 protease, and
the like);
modification by, for example, NaBH4, acetylation, formylation, oxidation and
reduction, metabolic synthesis in the presence of tunicamycin, and the like.
14
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Relaxin polypeptides, analogs and fragments can be purified from
natural sources by standard methods such as those described herein (e.g.,
inununoaffinity purification). Relaxin polypeptides, analogs and fragments can
also
be isolated and purified by standard methods including chromatography (e.g.,
ion
exchange, affinity, sizing column chromatography, high pressure liquid
chromatography), centrifugation, differential solubility, or by any other
standard
technique for the purification of polypeptides. Relaxin polypeptides can be
synthesized by standard chemical methods known in the art (see, e.g.,
Hunkapiller et
al., Nature 310:105-11(1984); Stewart and Young, Solid Phase Peptide
Synthesis, 2nd
Ed., Pierce Chemical Co., Rockford, IL, (1984)).
In addition, analogs of relaxin polypeptides can be chemically
synthesized. For example, a peptide corresponding to a fragment of a relaxin
polypeptide, which comprises a desired domain, or which mediates a desired
activity
in vivo, can be synthesized by use of chemical synthetic methods using, for
example,
an automated peptide synthesizer. Furthermore, if desired, nonclassical amino
acids or
chemical amino acid analogs can be introduced as a substitution or addition
into the
relaxin polypeptide sequence. Non-classical amino acids include, but are not
limited
to, the D-isomers of the common amino acids, a-amino isobutyric acid, 4-
aminobutyric acid, 2-amino butyric acid, E-amino hexanoic acid, 6-amino
hexanoic
acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline,
hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-
butylalanine,
phenylglycine, cyclohexylalanine, 13-a1sni11e, selenocysteine, fluoro-amino
acids,
designer amino acids such as [3-methyl amino acids, C a-methyl amino acids, N
a-
methyl amino acids, and amino acid analogs in general. Furthermore, the amino
acid
can be D (dextrorotary) or L (levorotary).
In another embodiment, the relaxin agonist is a chimeric, or fusion,
protein comprising a relaxin polypeptide, or fragment thereof (typically
consisting of
at least a domain or motif of the relaxin polypeptide, or at least 10
contiguous amino
acids of the relaxin polypeptide), joined at its amino- or carboxy-terminus
via a
peptide bond to an amino acid sequence of a different protein. In one
embodiment,
such a chimeric protein is produced by recombinant expression of a nucleic
acid
CA 02425712 2010-07-21
encoding the chimeric polypeptide. The chimeric product can be made by
ligating the
appropriate nucleic acid sequences, encoding the desired amino acid sequences,
to
each other in the proper reading frame and expressing the chimeric product by
methods commonly known in the art. Alternatively, the chimeric product can be
made
by protein synthetic techniques (e.g., by use of an automated peptide
synthesizer).
In a specific embodiment, the fusion protein is a relaxin-ubiquitin
fusion protein. For example, U.S. Patent No. 5,108,919 discloses methods for
preparing a
fusion protein of a relaxin chain and ubiquitin.
In preferred embodiments, the relaxin analog, or fragment is
functionally active (i.e., capable of exhibiting one or more functional
activities
associated with a full-length, wild-type relaxin polypeptide). As one example,
analogs
or fragments that retain a desired relaxin property of interest (e.g., binding
to a relaxin
binding partner (e.g., relaxin receptor) and/or modulation (e.g., inhibition)
of
apoptosis) can be used as inducers of such property and its physiological
correlates. A
specific embodiment relates to a relaxin analog or fragment that bind to a
relaxin
receptor and induces a relaxin-associated decrease in apoptosis. Analogs or
fragments
of relaxin can be tested for the desired activity by procedures known in the
art,
including but not limited to the functional assays described herein.
Relaxin Nucleic Acids:
The invention provides relaxin nucleic acid sequences for expression of
relaxin polypeptide, fragments and analogs in vivo or in vitro. The relaxin
nucleic acid
can be a vertebrate or mammalian relaxin, including, for example, human,
mouse, rat,
pig, cow, dog, or monkey relaxin. The relaxin nucleic acids can comprise
genomic
nucleic acids, cDNA, the relaxin coding region or a fragment thereof. Relaxin
nucleic
acids further include mRNAs corresponding to the relaxin locus. Relaxin
nucleic acids
can also include analogs (e.g., nucleotide sequence variants), such as those
encoding
other possible codon choices for the same amino acid or conservative amino
acid
substitutions thereof, such as naturally occurring allelic variants. Due to
the
degeneracy of nucleotide coding sequences, other nucleic acid sequences that
encode
substantially the same amino acid sequence as a relaxin cDNA or open reading
frame,
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can be used in the practice of the present invention. These nucleic acid
sequences
include, but are not limited to, nucleic acid sequences comprising all or
portions of a
relaxin gene which is altered by the substitution of different codons that
encode the
same or a functionally equivalent amino acid residue (e.g., a conservative
substitution)
within the sequence, thus producing a silent change.
The invention further provides relaxin nucleic acid fragments of at least
6 contiguous nucleotides (e.g., a hybridizable portion); in other embodiments,
the
nucleic acids comprise at least 8 contiguous nucleotides, at least contiguous
25
nucleotides, at least contiguous 50 nucleotides, at least 100 nucleotides, or
at least, 150
nucleotides, or more of a relaxin sequence. In another embodiment, the nucleic
acids
are smaller than 150 nucleotides in length. The relaxin nucleic acids can be
single or
double-stranded. As is readily apparent, as used herein, a "nucleic acid
encoding a
fragment of a relaxin polypeptide" is construed as referring to a nucleic acid
encoding
only the recited fragment or portion of the relaxin polypeptide and not the
other
contiguous portions of the relaxin polypeptide as a contiguous sequence.
Fragments of
relaxin nucleic acids encoding one or more relaxin domains are also provided.
Relaxin nucleic acids, or a functionally active analog or fragment
thereof, can be inserted into an appropriate vector, such as an expression
vector (i.e., a
vector which contains the necessary elements for the transcription and
translation of
the inserted polypeptide-coding sequence). The necessary transcriptional and
translational signals can also be supplied by the native relaxin gene and/or
its flanking
regions. A variety of host-vector systems can be utilized to express the
relaxin nucleic
acid sequences. These include but are not limited to, mammalian cell systems
infected
with virus (e.g., vaccinia virus, adenovirus, adeno-associated virus, and the
like),
insect cell systems infected with virus (e.g., baculovirus), microorganisms
such as
yeast containing yeast vectors, or bacteria transformed with bacteriophage,
DNA,
plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their
strengths and specificities. Depending on the host-vector system utilized, any
one of a
number of suitable transcription and translation elements can be used. In
specific
embodiments, human relaxin nucleic acids, or a nucleic acid sequence encoding
a
functionally active portion of human relaxin, is expressed in yeast or
bacteria. In yet
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another embodiment, a fragment of relaxin comprising a domain of the relaxin
polypeptide is expressed.
Any of the methods known in the art for the insertion of nucleic acids
into a vector can be used to construct expression vectors containing a
chimeric gene
consisting of appropriate transcriptional/translational control signals and
relaxin
nucleic acid sequences. These methods include in vitro recombinant DNA and
synthetic techniques and in vivo recombinants (genetic recombination).
Expression of
nucleic acid sequence encoding a relaxin polypeptide, analog or fragment can
be
regulated by a second nucleic acid sequence so that the relaxin polypeptide,
analog or
fragment is expressed in a host transformed with the recombinant DNA molecule.
For
example, expression of a relaxin polypeptide can be controlled by any
promoter/enhancer element known in the art. Promoters which can be used to
control
relaxin gene expression include, but are not limited to, the SV40 early
promoter region
(Benoist and Chambon, Nature 290:304-10 (1981)), the promoter contained in the
3' ,
long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-97
(1980)),
the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci.
USA
78:1441-45 (1981)), the regulatory sequences of the metallothionen gene
(Brinster et
al., Nature 296:39-42 (1982)), prokaryotic expression vectors such as the P -
1 act amase
promoter (Villa-Komaroff et al., Proc. NatL Acad. Sci. USA 75:3727-31 (1978))
or the
tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)),
plant
expression vectors including the cauliflower mosaic virus 35S RNA promoter
(Gardner et al., NucL Acids Res. 9:2871-88 (1981)), the promoter of the
photosynthetic
enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., Nature
310:115-20
(1984)), promoter elements from yeast or other fungi such as the Ga17 and Ga14
promoters, the ADH (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol
kinase) promoter, the alkaline phosphatase promoter, and the like.
The following animal transcriptional control regions, which exhibit
tissue specificity, have been utilized in transgenic animals: the elastase I
gene control
region which is active in pancreatic acinar cells (e.g., Swift et al., Cell
38:639-46
(1984); Omitz et al., Cold Spring Harbor Symp. Quant Biol. 50:399-409 (1986);
MacDonald, Hepatology 7(1 Suppl.):42S-51S (1987)); the insulin gene control
region
which is active in pancreatic beta cells (e.g., Hanahan, Nature 315:115-22
(1985)), the
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CA 02425712 2010-07-21
immunoglobulin gene control region which is active in lymphoid cells (e.g.,
Grosschedl et aL , Cell 38:647-58 (1984); Adams et aL, Nature 318:533-38
(1985);
Alexander et al., Mol. Cell. Biol. 7:1436-44 (1987)), the mouse mammary tumor
virus
control region which is active in testicular, breast, lymphoid and mast cells
(e.g., Leder
et al., Cell 45:485-95 (1986)), the albumin gene control region which is
active in liver
(e.g., Pinkert et al., Genes Dev. 1:268-76 (1987)), the alpha-fetoprotein gene
control
region which is active in liver (e.g., Krumlauf et al., MoL Cell. Biol. 5:1639-
48 (1985);
Hammer et al., Science 235:53-58 (1987)); the alpha 1-antitrypsin gene control
region
which is active in the liver (e.g., Kelsey et al., Genes and Devel. 1:161-71
(1987)); the
beta-globin gene control region which is active in myeloid cells (e.g., Magram
et aL,
Nature 315:338-40 (1985); Kollias et aL, Cell 46:89-94 (1986)); the myelin
basic
protein gene control region which is active in oligodendrocyte cells in the
brain (e.g.,
Readhead et al., Cell 48:703-12 (1987)); the myosin light chain-2 gene control
region
which is active in skeletal muscle (e.g., Simi, Nature 314:283-86 (1985)); and
the
gonadotropic releasing hormone gene control region which is active in the
hypothalamus (e.g., Mason et al., Science 234:1372-78 (1986)). In a preferred
embodiment, the tissue specific promoter is the prostate specific antigen
promoter.
(See, e.g., U.S. Patent No. 6,100,444).
In another embodiment, a vector is used that comprises a promoter
operably linked to a relaxin nucleic acid, one or more origins of replication,
and,
optionally, one or more selectable markers (e.g., an antibiotic or drug
resistance
marker). For example, an expression construct can be made by subcloning a
relaxin
nucleic acid into a restriction site of the pRSECT expression vector. Such a
construct
allows for the expression of a relaxin polypeptide, analog or fragment under
the
control of the T7 promoter with a histidine amino terminal flag sequence for
affinity
purification of the expressed polypeptide. In another specific embodiment, a
vector is
used that comprises the prostate specific antigen promoter operably linked to
a relaxin
nucleic acid, one or more origins of replication, and, optionally, one or more
selectable
markers (e.g., an drug resistance marker).
Expression vectors containing relaxin nucleic acids can be identified by
general approaches well known to the skilled artisan, including: (a) nucleic
acid
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hybridization, (b) the presence or absence of "marker" gene function, (c)
expression of
inserted sequences, (d) by polymerase chain reaction (PCR), and the like. In
the first
approach, the presence of a relaxin nucleic acid inserted in an expression
vector can be
detected by nucleic acid hybridization using probes comprising sequences that
are
homologous to an inserted relaxin nucleic acid. In the second approach, the
recombinant vector/host system can be identified and selected based upon the
presence
or absence of certain "marker" gene functions (e.g., thymidine kinase
activity,
resistance to antibiotics, transformation phenotype, occlusion body formation
in
baculovirus, colorimetric change, and the like) caused by the insertion of the
relaxin
nucleic acids into the vector. For example, if the relaxin nucleic acid is
inserted within
the marker gene sequence of the vector, recombinants containing the relaxin
nucleic
acid can be identified by the absence of the marker gene function.
In the third approach, recombinant expression vectors can be identified
by assaying the relaxin polypeptide, analog or fragment expressed by the
recombinant.
Such assays can be based, for example, on the physical or functional
properties of the
relaxin polypeptide, analog or fragment in in vitro assay systems (e.g.,
binding with
anti-relaxin antibody, binding to relaxin receptor, and the like). In a forth
approach,
recombinant expression vectors can be identified by polymerase chain reaction.
(See,
e.g., U.S. Patent Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllensten et al.,
Proc.
NatL Acad. Sci. USA 85:7652-56 (1988); Ochman et al., Genetics 120:621-23
(1988);
Loh et al., Science 243:217-20 (1989).) Once a particular recombinant vector
is
identified and isolated, several methods that are known in the art can be used
to
propagate it.
Once a suitable host system and growth conditions are established,
recombinant vectors can be propagated and prepared in quantity. As previously
explained, the vectors which can be used include, but are not limited to the
following
vectors or their analogs: human or animal viruses such as vaccinia virus or
adenovirus;
insect viruses such as baculovirus; yeast vectors; bacteriophage vectors
(e.g., lambda);
and plasmid and cosmid DNA vectors; to name but a few.
In addition, a host cell strain can be chosen that modulates the
expression of the inserted nucleic acids, or modifies or processes the
relaxin, relaxin
analog or fragment in the specific fashion desired. Expression from certain
promoters
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can be elevated in the presence of certain inducers; thus, expression of the
relaxin
polypeptide, analog or fragment can be controlled. Furthermore, different host
cells
having characteristic and specific mechanisms for the translational and post-
translational processing, and modification (e.g., glycosylation and/or
phosphorylation)
can be used. Appropriate cell lines or host systems can be chosen to ensure
the desired
modification and processing of the polypeptide, analog or fragment expressed.
For
example, expression in a bacterial system can be used to produce an
unprocessed core
protein product. Expression in mammalian cells can be used to ensure "native"
processing of mammalian relaxin polypeptides, or of analogs or fragments.
Furthermore, different vector/host expression systems can affect processing
reactions
to different extents.
Relaxin Antagonists
In another aspect of the invention, relaxin antagonists are provided for
the modulation of apoptosis. Relaxin antagonists can include, for example,
relaxin
binding agents, relaxin receptor binding agents, antisense nucleic acids, and
the like.
Relaxin Antibodies
Relaxin antagonists can comprise antibodies that immunospecifically-
recognize relaxin or a relaxin receptor polypeptide and that stimulate
apoptosis, and/or
reduce or inhibit relaxin-associated cell accumulation in cell populations or
tissues.
Anti-relaxin and anti-relaxin receptor antibodies include, but are not limited
to,
polyclonal antibodies, monoclonal antibodies, chimeric antibodies (e.g., fully
humanized antibodies or human chimeric antibodies), single chain antibodies,
antibody
fragments (e.g., Fab, F(ab'), F(ab')2, Fv, or hypervariable regions), single
heavy
chains, and an Fab expression library. In a specific embodiment, polyclonal
and/or
monoclonal antibodies to full length, vertebrate or mammalian relaxin or
relaxin
receptor polypeptide are produced and selected for those antibodies that
selectively
bind to relaxin or a relaxin receptor polypeptides, and thereby functionally
inactivate
such polypeptides. In another embodiment, antibodies to a domain of a
vertebrate
relaxin polypeptide, or a relaxin receptor polypeptide, are produced. In still
another
embodiment, fragments of a vertebrate relaxin polypeptide, or a relaxin
receptor
21
CA 02425712 2010-07-21
polypeptide, which are identified as hydrophilic, are used as immunogens for
antibody
production and selected for immunospecific binding to such a polypeptide and
inhibition of its biological activity.
Various procedures known in the art can be used for the production of
polyclonal antibodies to a relaxin or relaxin receptor polypeptide, or a
fragment or
analog thereof. For the production of such antibodies, various host animals
(including,
but not limited to, rabbits, mice, rats, sheep, goats, and the like) can be
immunized by
injection with the native relaxin or relaxin receptor polypeptide, or a
fragment or
analog thereof. Alternatively, transgenic animals having a human immune system
can
be immunized by injection with the native relaxin or relaxin receptor
polypeptide.
(See, e.g., U.S. Patent Nos. 6,114,598 and 6,111,166). Various adjuvants can
be used to
increase the immunological response, depending on the host species, including
but not
limited to Freund's adjuvant (complete or incomplete), mineral gels such as
aluminum
hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions,
peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful
human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium
parvum.
For preparation of monoclonal antibodies directed toward a relaxin or
relaxin receptor polypeptide, fragment, or analog thereof, any technique which
provides for the production of antibody molecules by continuous cell lines in
culture
can also be used. Such techniques include, for example, the hybridoma
technique
originally developed by Kohler and Milstein (Nature 256:495-97 (1975)), as
well as
the trioma technique, the human B-cell hybridoma technique (see, e.g., Kozbor
et al.,
Immunology Today 4:72 (1983)), and the EBV-hybridoma technique to produce
human monoclonal antibodies (see, e.g., Cole et al., In Monoclonal Antibodies
and
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Mammalian antibodies
can be
used and can be obtained by using hybridomas (see, e.g., Cote et al., Proc.
Natl. Acad.
Sci. USA 80:2026-30 (1983)) or by transforming human B cells with EBV virus in
vitro (see, e.g., Cole et al. (1985), supra). Selection of hybridomas
producing
antibodies with appropriate biological function are well known in the art or
are
described herein below. Human monoclonal antibodies can also be prepared by
preparing hybridomas from animals having a human immune system that have been
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CA 02425712 2010-07-21
immunized by injection with the native relaxin or relaxin receptor
polypeptide. (See,
e.g., U.S. Patent No. 6,114,598 and 6,111,166).
Further to the invention, "chimeric" or "humanized" antibodies (see,
e.g., Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-55 (1984); Neuberger
et al,
Nature 312:604-08 (1984); Takeda et al., Nature 314:452-54 (1985)) can be
prepared.
Such chimeric antibodies are typically prepared by splicing the non-human
genes for
an antibody molecule specific for a relaxin or receptor polypeptide together
with genes
from a human antibody molecule of appropriate activity. It can be desirable to
transfer
the antigen binding regions (e.g., an F(ab')2, F(ab'), Fv, or hypervaxiable
region(s)) of
non-human antibodies into the framework of a human antibody by recombinant DNA
techniques to produce a substantially human molecule. In a preferred
embodiment, the
antibodies are fully humanized.
Methods for producing such "chimeric" molecules are generally well
known and described in, for example, U.S. Patent Nos. 4,816,567; 4,816,397;
5,693,762; 5,712,120; 5,821,337; 6,054,297; International Patent Publications
WO
87/02671 and WO 90/00616; and European Patent Publication EP 0 239 400.
Alternatively, a human monoclonal antibody or portions thereof can be
identified by first
screening a human B-cell cDNA library for DNA molecules that encode antibodies
that
specifically bind to a relaxin or a relaxin receptor polypeptide according to
the method
generally set forth by Huse et al. (Science 246:1275-81 (1989)). The DNA
molecule can
then be cloned and amplified to obtain sequences that encode the antibody (or
binding
domain) of the desired specificity. Phage display technology offers another
technique for
selecting antibodies that bind to relaxin or relaxin receptor polypeptides,
fragments or
analogs thereof. (See, e.g., International Patent Publications WO 91/17271 and
WO
92/01047; and Huse et al., supra.)
According to another aspect of the invention, techniques for the
production of single chain antibodies (see, e.g., U.S. Patents Nos. 4,946,778
and
5,969,108) can be adapted to produce relaxin- or relaxin receptor-specific
single chain
antibodies. (See also Riechmann and Muyldermans, J 1111712117101. Methods
231:25-38
(1999); Muyldermans and Lauwereys, J. Mol. Recognit. 12:131-40 (1999)).
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An additional aspect of the invention utilizes the techniques described
for the construction of a Fab expression library (see, e.g., Huse et al.
(1989), supra) to
allow rapid and easy identification of monoclonal Fab fragments with the
desired
specificity for relaxin polypeptides, fragments, or analogs thereof and
biological
activity.
Antibody fragments which contain the idiotype of the molecule can be
generated by known techniques. For example, such fragments include, but are
not
limited to, an F(a13')2 fragment which can be produced by pepsin digestion of
the
antibody molecule, Fab' fragments which can be generated by reducing the
disulfide
bridges of the F(ab')2 fragment, Fab fragments which can be generated by
treating the
antibody molecule with papain and a reducing agent, and Fv fragments.
Recombinant
Fv fragments can also be produced in eukaryotic cells using, for example, the
methods
described in U.S. Patent No. 5,965,405.
In the production of antibodies, screening for the desired antibody can
be accomplished by techniques known in the art (e.g., ELISA (enzyme-linked
immunosorbent assay)). In one example, antibodies which recognize a specific
domain of a relaxin or relaxin receptor polypeptide can be used to assay
hybridomas
for a product (e.g., antibody) that binds to a relaxin or relaxin receptor
fragment
containing that domain. For selection of an antibody that specifically binds
to a first
relaxin or relaxin receptor polypeptide, but which does not specifically bind
a second,
different relaxin polypeptide, one can select on the basis of antibody-
positive binding
to the first polypeptide and a lack of antibody binding to the second,
different
polypeptide.
Soluble Relaxin Receptors
In another aspect of the invention, the relaxin antagonist is a relaxin
binding agent comprising a soluble relaxin receptor, or a fragment or analog
thereof,
that binds relaxin. The term "soluble relaxin receptor" refers to a relaxin
receptor
polypeptide that is not bound to a cell membrane. The relaxin receptor is
approximately 200 kilodaltons. (See Palejwala et al., Endocrinology
139(3):1208-12
(1998), the disclosure of which is incorporated by reference herein.) The
soluble form
of the relaxin receptor retains the ability to bind vertebrate relaxin, but
typically lacks
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transmembrane and/or cytoplasmic domains. Soluble relaxin receptors can
comprise
additional amino acid residues, such as affinity tags, that provide for a
means for
purification of the polypeptide or to provide sites for attachment of the
polypeptide to
another polypeptide, or to immunoglobulin sequences.
The soluble relaxin receptor can optionally contain a transmembrane
domain that cannot associate with a cell membrane. By "transmembrane domain"
is
meant a domain of the relaxin receptor polypeptide that contains a sufficient
number of
hydrophobic amino acids to allow the polypeptide to insert and anchor in a
cell
membrane. By "transmembrane domain that cannot associate with a cell membrane"
is meant a transmembrane domain that has been altered by mutation or deletion
such
that it is not sufficiently hydrophobic to allow insertion or other
association with a cell
membrane. Such a transmembrane domain does not preclude, for example, the
fusion
of the relaxin receptor polypeptide, or fragment thereof, with a secretion
signal
sequence useful for secretion of the polypeptide from the cell. Substitutions
or
alterations of the amino acid sequence useful to achieve an inactive
transmembrane
domain include, but are not limited to, deletion or substitution of amino
acids within
the transmembrane domain. Methods of making soluble receptors are known in the
art. (See, e.g., U.S. Patent Nos. 6,033,903; 6,037,450; and 5,925,549).
The soluble relaxin receptors include soluble, naturally-occurring
amino acid sequence variants of relaxin receptor. Soluble relaxin receptors
further
include those altered by substitution, addition or deletion of one or more
amino acid
residues that provide for functionally active relaxin receptor polypeptides.
Such
relaxin receptors include, but are not limited to, those containing as a
primary amino
acid sequence of all or part of the amino acid sequence of a relaxin receptor
polypeptide including sequences in which one or more functionally equivalent
amino
acid residues are substituted for residues within the sequence, resulting in a
silent
functional change (e.g., a conservative substitution).
In another aspect, the soluble relaxin receptor is a polypeptide
consisting of or comprising a fragment of a relaxin receptor polypeptide
having at least
10 contiguous amino acids of the relaxin receptor polypeptide. More typically,
the
fragment contains at least 20 or at least 50 contiguous amino acids of the
relaxin
CA 02425712 2010-07-21
receptor polypeptide. In other embodiments, the fragments are larger than 100
or even
200 amino acids.
The relaxin receptor polypeptide can be a polypeptide comprising
regions that are substantially similar to a relaxin receptor polypeptide or
fragments
thereof (e.g., in various embodiments, at least 60%, 70%, 75%, 80%, 90%, or
even
95% identity or similarity over an amino acid sequence of identical size), or
when
compared to an aligned sequence in which the alignment is done by a computer
sequence comparison/alignment program known in the art, or by visual
inspection.
(See, e.g., Smith and Waterman, Adv. App!. Math. 2:482 (1981); Needleman and
Wunsch, J. MoL Biol. 48:443 (1970); Pearson and Lipman, Proc. Natl. Acad. ScL
USA
85:2444 (1988); GAP, BESTFIT, PASTA, and TEASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI);
Ausubel et al. (supra)). Sequence identity or similarity can also be
determined by
identifying nucleic acids that are capable of hybridizing to a relaxin
receptor nucleic acid,
under high stringency, moderate stringency, or low stringency conditions.
(See, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring
Harbor
Laboratory Press, New York (2001); Ausubel et al., (1996), supra).
Soluble relaxin receptors and fragments thereof can be produced by
various methods known in the art. The manipulations which result in their
production
can occur at the gene or protein level. For example, cloned relaxin receptor
nucleic
acids can be modified by any of numerous strategies known in the art (see,
e.g.,
Sambrook et aL, supra; Ausubel et aL, supra), such as making conservative
substitutions, deletions, insertions, and the like. The sequence can be
cleaved at
appropriate sites with restriction endonuclease(s), followed by further
enzymatic
modification if desired, isolated, and ligated in vitro. In the production of
the relaxin
receptor nucleic acids, the modified nucleic acid typically remains in the
proper
translational reading frame, so that the reading frame is not interrupted by
translational
stop signals or other signals which interfere with the synthesis of the
soluble relaxin
receptor or fragment thereof. The relaxin receptor nucleic acid can also be
mutated in
vitro or in vivo to create and/or destroy translation initiation and/or
termination
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sequences. The relaxin receptor nucleic acid can also be mutated to create
variations
in coding regions (e.g., amino acid substitutions) and/or to form new
restriction
endonuclease sites or destroy preexisting ones and to facilitate further in
vitro
modification. Any technique for mutagenesis known in the art can be used,
including
but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis
(see, e.g.,
Hutchison et al., J. Biol. Chem. 253:6551-60 (1978)), the use of TAB linkers
(Pharmacia), and the like.
Manipulations of the relaxin receptor polypeptide sequence can also be
made at the polypeptide level. Included within the scope of the invention are
relaxin
receptor polypeptides that are differentially modified during or after
synthesis (e.g., in
vivo or by in vitro translation). Such modifications include conservative
substitution,
glycosylation, acetylation, phosphorylation, amidation, derivatization by
known
protecting/blocking groups, proteolytic cleavage, linkage to an antibody
molecule, a
protein or other cellular ligand, and the like. Any of numerous chemical
modifications
can be can-led out by known techniques, including, but not limited to,
specific
chemical cleavage (e.g., by cyanogen bromide), enzymatic cleavage (e.g., by
trypsin,
chymotrypsin, papain, V8 protease, and the like); modification by, for
example,
NaBH4, acetylation, formylation, oxidation and reduction, metabolic synthesis
in the
presence of tunicamycin, and the like.
Relaxin receptor polypeptides and fragments thereof can be purified
from natural sources by standard methods such as those described herein (e.g.,
immunoaffinity purification). Relaxin receptor polypeptides and fragments can
also be
isolated and purified by standard methods including chromatography (e.g., ion
exchange, affinity, sizing column chromatography, high pressure liquid
chromatography), centrifugation, differential solubility, or by any other
standard
technique for the purification of polypeptides. Relaxin receptor polypeptides
and
fragments thereof can be synthesized by standard chemical methods known in the
art
(see, e.g., Hunkapiller et al., Nature 310:105-11 (1984); Stewart and Young,
Solid
Phase Peptide Synthesis, 21 Ed., Pierce Chemical Co., Rockford, IL, (1984)).
Furthermore, if desired, nonclassical amino acids or chemical amino acid
analogs can
be introduced as a substitution or addition into the relaxin receptor
polypeptide
sequence. Non-classical amino acids include, but are not limited to, the D-
isomers of
27
CA 02425712 2010-07-21
the common amino acids, a-amino isobutyric acid, 4-aminobutyric acid, 2-amino
butyric acid, e-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric
acid,
3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline,
sarcosine,
citralline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, 13-alanine, selenocysteine, fluoro-amino acids, designer
amino acids
such as 13-methyl amino acids, C a-methyl amino acids, N a-methyl amino acids,
and
amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or
L (levorotary).
In another embodiment, the soluble relaxin receptor is a chimeric, or
fusion, protein comprising a relaxin receptor polypeptide, or fragment thereof
(typically consisting of at least a domain or motif of the relaxin receptor
polypeptide,
or at least 10 contiguous amino acids of the relaxin receptor polypeptide)
joined at its
amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a
different protein. In one embodiment, such a chimeric protein is produced by
. 15 recombinant expression of a nucleic acid encoding the chimeric
polypeptide. The
chimeric product can be made by ligating the appropriate nucleic acid
sequences,
encoding the desired amino acid sequences, to each other in the proper reading
frame
and expressing the chimeric product by methods commonly known in the art.
Alternatively, the chimeric product can be made by protein synthetic
techniques (e.g.,
by use of an automated peptide synthesizer). In a specific embodiment, the
fusion
protein is a relaxin-receptor-ubiquitin fusion protein.
Relaxin Analogs
The relaxin antagonist can further be a relaxin analog, such as a relaxin
polypeptide that binds to a relaxin receptor but fails to induce a response by
that
receptor. For example, the relaxin analog can be a competitive inhibitor of
relaxin
binding or a conventional antagonist of the relaxin receptor. The term
"relaxin" refers
to vertebrate relaxin polypeptides, including full length relaxin polypeptide
or a
portion of the relaxin polypeptide that retains biological activity. Relaxin
has been
well defined in its natural human form, animal form, and in its synthetic
form. In
particular, relaxin has been extensively described in U.S. Patent Nos.
5,179,195;
5,166,191; 5,023,321; 4,835,251; and 4,758,516. Methods of making relaxin and
its analogs
28
CA 02425712 2010-07-21
are known in the art (supra). Relaxin analogs can be prepared by modification
of relaxin
polypeptides such that the relaxin analog retains relaxin receptor binding
activity, but
does not induce a response by the relaxin receptor. For example, relaxin
analogs can
be amino acid sequence variants of relaxin that retain relaxin receptor
binding activity,
but that fail to induce a response by a relaxin receptor. Relaxin analogs
further include
relaxin polypeptides, altered by addition or deletion of one or more amino
acid
residues, that retain receptor-binding function but fail to induce a response
by relaxin
receptor.
In various aspects according to the present invention, the relaxin analog
is fragment of a relaxin polypeptide consisting of or comprising at least 10
contiguous
amino acids of the relaxin polypeptide. Alternatively, the fragment contains
at least 20
or 40 contiguous amino acids of the relaxin polypeptide. In other embodiments,
the
fragments are not larger than 35 amino acids.
The relaxin analog can be a polypeptide comprising regions that are
substantially similar to a relaxin polypeptide (e.g., in various embodiments,
at least
60%, 70%, 75%, 80%, 90%, or even 95% identity or similarity over an amino acid
sequence of identical size), or when compared to an aligned sequence in which
the
alignment is done by a computer sequence comparison/alignment program known in
the art, or which coding nucleic acid is capable of hybridizing to a relaxin
nucleic acid,
under high stringency, moderate stringency, or low stringency conditions. (See
supra.)
Relaxin analogs can be produced by various methods known in the art.
The manipulations which result in their production can occur at the gene or
protein
level. For example, cloned relaxin nucleic acids can be modified by any of
numerous
strategies known in the art (see, e.g., Sambrook et al., supra; Ausubel et aL,
supra),
such as by making conservative or non-conservative substitutions, deletions,
insertions, and the like. The sequence can be cleaved at appropriate sites
with
restriction endonuclease(s), followed by further enzymatic modification, if
desired,
isolated, and ligated in vitro. In the production of the relaxin analog
nucleic acids, the
modified nucleic acid typically remains in the proper translational reading
frame, so
that the reading frame is not interrupted by translational stop signals or
other signals
which interfere with the synthesis of the relaxin analog. The relaxin nucleic
acid can
29
CA 02425712 2010-07-21
be mutated in vitro or in vivo to create and/or destroy translation,
initiation and/or
termination sequences. The relaxin nucleic acid can also be mutated to create
variations in coding regions and/or to form new restriction endonuclease sites
or
de.stroy preexisting ones and to facilitate further in vitro modification. Any
technique
for mutagenesis known in the art can be used, including but not limited to,
chemical
mutagenesis, in vitro site-directed mutagenesis (see, e.g., Hutchison et al.,
J. Biol.
Chem. 253:6551-60 (1978)), the use of TAB linkers (Pharmacia), and the like.
In a specific embodiment, relaxin analogs are prepared from relaxin-
encoding nucleic acids that are altered to introduce aspartic acid codons at
specific
position(s) within at least a portion of the relaxin coding region. (See,
e.g., U.S. Patent
No. 5,945,402). The resulting analogs can be treated with dilute acid to
release a desired
analog, thereby rendering the protein more readily isolated and purified.
Manipulations of the relaxin polypeptide sequence can also be made at
the polypeptide level. Included within the scope of the invention are relaxin
analogs
that are differentially modified during or after synthesis (e.g., in vivo or
by in vitro
translation). Such modifications include amino acid substitution (either
conservative
or non-conservative), glycosylation, acetylation, phosphorylation, arnidation,
derivatization by known protecting/blocking groups, proteolytic cleavage,
linkage to
an antibody molecule, a protein or other cellular ligand, and the like. Any of
numerous chemical modifications can be carried out by known techniques,
including,
but not limited to, specific chemical cleavage (e.g., by cyanogen bromide),
enzymatic
cleavage (e.g., by trypsin, chymotrypsin, papain, V8 protease, and the like);
modification by, for example, NaBH4, acetylation, forraylation, oxidation and
reduction, metabolic synthesis in the presence of tunicamycin, and the like.
Relaxin analogs can be purified from natural sources by standard
methods such as those described herein (e.g., immunoaffinity purification).
Relaxin
analogs can also be isolated and purified by standard methods including
chromatography (e.g., ion exchange, affinity, sizing column chromatography,
high
pressure liquid chromatography), centrifugation, differential solubility, or
by any other
standard technique for the purification of polypeptides. Relaxin analogs can
be
synthesized by standard chemical methods known in the art (see, e.g.,
Hunkapiller et
CA 02425712 2010-07-21
al., Nature 310:105-11 (1984); Stewart and Young, Solid Phase Peptide
Synthesis, 2'd
Ed., Pierce Chemical Co., Rockford, IL, (1984)). Furthermore, if desired,
nonclassical
amino acids or chemical amino acid analogs can be introduced as a substitution
or
addition into the relaxin polypeptide sequence. Non-classical amino acids
include, but
are not limited to, the D-isomers of the common amino acids, a-amino
isobutyric acid,
4-aminobutyric acid, 2-amino butyric acid, s-amino hexanoic acid, 6-amino
hexanoic
acid, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline,
hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-
butylalanine,
phenylglycine, cyclohexylalanine, P-alanine, selenocysteine, fluoro-amino
acids,
designer amino acids such as P-methyl amino acids, C a-methyl amino acids, N
cc-
methyl amino acids, and amino acid analogs in general. Furthermore, the amino
acid
can be D (dextrorotary) or L (levorotary).
In another embodiment, the relaxin analog is a chimeric, or fusion,
protein comprising a relaxin polypeptide (typically consisting of at least a
domain or
motif of the relaxin polypeptide, or at least 10 contiguous amino acids of the
relaxin
polypeptide) joined at its amino- or carboxy-terminus via a peptide bond to an
amino
acid sequence of a different protein. In one embodiment, such a chimeric
protein is
produced by recombinant expression of a nucleic acid encoding the chimeric
polypeptide. The chimeric product can be made by ligating the appropriate
nucleic
acid sequences, encoding the desired amino acid sequences, to each other in
the proper
reading frame and expressing the chimeric product by methods commonly known in
the art. Alternatively, the chimeric product can be made by protein synthetic
techniques (e.g., by use of an automated peptide synthesizer).
In a specific embodiment, the fusion protein is a relaxin analog-
ubiquitin fusion protein. For example, U.S. Patent No. 5,108,919, discloses
methods for
preparing a fusion protein of a relaxin chain and ubiquitin.
Nucleic Acids:
The invention further provides nucleic acids for use as an antagonist, or
for expressing an antagonist, according to the present invention. Such nucleic
acids
include those encoding a soluble relaxin receptor or a relaxin analog for
synthesis of
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soluble relaxin receptor or relaxin analog, respectively. Antisense nucleic
acids are
provided for inhibition of relaxin or relaxin receptor expression. Nucleic
acids
encoding a relaxin polypeptide or a relaxin receptor polypeptide are also
provided for
the preparation of antibodies (see supra).
In one aspect, the invention provides nucleic acid sequences encoding a
relaxin receptor or relaxin analog for expression in vivo. The relaxin
receptor or
relaxin analog, or antisense nucleic acids, can be expressed in vivo for gene
therapy.
Relaxin receptor, relaxin analog or antisense nucleic acids can also be
expressed in
vivo or in vitro for the production of recombinant soluble relaxin receptor,
relaxin
analog or antisense nucleic acids for exogenous administration to a subject.
The nucleic acids can be vertebrate nucleic acid, including, for
example, human, mouse, rat, pig, cow, dog, or monkey relaxin receptor,
relaxin, or a
relaxin analog derived from a vertebrate relaxin. The nucleic acids can
comprise
genomic DNA, cDNA, or the coding region of the relaxin receptor, relaxin or a
relaxin
analog. The nucleic acids can further include mRNAs corresponding to the
relaxin
receptor locus or the relaxin locus. The nucleic acids also include nucleotide
sequence
variants, such as those encoding other possible codon choices for the same
amino acid
or conservative amino acid substitutions thereof, such as naturally occurring
allelic
variants. Due to the degeneracy of nucleotide coding sequences, other nucleic
acid
sequences which encode substantially the same amino acid sequence as a relaxin
receptor or relaxin coding sequence, can be used in the practice of the
present
invention. These nucleic acid sequences include, but are not limited to,
nucleotide
sequences comprising all or portions of a relaxin gene which is altered by the
substitution of different codons that encode the same or a functionally
equivalent
amino acid residue (e.g., a conservative substitution) within the sequence,
thus
producing a silent change.
The invention further provides nucleic acid fragments of at least 6
contiguous nucleotides (e.g., a hybridizable portion); in other embodiments,
the
nucleic acids comprise at least 8 contiguous nucleotides, at least contiguous
25
nucleotides, at least contiguous 50 nucleotides, at least 100 nucleotides, 150
nucleotides or more of a relaxin sequence. In another embodiment, the nucleic
acids
are smaller than 100 or 150 nucleotides in length. The nucleic acids can be
single or
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double-stranded. As is readily apparent, as used herein, a nucleic acid
encoding a
fragment of a relaxin or relaxin receptor polypeptide is construed as
referring to a
nucleic acid encoding only the recited fragment or portion of the relaxin
polypeptide
and not the other contiguous portions of the relaxin receptor or relaxin
polypeptide as a
contiguous sequence. Fragments of the nucleic acids encoding one or more
domains
of relaxin or relaxin receptor are also provided.
Relaxin receptor, relaxin analog or antisense nucleic acids can be
inserted into an appropriate vector (e.g., an expression vector which contains
the
necessary elements for the transcription or transcription and translation of
the inserted
polypeptide-coding sequence) in either the sense or antisense orientations, as
desired.
The necessary transcriptional and translational signals can also be supplied
by the
native relaxin or relaxin receptor gene and/or its flanking regions. A variety
of host-
vector systems can be utilized to express the polypeptide-coding sequence.
These
include but are not limited to, mammalian cell systems infected with virus
(e.g.,
vaccinia virus, adenovirus, adeno-associated virus, and the like), insect cell
systems
infected with virus (e.g., baculovirus), microorganisms such as yeast
containing yeast
vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or
cosmid
DNA. The expression elements of vectors vary in their strengths and
specificities.
Depending on the host-vector system utilized, any of a number of suitable
transcription
and translation elements can be used. In specific embodiments, the nucleic
acids are
expressed, or a nucleic acid sequence encoding a functionally active portion
of a
relaxin receptor or relaxin analog is expressed in mammalian cells, yeast or
bacteria.
In yet another embodiment, a fragment of a relaxin receptor or relaxin analog
comprising a domain of the respective polypeptide is expressed.
Any of the methods known in the art for the insertion of nucleic acids
into a vector can be used to construct expression vectors containing a
chimeric gene
having the appropriate transcriptional, translational control signals and/or
polypeptide
coding sequences. These methods include in vitro recombinant DNA and synthetic
techniques and in vivo recombinants (genetic recombination). Expression of
nucleic
acids encoding a relaxin receptor or relaxin analog can be regulated by a
second
nucleic acid sequence so that the nucleic acid or polypeptide is expressed in
a host
transformed with the recombinant DNA molecule. For example, expression of a
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nucleic acid of a relaxin receptor or relaxin analog can be controlled by any
promoter/enhancer element known in the art. Promoters which can be used to
control
expression include, but are not limited to, the SV40 early promoter region
(see, e.g.,
Benoist and Chambon, Nature 290:304-10 (1981)), the promoter contained in the
3'
long terminal repeat of Rous sarcoma virus (see, e.g., Yamamoto et al., Cell
22:787-97
. (1980)), the herpes thymidine kinase promoter (see, e.g., Wagner et al.,
Proc. NatL
Acad. Sci. USA 78:1441-45 (1981)), the regulatory sequences of the
metallothionen
gene (see, e.g., Brinster et al., Nature 296:39-42 (1982)), prokaryotic
expression
vectors such as the 13-lactamase promoter (see, e.g., Villa-Komaroff et al.,
Proc. NatL
Acad. Sci. USA 75:3727-31 (1978)) or the tac promoter (see, e.g., deBoer et
al., Proc.
Natl. Acad. Sci. USA 80:21-25 (1983)), plant expression vectors including the
cauliflower mosaic virus 35S RNA promoter (see, e.g., Gardner et al., NucL
Acids Res.
9:2871-88 (1981)), and the promoter of the photosynthetic enzyme ribulose
biphosphate carboxylase (see, e.g., Herrera-Estrella etal., Nature 310:115-20
(1984)),
promoter elements from yeast or other fungi such as the Ga17 and Ga14
promoters, the
ADH (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase)
promoter,
the alkaline phosphatase promoter, and the like.
The following animal transcriptional control regions, which exhibit
tissue specificity, have been utilized in transgenic animals: the elastase I
gene control
region which is active in pancreatic acinar cells (see, e.g., Swift et al.,
Cell 38:639-46
(1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986);
MacDonald, Hepatology 7(1 Suppl.):42S-51S (1987)); the insulin gene control
region
which is active in pancreatic beta cells (see, e.g., Hanahan, Nature 315:115-
22 (1985)),
the immuno globulin gene control region which is active in lymphoid cells
(see, e.g.,
Grosschedl etal., Cell 38:647-58 (1984); Adams etal., Nature 318:533-38
(1985);
Alexander et al., Mol. Cell. Biol. 7:1436-44 (1987)), the mouse mammary tumor
virus
control region which is active in testicular, breast, lymphoid and mast cells
(see, e.g.,
Leder et al., Cell 45:485-95 (1986)), the albumin gene control region which is
active in
liver (see, e.g., Pinkert et al., Genes Dev. 1:268-76 (1987)), the alpha-
fetoprotein gene
control region which is active in liver (see, e.g., Krumlauf et al., MoL Cell.
Biol.
5:1639-48 (1985); Hammer etal., Science 235:53-58 (1987)); the alpha 1-
antitrypsin
gene control region which is active in the liver (see, e.g., Kelsey et al.,
Genes and
34
CA 02425712 2010-07-21
Devel. 1:161-71 (1987)); the beta-globin gene control region which is active
in
myeloid cells (see, e.g., Magram et al., Nature 315:338-40 (1985); Kollias et
al., Cell
46:89-94 (1986)); the myelin basic protein gene control region which is active
in
oligodendrocyte cells in the brain (see, e.g., Readhead et al., Cell 48:703-12
(1987));
the myosin light chain-2 gene control region which is active in skeletal
muscle (see,
e.g., Shani, Nature 314:283-86 (1985)); and the gonadotropic releasing hormone
gene
control region which is active in the hypothalamus (see, e.g., Mason et al.,
Science
234:1372-78 (1986)). In a preferred embodiment, the tissue-specific promoter
is the
prostate specific antigen promoter. (See, e.g., U.S. Patent No. 6,100,444).
In another embodiment, a vector is used that comprises a promoter
operably linked to a nucleic acid, one or more origins of replication, and,
optionally,
one or more selectable markers (e.g., an antibiotic or drug resistance
marker). For
example, an expression construct can be made by subcloning a relaxin receptor
or
relaxin analog nucleic acid into a restriction site of the pRSECT expression
vector.
Such a construct allows for the expression of the relaxin analog or relaxin
receptor
polypeptide under the control of the T7 promoter with a histidine amino
terminal flag
sequence for affinity purification of the expressed polypeptide. In another
specific
embodiment, a vector is used that comprises the prostate specific antigen
promoter
operably linked to a relaxin analog nucleic acid, one or more origins of
replication,
and, optionally, one or more selectable markers (e.g., a drug resistance
marker).
Expression vectors containing such nucleic acids can be identified by
general approaches well known to the skilled artisan, including: (a) nucleic
acid
hybridization or polymerase chain reaction, (b) the presence or absence of
"marker"
gene function, (c) expression of inserted sequences, or polymerase chain
reaction
(PCR). In the first approach, the presence of a nucleic acid inserted in an
expression
vector can be detected by nucleic acid hybridization or polymerase chain
reaction
using probes comprising sequences that are homologous to an inserted nucleic
acid. In
the second approach, the recombinant vector/host system can be identified and
selected based upon the presence or absence of certain "marker" gene functions
(e.g.,
thymidine kinase activity, resistance to antibiotics, transformation
phenotype,
occlusion body formation in baculovirus, and the like) caused by the insertion
of a
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vector containing the relaxin receptor, relaxin or relaxin analog nucleic
acids. For
example, if the nucleic acid is inserted within the marker gene sequence of
the vector,
recombinants containing the nucleic acid can be identified by the absence of
the
marker gene function.
In the third approach, recombinant expression vectors can be identified
by assaying the polypeptide expressed by the recombinant. Such assays can be
based,
for example, on the physical or functional properties of the polypeptide in in
vitro
assay systems (e.g., binding with anti-relaxin antibody, binding relaxin or a
relaxin
analog, binding to relaxin receptor, and the like). Once a particular
recombinant
vector is identified and isolated, several methods that are known in the art
can be used
to propagate it. Once a suitable host system and growth conditions are
established,
recombinant expression vectors can be propagated and prepared in quantity. As
previously explained, the expression vectors which can be used include, but
are not
limited to the following vectors or their derivatives: human or animal viruses
such as
vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast
vectors;
bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to
name
but a few. In the fourth approach, PCR is used to detect the nucleic acid in
the vector
(see supra).
In addition, a host cell strain can be chosen that modulates the
expression of the inserted sequences, or modifies or processes the gene
product in the
specific fashion desired. Expression from certain promoters can be elevated in
the
presence of certain inducers; thus, expression of the polypeptide can be
controlled.
Furthermore, different host cells having characteristic and specific
mechanisms for the
translational and post-translational processing, and modification (e.g.,
glycosylation,
phosphorylation) of polypeptides can be used. Appropriate cell lines or host
systems
can be chosen to ensure the desired modification and processing of the foreign
protein
expressed. For example, expression in a bacterial system can be used to
produce an
unprocessed core protein product. Expression in mammalian cells can be used to
ensure "native" processing of mammalian receptor polypeptide. Furthermore,
different vector/host expression systems can affect processing reactions to
different
extents.
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CA 02425712 2010-07-21
Functional Assays for Relaxin and Relaxin Receptor Agonists and Antagonists
The activity of relaxin agonists and antagonists, and of relaxin receptor
agonists and antagonists, can be determined by standard assays for relaxin
and/or
relaxin receptor activity. In one aspect of the invention, the activity of a
relaxin
agonist is assayed. For example, the ability of a relaxin agonist to decrease
apoptosis,
or to stimulate maturation of tissue, is assayed.
In another aspect of the invention, the ability of a relaxin antagonist to
inhibit relaxin functional activity by binding to relaxin is assayed.
Similarly, the
ability of a relaxin antagonist to inhibit relaxin function, or relaxin
receptor function,
can be assayed by, for example, adding a relaxin antagonist to a relaxin
receptor assay
and determining the inhibition, as compared with a control without the relaxin
antagonist. Suitable measurements of relaxin antagonist activity include
measuring
percent inhibition, IC50, and the like.
Suitable assays for measuring relaxin or relaxin receptor agonist or
antagonist activity, include, for example, those described in the following
references:
MacLennan et al., Ripening of the Human Cervix and Induction of Labor with
Intracervical Purified Porcine Relaxin, Obstetrics & Gynecology 68:598-601
(1986);
Poisner et al., Relaxin Stimulates the Synthesis and Release of Prorenin From
Human
Decidual Cells: Evidence For Autocrine/Paracrine Regulation, J Clinical
Endocrinology
and Metabolism 70:1765-67 (1990); O'Day-Bowman et al., Hormonal Control of the
Cervix in Pregnant Gilts. III. Relaxin's Influence on Cervical Biochemical
Properties in
Ovariectomized Hormone-Treated Pregnant Gilts, Endocrinology 129:1967-76
(1991);
Saugstad, Persistent Pelvic Pain and Pelvis Joint Instability, Eur. J
Obstetrics &
Gynecology and Reproductive Biology 41:197-201 (1991).
Other assays include those disclosed by Bullesbach et al., The
Receptor-Binding Sites of Human Relaxin II, J. Biol. Chem. 267:22957-60
(1992);
Hall et al., Influence of Ovarian Steroids on Relaxin-Induced Uterine Growth
in
Ovariectomized Gilts, Endocrinology 130:3159-66 (1992); Kibblewhite et al.,
The
Effect of Relaxin on Tissue Expansion, Arch. Otolwyngol. Head Neck Surg.
118:153-
56 (1992); Lee et al., Monoclonal Antibodies Specific for Rat Relaxin. VI.
Passive
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Immunization with Monoclonal Antibodies Throughout the Second Half of
Pregnancy
Disrupts Histological Changes Associated with Cervical Softening at
Parturition in
Rats, Endocrinology 130:2386-91 (1992); Bell et al., A Randomized, Double-
Blind
Placebo-Controlled Trial of the Safety of Vaginal Recombinant Human Relaxin
for
Cervical Ripening, Obstetrics & Gynecology 82:328-33 (1993); Bryant-Greenwood
et
al., Sequential Appearance of Relaxin, Prolactin and IGFBP-1 During Growth and
Differentiation of the Human Endometrium, Molecular and Cellular Endocrinology
95:23-29 (1993); Chen et al., The Pharmacokinetics of Recombinant Human
Relaxin
in Nonpregnant Women After Intravenous, Intravaginal, and Intracervical
Administration, Pharmaceutical Research 10:834-38 (1993); Huang et al.,
Stimulation
of Collagen Secretion by Relaxin and Effect of Oestrogen on Relaxin Binding in
Uterine Cervical Cells of Pigs, Journal of Reproduction and Fertility 98:153-
58
(1993);
Additional assays are disclosed in Saxena et al., Is the Relaxin System a
Target for Drug Development? Cardiac Effects of Relaxin, TiPS 14:231 (June
1993,
letter); Winn et al., Hormonal Control of the Cervix in Pregnant Gilts. IV.
Relaxin
Promotes Changes in the Histological Characteristics of the Cervix that are
Associated
with Cervical Softening During Late Pregnancy in Gifts, Endocrinology 133:121-
28
(1993); Colon et al., Relaxin Secretion into Human Semen Independent of
Gonadotropin Stimulation, Biology of Reproduction 50:187-92 (1994); Golub et
al.,
Effect of Short-Term Infusion of Recombinant Human Relaxin on Blood Pressure
in
the Late-Pregnant Rhesus Macaque (Macaca Mulatta), Obstetrics & Gynecology
83:85-88 (1994); Jauniaux et al., The Role of Relaxin in the Development of
the
Uteroplacental Circulation in Early Pregnancy, Obstetrics & Gynecology 84:338-
342
(1994); Johnson et al., The Regulation of Plasma Relaxin Levels During Human
Pregnancy, J. Endocrinology 142:261-65 (1994); Lane et aL, Decidualization of
Human Endometrial Stromal Cells in Vitro: Effects of Progestin and Relaxin on
the
Ultrastructure and Production of Decidual Secretory Proteins, Human
Reproduction
9:259-66 (1994); Lanzafame et al., Pharmacological Stimulation of Sperm
Motility,
Human Reproduction 9:192-99 (1994); Petersen et al., Normal Serum Relaxin in
Women with Disabling Pelvic Pain During Pregnancy, Gynecol. Obstet. Invest.
38:21-
23 (1994); Tashima et al., Human Relaxins in Normal, Benign and Neoplastic
Breast
38
CA 02425712 2003-04-08
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PCT/US01/42484
Tissue, J. Mol. Endocrinology 12:351-64 (1994); Winn etal. Individual and
Combined
Effects of Relaxin, Estrogen, and Progesterone in Ovariectomized Gilts. I.
Effects on
the Growth, Softening, and Histological Properties of the Cervix,
Endocrinology
135:1241-49 (1994); Winn etal., Individual and Combined Effects of Relaxin,
Estrogen, and Progesterone on Ovariectomized Gilts. II. Effects on Mammary
Development, Endocrinology 135:1250-55 (1994); Bryant-Greenwood etal., Human
Relaxins: Chemistry and Biology, Endocrine Reviews 15:5-26 (1994); Johnson et
al.,
Relationship Between Ovarian Steroids, Gonadotrophins and Relaxin During the
Menstrual Cycle, Acta Endocrinilogica 129:121-25 (1993).
In yet another aspect of the invention, the activity of an agonist or
antagonist is determined by measuring the ability of the agonist or antagonist
to
compete with wild-type relaxin polypeptide, or relaxin receptor polypeptide,
for
binding to anti-relaxin antibody. Various immunoassays known in the art can be
used.
Such assays include, but are not limited to, competitive and non-competitive
assay
systems using techniques such as radioimmunoassays, ELISA (enzyme linked
immunosorbent assay) "sandwich" immunoassays, immunoradiometric assays, gel
diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays
(using
colloidal gold, enzyme or radioisotope labels, and the like), Western blots,
precipitation reactions, agglutination assays (e.g., gel agglutination assays
or
hemagglutination assays), complement fixation assays, immunofluorescence
assays,
protein A assays, immunoelectrophoresis assays, and the like. Antibody binding
can
be detected by measuring the amount of label on the primary antibody that is
bound, or
prevented from binding to, a substrate. Alternatively, primary antibody
binding is
detected by measuring binding of a secondary antibody or reagent to the
primary
antibody. The secondary antibody can also be directly labeled. Many means are
known in the art for detecting binding in an immunoassay and are considered
within
the scope of the present invention.
The functional activity of an agonist or antagonist can also be
deteimined in an in vivo system. For example, the ability of relaxin agonists
or
antagonists to bind, or to compete for binding to, a relaxin receptor, or to
modulate
apoptosis in a cell population and/or tissues can be measured. The assays
described
above can be used to determine the activity resulting from expression of
relaxin
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PCT/US01/42484
agonist or antagonists in vertebrate cells. Alternatively, relaxin agonist or
antagonist
can be expressed in a heterologous system and the activity of the relaxin
agonist or
antagonist can be assayed as a modulator of a physiological change in that
system. For
example, the ability of a relaxin agonist or antagonist to modulate apoptosis
can be
tested in vertebrate cells (e.g., transfected mammalian cells).
Administration of Relaxin Agonists and Antagonists
The invention provides methods for the administration to a subject of an
effective amount of a relaxin agonist or antagonist (also referred to
collectively as an
"active agent"). Typically, the active agent is substantially purified prior
to
formulation. The subject can be a human or non-human animal, a vertebrate, and
is
typically an animal, including but not limited to, cows, pigs, horses,
chickens, cats,
dogs, and the like. More typically, the subject is a mammal, and in a
particular
embodiment, human.
Various delivery systems are known and can be used to administer a
active agent, such as, for example, by infusion, injection (e.g., intradennal,
intramuscular or intraperitoneal), oral delivery, nasal delivery,
intrapulmonary
delivery, rectal delivery, transdermal delivery, interstitial delivery or
subcutaneous
delivery. In a specific embodiment, it can be desirable to administer the
active agent
locally to the area in need of treatment; this administration can be achieved
by, for
example, and not by way of limitation, local infusion, topical application, by
injection
(e.g., intratesticular or intraprostatic), by means of a catheter, or by means
of an
implant, the implant being for example, a porous, non-porous, gelatinous or
polymeric
material, including membranes such as silastic membranes or fibers. In one
embodiment, administration can be by direct injection at the target site.
Pharmaceutical compositions containing the active agent can be
formulated according to the desired delivery system. Such pharmaceutical
compositions typically comprise a therapeutically effective amount of active
agent and
a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable"
means
approved by a regulatory agency of the Federal or a state government or listed
in the
U.S. Pharmacopeia or other generally recognized pharmacopeia for use in
vertebrates,
typically animals, and more typically in humans. The term "carrier" refers to
a diluent,
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PCT/US01/42484
adjuvant, excipient, stabilizer, preservative, viscogen, or vehicle with which
the active
agent is formulated for administration. Phatinaceutical carriers can be
sterile liquids,
such as water and oils, including those of petroleum, animal, vegetable or
synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the
like. Suitable
excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim
milk, glycerol, propylene glycol, ethanol, and the like. The composition, if
desired,
can also contain minor amounts of wetting or emulsifying agents, or pH
buffering
agents.
Suitable preservatives include, for example, sodium benzoate,
quaternary ammonium salts, sodium azide, methyl paraben, propyl paraben,
sorbic
acid, ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,
chlorobutanol, dehydroacetic acid, ethylenediamine, potassium benzoate,
potassium
metabisulfite, potassium sorbate, sodium bisulfite, sulfur dioxide, organic
mercurial
salts, phenol and ascorbic acid. Suitable viscogens include, for example,
carboxymethylcellulose, sorbitol, dextrose, and polyethylene glycols. Other
examples
of suitable pharmaceutical carriers are described in, for example, Remington
's
Pharmaceutical Sciences (Gennaro (ed.), Mack Publishing Co., Easton,
Pennsylvania
(1990)).
The active agents can also be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with free amino groups
such as
those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids,
and the like,
and those formed with free carboxyl groups such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-
ethylamino ethanol, histidine, procaine, and the like.
In one embodiment, the active agent is formulated in accordance with
routine procedures as a pharmaceutical composition adapted for intravenous
administration to human beings. For intravenous delivery, water is a typical
carrier.
Saline, aqueous dextrose and glycerol solutions can also be employed as liquid
carriers, particularly for injectable solutions. Typically, compositions for
intravenous
administration are solutions in sterile isotonic aqueous buffer. Where
necessary, the
composition can also include a solubilizing agent and a local anesthetic to
ease pain at
41
CA 02425712 2010-07-21
the site of the injection. Generally, the ingredients are supplied either
separately or
mixed together in unit dosage form, for example, as a dry lyophilized powder
or water-
free concentrate in a hermetically sealed container such as an ampoule or
sachet
indicating the quantity of active agent. Where the composition is to be
administered
by infusion, it can be dispensed with an infusion bottle containing sterile
pharmaceutical grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can be provided
so that the
ingredients can be mixed prior to administration.
Orally deliverable compositions can take the form of solutions,
suspensions, emulsions, tablets, pills, capsules, powders, sustained-release
foimulations, and the like. Oral formulations can include standard carriers
such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, and the like.
For rectal administration, the compositions are formulated according to
standard pharmaceutical procedures. Typically, the composition is formed as a
meltable composition, such as a suppository. Suppositories can contain
adjuvants
which provide the desired consistency to the composition. They can also
contain
water-soluble carriers, such as polyethylene glycol, polypropylene glycol,
glycerogelatine, methylcellulose or carboxymethylcellulose. Wetting agents,
such as
fatty acids, fatty acid glycerides, polyoxyethylene sorbitan fatty acid
esters,
polyoxyethylene sorbitol fatty acid esters, polyoxyethylene fatty acid esters,
as well as
higher alcohol esters of polyoxyethylene and esters of lower alkylsulfonic
acids. The
suppositories can also contain suitable emulsifying and dispersing components
as well
as components for adjusting the viscosity and coloring substances.
Nasal administration is typically performed using a solution as a nasal
spray and can be dispensed by a variety of methods known to those skilled in
the art.
Systems for intranasally dispensing liquids as a spray are well known (see,
e.g., U.S.
Patent No. 4,511,069). Preferred nasal spray solutions comprise the active
agent in a
liquid carrier that optionally includes a nonionic surfactant for enhancing
absorption of the
drug and one or more buffers or other additives to minimize nasal irritation.
In some
embodiments, the nasal spray
42
CA 02425712 2010-07-21
solution further comprises a propellant. The pH of the nasal spray solution is
typically
between about pH 6.8 and 7.2.
For intranasal administration, ingredients which improve the absorption
of nasally administered active agent and reduce nasal irritation, especially
when used
in a chronically administered treatment protocol, are desirable. In this
context, the
utilization of surfactants to enhance absorption of the active agent is
preferred. (See,
e.g., Hirai et al., Int. J. Pharmaceutics 1:173-84 (1981); Great Britain
Patent
Specification 1 527 605). Nasal administration of drugs enhanced by
surfactants,
however, can cause nasal irritation, including stinging, congestion and
rhinorrhea. Thus, compositions which enhance absorption through the nasal
mucosa with reduced irritation are desirable, such as, for example, nonionic
surfactants such as nonoxyno1-9, laureth-9, poloxamer-124,
octoxyno1-9 and lauramide DEA. Nonoxyno1-9 (N-9) is an ethoxylated alkyl
phenol,
the polyethyleneoxy condensate of nonylphenol with 9 moles of ethylene oxide.
This
surfactant has been used in detergent products and is sold under trade names
such as
SURFONIC N-95 (Jefferson), NEUTRONYX 600 (Onyx) and IGEPAL (C0-630
(GAF). N-9 is considered to be a hard detergent, and has been used as a
spermatocide.
(See The Merck Index, 10th Ed., Entry 6518). To minimize irritation attributed
to
employment of surfactants, one or more anti-irritant additives are included in
the
emulsion. In one example, polysorbate-80 has been shown to reduce the
irritation
caused by intranasally administered drugs where delivery was enhanced by use
of a
nonionic surfactant (see, e.g., U.S. Patent No. 5,902,789).
Intrapulmonary dosage forms containing the active agent can be
administered to the respiratory tract intranasally or by breathing a spray or
aerosol
containing the active agent. The active agent is typically delivered directly
into the
lungs in a small particle aerosol, which is specifically targeted to the
smallest air
passages and alveoli.
Intrapulmonary dosage forms are typically formed as particulate
dispersed forms. This can be accomplished by preparing an aqueous aerosol of
solid
particles which contain the composition. Typically, an aqueous aerosol is made
by
formulating an aqueous solution or suspension of the composition together with
43
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conventional pharmaceutically acceptable carriers and stabilizers. The
carriers and
TM TM
stabilizers typically include nonionic surfactants (e.g., Tweens, Pluronics or
polyethylene glycol), innocuous proteins such as serum albumin, sorbitan
esters, oleic
acid, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.
The
formulations can also include mucolytic agents, such as those described in
U.S. Patent
No. 4,132,803 (which is incorporated by reference herein), as well as broncho-
dilating
agents. The formulations are preferably sterile. Aerosols are generally
prepared from
isotonic solutions. The particles optionally include normal lung surfactant
proteins.
The aerosol of particles can be formed in aqueous or nonaqueous (e.g.,
fluorocarbon propellant) suspensions. The aerosols are preferably free of lung
irritants
(i.e., substances that cause acute bronchoconstriction, coughing, pulmonary
edema or
tissue destruction). Nonirritating, absorption enhancing agents are also
suitable for use
herein.
Sonic nebulizers can be used to prepare aerosols. Sonic nebulizers
minimize exposure of the composition to shear, which can result in
degradation. A
suitable device is the Bird Micronebulizer. Other suitable atomizing or
nebulizing
systems or intratracheal delivery systems include, for example, those
disclosed in U.S.
Patent No. 3,915,165; European Patent No. 0 166 476; the jet nebulizers
described by
Newman et al. (Thorax 40:671-76 (1985)); metered dose inhalers (see, e.g.,
Berenberg,
J. Asthma-USA 22:87-92 (1985)); the endotracheal catheter assembly of Braurmer
(U.S. Patent No. 5,803,078), or other devices (see, e.g., Sears et al., N.Z.
Med. J
96:74311 (1983); O'Reilly et al., Br. Med. J. 286:6377 (1983); or Stander et
al.,
Respiration 44:237-40 (1982)), so long as they are compatible with the
composition to
be administered and are capable of delivering particles of the desired size.
The particulate aerosol suspensions are typically fine dry powders
containing the active agent. Particulate aerosol suspension are prepared by
any
number of conventional procedures. The simplest method of preparing such
suspensions is to micronize the active agent (e.g., as crystals or
lyophilization cakes),
and suspend the particles in dry fluorocarbon propellants. In these
formulations the
active agent is preferably suspended in the fluorocarbon. In an alternate
embodiment,
the active agent is stored in a compartment separate from the propellant.
Discharge of
44
CA 02425712 2010-07-21
the propellant withdraws a predetermined dose from the storage compartment.
The
devices used to deliver active agents in this manner are known as metered dose
inhalers (MDIs) (see, e.g., Byron, Drug Development and Industrial Pharmacy
12:993
(1986)).
The size of the aerosols or particles generally will range about from 0.5
to about 5 gm, typically about 2 gm to 5 gm, preferably about 2 to about 4 p.m
or
about 4 to about 5 p.m. In some aspects, smaller particles are less acceptable
because
they tend not to be deposited, but instead are exhaled. Similarly, in other
aspects,
larger particles are not preferred because they are less likely to be
deposited in the
alveoli, being removed by impaction within the nasopharyngeal or oral cavities
(see,
e.g., Byron, J. Pharm. Sci. 75:433 (1986)). The aerosol or particulate
compositions
can be heterogeneous in size distribution, although heterogeneity can be
reduced by
known methods (e.g., the screening unit described in EP 0 135 390).
Heterogeneity is
typically not disadvantageous unless the proportion of particles having an
average
mean diameter in excess of about 4 gm is so large as to impair the delivery of
a
therapeutic dose by pulmonary inhalation. Suspensions containing greater than
about
15% of particles within the 0.5-5 gm range can be used, but generally the
proportion
of particles having an average mean diameter larger than 5 gm is typically
less than
about 25%, and preferably not greater than 10%, of the total number of
particles. The
diameters recited refer to the particle diameters as introduced into the
respiratory tract.
The amount of the active agent which will be effective in the treatment
of a particular subject will depend on the specific abnormality being treated,
and can
be determined by standard clinical techniques. In addition, in vitro assays
can
optionally be employed to help identify optimal dosage ranges. The precise
dose of
the active agent to be employed in the formulation will also depend on the
route of
administration, and the seriousness of the condition, and should be decided
according
to the judgment of the practitioner and each subject's circumstances. Suitable
dosage
ranges for administration are generally about 0.001 mg/kg to about 100 mg/kg
of
active agent per kilogram body weight. Effective doses can also be
extrapolated from
dose response curves derived from in vitro or animal model test systems.
CA 02425712 2010-07-21
Suppositories generally contain active ingredient in the range of 0.5% to 10%
by
weight; oral formulations typically contain 10% to 95% active ingredient.
The invention also provides a pharmaceutical pack or kit comprising
one or more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Optionally associated with such container(s)
can be a
notice in the form prescribed by a governmental agency regulating the
manufacture,
use or sale of pharmaceuticals or biological products, which notice reflects
approval by
the agency of manufacture, use or sale for human administration.
In yet another embodiment, the active agent can be delivered in a
controlled release system. In one embodiment, a pump can be used (see, e.g.,
Langer,
supra; Sefton, Crit. Ref Biomed. Eng. 14:201-40 (1987); Buchwald et al.,
Surgeiy
88:507-16 (1980); Saudek et aL , N. Engl. J. Med. 321:574-79 (1989)). In
another
embodiment, polymeric materials can be used (see Medical Applications of
Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974);
Controlled
Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball
(eds.),
Wiley, New York (1984); Ranger and Peppas, I Macromol. Sci. Rev. Macromol.
Chem. 23:61 (1983); see also Levy et al., Science 228:190-92 (1985); During et
al.,
Ann. Neurol. 25:351-56 (1989); Howard et al., J. Neurosurg. 71:105-12 (1989)).
In yet another embodiment, a controlled release system can be placed in
proximity of the therapeutic target, thus requiring only a fraction of the
systemic dose
(see, e.g., Goodson, Medical Applications of Controlled Release, supra, Vol.
2, pp.
115-38 (1984)). Other controlled release systems are discussed in, for
example, the
review by Langer (Science 249:1527-33 (1990)).
Administration of Nucleic Acids
The invention provides further methods for the administration of
nucleic acids (e.g., nucleic acids encoding relaxin agonists or antagonists,
such as
30 relaxins, relaxin analogs, soluble relaxin receptor, relaxin binding
agents, relaxin
receptor binding agents, relaxin antisense nucleic acids and/or relaxin
receptor
antisense nucleic acids) to modulate apoptosis. Nucleic acids, both sense and
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PCT/US01/42484
antisense, can be used in the process of gene therapy. Gene therapy refers to
the
process of providing for the expression of nucleic acids of exogenous origin,
including
antisense nucleic acids or those encoding relaxin agonist or antagonist in a
subject for
the modulation of apoptosis (e.g. to treat a tissue abnormality) within the
subject. In
specific embodiments, nucleic acids encoding a relaxin or a relaxin analog are
administered to decrease apoptosis in cells have a relaxin receptor. In other
specific
embodiments, nucleic acids encoding a relaxin binding agent (e.g., a relaxin a
soluble
relaxin receptor) or a relaxin receptor binding agent (e.g., a relaxin analog)
is
administered to increase apoptosis in a cell population expressing a relaxin
receptor.
Any of the methods for gene therapy available in the art can be used according
to the
present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Steinberg and
Raso
Pharm. Pharm. Sci. 1:48-59 (1998)); Pantuck et al. (World J. UroL 18:143-47
(2000)); Prince (Pathology 30:335-47 (1998)); Ledley (Curr. Opin. BiotechnoL
5:626-
36 (1994)); Goldspiel et al. (Clin. Pharm. 12:488-505 (1993)); Wu and Wu
(Biotherapy 3:87-95 (1991)); Tolstoshev (Ann. Rev. PharmacoL ToxicoL 32:573-96
(1993)); Mulligan (Science 260:926-32 (1993)); Morgan and Anderson (Ann. Rev.
Biochem. 62:191-217 (1993)); and May (TIBTECH 11:155-215 (1993)).
Methods commonly known in the art of recombinant DNA technology
that can be used include those described in Ausubel et al. (1996, supra) and
Kriegler
(Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY
(1990)).
In one embodiment, the nucleic acid comprises a sense nucleic acid (e.g.,
encoding a
relaxin analog, soluble relaxin receptor, and the like) that is part of a
vector that
expresses the nucleic acid in a suitable host. In particular, such a nucleic
acid has a
promoter operably linked to the coding region (e.g., relaxin or relaxin
receptor) in a
sense orientation, the promoter being inducible or constitutive, and,
optionally, tissue-
specific. In another embodiment, the nucleic acid comprises an antisense
nucleic acid
(e.g., a relaxin antisense nucleic acid or relaxin receptor antisense nucleic
acid) that is
part of a vector that expresses the nucleic acid in a suitable host. In
particular, such a
nucleic acid has a promoter operably linked to the coding region (e.g.,
relaxin or
relaxin receptor) in an antisense orientation, the promoter being inducible or
constitutive, and, optionally, tissue-specific.
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CA 02425712 2010-07-21
In another particular embodiment, a nucleic acid (e.g., a sense nucleic
acid encoding a relaxin analog, soluble relaxin receptor, and the like, or an
antisense
nucleic acid encoding a relaxin antisense nucleic acid, a relaxin receptor
antisense
nucleic acid, and the like) used in which the nucleic acid and any other
desired
sequences are flanked by regions that promote homologous recombination at a
desired
site in the genome, thus providing for intrachromosomal expression of the
nucleic acid
(see, e.g., Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-35 (1989);
Zijlstra
et al., Nature 342:435-38 (1989); U.S. Patent Nos. 5,631,153; 5,627,059;
5,487,992;
and 5,464,764).
For any of these embodiments, delivery of the nucleic acid into a
subject can be either direct, in which case the subject is directly exposed to
the nucleic
acid or nucleic acid-carrying vector, or indirectly, in which case cells are
first
transformed with the nucleic acid in vitro, and then transplanted into the
subject.
These two approaches are known, respectively, as in vivo or ex vivo gene
therapy. In a
specific embodiment, the nucleic acids are directly administered in vivo,
where they
are expressed to produce the encoded product. This can be accomplished by any
of
numerous methods known in the art (e.g., by constructing it as part of an
appropriate
nucleic acid expression vector and administering it so that it becomes
intracellular, for
example, by infection using a defective or attenuated retroviral or other
viral vector
(infra), by direct injection of naked DNA (see, e.g., Asahara et al., Semin.
Interv.
CardioL 1:225-32 (1996); Prazeres etal., Trends BiotechnoL 17:169-74 (1999)),
electroporation (see, e.g., Muramatsu et al., Int. J MoL Med. 1:55-62 (1998)),
or by use of
microparticle bombardment, such as a gene gun (BIOLISTICTm, Dupont); see,
e.g.,
Biewenga et al., J Neurosci. Methods 71:67-75 (1997)). Nucleic acids can also
be
inserted into cells by coating naked nucleic acids with lipids or cell-surface
receptors or
transfection agents, encapsulation in liposomes, derivatized liposomes,
microparticles, or
microcapsules (see, e.g., De Smedt etal., Pharm. Res. 17:113-26 (2000); Maurer
etal.,
MoL Membr. Biol. 16:129-40 (1999); Tarahovsky and Ivanitsky, Biochemistry
63:607-18
(1998); Lasci, Trends BiotechnoL 16:307-21 (1998); Gao and Huang, Gene Ther.
2:710-22
(1995)).
48
CA 02425712 2010-07-21
Nucleic acids can also be administered in linkage to a peptide which is known
to enter the
cell or by administering the nucleic acids in linkage to a ligand subject to
receptor-
mediated endocytosis, which can be used to target cell types specifically
expressing the
receptors, and the like. (See, e.g., Liang et al., Pharmazie 54:559-66 (1999);
Cristiano,
Front. Biosci. 15:D1161-70 (1998); Guy et al., Mol. Biotechnol. 3:237-48
(1995); Wu and
Wu, J. Biol. Chem. 262:4429-32 (1987)). In another embodiment, a nucleic acid-
ligand
complex can be formed in which the ligand comprises a fusogenic viral peptide
to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal degradation. (See,
e.g., Phillips,
Biologicals 23:13-16 (1995)).
In yet another embodiment, the antisense nucleic acid can be targeted in
vivo for cell specific uptake and expression by targeting a specific receptor
(see, e.g.,
Phillips, Biologicals 23:13-16 (1995); International Patent Publications WO
92/06180;
WO 92/22635; WO 92/20316; WO 93/14188, and WO 93/20221).
In a specific embodiment, a viral vector is used that contains the nucleic
acid (e.g., a sense nucleic acid encoding a relaxin analog, soluble relaxin
receptor, and the
like, or an antisense nucleic acid encoding a relaxin antisense nucleic acid,
a relaxin
receptor antisense nucleic acid, and the like). For example, a retroviral
vector can be used
(see, e.g., Palu et al., Rev. Med. Virol. 10:185-202 (2000); Buchschacher and
Wong-Staal,
Blood 15:2499-504 (2000); Miller et al., Meth. Enzymol. 217:581-99 (1993)).
These
retroviral vectors are typically modified to delete retroviral sequences that
are not
necessary for packaging of the viral genome and integration into host cell
DNA. The
antisense nucleic acid to be used in gene therapy is cloned into the vector,
which facilitates
delivery of the antisense nucleic acid into the subject. Lentiviral vectors
can also be used.
(See, e.g., Buchschacher and Wong-Staal, supra; Naldini et al., Science
272:263-67
(1996)). Other references illustrating the use of viral vectors in gene
therapy are by
Lundstrom (J. Recept. Signal. Transduct. Res. 19:673-86 (1999)); Clowes etal.
(J. Clin.
Invest. 93:644-51 (1994)); Kiem etal. (Blood 83:1467-73 (1994)); Salmons and
Gunzberg
49
CA 02425712 2010-07-21
(Hum Gene 77zer. 4:129-41(1993)); and Grossman and Wilson (Curr. Opin. Genet
Dev. 3:110-14 (1993)).
Adenoviruses can also be used in gene therapy. Adenoviruses are
especially attractive vehicles for delivering genes to prostate, liver, the
central nervous
system, endothelial cells, and muscle. Adenoviruses have the advantage of
being capable
of infecting non-dividing cells. Kozarsky and Wilson (Curr. Opin. Genet Dev.
3:499-503
(1993)) present a review of adenovirus-based gene therapy. Herman et al.
(Human Gene
Therapy 10:1239-49 (1999)) describe the intraprostatic injection of a
replication-deficient
adenovirus containing the herpes simplex thymidine kinase gene into human
prostate,
followed by intravenous administration of the prodrug ganciclovir in a phase I
clinical
trial. Other instances of the use of adenoviruses in gene therapy can be found
in Rosenfeld
et al. (Science 252:431-34 (1991)); Rosenfeld et al. (Cell 68:143-55 (1992));
Mastrangeli
et al. (.1 Clin. Invest. 91:225-34 (1993)); and Thompson (Oncol. Res. 11:1-8
(1999)).
Adeno-associated virus (AAV) can also be used in gene therapy (see, e.g.,
Rabinowitz and
Samulski, Curr. Opin, Biotechnol. 9:475-85 (1988); Carter and Samulski, Int. J
Mol. Med.
6:17-27 (2000); Tal, J. Biomed. Sci. 7:279-91 (2000); Ali et al., Gene Therapy
1:367-84
(1994); U.S. Patent Nos. 4,797,368 and 5,139,941; Walsh et al., Proc. Soc.
Exp. Biol.
Med. 204:289-300 (1993); Grimm et al., Human Gene Therapy 10:2445-50 (1999)).
Another approach to gene therapy involves transferring a nucleic acid
to cells in tissue culture by methods such as electroporation, lipofection,
calcium
phosphate mediated transfection, or viral infection. Typically, the method of
transfer
includes the transfer of a selectable marker to the cells. The cells are then
placed
under selection to isolate those cells that have taken up and are expressing
the nucleic
acid. The selected cells are then delivered to a subject.
In one embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be
carried out by any method known in the art, including but not limited to
transfection,
electroporation, microinjection, infection with a viral or bacteriophage
vector
containing the nucleic acid, cell fusion, chromosome-mediated gene transfer,
CA 02425712 2010-07-21
microcell-mediated gene transfer, and the like. Numerous techniques are known
in the
art for the introduction of foreign genes into cells (see, e.g., Muramatsu et
al., Int. J.
MoL Med. 1:55-62 (1998); Liang et al., Pharmazie 54:559-66 (1999); Loeffler
and
Behr, Meth. Enzymol. 217:599-618 (1993); Cotten et al., Meth. EnzymoL 217:618-
44
(1993); Cline, Pharmacol. Ther. 29:69-92 (1985)) and can be used in accordance
with the
present invention. The technique typically provides for the stable transfer of
the nucleic
acids to the cell, so that the nucleic acids are expressible by the cell and
is heritable and
expressible by its cell progeny.
The resulting recombinant cells can be delivered to a subject by various
methods known in the art. Typically, cells are injected subcutaneously. In
another
embodiment, recombinant skin cells can be applied as a skin graft onto the
subject.
The amount of cells required for use depends on the desired effect, the
subject's
condition, and the like, and can be determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene
therapy encompass any desired, available cell type, and include but are not
limited to
cells or populations of cells of the male reproductive tract (e.g., prostate
cells, cells of
the testes, seminiferous cells, or epididymal cells), female reproductive
tract (e.g.,
uterus, cervix, the interpubic ligament, connective tissues within the pelvic
girdle, and
the like), liver, kidney, spleen, thymus, brain, heart, intestine, skin, lung,
and the like.
Suitable cells further include epithelial cells, endothelial cells,
keratinocytes,
fibroblasts, muscle cells, and stem or progenitor cells. The cells used for
gene therapy
generally are autologous to the subject, but heterologous cells that can be
typed for
compatibility with the subject can be used.
In another aspect, nucleic acids (e.g., a sense nucleic acid encoding a
relaxin analog, soluble relaxin receptor, and the like, or an antisense
nucleic acid
encoding a relaxin antisense nucleic acid, a relaxin receptor antisense
nucleic acid, and
the like) are administered directly to cells. The nucleic acids are at least
six
nucleotides and are typically oligonucleotides (ranging from 6 to about 50
nucleotides
or more). In specific aspects, the oligonucleotide is at least 10 nucleotides,
at least 15
nucleotides, at least 100 nucleotides, or can be at least 200 nucleotides. The
oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or
analogs
51
CA 02425712 2010-07-21
=
thereof, and can be single-stranded or double-stranded. The oligonucleotide
can be
modified at the base moiety, sugar moiety, or phosphate backbone. The
oligonucleotide can include other appending groups such as peptides, or agents
facilitating transport across the cell membrane (see, e.g., Nielsen,
Pharmacol. Toxicol.
86:3-7 (2000); Soomets et al., Front. Biosci. 1:D782-86 (1999); Galderisi et
al., J. Cell
Physiol. 181:251-57 (1999); Letsinger etal., Proc. Natl. Acad. Sci. USA
86:6553-56
(1989); Lemaitre etal., Proc. Natl. Acad. Sci. USA 84:648-52 (1987);
International
Patent Publication WO 88/09810), hybridization-triggered cleavage agents (see,
e.g.,
Krol et al., BioTechilignes 6:958-76 (1988)) or intercalating agents (see,
e.g., Zon,
Phann. Res. 5:539-49 (1988)).
In one embodiment, antisense oligonucleotides are provided as single-
stranded DNA. The oligonucleotides can be modified at any position on its
structure
with substituents generally known in the art. The oligonucleotides can
comprise at
least one modified base moiety, such as, for example, 5-fluorouracil, 5-
bromouracil, 5-
chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-
(carboxy-
hydroxylmethyl) uracil, 5-carboxymethylaminomethyl -2-thiouridine, 5-
carboxymethylamino-methyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-
methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine, 7-
methylguanine, 5-methylaminomethyluracil, 5-methoxyamin.omethy1-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-
methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v) , pseudouracil,
queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid
(v), 5-
methy1-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, 2,6-
diaminopurine, and
the like. In another embodiment, the oligonucleotides comprise at least one
modified
sugar moiety, such as, for example, arabinose, 2-fluoroarabinose, xylulose,
and
hexose.
In yet another embodiment, the antisense oligonucleotides comprise at
least one modified phosphate backbone, such as, for example, a
phosphorothioate, a
phosphorodithioate, a phosphormidothioate, a phosphoramidate, a
52
CA 02425712 2010-07-21
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a
formacetal
or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an a-
anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-
stranded hybrids with complementary RNA in which, contrary to the usual 13-
units, the
strands run parallel to each other (see Gautier et al., NucL Acids Res.
15:6625-41
(1987)). The oligonucleotide can be conjugated to another molecule (e.g., a
peptide,
hybridization triggered cross-linking agent, transport agent, hybridization-
triggered
cleavage agent, and the like).
In a specific embodiment, the antisense oligonucleotide comprises
catalytic RNA, or a ribozyme (see, e.g., Welch etal., Curr. Opin. Biotechnol.
9:486-96
(1998); Norris et al., Adv. Exp Med. Biol. 465:293-301 (2000); International
Patent
Publication WO 90/11364; Sarver etal., Science 247:1222-25 (1990)). In another
embodiment, the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al.,
Nucl.
Acids Res. 15:6131-48 (1987)), or a chimeric RNA-DNA analogue (Inoue et al.,
FEBS
Lett. 215:327-30 (1987)).
In another specific embodiment, double-stranded RNA directs the
sequence-specific degradation of mRNA by RNA interference. (See generally
Hunter,
Curr. Biol. 10:R137-40 (2000); Bosher and Labouesse, Nat. Cell. Biol. 2:e31-36
(2000); the disclosures of which are incorporated by reference herein.)
Briefly,
double-stranded nucleic acids are introduced into a cell to selectively
inhibit gene
expression by causing degradation of the mRNA. (See, e.g., Zamore et al., Cell
101:25-33 (2000)).
Nucleic acids according to the present invention can be synthesized by
standard methods known in the art. Enzymatic methods for the synthesis of
nucleic
acids frequently employ Klenow, T7, T4, Taq or Escherichia coli DNA
polymerases,
as described in Sambrook et al. (supra). Enzymatic methods of RNA nucleic
acids
frequently employ SP6, T3 or T7 RNA polymerases, as described in Sambrook et
al.
(supra). Reverse transcriptase can also be used to synthesize DNA from RNA
(Sambrook et al., supra). Nucleic acids are typically prepared enzymatically
using a
template nucleic acid which can either be synthesized chemically, or be
obtained as
mRNA, genomic DNA, cloned genomic DNA, cloned cDNA or other nucleic acid.
53
CA 02425712 2010-07-21
=
Some enzymatic methods of DNA nucleic acid synthesis can require an additional
primer which can be synthesiied chemically. Finally linear nucleic acids can
be
prepared by polymerase chain reaction (PCR) techniques as described, for
example, by
Saiki et al. (Science 239:487 (1988)).
Chemical methods can also be used to synthesize nucleic acids (e.g.,
antisense oligonucleotides), such as by use of a commercially available
automated
DNA synthesizer). As examples, phosphorothioate nucleic acids can be
synthesized
by the method of Stein etal. (Nucl. Acids Res. 16:3209-21 (1988)),
methylphosphonate nucleic acids can be prepared by use of controlled pore
glass
polymer supports (see, e.g., Sarin etal., Proc. Natl. Acad. Sci. USA 85:7448-
51
(1988)), and the like. Other methods include those disclosed by Usman etal.
(J. Am.
Chem. Soc. 109:7845-54 (1987)), Scaringe etal. (Nucleic Acids Res. 18:5433-41
(1990));
Caruthers (Oligonucleotides: Antisense Inhibitors of Gene Expression, pp. 7-
24, Cohen,
(ed.), CRC Press, Inc. Boca Raton, Fla., 1989)); Oligonucleotide Synthesis, A
Practical
Approach (Gait (ed.), IRL Press, 1984); Oligonucleotides and Analogues, A
Practical
Approach (Eckstein, IRL Press, 1991); and in U.S. Patent Nos. 4,415,732;
4,458,066;
4,500,707; 4,668,777; 4,973,679; 5,026,838; 5,132,418; and Re. 34,069.
Small Molecule Effectors or Antagonists
Relaxin nucleic acids, polypeptides, analogs and fragments, and relaxin
receptor nucleic acids, polypeptides, analogs and fragments and analogs, also
have
uses in screening assays to detect candidate compounds that specifically bind
to
relaxin polypeptides, or to relaxin receptor, and modulate apoptosis. Such
candidate
compounds are typically small molecule effectors (agonists) or antagonists,
and can be
identified by in vitro and/or in vivo assays. Such assays can be used to
identify small
molecule effectors or antagonists that are therapeutically effective as
relaxin agonists
or antagonists or as lead compounds for drug development. The invention thus
provides assays to detect compounds that specifically affect the activity or
expression
of relaxin nucleic acids, relaxin polypeptides, relaxin receptor nucleic
acids, relaxin
receptor, and the like.
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In a typical in vivo assay, recombinant cells expressing relaxin or
relaxin receptor nucleic acids can be used to screen candidate compounds for
those
that affect relaxin or relaxin receptor nucleic acid expression. Agonistic or
antagonistic effects on relaxin or relaxin receptor expression can include
stimulation or
inhibition (e.g., up or down regulation) of transcription of mRNA, a increase
or
decrease in mRNA stability, translation of the mRNA, synthesis of relaxin or
relaxin
receptor polypeptides, relaxin or relaxin receptor polypeptide function (e.g.,
binding to
relaxin receptor), and/or effects on relaxin or relaxin receptor polypeptide
stability or
localization. Such effects on expression can be identified as physiological
changes,
such as, for example, changes in relaxin-responsive tissue growth rate,
division,
viability, collagen deposition, apoptosis, and the like. In one embodiment,
candidate
compounds are administered to recombinant cells expressing a relaxin or
relaxin
receptor polypeptide to identify those compounds that produce a physiological
change
(e.g., stimulate or inhibit relaxin or relaxin receptor polypeptide function).
Candidate compounds can also be identified by in vitro screening
methods. For example, recombinant cells expressing a relaxin or a relaxin
receptor
nucleic acid can be used to recombinantly produce relaxin or relaxin receptor
polypeptide for in vitro assays to identify candidate compounds that bind to
relaxin or
relaxin receptor polypeptide. Candidate compounds (such as small molecules)
are
contacted with the polypeptide (or a fragment or analog thereof) under
conditions
conducive to binding, and then candidate compounds that specifically bind to
the
polypeptide are identified. Methods that can be used to carry out the
foregoing are
commonly known in the art, and include diversity libraries, such as random or
combinatorial peptide or non-peptide libraries that can be screened for
candidate
compounds that specifically bind to relaxin or relaxin receptor polypeptide.
Many
libraries are known in the art that can be used, for example, include
chemically
synthesized libraries, recombinant phage display libraries, and in vitro
translation-
based libraries.
Examples of chemically synthesized libraries are described in Fodor et
al. (Science 251:767-73 (1991)), Houghten et al. (Nature 354:84-86 (1991)),
Lam et
al. (Nature 354:82-84 (1991)), Medynski (BioTechnology 12:709-10 (1994)),
Gallop
et al. (J. Med. Chenz. 37(9):1233-51 (1994)), Ohlmeyer et al. (Proc. Natl.
Acad. Sci.
CA 02425712 2010-07-21
USA 90:10922-26 (1993)), Erb et al. (Proc. Natl. Acad. Sci. USA 91:11422-26
(1994)),
Houghten et al. (Biorechniques 13:412-21(1992)), Jayawickreme et al. (Proc.
Natl.
Acad. Sci. USA 91:1614-18 (1994)), Salmon et al. (Proc. Natl. Acad. Sci. USA
90:11708-12 (1993)), International Patent Publication WO 93/20242, and Brenner
and
Lerner (Proc. Nall. Acad. Sci. USA 89:5381-83 (1992)).
Examples of phage display libraries are described in Scott and Smith
(Science 249:386-90 (1990)), Devlin et al. (Science 249:404-06 (1990)),
Christian et
al. (J. MoL Biol. 227:711-18 (1992)), Lenstra (J. 1772111147201. Meth. 152:149-
57 (1992)),
Kay et al. (Gene 128:59-65 (1993)), and International Patent Publication WO
94/18318.
In vitro translation-based libraries include, but are not limited to, those
described in International Patent Publication WO 91/05058, and Mattheakis et
al.
(Proc. NatL Acad. Sci. USA 91:9022-26 (1994)). By way of examples of
nonpeptide
libraries, a benzodiazepine library (see, e.g., Bunin et al., Proc. Natl.
Acad. ScL USA
91:4708-12 (1994)) can be adapted for use. Peptide libraries (see, e.g., Simon
etal.,
Proc. Natl. Acad. Sci. USA 89:9367-71(1992)) can also be used. Another example
of a
library that can be used, in which the amide functionalities in peptides have
been
permethylated to generate a chemically transformed combinatorial library, is
described
by Ostresh et al. (Proc. Natl. Acad. Sci. USA 91:11138-42 (1994)).
Screening the libraries can be accomplished by any of a variety of
commonly known methods. See, for example, the following references, which
disclose screening of peptide libraries: Parmley and Smith (Adv. Exp. Med.
Biol.
251:215-18 (1989)); Scott and Smith (1990, supra); Fowlkes etal.
(BioTechniques
1.3:422-28 (1992)); Oldenburg etal. (Proc. Natl. Acad. Sci. USA 89:5393-97
(1992));
Yu et al. (Cell 76:933-45 (1994)); Staudt et al. (Science 241:577-80 (1988));
Bock et
al. (Nature 355:564-66 (1992)); Tuerk et al. (Proc. NatL Acad. Sci. USA
89:6988-92
(1992)); Ellington et al. (Nature 355:850-52 (1992)); U.S. Patent Nos.
5,096,815,
5,223,409, and 5,198,346; Rebar and Pabo (Science 263:671-73 (1994)); and
International Patent Publication WO 94/18318.
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In a specific embodiment, screening can be carried out by contacting
the library members with relaxin, a relaxin analog, or a relaxin receptor
immobilized
on a solid phase and harvesting those library members that bind to the
relaxin, relaxin
analog or relaxin analog. Examples of such screening methods, termed "panning"
techniques are described by way of example in Parmley and Smith (Gene 73:305-
18
(1988)); Fowlkes et al. (1992, supra); International Patent Publication WO
94/18318;
and in references cited herein.
Identing a Subject with a Relaxin-Associated Abnormality
Relaxin nucleic acids (both sense and antisense), and fragments and
analogs thereof, and anti-relaxin antibodies, also have utility to identify
subjects with a
relaxin-associated abnormality. Such molecules can be used in assays, such as
hybridization or immunoassays, to detect, prognose, diagnose, or monitor
various
abnormalities, to determine whether relaxin expression, or the response to
relaxin or a
relaxin analog is affected. Similarly, such molecules have utility to monitor
the
treatment of the cell or tissue abnormalities. In particular, methods, such as
an
immunoassay, can be carried out by steps comprising contacting a sample
derived
from a subject with an anti-relaxin antibody under conditions conducive to
immunospecific binding, and detecting or measuring the amount of any
immunospecific binding of protein by the antibody. Binding of antibody to
relaxin or
relaxin receptor polypeptide, in tissue sections or from seminal fluid, can be
used to
detect aberrant (e.g., low, absent or elevated) levels of relaxin and/or
relaxin receptor
polypeptide. In a specific embodiment, antibody to relaxin or relaxin receptor
polypeptide can be used to assay a subject's tissue or seminal fluid for the
presence of
relaxin or relaxin receptor polypeptide, where an aberrant level of relaxin is
an
indication of a relaxin-associated abnormality. By "aberrant levels" is meant
increased
or decreased levels relative to that present, or to a standard level
representing that
present, in an analogous sample from a portion of the body or from a subject
not
having the abnormality.
The immunoassays which can be used include, but are not limited to,
competitive and non-competitive assay systems using techniques such as Western
blot,
radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich"
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immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion
precipitin
reaction, immunodiffusion assay, agglutination assay, complement-fixation
assay,
immunoradiometric assay, fluorescent immunoassay, protein A immunoassay, and
the
like.
Relaxin and relaxin receptor nucleic acids (both sense and antisense),
including fragments and analogs thereof, can also be used in hybridization
assays.
Such nucleic acids, comprising or consisting of at least contiguous 8
nucleotides, can
be used as hybridization probes or for polymerase chain reaction detection.
Hybridization assays can be used to detect, prognose, diagnose, or monitor
diseases or
conditions associated with aberrant relaxin or relaxin receptor expression
and/or
activity, as described supra. In particular, a hybridization assay can be
carried out by a
method comprising contacting a sample containing polynucleotides with a
nucleic acid
probe capable of hybridizing to relaxin or relaxin receptor DNA or RNA, under
conditions such that hybridization can occur, and detecting or measuring any
resulting
hybridization.
In a specific embodiment, abnormalities associated with over- or under-
expression of relaxin can be diagnosed, or their suspected presence can be
screened
for, or a predisposition to develop such abnormalities can be identified by
detecting
decreased or increased levels of relaxin polypeptide, relaxin RNA, or relaxin
functional activity. Additionally, over-expression of relaxin or increased
relaxin
functional activity can be diagnosed by detecting mutations in relaxin RNA or
DNA or
relaxin polypeptide (e.g., translocations in relaxin nucleic acids,
truncations of the
relaxin gene or relaxin polypeptide, changes in the nucleotide or amino acid
sequence
relative to wild-type relaxin, respectively) that cause increased expression
or activity
of relaxin polypeptide.
By way of example, levels of relaxin polypeptide in a biopsy or from
seminal fluid can be detected by immunoassay of tissues; levels of relaxin RNA
can be
detected by hybridization assays (e.g., Northern blot or dot blot).
Translocations and
point mutations in relaxin or relaxin receptor nucleic acids can be detected
by Southern
blot, RFLP analysis, PCR using primers that detect point mutations, deletions
or
insertions, sequencing of the relaxin genomic DNA or cDNA obtained from the
sample, and the like.
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In another embodiment, levels of relaxin or relaxin receptor mRNA or
polypeptide in a sample of tissue isolated from a subject are detected or
measured, in
which increased levels indicate that the subject has, or has a predisposition
to a
relaxin-associated tissue abnormality.
In yet another embodiment, abnormal relaxin receptor activity in a
tissue is detected or measured using any of the functional assays described
above.
Abnormal relaxin receptor activity can include, for example, an increased or
decreased
number of a relaxin receptors on relaxin-responsive cells, the presence of
relaxin
receptors on cells that are not normally responsive to relaxin, increased or
decreased
relaxin receptor response time, an increased or decreased binding affinity for
relaxin or
relaxin receptor, or a change in the dissociation constant of relaxin from a
relaxin
receptor, and the like.
Kits for diagnostic and/or prognostic use are also provided that
comprise, in one or more containers, a relaxin agonist or antagonist and,
optionally, a
labeled binding partner to an antibody. Alternatively, an antibody can be
labeled with
a detectable marker (e.g., a chemiluminescent, enzymatic, fluorescent, a
radioactive
moiety, and the like). A kit is also provided that comprises, in one or more
containers,
a nucleic acid probe capable of hybridizing to relaxin or relaxin receptor
raRNA or
DNA.
In another embodiment, the kit can comprise in one or more containers
a pair of primers (e.g., each in the size range of 6-30 nucleotides or more)
that are
capable of priming amplification (e.g., by polymerase chain reaction (see,
e.g., Innis et
aL, PCR Protocols, Academic Press, Inc., San Diego, CA (1989)), ligase chain
reaction (see, e.g., EP 0 320 308), use of Q13 replicase, cyclic 5' probe
reaction, or
other methods known in the art) under appropriate reaction conditions such
that at least
a portion of a relaxin nucleic acid is amplified. A kit can optionally further
comprise
in a conthiner a predetermined amount of a purified relaxin or relaxin
receptor nucleic
acid, for example, for use as a standard or control.
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EXAMPLE 1
The effect of inactivation of one or both alleles of the mouse RLX genes
was examined. The following methods and materials were followed.
Animals:
Wild-type, heterozygous and homozygous relaxin knockout mice were
obtained from the Howard Florey Institute of Experimental Physiology and
Medicine
(Parkville, Victoria, 3052, Australia) and used to establish a breeding
program.
Subsequent generations of RLX +1+ (wild-type), RLX +/- (heterozygous) and rlx -
/-
(null mutant) mice were generated from RLX +/- parents. All animals were
housed in
a controlled environment and maintained on a 14 hours light, 10 hours dark
schedule
with access to Labdiet rodent lab chow (Deans Animal Feed, San Bruno, CA) and
water. These experiments were approved by the Institute's Animal Experimental
Ethics Committee, which adheres to the NIH Code of Practice for the care and
use of
laboratory animals.
Gencnyping by PCR
Mouse DNA was isolated by lysing tail tissue (5-7 millimeters) in 400
jtl of PCR lysis buffer, containing 50 mM Tris-HC1, pH 8.0, 0.5% SDS, 0.1 M
EDTA
and 1 mg/ml proteinase K (Gibco BRL, Gaithersburg, MD) at 50-55 C overnight.
Digested samples were then mixed with 3M sodium acetate (40 1), buffer
saturated
phenol (200 1) and chloroform (200 1) in serum vaccutainer tubes, before
samples
were centrifuged at 3000 rpm for 10 minutes. The DNA (contained in the upper
aqueous phase) was decanted into separate microcentrifuge tubes containing
isopropyl
alcohol (240 pil) to precipitate the DNA before samples were vortexed, spun
and the
supernatant discarded. The remaining DNA pellet was dissolved in 40-50111 of
sterile
water. For PCR, each DNA template (1111) was used in a 30 1 reaction mixture
containing PCR buffer (10 mM Tris-HC1, pH 8.3, 50 mM KC1, 1.5 mM MgCl2), 2.5
mM dNTPs, 2.5 U Taq Polymerase (PGC Scientific, Gaithersburg, MD) and 150 ng
of
each of the RLX +/+ and rlx -/- primers, designed by Zhao and colleagues (Zhao
CA 02425712 2010-07-21
Endocrinology 140:445-53 (1999)). The amplification protocol consisted of an
initial
denaturation step at 94 C (3 minutes) followed by 35 sequential cycles of 94 C
(60
seconds), 55 C (60 seconds) and 72 C (90 seconds) and concluded by an
additional 10
minute extension at 72 C. 15 p.1 of each sample were then analyzed by
electrophoresis
on a 2% (w/v) agarose gel, and stained with ethidium bromide. A 235 bp product
was
generated by primers designed from the wild-type allele, while a 170 bp
product was
generated from the mutant allele primers (Zhao (1999), supra).
Tissue Collection and Histology
Relaxin wildtype, heterozygous and homozygous males and females (n
> 20 in each of the 6 groups) were obtained at 1 week, 1 month, 2 months and 3
months of age and weighed. RLX +/+ and rlx -/- male mice were then sacrificed
under
anesthesia with carbon dioxide for tissue collection. The male reproductive
tract
(including testis, epididymis, prostate, seminal vesicle and attached fat)
were collected
from each animal at 1 week of age (n=10 RLX +/+ males, n=11 -/- males); 1
month of
age (n=10 RLX +/+ males, n=10 -1- males); and 3 months of age (n=10 RLX +/+
males, n=10 rlx -/- males. After weighing each tissue, they were placed in 10%
formalin for histological analysis.
The collected tissues were processed (sequentially dehydrated) from
70% alcohol to paraffin before being embedded and cut (4 gm sections), using
an AO
Spencer 820 microtome and placed on poly-L-lysine coated glass slides.
Consecutive
sections from each tissue were stained with H & E (hematoxylin and eosin) and
for
collagen, with Masson trichrome (staining kit, Richard-Allan Scientific,
Kalamazoo,
MI) as described by the manufacturer. The stained slides were viewed using a
Zeiss
TM
Axioplan-2 microscope, the images captured by digital camera (Hamamatsu) and
stored for retrieval and analysis. The images were digitally enhanced for
maximum
contrast and brightness using Adobe Photosh; (Adobe Systems Inc, Mountain
View,
CA).
Antibody staining of paraffin-embedded tissue sections
The tissues from the reproductive tract of one and three month old male
mice were mounted on precoated slides and deparaffinized by heating at 58 C
(about
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30 minutes), then washed three times in xylene, twice in absolute ethanol and
twice in
95% ethanol, before being briefly soaked in water. The samples were then
stained
using an Immunocruz staining system utilizing a horseradish peroxidase (HRP)-
streptavidin complex (Santa Cruz Biotechnology Inc, Santa Cruz, CA) in a
humidified
atmosphere. Tissue sections were initially treated with a peroxidase blocker
(to
quench endogenous peroxidase activity) (5 minutes) before being preblocked in
goat
serum (20 minutes). Serial sections from each RLX +/+ and rlx -/- tissue
sample were
then incubated with either a Bax monoclonal IgG primary antibody (4
g/m1)(Santa
Cruz Biotechnology Inc), a caspase-9 polyclonal IgG antibody (4.5 1.tg/m1)
(Santa Cruz
Biotech.) or a proliferating cell nuclear antigen (PCNA)(Santa Cruz Biotech.)
monoclonal IgG antibody (4 g/ml) (2 hours). Depending on the type of antibody
used, either a mouse IgG or rabbit IgG control (Santa Cruz Biotech.) stain of
tissues (2
hours) was also used in all experiments performed. Samples were washed in PBS
(2
minutes), subjected to the appropriate secondary antibody (goat anti-mouse IgG
or
goat anti-rabbit IgG) (30 minutes), washed as above (2 minutes), treated with
a HRP-
streptavidin complex (30-45 minutes) and incubated with a diaminobenzidine
chromagen substrate (2-10 minutes) that was prepared in accordance with the
manufacturer's instructions. The slides were then washed in distilled water (2
minutes) before being dehydrated from 95% alcohol to xylene and mounted, then
photographed as described above.
Statistical analysis:
The results were analyzed using a one-ANOVA test. All data in this
paper are presented as the mean SEM, with p <0.05 considered statistically
significant.
EXAMPLE 2
The effects of relaxin gene knockout on the growth of mice:
The body weights of male and female relaxin wildtype (+1+),
heterozygous (+/-) and null mutant (-/-) mice were measured at 1 week, 1
month, 2
months and 3 months of age (n=20-21 for each group). No significant
differences in
mean body weight were noted in male (RLX +/+: 3.870.09g; rlx -/-: 3.6 0.13g)
or
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female (RLX +/+: 3.52 0.09g; rlx -/-: 3.37 0.08g) mice at 1 week of age.
However, at
the time the mice were 1 month of age, mean body weights of both rlx -/- males
(17.05 0.65g) and females (14.77 0.42g) were significantly less (p<0.05) than
their
respective RLX wildtype counterparts (RLX +1+ M: 18.92 0.61g; RLX +1+ F:
16.34 0.51g). Male and female null mutant mice continued to be significantly
smaller
(p<0.05) than RLX wildtype animals at 2 months of age (RLX +1+ M: 25.42 0.41g;
rlx -/- M: 24.23 0.42g; RLX +/+ F: 20.96 0.32g; rlx -/- F: 19.940.22g);
however, the
differences in size between the two groups were less than that observed at 1
month of
age. By adulthood (3 months of age), the mean weight of rlx -/- mice (rlx -/-
M:
26.570.47g; rlx -/- F: 22.54 0.31g) was still slightly less than the average
weight of
adult RLX +1+ mice (RLX +/+ M: 27.30 0.32g; RLX +/+ F: 23.03 0.71g), but this
difference was no longer significant. The mean weight of the RLX +/- mice was
between that of the average weight of RLX +/+ and rlx -/- mice. No significant
differences were observed between the average weight of RLX +1+ and RLX +/-
animals, or between RLX +/- and rlx -/- mice.
The effects of relaxin gene knockout on the size of the male reproductive
tract:
No significant difference was observed in the weight or size of the male
reproductive tract at 1 week of age, which was represented primarily by the
testis
(Table 1). By 1 month of age the collected male genital tract was composed of
the
testis, epididymis, prostate, seminal vesicle, ductus deference and attached
fat.
Although no difference in the overall weight of the reproductive tract was
observed at
one month of age, differences in size of the testis and prostate, derived from
1 month
null mutant mice, were approximately 20% and 30% smaller, respectively, than
the
corresponding tissues of RLX wildtype animals, while the size of the
epididymis,
derived from rlx -/- mice was significantly (p<0.05) smaller than that
obtained from
RLX +/+ animals. No differences were noted, however, in the size of the
seminal
vesicle between tissue samples collected from RLX +/+ and rlx -/- mice.
By the time the mice had reached adulthood (3 months of age), a
significant (p<0.05) difference in the overall weight of the male reproductive
tract as
well as in the size of individual organs was observed (Table 1). The weight of
the
reproductive tract in normal mice increased 248.6% from 1 month to adulthood
and
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represented 3.37% of the total body weight. During this time, the weight of
the
reproductive tract from male RLX null mutant mice only increased by 187.9% and
represented 2.33% of the total body. This latter finding implied that the
reproductive
tract of male rlx -/- mice represented a 31% decrease in % body weight. The
size of
the testis, epididymis, prostate and seminal vesicle from rlx -/- mice were
all smaller
(p<0.05) than their respective counterparts, derived from RLX +/+ mice.
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Table 1: The weight and size of the male reproductive tract from 1
week of age to adulthood (6.5 months).
1 Week of Age:
RLX +1+ % of Body rlx -/- % of Body
[Mean SE(n)1 Weight [Mean 1 SE(n)]
Weight
Weight (g):
Overall 0.0171 0.001(10) 0.43 0.0181
0.002(15) 0.53
Size(Area; mm2):
Testis 1.30 1 0.30 (5)1 -- 1.41 .1 0.23 (5)1
--
1 Month of Age:
RLX +/+ % of Body rlx -/- % of Body
[Mean 1 SE(n)] Weight [Mean 1 SE(n)]
Weight
Weight (g):
Overall 0.37 0.02(11) 1.81 0.33 0.01 (11)
1.77
Size (Area;mm2):
Testis 16.36 1 1.58 (9)1 - 12.91 0.96 (8)1
-
Epididymis 10.731 2.22 (7) 2 - 4.62 1
0.64 (7)2* -
Prostate 9.20 1.20 (4)3 - 6.30 10.54 (4)3
-
Seminal Vesicle 1.50 0.25 (4)4 - 1.69 1 0.22 (4)4
-
3 Months of Age:
RLX +/-k % of Body rlx -/- % of Body
[Mean SE(n)] Weight [Mean 1 SE(n)]
Weight
Weight (g):
Overall 0.92 1 0.06(14) 3.37 0.62 1 0.03 (11)*
2.33
Size (Area;mm2):
Testis 27.21 11.52 (12)1 - 17.31 12.28 (10)1
-
Epididymis 23.621 3.19 (11)2 -
13.7112.74(10)2* -
Prostate 20.89 1 1.20 (6)3 - 15.03 11.60 (6)3*
-
Seminal Vesicle 25.75 1 3.58 (7)4 - 14.6 1 2.88 (5)4*
-
The size of each 'testis and 3prostate was measured by its area, multiplying
the tissue
length by width. The size (area) of the epididymis and seminal vesicle could
not be
measured accurately, so a close approximation of each tissue was calculated as
follows: epididymis (by measuring the length of the tissue by the width of the
epididymis head2); seminal vesicle (by measuring the length of each organ by
the
average width of the tissue4). * denotes p<0.05.
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The effects of relaxin gene knockout on histology of the male reproductive
tract:
H&E & Masson Trichrome Staining: Male reproductive tissue sections
from RLX +/+ and rlx -/- mice, were first observed for differences in sperm
maturation, tubule size/compactness (testis, epididymis) and collagen.
Testis: At 1 week of age, the seminiferous tubules (the compartments
containing germ cells/spermatocytes, Sertoli cells and where sperm maturation
occurs)
of relaxin wildtype animals were smaller, more cylindrical in shape (Table 2)
and
mainly supported by a thin layer of collagen surrounding the tunica albuginea
(the
membrane that covers the oval body) of each organ. In comparison, tubules
derived
from rlx -/- mouse testes were much larger and elongated, but were completely
surrounded and supported by collagen within the testis. This immediately
suggested a
difference in the internal organization of the testis in the absence of
relaxin, even just
after birth. No immature sperm though were detected in either group of tissue
sections
at one week of age. By one month of age, testis tubules derived from RLX +/+
mice
were larger in size (compared to 1 week tubule size), were slightly less
compact and
contained mainly immature sperm. In comparison, the tubules derived from 1
month
old RLX homozygous mice were no different to tubule sizes measured from 1 week
old knockout mice and contained less immature sperm compared to samples
derived
from RLX +/+ mice. This further suggested that relaxin null mutant mice were
undergoing a process of delayed sperm maturation which was further confirmed
when
comparing three month tissue sections; sections derived from RLX +/+ mouse
tissues
contained larger tubules (compared to 1 month tubule size) with mainly mature
sperm.
Conversely, sections derived from rlx -/- mice contained slightly smaller
tubules
(which were indifferent to tubule sizes derived from 1 week/1 month rlx -/-
mice) with
less mature sperm (compared to 3 month RLX +/+ tissue sections) and still some
immature sperm. The level of sperm maturity in testes derived from RLX +/+
mice
was shown to be significantly (p<0.05) greater than the level of sperm
maturation
observed in the testes of rlx -/- mice (see Table 2 for grading scale used).
It was also
noted that the level of sperm maturation in adult RLX knockout mouse testes
resembled that observed in immature (1 month) RLX wildtype mouse tissues. From
1-
3 months of age, the testis of normal mice continued to be surrounded by a
thin layer
of collagen, covering the tunica albuginea; however, in some tissues a
scattered thin
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lining of collagen was also observed to support the seminiferous tubules. The
internal
layering of collagen though, was found to decrease with age. In rlx -/- mice,
the
collagen detected in-between tubular structures had regressed due to the
larger tubular
sized structures within the testis, but was still more dense and consistent
compared to
that observed in tissue samples derived from wildtype animals. It is
postulated that the
increased collagen observed in the reproductive tract of RLX null mutant mice
at a
very young age may cause tissues to be more rigid and firm, which may be
linked to
the changes in tubular composition and sperm maturation that are discussed
above.
Interestingly, the presence of relaxin in normal adult mice also induced a
loosening
(increased interstitial spacing) of the seminiferous testis tubular structures
compared to
that observed from normal immature (1 month) mice. In comparison, there was no
difference in tubule organization (size/compactness) from testes derived from
1 month
and 3 month rlx -/- mice. Additionally, 1 month, and to an extent 3 month,
testis
tubules derived from rlx -/- mice appeared to contain an increased number of
dead
cells, compared to tubular cells derived from RLX wildtype animals. The
increased
number of dead cells seen in immature tissues were variable between animals
but were
consistently detected as the mice matured with age.
Epididymis: No significant differences in tubule size were noted in the
epididymis of RLX +/+ and rlx -/- reproductive tracts at 1 month and 3 months
of age.
By adulthood however, tubule compactness was more evident in tissues derived
from
RLX homozygous mice as compared to tissues derived from RLX wildtype animals.
The epididymis tubules of rlx -/- mice were also supported to a greater extent
by
connective tissue. Tubular structures derived from 3 month RLX +/+ mouse
tissues
were loose and only partially maintained by collagen. Conversely, tissue
sections
obtained from RLX knockout animals were shown to have more compact tubules
that
were fairly well enclosed by thin layers of collagen. As in the case of the
testis, tissues
obtained from rlx -/- mice were upheld by an increased concentration of
collagen, at all
ages observed, compared to tissue samples derived from RLX +/+ mice. As shown
in
Table 2, it was also noted that the density of mature sperm (in epididymis
tubules) was
less in tissue sections derived from RLX null mutant mice at 1 month and 3
months of
age, compared to that obtained from RLX wildtype mice, at each respective age
group.
This difference, however, was statistically insignificant. This decrease in
mature
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sperm in the epididymis of mice lacking relaxin was most likely attributed to
the
delayed sperm maturation process that took place in the testis of these
animals (Table
2).
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Table 2: The effects of relaxin gene knockout on tissue compartment
and sperm maturation, during murine growth and development
Age RLX +/+ rlx
Tissue [months] [Mean SE (n)] [Mean SE (n)]
Testis:
Tubule size' [0.23] 2.20 0.32 (5) 2.60 0.20
(5)
[1] 2.92 1 0.17 (9) 2.70 0.13
(8)
[3] 3.64 0.08 (9) 2.86 0.18
(10)*
[6.5] 3.17 1 0.17 (3) 3.08 0.08
(3)
Tubule compactnessb [0.23] 3.90 0.10 (5) 3.45 0.20
(5)
[1] 3.56 0.15 (9) 2.66 0.32
(8)
[3] 1.75 0.28 (9) 2.60 0.17
(8)*
[6.5] 2.33 0.08 (3) 3.66 0.09
(4)*
Sperm maturationc [0.23] 0 (5) 0 (5)
[1] 1.78 0.36 (9) 0.94 0.20
(8)
[3] 3.78 1 0.15 (9) 2.20 0.29
(10)*
[6.5] 3.75 (3) 3.56 0.12 (4)
Epididymis:
Tubule sizea [1] 2.48 0.24 (7) 2.74 0.36
(7)
[3] 2.47 0.22 (8) 2.92 0.23
(7)
[6.5] 2.67 0.17 (3) 2.88 1 0.13
Tubule compactnesSb [1] 3.16 1 0.28 (7) 3.01 0.25
(7)
[3] 2.68 0.28 (8) 3.24 0.28
(7)
[6.5] 2.33 0.44 (3) 3.00 0.18
(4)
Sperm maturation' [1] 0.94 0.06 (8) 0.75 1 0.10
(8)
[3] 3.56 1 0.13 (9) 3.05 0.17
(10)
[6.5] 3.83 0.08 (3) 3.63 I 0.07
(4)
Table 2: Tissue sections were stained with H & E and graded as follows:
aTubule size - 1 = all tubules small and circular in shape; 2 = most tubules
slightly
elongated compared to 1 but some smaller circular tubules are observed; 3 =
tubules
grossly elongated compared to 1, but some smaller circular and mid-sized
tubules are
observed; 4 = all tubules grossly elongated compared to 1 and 2.
bTubule compactness - 1 = all tubules loose; 2= proportion of loose tubules
greater
than proportion of compact tubules; 3 --- proportion of compact tubules
greater than
proportion of loose tubules; 4 = all tubules compact.
Sperm maturation - 0 = no sperm detected; 1 = only immature sperm detected; 2
=
proportion of immature sperm greater than proportion of mature sperm; 3 =
proportion
of mature sperm greater than proportion of immature sperm; 4 = only mature
sperm
detected.
The numbers presented are the mean SE (n) of the grading scale used for each
parameter measured. * denotes p <0.05.
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Table 3: The effects of relaxin gene knockout on collagen maturation, during
murine growth and development
Age RLX +/+ rlx
Tissue [months' [Mean SE (0] [Mean SE (n)*1
Testis':
[0.23] 1 (4) 2.00 27 (4)
[1] 1.09 0.07 (8) 1.61 0.25 (7)
[3] 1.06 0.06 (9) 1.28 0.09 (8)
[6.5] 1.75 0.14 (3) 2.31 0.12 (4)
Epididymisa:
[1] 2.41 0.33 (7) 2.57 0.39 (7)
[3] 1.82 0.21 (8) 2.86 0.29 (8)*
[6.5] 2.92 0.08 (3) 3.06 0.19 (4)
Prostate':
[1] 1(3) 2.33 0.17 (3)
[3] 1.25 0.17 (6) 2.25 0.28 (6)
[6.5] 1.5 (3) 2.69 0.37 (4)*
Seminal Vesicle':
[1] 1(3) 1.50 0.25 (4)
[3] 1.14 0.09 (7) 1.70 0.19 (5)
Table 3: Tissue sections were stained with Masson trichrome stain and graded
as
follows:
aCollagen - 1 = only thin lining of collagen surrounding the outer layer of
structures; 2
= additional lining of collagen surrounding internal components of tissue; 3 =
thicker
lining of collagen surrounding outer layer and internal components of tissues;
4 = 3 +
spacing between tubules/components filled with collagen.
The numbers presented are the mean SE (n) of the grading scale. * denotes p
<0.05
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Prostate: At 1 month of age, no significant differences were noted in the
structure of the prostate between RLX +1+ and rlx -/- derived tissues.
However, the
spacing between the ducts of prostate glands from normal animals was
associated with
only trace amounts of collagen. In contrast, the ducts of prostate glands from
rlx -/-
mice were intervened by layers of loose connective tissue. By adulthood (3
months),
rlx -/- mice contained smaller prostates with smaller glands and ducts. The
ducts of
tissues derived from RLX knockout mice were more compact and completely
supported by connective tissue, while the ducts obtained from RLX wildtype
mice
were more spread out (loose) and still supported by little collagen.
Furthermore, the
ducts of the adult prostate obtained from rlx -/- mice appeared to have
smaller
epithelium, containing less cells, while the ducts of RLX +/+ prostate samples
contained larger epithelial layers/glandular tissue. These results added to
our initial
findings on the testis and epididymis in implying that a delayed maturation
process of
male reproductive tissues was taking place in mice lacking a functionally
active
relaxin gene and involved an accumulation of collagen within these tissues.
Detection of Cell Apoptosis by Antibody Staining:
Based on observations made from H&E staining, whereby tissues
derived from rlx -/- mice underwent decreased sperm maturation and increased
cell
death (testis) and contained decreased epithelial (cell) layers (prostate), it
was decided
to investigate whether the increased cell death observed were the result of
apoptotic
pathways.
Bax Antibody Staining: Overexpression of Bax accelerates apoptotie
death induced by cytokine deprivation and also counters the death repressor
activity of
Bc1-2 ('Krajewski et al., Am. I. Pathol. 145:1323-36 (1994)). Using a Bax
monoclonal
antibody, the in vivo distribution of the Bax protein was evaluated in the
male
reproductive tract. Testis: Seminiferous tubules derived from immature
(1 month) wildtype animals showed weak immunostaining for Bax (dark brown cell
staining), which was primarily observed in the germinal cells near the
basement
membrane. These findings are consistent with those of previous reports (Ben-
Hur et
al., Cakif. Tiss. Int. 53:91-96 (1996)). In comparison, an increased number of
cells
were stained positive for Bax in testes derived from rlx -/- mice. The number
of Bax
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positive cells were counted using a hemacytometer and shown to be
significantly
greater in the testis of rlx -/- mice (32.7 4.8 cells / testis (n=6))
compared to the
number of apoptotic cells observed from RLX +/+ mouse-derived tissues (13.1
3.5
cells / testis (n=6)). While increased Bax staining was consistently observed
in rlx -/-
tissues, the number of tubules stained positive for Bax varied between tissue
samples,
and some tubules were also observed to be Bax-free. As the male reproductive
tract
matured, adult RLX wildtype mice testes showed decreased staining for Bax (3.4
1.4
cells / testis (n=6)) which correlated with the increased level of sperm and
tissue
maturity that these animals underwent. Conversely, the slower rate of tissue
maturity
observed in the rlx -/- mouse reproductive tract correlated with a further
increase in
Bax staining in 3 month RLX knockout mice (44.4 8.1 cells / testis (n=6)).
Many of
the observed cells appeared to be at the final stages of apoptosis, perhaps
representing
apoptotic bodies. Bax staining was not associated with mature sperm cells
though.
Epididymis: A more intense Bax staining was present in the intracellular
membrane of
epithelial cells of epididymis tubules derived from 1 month RLX +/+ mouse
tissues.
The same tubules derived from 1 month rlx -/- mouse tissues contained a
relatively
stronger level of Bax staining, which was further maintained, if not increased
in 3
month tissue sections. In contrast, the epididymis of adult wildtype mice
contained a
weaker expression of Bax staining, compared to tissues derived from 1 month
normal
mice, as the tissue matured with age. Prostate: The epithelial cells of the
prostate of
normal 1 month and 3 month animals showed weak positive staining for Bax. As
with
the other tissues studied, cells specifically within the epithelial layer (of
prostate ducts)
of rlx -/- animals showed increased staining for Bax in both immature (1
month) and
adult (3 month) tissues. These findings suggested that relaxin may have played
a
novel role in the regulation of cell apoptosis within the male reproductive
tract,
however, further work using a separate antibody to detect cell apoptosis was
conducted
to confirm the actions of relaxin.
Caspase-9, a central death protease, belongs to a unique family of
cysteine proteases that differ in sequence, structure and substrate
specificity to other
described protease families. The caspase family members (which are usually
involved
in a cascade of proteolytic cleavage events) function as key components of the
apoptotic machinery by acting to destroy specific target proteins which are
critical to
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cellular activity. Testis: Moderate caspase-9 staining was observed in 1 month
RLX
+/+ testes (28.1 5.6 cells/testis (n=7)), which appeared to be primarily
associated
with the germinal cells within the seminiferous tubules, rather than with
spermatagonia
or spermatocytes. As with Bax, a significantly (p<0.05) increased level of
caspase-9
staining was detected in 1 month rlx -/- mouse testes (76.6 1 10.2 cells /
testis (n=6)),
although heavy caspase-9 staining was associated with few tubules, while many
tubules were observed to contain little or no caspase-9 protein. With age, the
level of
caspase-9 detected in normal immature tissues was consistently found in older
tissues
at 3 months of age (26.9 4 cells / testis (n=6)). However, as the testis
increased in
size, the number of apoptotic cells detected represented a smaller fraction of
the tissue
with age. Testis tubules derived from rlx -/- mice contained slightly fewer
apoptotic
cells at 3 months (61.6 9.8 cells /testis (n=5)), compared to the density of
caspase-9
stained cells in 1 months tissues. The number of positively stained apoptotic
cells
from RLX knockout mouse tissues though, remained significantly (p<0.05)
greater
than their wildtype counterparts at all ages studied. Epididymis: No staining
for
caspase-9 was detected from 1 month RLX +/+ epididymis tubules, while trace
amounts of positive cells were observed in rlx -/- tissue sections. However,
the
positively stained cells were sparsely scattered and were detected in the
epithelial layer
of the tubules. With age, clear staining for caspase-9 was not detected in 3
month RLX
+/+ and rlx -/- mouse tissues. Prostate: No staining for caspase-9 was
identified in the
prostate of RLX +/+ and rlx -/- tissues at all ages (1-3 months) investigated.
Nevertheless, the obtained results suggested for the first time, that relaxin
is linked to
the regulation of cell apoptosis. Additional work though was required to
establish
whether relaxin was involved in cell proliferation pathways.
Detection of Cell Proliferation by Antibody Staining:
PCNA staining: The proliferating cell nuclear antigen (PCNA) antibody
was used to detect cell proliferation in the male reproductive tract based on
its ability
to associate with nuclear regions where DNA synthesis is occurring. Testis:
PCNA
was detected in immature and mature seminiferous tubules, although no
significant
differences in PCNA staining were detected in testis tubules derived from RLX
+/+
and rlx -/- mice at 1 month and 3 months of age. In immature tissues, PCNA
staining
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is usually highly expressed in mitotically active spermatogonia and
occasionally in
some Sertoli cells, which corresponds to proliferative activity. In this
study, these
cells were more uniformly and continuously labeled for PCNA even well into
murine
adulthood, which perhaps reflects the ability of both groups to be able to
initiate
reproduction, via the constant activation of these proliferative cells.
Epididymis: No
staining for PCNA was detected in the epididymis derived from either RLX +/+
or rlx
-/- mice at 1 month and 3 months of age. These findings are consistent with
the role of
the epididymis in acting as a reservoir for mature sperm. Prostate: Weak
staining for
PCNA was associated with the epithelial cells of prostate ducts derived from 1
month
RLX +1+ and rlx -/- mice. However, no staining for PCNA was detected from
either
group at 3 months of age. While cell proliferation studies were limited to
some
reproductive tissues, the accumulated findings perhaps confirmed that relaxin
was
most likely to play an influential role in the regulation of cell apoptosis,
rather than on
cell proliferation.
The effects of relaxin gene knockout on histology of other body tissues
RLX wildtype (RLX +/+) and RLX gene knockout (rlx -/-) male and
female mice were generated from RLX heterozygous (RLX +/-) parents, as
described
above. Mice were weighed and sacrificed at 1 week, 1 month and 3 months of age
for
tissue collection. The following tissues, including the brain, heart, liver,
kidneys,
thymus, spleen and male reproductive tract were collected, weighed and placed
into
10% formalin for detailed histological analysis. A summary of the weights of
these
tissues are shown in Tables 4-8.
At 1 week of age, male rlx -/- mice contained significantly (p<0.05)
smaller livers, kidneys and spleens compared to RLX +/+ animals, but these
weight
differences were no longer apparent at 1 month of age. Instead, the thymus of
1 month
old rlx -/- male mice was smaller (p<0.05) than their RLX +/+ counterparts.
This
weight difference was no longer apparent at 3 months of age. In the case of
the male
reproductive tract, no significant differences were noted at 1 week or 1 month
of age
between RLX +/+ and rlx -/- groups, however by 3 months of age, the
reproductive
tract of rlx -/- mice was significantly (p<0.05) smaller than that obtained
from RLX
+/+ animals.
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No notable differences were observed between 1 week old RLX +/+
and rlx -/- female mice, however, by 1 month of age, rlx -/- female mice had
significantly smaller livers than their RLX +/+ counterparts. By three months
of age,
this weight difference was no longer observed and no other significant
differences of
the other organs were noted.
To determine if the organ and tissue differences in RLX-/- mice
(derived from RLX +/- parents) would be accentuated further if rlx -/-
offspring were
obtained from RLX-/- parents, RLX +1+ and rlx -/- mice ere obtained from the
corresponding set of parents and were sacrificed at 1 week, 1 month and 3
months of
age. The brain, heart, liver, kidneys, thymus, spleen, male reproductive
tract, intestine
and lung were collected and weighed. The weights of these organs are shown in
Tables 4-8.
It was further noted that, although the weights of organ or tissue
appeared to normalize as the mice become older, normal weight alone does not
equate
with a normal organ or tissue. The data show that a discrepancy in organ
(tissue)
weight to body weight ratio at any time in the growth cycle indicates a change
in
underlying organ or tissue development or cellular architecture. As one
example, a
small liver that is infiltrated (altered) by fibrosis will weigh the same or
more than a
larger normal liver or one filled with fat. Thus, tissues from brain, heart,
liver,
kidneys, thymus, spleen, intestine and lung of male and female RLX-/- mice
(developed in the absence of relaxin) show evidence of increased apoptosis and
extracellular matrix (collagen) accumulation.
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Table 4: Tissue Comparison of 1 week old (from RLX +/+ & rlx -/- parents,
respectively), Represented as Mean SE (n):
RLX +/+ Male % body rlx -/- Male % body
wt wt
Body wt (g) 3.85 0.14 (10) 3.32 0.14 (15)
Rep. Organ (g) 0.017 0.001 (10) 0.43 0.018
0.001 (15) 0.53
Brain (g) 0.24 0.01 (10) 6.31 0.21 0.01
(15) 6.40
Heart (g) 0.023 0.002 (10) 0.58 0.020
0.001 (15) 0.60
Liver (g) 0.12 0.01 (10) 3.13 0.094 0.01
(15)* 2.82
L. Kidney (g) 0.023 0.001 (10) 0.59 0.018 0.001
(15)* 0.54
R. Kidney (g) 0.023 0.001 (10) 0.59 0.019
0.001(15)* 0.56
Spleen (g) 0.033 0.002 (10) 0.59 0.018 0.001
(15)* 0.54
Thymus (g) 0.020 0.001 (10) 0.51 0.018 0.001
(15) 0.54
RLX +/+ Female % body rlx -/- Female % body
wt wt
Body wt (g) 3.45 0.19(9) 3.51 0.15(9)
Brain (g) 0.22 0.01 (9) 6.23 0.22 0.01 (9) 6.11
Heart (g) 0.023 0.002 (9) 0.67 0.02 0.001
(9) 0.57
Liver (g) . 0.085 0.01 (9) 2.46 0.10
0.01 (9) 2.85
L. Kidney (g) 0.019 0.001 (9) 0.55 0.20 0.001
(9) 0.56
R. Kidney (g) 0.020 0.001 (9) 0.57 0.20 0.001
(9) 0.57
Spleen (g) 0.020 0.002 (9) 0.57 0.017
0.002 (9) 0.48
Thymus (g) 0.017 0.002 (9) 0.48 0.017
0.001 (9) 0.47
*=> p < 0.05
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Table 5: Tissue Comparison 1 month old mice (from RLX+/- parents), Represented
as
Mean SE (n):
RLX +/+ Male rlx -/- Male % body
body wt
wt
Body wt (g) 20.54 0.82 (10) 18.29 0.84 (10)
Rep. Organ (g) 0.37 0.02 (11) 1.81 0.33 0.01 (11) 1.78
Brain (g) 0.37 0.01 (10) 1.81 0.37 0.01 (10) 2.03
Heart (g) 0.13 0.004 (10) 0.62 0.13 0.01 (10) 0.69
Liver (g) 1.17 0.05 (10) 5.70 1.10 I 0.05 (10) 5.99
L. Kidney (g) 0.16 0.01 (10) 0.77 0.14 0.01 (10) 0.78
R. Kidney (g) 0.17 0.01 (10) 0.82 0.14 0.01 (10) 0.78
Spleen (g) 0.12 0.01 (10) 0.57 0.12 0.01 (10) 0.66
Thymus (g) 0.06 0.01 (10) 0.28 0.075 0.005 (10) 0.41
RLX +/+ Female rlx -/- Female % body
body wt
wt
Body wt (g) 16.93 0.45 (10) 16.04 0.61 (9)
Brain (g) 0.36 0.01 (9) 2.14 0.36 0.01 (9) 2.23
Heart (g) 0.09 0.003 (10) 0.53 0.10 0.003 (9) 0.60
Liver (g) 0.90 0.02 (10) 5.32 0.78 0.02 (9)* 4.84
L. Kidney (g) 0.13 0.005 (10) 0.78 0.12 0.003 (9) 0.76
R. Kidney (g) 0.13 0.004 (10) 0.78 0.13 0.01(9) 0.80
Spleen (g) 0.08 0.004 (10) 0.45 0.08 0.01 (9) 0.50
Thymus (g) 0.07 0.01 (10) 0.39 0.07 0.01 (9) 0.42
*=>p<0.05
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Table 6: Tissue Comparison of 3 month old male mice (from RLX +/+ & rlx -/-
parents, respectively), Represented as Mean SE (n):
RLX +1+ Male % body rlx -/- Male %
body
wt wt
Body wt (g) 27.41 0.39 (14) 26.57 0.51 (11)
Rep. Organ (g) 0.92 0.06 (14) 3.37 0.62
0.02 (11) 2.33
Brain (g) 0.37 0.01 (10) 1.34 0.37 0.01
(10) 1.40
Heart (g) 0.15 0.003 (10) 0.56 0.14
0.01 (10) 0.54
Liver (g) 1.32 0.08 (10) 4.80 1.28 1
0.04 (10) 4.82
L. Kidney (g) 0.24 0.01 (10) 0.87 0.21
0.01 (10) 0.81
R. Kidney (g) 0.23 0.01 (10) 0.83 0Ø22
0.01 (10) 0.82
Spleen (g) 0.08 0.01 (10) 0.29 0.08 0.003 (10)
0.29
Thymus (g) 0.056 0.01(10) 0.20 0.051 .1 0.003
(10) 0.19
RLX +/+ Female rlx -/- Female % body
body wt
wt
Body wt (g) 23.14 1.08 (10) - 21.62 0.39(9)
Brain (g) 0.38 0.01 (10) 1.64 0.39
0.01(9) 1.80
Heart (g) 0.10 0.004 (10) 0.44 0.11 0.004
(9) 0.49
Liver (g) 1.07 0.05 (10) 4.61 1.01 0.004
(9) 4.66
L. Kidney (g) 0.16 0.004 (10) 0.67 0.15 0.006
(9) 0.69
R. Kidney (g) 0.16 0.005 (10) 0.70 0.15 0.003
(9) 0.70
Spleen (g) 0.09 0.01 (10) 0.37 0.09
0.01 (9) 0.41
Thymus (g) 0.06 0.003 (10) 0.26 0.06 .1 0.003
(9) 0.29
*=> p < 0.05
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Table 7: Tissue Comparison of 1 month old mice (from RLX+/+ and RLX-/-
parents,
respectively), represented as Mean SE (n):
RLX +1+ Male % rlx -/- Male % body
body wt
wt
Body wt (g) 19.92 0.51 (11) - 14.90 1.02(4)* -
Rep. Organ (g) 0.48 0.02 (11) 2.42 0.27 0.04 (4)
1.78
Brain (g) 0.41 0.01 (11) 2.06 0.40 0.01 (4)
2.70
Heart (g) 0.12 0.004 (11) 0.61 0.12 0.01 (4)
0.81
Liver (g) 1.11 0.04 (11) 5.58 0.72 0.06(4)* 4.85
L. Kidney (g) 0.14 1 0.01 (11) 0.68 0.10 0.01 (4)
0.65
R. Kidney (g) 0.13 0.01 (10) 0.67 0.10 1 0.01(4)
0.65
Spleen (g) 0.11 0.01 (11) 0.53 0.11 0.02 (4)
0.72
Thymus (g) 0.08 0.01 (11) 0.42 0.085 0.003 (4) 0.57
Gut (g) 0.48 1 0.03 (11) 2.40 0.56 1 0.07 (4)
3.78
Lung (g) 0.19 0.01 (11) 0.97 0.16 0.01 (4)
1.06
RLX +/+ Female % rlx -/- Female % body
body wt
wt
Body wt (g) 16.68 0.38 (11) - 13.15 1.40(5)* -
Brain (g) 0.41 0.01 (11) 2.48 0.40 0.02 (5)
3.07
Heart (g) 0.10 0.003 (11) 0.59 0.10 0.003 (5)
0.76
Liver (g) 0.90 0.03 (11) 5.39 0.57 0.07 (5)*
4.32
L. Kidney (g) 0.10 0.004 (11) 0.61 0.10 0.003 (5)
0.73
R. Kidney (g) 0.10 0.003 (11) 0.62 0.10 0.01 (5)
0.75
Spleen (g) 0.10 0.01 (11) 0.57 0.09 0.01 (5)
0.68
Thymus (g) 0.10 0.010 (11) 0.59 0.08 0.003 (5)
0.61
Gut (g) 0.50 0.04 (11) 2.97 0.51 0.03 (5)
3.86
Lung (g) 0.20 0.01 (6) 1.18 0.12 0.01 (4)
0.93
*---->p<0.05
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Table 8: Tissue Comparison of 3 month old mice (from RLX+/+ and RLX-/-
parents,
respectively), represented as Mean SE (n):
RLX +/+ Male % body rlx -/- Male %
body
wt wt
Body wt (g) 26.10 0.51 (12) 24.37 (1)
Rep. Organ (g) 0.90 0.03 (12) 3.45 0.82 (1)
Brain (g) 0.41 0.01 (12) 1.56 0.40 (1)
Heart (g) 0.16 0.003 (12) 0.60 0.14 (1)
Liver (g) 1.25 0.04 (8) 4.79 1.24 (1)
L. Kidney (g) 0.20 0.005 (12) 0.75 0.17 (1)
R. Kidney (g) 0.20 0.004 (12) 0.75 0.18(1)
Spleen (g) 0.085 0.003 (12) 0.33 0.08 (1)
Thymus (g) 0.058 0.004 (12) 0.22 0.011 (1)
Gut (g) 0.62 0.05 (12) 2.36 0.5 (1)
Lung (g) 0.25 0.02 (7) 0.95 0.2 (1)
RLX +1+ Female rlx -/- Female % body
body wt
wt
Body wt (g) 22.30 1.63 (12) - 23.73 0.45
(5)
Brain (g) 0.41 ' 0.01 (12) 1.86 0.44
0.01(5) 1.86
Heart (g) 0.13 0.003 (12) 0.59 0.14 0.002 (5)
0.61
Liver (g) 1.15 0.05 (10) 5.16 1.11
0.003 (5) 4.67
L. Kidney (g) 0.14 0.004 (12) 0.62 0.14 0.003 (5)
0.60
R. Kidney (g) 0.16 0.005 (12) 0.62 0.14 0.01 (5)
0.61
Spleen (g) 0.09 0.004 (12) 0.40 0.10 0.01 (5)
0.41
Thymus (g) 0.07 0.003 (12) 0.29 0.08 0.010 (5)
0.33
Gut (g) 0.55 0.04 (12) 2.45 0.68 0.04 (5)
2.86
Lung (g) 0.21 0.01 (6) 0.96 0.21 0.005 (5)
0.86
*=> p <0.05
The effects of relaxiit gene knockout on the histology of skin
The role of relaxin in the regulation of tissue remodeling was examined by
studying the skin histology in rlx-null mice (rlx-/-). These mice were the
progeny of rlx-/-
male and female parents. Sequential skin samples from the ear and dorsum of
the back of
at least 5 male and 5 female mice were stained with H&E and Masson's trichrome
stain
and examined at each time point. Similar samples at the same time points were
obtained
from Rlx+/+ mice of the same strain. Upon histological examination of rlx-null
mice, the
dermis was found to have thickened progressively with time and had increased
fibrosis
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throughout the dermis. Dermal samples of rlx-null mice were normal at one week
of age;
however, by one month early dermal fibrosis was evident. By 3 months of age
there was a
marked increase in dermal fibrosis that increased in density by 6 months of
age. These
dermal findings were similar in male and female rlx-null mice.
In these mice, the epidermis was normal, and hair was not altered, except
for an initial lighter coat color in rlx-null mice that became
indistinguishable from Rix +1+
mice by one month of age. Examination of serum chemistries, hematological
parameters
and urine from the rlx-null mice were unremarkable.
These findings support previous observations that relaxin influences matrix
turnover in vitro and in vivo by altering key matrix molecules, matrix-
degrading enzymes
and growth factors. Many stromal cells that produce interstitial collagens
have relaxin
receptors, whether derived from male or female sources. Antibody-based assays
can
detect relaxin in serum at the low picogam level in females during the luteal
phase of the
menstrual cycle, and these levels rise during pregnancy, a time when tissue
remodeling is
most evident. In contrast, serum relaxin is reported to be undetectable in
males.
Rix-null mice offer the first direct evidence that links relaxin to a
generalized alteration of collagen turnover in normal skin. These results
indicate that
relaxin may be produced and circulate at biologically relevant concentrations
below
current levels of detection in males and females. Relaxin participates in the
ordered
maintenance of matrix turnover in concert with other matrix-regulating
molecules.
Relaxin can influence tissue remodeling, promotion of blood vessel formation
and
stimulation of vasodilatation. These results also indicate that the relaxin
synthetic
pathway, and/or relaxin receptor, is associated with fibrotic conditions, such
as
scleroderma.
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