U.S. patent application number 10/398553 was filed with the patent office on 2005-02-10 for methods of modulating apoptosis by administration of relaxin agonists or antagonists.
Invention is credited to Amento, Edward P., Samuel, Chrishan S..
Application Number | 20050032683 10/398553 |
Document ID | / |
Family ID | 34118243 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032683 |
Kind Code |
A1 |
Amento, Edward P. ; et
al. |
February 10, 2005 |
Methods of modulating apoptosis by administration of relaxin
agonists or antagonists
Abstract
The present invention relates to the discovery that relaxin is
associated with the development or maturation of body tissues.
Knockouts of the gene encoding relaxin result in various
abnormalities in the development of various tissues. The present
invention provides methods of modulating apoptosis by administering
a relaxin agonist or antagonist to a subject.
Inventors: |
Amento, Edward P.; (Portola
Valley, CA) ; Samuel, Chrishan S.; (Glen Waverly,
AU) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
34118243 |
Appl. No.: |
10/398553 |
Filed: |
September 22, 2003 |
PCT Filed: |
October 4, 2001 |
PCT NO: |
PCT/US01/42484 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60238232 |
Oct 4, 2000 |
|
|
|
Current U.S.
Class: |
514/12.7 ;
514/16.5; 514/18.9; 514/19.3 |
Current CPC
Class: |
A01K 67/0276 20130101;
A61P 15/00 20180101; A61P 1/16 20180101; A61P 1/00 20180101; A61P
43/00 20180101; C07K 14/64 20130101; A61K 38/2221 20130101; A61P
13/12 20180101; A61P 35/00 20180101; A61K 38/22 20130101; A61P
13/08 20180101; A61P 5/00 20180101; A61P 25/00 20180101; A01K
2227/105 20130101; A61P 11/00 20180101; A61P 9/00 20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17 |
Claims
What is claimed is:
1. A method of modulating apoptosis, comprising: 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.
2. The method of claim 1, wherein the cells are from the liver,
kidney, spleen, thymus, brain, heart, intestine, skin, lung, the
male reproductive tract, or the female reproductive tract.
3. The method of claim 2, wherein the male reproductive tract
tissue is prostate, epididymis, seminal vesicles or testes.
4. The method of claim 2, wherein the female reproductive tract
tissue is uterus, cervix, the interpubic ligament, or connective
tissues within the pelvic girdle.
5. The method of claim 2, wherein the number of apoptotic cells
decreases.
6. The method of claim 1, wherein tissue maturation is
stimulated.
7. The method of claim 6, wherein fibrosis is reduced.
8. The method of claim 6, wherein the tissue is male reproductive
tract tissue.
9. The method of claim 8, wherein the tissue is prostatic tissue,
epididymal tissue, seminiferous tissue, testicular tissue or
sperm.
10. The method of claim 9, wherein the tissue is testicular
tissue.
11. The method of claim 10, wherein the maturation is an increase
in cell number in an under-developed testes.
12. The method of claim 9, wherein the number of viable sperm cells
increases.
13. The method of claim 1, wherein the subject has a
relaxin-deficient condition.
14. The method of claim 13, wherein the relaxin-deficient condition
is immature tissue, excessive collagen deposition, or low sperm
count.
15. The method of claim 14, wherein the immature tissue is immature
male productive tract tissue.
16. The method of claim 15, wherein the immature male reproductive
tract tissue is underdeveloped testes.
17. The method of claim 1, wherein excessive collagen deposition is
reduced.
18. The method of claim 1, wherein the relaxin agonist is relaxin,
a relaxin analog, a small molecule relaxin effector, or a relaxin
nucleic acid.
19. The method of claim 18, wherein the relaxin is vertebrate
relaxin.
20. The method of claim 19, wherein the relaxin is human
relaxin.
21. The method of claim 1, wherein the administering is by
infusion, injection, oral delivery, nasal delivery, intrapulmonary
delivery, rectal delivery, transdermal delivery, interstitial
delivery or subcutaneous delivery.
22. The method of claim 21, wherein the administering is by delayed
release delivery.
23. The method of claim 21, wherein the subcutaneous delivery is by
infusion or injection.
24. The method of claim 21, wherein the administering is by
intrapulmonary, subcutaneous or transdermal delivery.
25. The method of claim 1, wherein the administering comprises
delivery of a vector encoding the relaxin agonist.
26. The method of claim 1, wherein the vector is an expression
vector, and whereby the relaxin agonist is expressed in the
cells.
27. The method of claim 1, wherein the administering comprises
delivery of a relaxin or relaxin analog nucleic acid.
28. The method of claim 1, wherein the subject is a post-pubescent
subject.
29. The method of claim 1, wherein the subject is a pre-pubescent
subject.
30. A method of modulating apoptosis in a population of cells
expressing a relaxin receptor, comprising: administering to a
subject in need thereof an effective amount of an antagonist of
relaxin for a period of time sufficient to increase apoptosis in
the cell population expressing the relaxin receptor.
31. The method of claim 30, wherein the relaxin antagonist inhibits
binding of relaxin to relaxin receptor.
32. The method of claim 30, wherein the relaxin antagonist reduces
relaxin-associated tissue remodeling.
33. The method of claim 30, wherein the cell population is from
heart, brain, liver, kidney, spleen, thymus, skin, female
reproductive tract tissue or male reproductive tract tissue.
34. The method of claim 33, wherein the male reproductive tract
tissue is prostate, epididymis, seminal vesicles or testes.
35. The method of claim 34, wherein the tissue is prostatic
tissue.
36. The method of claim 33, wherein the male reproductive tract
tissue is mature.
37. The method of claim 33, wherein the tissues of the female
reproductive tract are the uterus, cervix, the interpubic ligament,
and connective tissues within the pelvic girdle.
38. The method of claim 30, wherein the cell population comprises
fibroblasts, osteoblasts, monocytes, epithelial cells or
endothelial cells.
39. The method of claim 30, wherein the relaxin antagonist is a
relaxin binding agent, a relaxin receptor binding agent, or a
relaxin antisense nucleic acid.
40. The method of claim 39, wherein the relaxin binding agent is an
anti-relaxin antibody, a soluble relaxin receptor, or a small
molecule relaxin antagonist.
41. The method of claim 40, wherein the antibody is a monoclonal
antibody, a polyclonal antibody, a single chain antibody, an Fab,
Fab', an F(ab').sub.2, an Fv, a single heavy chain, or a chimeric
antibody.
42. The method of claim 39, wherein the relaxin receptor binding
agent is an anti-relaxin receptor antibody, a relaxin analog, or a
small molecule relaxin receptor antagonist.
43. The method of claim 42, wherein the antibody is a monoclonal
antibody, a polyclonal antibody, a single chain antibody, an Fab,
Fab', an F(ab').sub.2, an Fv, a single heavy chain, or a chimeric
antibody.
44. The method of claim 30, wherein the administering is by
infusion, injection, oral delivery, nasal delivery, intrapulmonary
delivery, rectal delivery, transdermal delivery, interstitial
delivery, or subcutaneous delivery.
45. The method of claim 44, wherein the administering is by delayed
release delivery.
46. The method of claim 44, wherein the subcutaneous delivery is by
infusion or injection.
47. The method of claim 44, wherein the administering is by
intrapulmonary, subcutaneous or transdermal delivery.
48. The method of claim 30, wherein the administering comprises
delivery of a vector encoding the relaxin antagonist.
49. The method of claim 30, wherein the vector is an expression
vector, and whereby the relaxin antagonist is expressed in the cell
population.
50. The method of claim 30, wherein the administering comprises
delivery of a relaxin or relaxin receptor antisense nucleic
acid.
51. The method of claim 30, wherein increased apoptosis reduces
unwanted cell accumulation.
52. The method of claim 51, wherein the unwanted cells accumulation
is hyperplasia, hypertrophy, cancer or neoplasia.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority from Provisional
Application U.S. Ser. No. 60/238,232 filed Oct. 4, 2000,
Provisional Application U.S. Ser. No. 60/241,991 filed Oct. 20,
2000, and Provisional Application U.S. Ser. No. 60/242,037, filed
Oct. 20, 2000, the disclosure of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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 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.
[0004] 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.
[0005] 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.
[0006] 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
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] Compositions comprising a relaxin agonist useful in the
methods according to the present invention can be formulated for
administration, for example, 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.
[0013] 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.
[0014] 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.
[0015] 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, 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').sub.2, an Fv, a
single heavy chain, a chimeric antibody, and the like.
[0016] Compositions comprising a relaxin antagonist useful in the
methods of the present invention can be formulated 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.
[0017] 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
[0018] 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.
[0019] 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 10.sup.9
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 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".
[0020] Nucleic acids include RNA, mRNA, cDNA, genomic DNA,
synthetic forms, 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,
internucleotide 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.
[0021] 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)).
[0022] 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 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 (1), Leucine (L), Methionine (M), Valine (V); and 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also
Creighton, Proteins, W. H. Freeman and Company (1984), which is
incorporated by reference herein.) 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."
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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 part of an organ or bone or it
can be a loose association of cells, such as cells of the immune
system.
[0030] 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.
[0031] The term "mature," in the context of body tissue, refers to
the achievement of full development, differentiation and/or growth
of the tissue.
[0032] The term "immature," in the context of body tissue, refers
to a tissue that has not fully developed and/or differentiated.
[0033] The term "pre-pubescent male" refers to a male that has not
completed the process of puberty.
[0034] The term "post-pubescent male" refers to a male that has
completed the process of puberty.
[0035] 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.
[0036] 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.
[0037] The terms "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.
[0038] 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.
[0039] 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.
[0040] 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, by an increase or
decrease in functionally active relaxin levels, or in functionally
active relaxin receptor.
[0041] The terms "hyperplasia" or "hyperplastic tissue" refer to an
increase in the number of cells in a tissue.
[0042] The terms "hypertrophy" or "hypertrophic" refer to an
increase in the size of a tissue.
[0043] The terms "cancer" and "malignancy" generally refer to the
various types of malignant neoplasms, most of which invade
surrounding tissues.
[0044] The terms "cancerous" and "malignant" relate to cells or
tissue having properties of cancer or a malignancy.
[0045] 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.
[0046] The term "apoptosis" refers to a regulated network of
biochemical events which lead to a selective form of cell silicide,
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 mitochondrial
cristae, opening of the mitochondrial permeability transition pores
and/or dissipation of the mitochondrial proton gradient.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0047] 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.
[0048] 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 abnormal 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.
[0049] 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 prostatic, 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.
[0050] 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.
[0051] 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, 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.
[0052] 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).
[0053] Relaxin Agonists
[0054] 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.
[0055] Relaxin and Relaxin Analogs
[0056] 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.
[0057] 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. Pat. Nos. 5,166,191 and
4,835,251 (both of which are hereby incorporated by reference). In
this application, "relaxin" generally refers to the terms
"relaxin," "human relaxin," "native relaxin," and "synthetic
relaxin" as defined in U.S. Pat. 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. Pat. Nos. 5,179,195;
5,023,321; and 4,758,516 (the disclosures of which are incorporated
by reference herein). "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).
[0058] 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. Pat. No. 5,166,191, including the following references:
U.S. Pat. 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
(all of which are incorporated by reference herein).
[0059] Additional methods of making relaxin are described in U.S.
Pat. No. 5,464,756, and PCT/US94/06997 (the disclosures of which
are incorporated by reference herein). 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, the disclosure of which is incorporated herein by
reference). 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.
[0060] 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(1329 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.
[0061] 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).
[0062] 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.
[0063] 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/aligmnent 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. Sci. USA 85:2444
(1988); GAP, BESTFIT, FASTA, and TEASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, 575 Science Dr.,
Madison, Wis.); Ausubel et al (eds.), Current Protocols in
Molecular Biology, 4th ed., John Wiley and Sons, New York (1999);
the disclosures of which are incorporated by reference herein).
Relaxin agonists further comprise functionally active relaxin
polypeptides, analogs or fragments that bind to a relaxin
receptor.
[0064] 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 Laboratory
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); which are incorporated
by reference herein), 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 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.RTM. linkers (Pharmacia), and the like. (See generally
Sambrook et al., supra; Ausubel et al., supra.)
[0065] 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. Pat. 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. Pat. Nos. 4,656,249; 5,179,195; 5,945,402; 5,811,395; and
5,795,807 (the disclosures of which are incorporated by reference
herein).
[0066] 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 trypsin;
chymotrypsin, papain, V8 protease, and the like); modification by,
for example, NaBH.sub.4, acetylation, formylation, oxidation and
reduction, metabolic synthesis in the presence of tunicamycin, and
the like.
[0067] Relaxin polypeptides, analogs and fragments can be purified
from natural sources by standard methods such as those described
herein (e.g., immunoaffinity 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, 2.sup.nd
Ed., Pierce Chemical Co., Rockford, Ill., (1984); the disclosures
of which are incorporated by reference herein).
[0068] 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,
.alpha.-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric
acid, .epsilon.-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,
.beta.-alanine, selenocysteine, fluoro-amino acids, designer amino
acids such as .beta.-methyl amino acids, C .alpha.-methyl amino
acids, N .alpha.-methyl amino acids, and amino acid analogs in
general. Furthermore, the amino acid can be D (dextrorotary) or t
(levorotary).
[0069] 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 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).
[0070] In a specific embodiment, the fusion protein is a
relaxin-ubiquitin fusion protein. For example, U.S. Pat. No.
5,108,919 (the disclosure of which is incorporated herein by
reference) discloses methods for preparing a fusion protein of a
relaxin chain and ubiquitin.
[0071] 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.
[0072] Relaxin Nucleic Acids:
[0073] 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, 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.
[0074] 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.
[0075] 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 another
embodiment, a fragment of relaxin comprising a domain of the
relaxin polypeptide is expressed.
[0076] 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 .beta.-lactamase
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 Gal7 and Gal4
promoters, the ADH (alcohol dehydrogenase) promoter, the PGK
(phosphoglycerol kinase) promoter, the alkaline phosphatase
promoter, and the like.
[0077] 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 Syrup. Quant. Biol. 50:399-409
(1986); MacDonald, Hepatology 7(1 Suppl.):4.sup.2S-51S (1987)); the
insulin gene control region which is active in pancreatic beta
cells (e.g., Hanahan, Nature 315:115-22 (1985)), the 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., Krumlaufet 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., Shani, 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. Pat. No. 6,100,444, the disclosure of which is
incorporated by reference herein.)
[0078] 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).
[0079] Expression vectors containing relaxin nucleic acids can be
identified by general approaches well known to the skilled artisan,
including: (a) nucleic acid 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.
[0080] 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.
Pat. 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.
[0081] 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.
[0082] 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 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.
[0083] Relaxin Antagonists
[0084] 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.
[0085] Relaxin Antibodies
[0086] 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').sub.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 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.
[0087] 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. Pat. Nos. 6,114,598 and
6,111,166, which are incorporated by reference herein.) 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.
[0088] 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
immunized by injection with the native relaxin or relaxin receptor
polypeptide. (See, e.g., U.S. Pat. Nos. 6,114,598 and 6,111,166,
which are incorporated by reference herein.)
[0089] 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').sub.2, F(ab'), Fv, or
hypervariable 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.
[0090] Methods for producing such "chimeric" molecules are
generally well known and described in, for example, U.S. Pat. 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 (the disclosures of which
are incorporated by reference herein). 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.)
[0091] According to another aspect of the invention, techniques for
the production of single chain antibodies (see, e.g., U.S. Pat.
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 Muylderrnans, J. Immunol. Methods 231:25-38 (1999);
Muyldermans and Lauwereys, J. Mol. Recognit. 12:131-40 (1999)).
[0092] 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.
[0093] 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(ab').sub.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').sub.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. Pat. No. 5,965,405.
[0094] 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.
[0095] Soluble Relaxin Receptors
[0096] 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
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.
[0097] 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. Pat. Nos. 6,033,903; 6,037,450; and 5,925,549; the
disclosures of which are incorporated by reference herein.)
[0098] 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).
[0099] 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 receptor polypeptide. In other embodiments, the fragments
are larger than 100 or even 200 amino acids.
[0100] 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. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Biol.
48:443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA
85:2444 (1988); GAP, BESTFIT, FASTA, and TEASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.); Ausubel et al. (supra); the disclosures of
which are incorporated by reference herein). 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; the disclosures of
which are incorporated by reference herein).
[0101] 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 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.RTM. linkers (Pharmacia),
and the like.
[0102] 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 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,
NaBH.sub.4, acetylation, formylation, oxidation and reduction,
metabolic synthesis in the presence of tunicamycin, and the
like.
[0103] 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, 2.sup.nd Ed., Pierce
Chemical Co., Rockford, Ill., (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 the common amino acids,
.alpha.-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric
acid, .epsilon.-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,
.beta.-alanine, selenocysteine, fluoro-amino acids, designer amino
acids such as .beta.-methyl amino acids, C .alpha.-methyl amino
acids, N .alpha.-methyl amino acids, and amino acid analogs in
general. Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0104] 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 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.
[0105] Relaxin Analogs
[0106] 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. Pat.
Nos. 5,179,195; 5,166,191; 5,023,321; 4,835,251; and 4,758,516 (the
disclosures of which are hereby incorporated by reference). Methods
of making relaxin and its analogs 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.
[0107] 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.
[0108] 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.)
[0109] 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 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.RTM. linkers (Pharmacia), and the like.
[0110] 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. Pat. 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.
[0111] 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, 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 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, NaBH.sub.4, acetylation, formylation,
oxidation and reduction, metabolic synthesis in the presence of
tunicamycin, and the like.
[0112] 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 al.,
Nature 310:105-11 (1984); Stewart and Young, Solid Phase Peptide
Synthesis, 2.sup.nd Ed., Pierce Chemical Co., Rockford, Ill.,
(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, .alpha.-amino isobutyric acid, 4-aminobutyric acid,
2-amino butyric acid, .epsilon.-amino hexanoic acid, 6-amino
hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid,
omithine, norleucine, norvaline, hydroxyproline, sarcosine,
citrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine, cyclohexylalanine, .beta.-alanine, selenocysteine,
fluoro-amino acids, designer amino acids such as .beta.-methyl
amino acids, C .alpha.-methyl amino acids, N .alpha.-methyl amino
acids, and amino acid analogs in general. Furthermore, the amino
acid can be D (dextrorotary) or L (levorotary).
[0113] 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).
[0114] In a specific embodiment, the fusion protein is a relaxin
analog-ubiquitin fusion protein. For example, U.S. Pat. No.
5,108,919 (the disclosure of which is incorporated herein by
reference), discloses methods for preparing a fusion protein of a
relaxin chain and ubiquitin.
[0115] Nucleic Acids:
[0116] 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
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).
[0117] 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.
[0118] 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.
[0119] 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 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.
[0120] 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.
[0121] 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 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
.beta.-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 et al., Nature
310:115-20 (1984)), promoter elements from yeast or other fungi
such as the Gal7 and Gal4 promoters, the ADH (alcohol
dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter,
the alkaline phosphatase promoter, and the like.
[0122] 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 et al., Cold Spring Harbor Synip. 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 immunoglobulin gene control region which is active in
lymphoid cells (see, 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 (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 et al., 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
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. Pat. No. 6,100,444, the
disclosure of which is incorporated by reference herein.)
[0123] 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).
[0124] 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 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.
[0125] 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).
[0126] 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.
[0127] Functional Assays for Relaxin and Relaxin Receptor Agonists
and Antagonists
[0128] 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.
[0129] 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,
IC.sub.50, and the like.
[0130] Suitable assays for measuring relaxin or relaxin receptor
agonist or antagonist activity, include, for example, those
described in the following references (which are incorporated by
reference herein): 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).
[0131] Other assays include those disclosed by Bullesbach et al.,
The Receptor-Binding Sites of Human Relaxin II, J. Biol. Clem.
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. Otolaryngol. Head Neck Surg.
118:153-56 (1992); Lee et al., Monoclonal Antibodies Specific for
Rat Relaxin. VI. Passive 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);
[0132] 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 Gilts,
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
Tissue, J. Mol Endocrinology 12:351-64 (1994); Winn et al.
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 et al., Individual and Combined Effects of
Relaxin, Estrogen, and Progesterone on Ovariectomized Gilts. II.
Effects on Mammary Development, Endocrinology 135:1250-55 (1994);
Bryant-Greenwood et al., 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).
[0133] 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.
[0134] The functional activity of an agonist or antagonist can also
be determined 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 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).
[0135] Administration of Relaxin Agonists and Antagonists
[0136] 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.
[0137] Various delivery systems are known and can be used to
administer a active agent, such as, for example, by infusion,
injection (e.g., intradermal, 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.
[0138] 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, adjuvant,
excipient, stabilizer, preservative, viscogen, or vehicle with
which the active agent is formulated for administration.
Pharmaceutical 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.
[0139] 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, Pa. (1990)).
[0140] 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.
[0141] 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 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.
[0142] Orally deliverable compositions can take the form of
solutions, suspensions, emulsions, tablets, pills, capsules,
powders, sustained-release formulations, 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.
[0143] 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.
[0144] 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. Pat. No.
4,511,069, which is incorporated by reference herein). 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
solution further comprises a propellant. The pH of the nasal spray
solution is typically between about pH 6.8 and 7.2.
[0145] 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, each of which
is incorporated by reference herein.) 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 nonoxynol-9, laureth-9, poloxamer-124,
octoxynol-9 and lauramide DEA. Nonoxynol-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 SURFONC.RTM. N-95
(Jefferson), NEUTRONYX.RTM. 600 (Onyx) and IGEPAL.RTM. (CO-630
(GAF). N-9 is considered to be a hard detergent, and has been used
as a spermatocide. (See The Merck Index, 10.sup.th 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. Pat. No. 5,902,789, which is incorporated by reference
herein).
[0146] 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.
[0147] 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
conventional pharmaceutically acceptable carriers and stabilizers.
The carriers and 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. Pat. 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.
[0148] 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.
[0149] 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. Pat. 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
Braunner (U.S. Pat. No. 5,803,078), or other devices (see, e.g.,
Sears et al., N.Z. Med. J 96:743II (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 disclosures of these references are incorporated by
reference herein.)
[0150] 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 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), which is incorporated by reference
herein).
[0151] The size of the aerosols or particles generally will range
about from 0.5 to about 5 .mu.m, typically about 2 .mu.m to 5
.mu.m, preferably about 2 to about 4 .mu.m or about 4 to about 5
.mu.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 .mu.m 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 .mu.m range can be used, but generally
the proportion of particles having an average mean diameter larger
than 5 .mu.m 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.
[0152] 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. Suppositories generally contain
active ingredient in the range of 0.5% to 10% by weight; oral
formulations typically contain 10% to 95% active ingredient.
[0153] 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.
[0154] 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., Surgery 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,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J. 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)) (the disclosures of which are incorporated by
reference herein).
[0155] 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), which is
incorporated by reference herein).
[0156] Administration of Nucleic Acids
[0157] The invention provides further methods for the
administration of nucleic acids (e.g., nucleic acids encoding
relaxin agonists or antagonists, such as 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 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.
[0158] For general reviews of the methods of gene therapy, see
Steinberg and Raso (J. Pharm. Pharm. Sci. 1:48-59 (1998)); Pantuck
et al. (World J. Urol. 18:143-47 (2000)); Prince (Pathology
30:335-47 (1998)); Ledley (Cur. 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)).
[0159] 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, New York (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.
[0160] 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. Pat. Nos.
5,631,153; 5,627,059; 5,487,992; and 5,464,764; the disclosures of
which are incorporated by reference herein).
[0161] 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 et al.,
Trends Biotechnol. 17:169-74 (1999); the disclosures of which are
incorporated by reference herein), electroporation (see, e.g.,
Muramatsu et al., Int. J. Mol. Med. 1:55-62 (1998), the disclosure
of which is incorporated by reference herein), or by use of
microparticle bombardment, such as a gene gun (BIOLISTIC.TM.,
Dupont); see, e.g., Biewenga et al., J. Neurosci. Methods 71:67-75
(1997), the disclosure of which is incorporated by reference
herein). 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 et
al., Pharm. Res. 17:113-26 (2000); Maurer et al., 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); the disclosures of which are
incorporated by reference herein). 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); the disclosures of which are incorporated by reference
herein). 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), the
disclosure of which is incorporated by reference herein.) 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; the disclosures of which
are incorporated by reference herein).
[0162] 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), the disclosures of which are incorporated by reference
herein). 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), the disclosures of which are incorporated by
reference herein). Other references illustrating the use of viral
vectors in gene therapy are by Lundstrom (J. Recept. Signal.
Transduct. Res. 19:673-86 (1999)); Clowes et al. (J. Clin. Invest.
93:644-51 (1994)); Kiem et al. (Blood 83:1467-73 (1994)); Salmons
and Gunzberg (Hum Gene Ther. 4:129-41 (1993)); and Grossman and
Wilson (Curr. Opin. Genet Dev. 3:110-14 (1993)) (the disclosures of
which are incorporated by reference herein).
[0163] 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), the disclosure of which is
incorporated by reference herein) present a review of
adenovirus-based gene therapy. Herman et al. (Human Gene Therapy
10:1239-49 (1999), the disclosure of which is incorporated by
reference herein) 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. (J.
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. Pat. 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)) (the
disclosures of which are incorporated by reference herein).
[0164] 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.
[0165] 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, 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); the disclosures of
which are incorporated by reference herein) 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.
[0166] 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.
[0167] 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.
[0168] 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 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. I:D782-86 (1999); Galderisi et al., J. Cell
Physiol. 181:251-57 (1999); Letsinger et al., Proc. Natl. Acad.
Sci. USA 86:6553-56 (1989); Lemaitre et al., 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., BioTechniques 6:958-76 (1988)) or intercalating agents
(see, e.g., Zon, Pharm. Res. 5:539-49 (1988)). (The disclosures of
these references are incorporated herein.)
[0169] 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-galactosylqueosi- ne, 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-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N-6-isopente- nyladenine, 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-methyl-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.
[0170] In yet another embodiment, the antisense oligonucleotides
comprise at least one modified phosphate backbone, such as, for
example, a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0171] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-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).
[0172] In a specific embodiment, the antisense oligonucleotide
comprises catalytic RNA, or a ribozyme (see, e.g., Welch et al.,
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 et al., 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)).
[0173] 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), the
disclosure of which is incorporated by reference herein.) 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. Some
enzymatic methods of DNA nucleic acid synthesis can require an
additional primer which can be synthesized 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)).
[0174] 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 et al. (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 et al.,
Proc. Natl. Acad. Sci. USA 85:7448-51 (1988)) (the disclosures of
which are incorporated by reference herein), and the like. Other
methods include those disclosed by Usman et al. (J. Am. Chem. Soc.
109:7845-54 (1987)), Scaringe et al. (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. Pat.
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. All of the foregoing
references are incorporated by reference herein.
[0175] Small Molecule Effectors or Antagonists
[0176] 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.
[0177] 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).
[0178] 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.
[0179] 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.
Chem. 37(9):1233-51 (1994)), Ohlmeyer et al. (Proc. Natl. Acad.
Sci. USA 90:10922-26 (1993)), Erb et al. (Proc. Nat. Acad. Sci. USA
91:11422-26 (1994)), Houghten et al. (BioTechniques 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. Natl. Acad. Sci. USA 89:5381-83 (1992)). (The
disclosures of these references are incorporated herein.)
[0180] 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. Immunol. Meth. 152:149-57 (1992)), Kay et al.
(Gene 128:59-65 (1993)), and International Patent Publication WO
94/18318, the disclosures of which are incorporated by reference
herein.
[0181] 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.
Sci. USA 91:4708-12 (1994)) can be adapted for use. Peptide
libraries (see, e.g., Simon et al., 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)).
[0182] 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 et al. (BioTechniques 13:422-28 (1992));
Oldenburg et al. (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. Pat. 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, the disclosures of
which are incorporated by reference herein.
[0183] 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.
[0184] Identifying a Subject with a Relaxin-Associated
Abnormality
[0185] 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.
[0186] 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" 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.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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 mRNA or
DNA.
[0193] 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, Calif. (1989)), ligase
chain reaction (see, e.g., EP 0 320 308), use of Q.beta. 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 container a predetermined amount of a purified
relaxin or relaxin receptor nucleic acid, for example, for use as a
standard or control.
[0194] The following examples are provided merely as illustrative
of various aspects of the invention and shall not be construed to
limit the invention in any way.
EXAMPLE 1
[0195] The effect of inactivation of one or both alleles of the
mouse RLX genes was examined. The following methods and materials
were followed.
[0196] Animals:
[0197] 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
+/+ (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, Calif.) 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.
[0198] Genotyping by PCR
[0199] Mouse DNA was isolated by lysing tail tissue (5-7
millimeters) in 400 .mu.l of PCR lysis buffer, containing 50 mM
Tris-HCl, pH 8.0, 0.5% SDS, 0.1 M EDTA and 1 mg/ml proteinase K
(Gibco BRL, Gaithersburg, Md.) at 50-55.degree. C. overnight.
Digested samples were then mixed with 3M sodium acetate (40 .mu.l),
buffer saturated phenol (200 .mu.l) and chloroform (200 .mu.l) 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 .mu.l) to precipitate the DNA before samples
were vortexed, spun and the supernatant discarded. The remaining
DNA pellet was dissolved in 40-50 .mu.l of sterile water. For PCR,
each DNA template (1 .mu.l) was used in a 30 .mu.l reaction mixture
containing PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 MM
MgCl.sub.2), 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 Endocrinology
140:445-53 (1999)). The amplification protocol consisted of an
initial denaturation step at 94.degree. C. (3 minutes) followed by
35 sequential cycles of 94.degree. C. (60 seconds), 55.degree. C.
(60 seconds) and 72.degree. C. (90 seconds) and concluded by an
additional 10 minute extension at 72.degree. C. 15 .mu.l 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).
[0200] Tissue Collection and Histology
[0201] Relaxin wildtype, heterozygous and homozygous males and
females (n .gtoreq.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 -/- 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.
[0202] The collected tissues were processed (sequentially
dehydrated) from 70% alcohol to paraffin before being embedded and
cut (4 .mu.m 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, Mich.) as described by the
manufacturer. The stained slides were viewed using a Zeiss
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
Photoshop (Adobe Systems Inc, Mountain View, Calif.).
[0203] Antibody Staining of Paraffin-Embedded Tissue Sections
[0204] 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.degree. C. (about 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, Calif.) 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 .mu.g/ml)(Santa Cruz
Biotechnology Inc), a caspase-9 polyclonal IgG antibody (4.5
.mu.g/ml) (Santa Cruz Biotech.) or a proliferating cell nuclear
antigen (PCNA)(Santa Cruz Biotech.) monoclonal IgG antibody (4
.mu.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.
[0205] Statistical Analysis:
[0206] 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
[0207] The Effects of Relaxin Gene Knockout on the Growth of
Mice:
[0208] The body weights of male and female relaxin wildtype (+/+),
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 +/+: 3.87.+-.0.09 g; rlx -/-: 3.6.+-.0.13 g) or female
(RLX +/+: 3.52.+-.0.09 g; rlx -/-: 3.37.+-.0.08 g) 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.+-.0.65 g) and females
(14.77.+-.0.42 g) were significantly less (p<0.05) than their
respective RLX wildtype counterparts (RLX +/+ M: 18.92.+-.0.61g;
RLX +/+ F: 16.34.+-.0.51 g). Male and female null mutant mice
continued to be significantly smaller (p<0.05) than RLX wildtype
animals at 2 months of age (RLX +/+ M: 25.42.+-.0.41 g; rlx -/- M:
24.23.+-.0.42 g; RLX +/+ F: 20.96.+-.0.32 g; rlx -/- F:
19.94.+-.0.22 g); 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:
26.57.+-.0.47 g; rlx -/- F: 22.54.+-.0.31 g) was still slightly
less than the average weight of adult RLX +/+ mice (RLX +/+ M:
27.30.+-.0.32 g; RLX +/+ F: 23.03.+-.0.71 g), 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 +/+ and RLX +/- animals, or between RLX +/- and rlx -/-
mice.
[0209] The Effects of Relaxin Gene Knockout of the Size of the Male
Reproductive Tract:
[0210] 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.
[0211] 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
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, epididyrnis, prostate and seminal vesicle from rlx -/-
mice were all smaller (p<0.05) than their respective
counterparts, derived from RLX +/+ mice.
1TABLE 1 The weight and size of the male reproductive tract from 1
week of age to adulthood (6.5 months). RLX +/+ % of Body rlx -/- %
of [Mean .+-. SE(n)] Weight [Mean .+-. SE(n)] Body Weight 1 Week of
Age: Weight (g): Overall 0.017 .+-. 0.001(10) 0.43 0.018 .+-.
0.002(15) 0.53 Size (Area; mm.sup.2): Testis 1.30 .+-. 0.30
(5).sup.1 -- 1.41 .+-. 0.23 (5).sup.1 -- 1 Month of Age: Weight
(g): Overall 0.37 .+-. 0.02(11) 1.81 0.33 .+-. 0.01 (11) 1.77 Size
(Area; mm.sup.2): Testis 16.36 .+-. 1.58 (9).sup.1 -- 12.91 .+-.
0.96 (8).sup.1 -- Epididymis 10.73 .+-. 2.22 (7).sup.2 -- 4.62 .+-.
0.64 (7).sup.2* -- Prostate 9.20 .+-. 1.20 (4).sup.3 -- 6.30 .+-.
0.54 (4).sup.3 -- Seminal Vesicle 1.50 .+-. 0.25 (4).sup.4 -- 1.69
.+-. 0.22 (4).sup.4 -- 3 Months of Age: Weight (g): Overall 0.92
.+-. 0.06(14) 3.37 0.62 .+-. 0.03 (11)* 2.33 Size (Area; mm.sup.2):
Testis 27.21 .+-. 1.52 (12).sup.1 -- 17.31 .+-. 2.28 (10).sup.1 --
Epididymis 23.62 .+-. 3.19 (11).sup.2 -- 13.71 .+-. 2.74(10).sup.2*
-- Prostate 20.89 .+-. 1.20 (6).sup.3 -- 15.03 .+-. 1.60 (6).sup.3*
-- Seminal Vesicle 25.75 .+-. 3.58 (7).sup.4 -- 14.6 .+-. 2.88
(5).sup.4* -- The size of each .sup.1testis and .sup.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 head.sup.2); seminal vesicle
(by measuring the length of each organ by the average width of the
tissue.sup.4), *denotes p < 0.05.
[0212] The Effects of Relaxin Gene Knockout on Histology of the
Male Reproductive Tract:
[0213] 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.
[0214] 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/i 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 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.
[0215] 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 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).
2TABLE 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.sup.a [0.23] 2.20 .+-. 0.32 (5) 2.60 .+-. 0.20
(5) [1] 2.92 .+-. 0.17 (9) 2.70 .+-. 0.13 (8) [3] 3.64 .+-. 0.08
(9) 2.86 .+-. 0.18 (10)* [6.5] 3.17 .+-. 0.17 (3) 3.08 .+-. 0.08
(3) Tubule compactness.sup.b [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 maturation.sup.c [0.23] 0 (5) 0 (5) [1] 1.78 .+-.
0.36 (9) 0.94 .+-. 0.20 (8) [3] 3.78 .+-. 0.15 (9) 2.20 .+-. 0.29
(10)* [6.5] 3.75 (3) 3.56 .+-. 0.12 (4) Epididymis: Tubule
size.sup.a [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 .+-. 0.13
Tubule compactness.sup.b [1] 3.16 .+-. 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.sup.c [1] 0.94 .+-. 0.06 (8)
0.75 .+-. 0.10 (8) [3] 3.56 .+-. 0.13 (9) 3.05 .+-. 0.17 (10) [6.5]
3.83 .+-. 0.08 (3) 3.63 .+-. 0.07 (4) Table 2: Tissue sections were
stained with H & E and graded as follows: .sup.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. .sup.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. .sup.cSperm 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.
[0216]
3TABLE 3 The effects of relaxin gene knockout on collagen
maturation, during murine growth and development Age RLX +/+ rlx
-/- Tissue [months] [Mean .+-. SE (n)] [Mean .+-. SE (n)]
Testis.sup.a: [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) Epididymis.sup.a: [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.sup.a: [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.sup.a: [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: .sup.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
[0217] Prostate: At 1 month of age, no significant differences were
noted in the structure of the prostate between RLX +/+ 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.
[0218] Detection of Cell Apoptosis by Antibody Staining:
[0219] 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.
[0220] Bax Antibody Staining: Overexpression of Bax accelerates
apoptotic death induced by cytokine deprivation and also counters
the death repressor activity of Bcl-2 (Krajewski et al., Am. J.
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., Calcif.
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 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.
[0221] 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 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.+-.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.
[0222] Detection of Cell Proliferation by Antibody Staining:
[0223] 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 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 +/+ 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.
[0224] The Effects of Relaxin Gene Knockout on Histology of Other
Body Tissues
[0225] 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.
[0226] 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.
[0227] 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.
[0228] 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 +/+ 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.
[0229] 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.
4TABLE 4 Tissue Comparison of 1 week old (from RLX +/+ & rlx
-/- parents, respectively), Represented as Mean .+-. SE (n): % %
body body RLX +/+ Male wt rlx -/- Male 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 % % body body RLX +/+ Female wt rlx -/-
Female 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
[0230]
5TABLE 5 Tissue Comparison 1 month old mice (from RLX+/- parents),
Represented as Mean .+-. SE (n): % % body body RLX +/+ Male wt rlx
-/- Male 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 .+-. 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 % % body body RLX +/+
Female wt rlx -/- Female 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
[0231]
6TABLE 6 Tissue Comparison of 3 month old male mice (from RLX +/+
& rlx -/- parents, respectively), Represented as Mean .+-. SE
(n): % % body body RLX +/+ Male wt rlx -/- Male 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 .+-. 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 0.0.22 .+-. 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 .+-. 0.003 (10) 0.19 % % body body RLX +/+ Female wt rlx -/-
Female 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 .+-. 0.003 (9) 0.29 *=> p < 0.05
[0232]
7TABLE 7 Tissue Comparison of 1 month old mice (from RLX+/+ and
RLX-/- parents, respectively), represented as Mean .+-. SE (n): %
body % body RLX +/+ Male wt rlx -/- Male 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 .+-. 0.01 (11) 0.68 0.10
.+-. 0.01 (4) 0.65 R. Kidney (g) 0.13 .+-. 0.01 (10) 0.67 0.10 .+-.
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 .+-. 0.03 (11) 2.40 0.56 .+-. 0.07 (4) 3.78 Lung
(g) 0.19 .+-. 0.01 (11) 0.97 0.16 .+-. 0.01 (4) 1.06 % body % body
RLX +/+ Female wt rlx -/- Female 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
[0233]
8TABLE 8 Tissue Comparison of 3 month old mice (from RLX+/+ and
RLX-/- parents, respectively), represented as Mean .+-. SE (n): %
body % body RLX +/+ Male wt rlx -/- Male 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) % body % body RLX +/+ Female wt rlx -/- Female 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
[0234] The Effects of Relaxin Gene Knockout on the Histology of
Skin
[0235] 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 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.
[0236] 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 Rlx +/+ mice by one month of
age. Examination of serum chemistries, hematological parameters and
urine from the rlx-null mice were unremarkable.
[0237] 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
picogram 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.
[0238] Rlx-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.
[0239] The previous examples are provided to illustrate but not to
limit the scope of the claimed inventions. Other variants of the
inventions will be readily apparent to those of ordinary skill in
the art and encompassed by the appended claims. All publications,
patents, patent applications and other references cited herein are
hereby incorporated by reference.
* * * * *