U.S. patent application number 10/491919 was filed with the patent office on 2005-02-03 for human 3 relaxin.
This patent application is currently assigned to AstraZeneca AB. Invention is credited to Bathgate, Ross Alexander David, Burazin, Tanya Christine, Gundlach, Andrew Lawrence, Samuel, Chrishan Surendran, Tregear, Geoffrey, Wade, John Desmond.
Application Number | 20050026822 10/491919 |
Document ID | / |
Family ID | 3831972 |
Filed Date | 2005-02-03 |
United States Patent
Application |
20050026822 |
Kind Code |
A1 |
Tregear, Geoffrey ; et
al. |
February 3, 2005 |
Human 3 relaxin
Abstract
Human H3 preprorelaxin, human H3 prorelaxin, human H3 relaxin,
human relaxin analogues having a modified A chain and/or a modified
B chain are described. Also described are nucleic acid sequences
encoded human H3 preprorelaxin, human H3 prorelaxin, human H3
relaxin, human relaxin analogues. Also described are methods for
the treatment of conditions responsive to administration of H3
relaxin or analogues thereof.
Inventors: |
Tregear, Geoffrey; (Hawthorn
Victoria, AU) ; Bathgate, Ross Alexander David; (West
Victoria, AU) ; Samuel, Chrishan Surendran; (Waverley
Victoria, AU) ; Burazin, Tanya Christine; (Waverley
Victoria, AU) ; Gundlach, Andrew Lawrence; (Hawthorn
Victoria, AU) ; Wade, John Desmond; (Canterbury
Victoria, AU) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
AstraZeneca AB
R & D HEADQUARTERS GLOBAL INTELLECTUAL PROPERTY
PATENTS
SODERTALJE
SE
15185
|
Family ID: |
3831972 |
Appl. No.: |
10/491919 |
Filed: |
September 29, 2004 |
PCT Filed: |
October 2, 2002 |
PCT NO: |
PCT/AU02/01338 |
Current U.S.
Class: |
514/4.7 ;
514/10.9; 514/12.7; 514/15.4; 514/15.7; 514/18.2; 514/19.3;
514/19.6 |
Current CPC
Class: |
A61P 25/18 20180101;
A61P 25/22 20180101; A61P 17/00 20180101; A61P 17/14 20180101; A61P
9/14 20180101; A61P 29/00 20180101; A61P 5/00 20180101; A61P 9/06
20180101; A61K 38/2221 20130101; A61P 15/06 20180101; A61P 9/08
20180101; A61P 25/28 20180101; A61P 27/02 20180101; A61P 25/30
20180101; A61P 37/00 20180101; C07K 14/64 20130101; A61P 43/00
20180101; A61P 7/00 20180101; A61P 25/32 20180101; A61P 17/02
20180101; A61P 25/00 20180101; A61P 5/38 20180101; A61P 15/00
20180101; A61P 35/00 20180101; A61P 39/02 20180101; A61P 25/24
20180101; A61P 5/24 20180101; A61P 13/12 20180101; A61P 3/00
20180101; A61P 1/00 20180101; A61P 25/14 20180101; A61P 9/00
20180101; A61P 15/04 20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 8, 2001 |
AU |
PR 8144 |
Claims
1. A method for the treatment of one or more of: vascular disease;
treatment of arterial hypertension; diseases related to
uncontrolled or abnormal collagen or fibronectin formation; kidney
disease; psychiatric disorders; depression or depressive disorders;
neurologic or neurodegenerative diseases; disorders of learning,
attention and motivation; addictive disorders; movement and
locomotor disorders; immunological disorders; breast disorders;
endometrial disorders; endocrine disorders; delayed onset of
labour, impaired cervical ripening, and prevention of prolonged
labour due to fetal dystocia; sinus bradycardia; hair loss;
alopecia; disorders of water balance including impaired or
inappropriate secretion of vasopressin; or placental insufficiency;
which comprises administering to a subject in need of any such
treatments a therapeutically effective amount of human H3 relaxin,
or an analogue thereof as herein defined, optionally in association
with one or more pharmaceutically acceptable carriers and/ diluents
and/or excipients.
2. A method according to claim 1 wherein the H3 relaxin or analogue
thereof is human H3 relaxin, human H3 prorelaxin, human H3
preprorelaxin, or the constitutive A, B or C peptide chains
thereof.
3. A method according to claim 2 wherein the human H3 relaxin or a
human H3 relaxin analogue thereof comprises an A chain and a B
chain, the A chain having the amino acid sequence:
31 Asp Val Leu Ala Gly Leu Ser Ser (SEQ ID NO: 4) 1 5 Ser Cys Cys
Lys Trp Gly Cys Ser 10 15 Lys Ser Glu Ile Ser Ser Leu Cys 20
or an amino acid sequence truncated by up to about 9 amino acids
from N-terminus, the B chain having the amino sequence:
32 Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly Arg Glu Phe Ile Arg
1 5 10 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg Trp 20 25
or an amino acid sequence truncated by up to 9 amino acids from the
amino-terminus and/or up to about 5 amino acids from the
carboxyl-terminus, the A and B chains being linked by disulphide
bonds between A11-B10 and A24-B22, and wherein the human H3 relaxin
or analogue thereof has relaxin bioactivity.
4. A method according to claim 1 wherein a human H3 relaxin
analogue comprises a modified A chain and/or a modified B chain,
the H3 relaxin A chain having the amino acid sequence:
33 Asp Val Leu Ala Gly Leu Ser Ser Ser Cys Cys Lys Trp Gly Cys Ser
(SEQ ID NO: 4) 1 5 10 15 Lys Ser Glu Ile Ser Ser Leu Cys 20
wherein the carboxyl-terminus is an amide derivative and/or Lys at
position 12 is replaced with Glu, and/or Glu at position 19 is
replaced with Gln, the H3 relaxin B chain having the amino acid
sequence:
34 Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly Arg Glu Phe Ile Arg
(SEQ ID NO: 2) 1 5 10 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg
Trp 20 25
wherein the carboxyl-terminus is an amide derivative, and/or Ala at
position 2 is replaced with Pro, and/or Arg at position 8 is
replaced with Lys, the A and B chains being linked by disulphide
bonds between A11-B10 and A24-B22, and wherein the human H3 relaxin
analogue has relaxin bioactivity.
5. A method according to claim 1 which is a method for the
treatment of arterial hypertension.
6. A method according to claim 1 which is a method for the
treatment of peripheral vascular disease including coronary artery
disease, peripheral vascular disease, vasospasm including Raynaud's
phenomenon, microvascular disease involving the central and
peripheral nervous system, kidney, eye and other organs.
7. A method according to claim 1 which is a method for the
treatment of kidney disease including vascular disease,
interstitial fibrosis, glomerulosclerosis, or other kidney
disorders.
8. A method according to claim 1 which is a method for the
treatment of psychiatric disorders including anxiety states
including panic attack, agoraphobia, global anxiety, phobic
states.
9. A method according to claim 1 which is a method for the
treatment of depression or depressive disorders including major
depression, dysthymia, bipolar an dunipolar depression; neurologic
or neurodegenerative diseases (including memory loss or other
memory disorders, dementias, Alzheimer's disease).
10. A method according to claim 1 which is a method for the
treatment of disorders of learning, attention and motivation
including Attention Deficit Hyperacitvity Disorder, Tourette's
disease, impulsivity, antisocial and personality disorders,
negative symptoms of psyochoses including those due to
schizophrenia, acquired brain damage and frontal lobe lesions).
11. A method according to claim 1 which is a method for the
treatment of hair loss including drug, alcohol and nicotine
addiction.
12. A method according to claim 1 which is a method for the
treatment of neurologic or neurodegenerative diseases including
memory loss or other memory disorders, dementias, Alzheimer's
disease.
13. A method according to claim 1 which is a method for the
treatment of movement and locomotor disorders including Parkinson's
disease, Huntington's disease, and motor deficits after stoke, head
injury, surgery, tumour or spinal cord injury.
14. A method according to claim 1 which is a method for the
treatment of diseases related to uncontrolled or abnormal collagen
or fibronectin formation including fibrosis of lung, heart and
cardiovascular system, kidney and genitourinary tract,
gastrointestinal system, cutaneous, rheumatologic and hepatobiliary
systems.
15. A method according to claim 1 which is a method for the
treatment of delayed onset of labour, impaired cervical ripening,
and prevention of prolonged labour due to fetal dystocia.
16. A method according to claim 1 which is a method for the
treatment of endocrine disorders including adrenal, ovarian and
testicular disorders related to steroid or peptide hormone
production.
17. A method according to claim 1 which is a method for the
treatment of breast disorders including fibrocystic disease,
impaired lactation, and cancer.
18. A method according to claim 1 which is a method for the
treatment of immunological disorders including immune deficiency
states, haematological and reticuloendothelial malignancy.
19. A method according to claim 1 which is a method for the
treatment of endometrial disorders including infertility due to
impaired implantation.
20. A method according to claim 1 which is a method for the
treatment of endocrine disorders including adrenal disorders,
ovarian disorders, and testicular disorders related to steroid or
peptide hormone production.
21. A method according to claim 1 which is a method for the
treatment of diseases associated with water balance including
impaired or inappropriate secretion of vasopressin.
22. A method according to claim 1 which is a method for the
treatment of placental insufficiency.
23-44. Canceled.
Description
FIELD OF THE INVENTION
[0001] This invention relates to human 3 relaxin (hereafter
referred to as "H3 relaxin"). More specifically, the invention
relates to H3 relaxin, pro- and prepro-H3 relaxin, the individual
peptide chains which comprise these sequences, analogues of H3
relaxin, compositions including pharmaceutical compositions, as
well as therapeutic uses and methods of treatment. Further, the
invention relates to nucleic acids encoding H3 relaxin, H3 pro- and
prepro-relaxin, H3 relaxin analogues, and individual peptide chains
which comprise these sequences.
BACKGROUND OF THE INVENTION
[0002] Pioneering work by Hisaw 1926 first suggested an important
role for the peptide hormone relaxin in animals through its effect
in dilating the pubic symphsis, thus facilitating the birth
process. Relaxin is synthesised in the corpora lutea of ovaries
during pregnancy, and is released into the blood stream prior to
parturition. The availability of ovarian tissue has enabled the
isolation and amino acid sequence determination of relaxin from the
pig (James et al (1977), Nature, 267, 554-546), the rat (John et al
(1981) Endocrinology 108, 726-729), and the shark (Schwabe et al
(1982) Ann. N.Y. Acad. Sci. 380, 6-12).
[0003] Relaxin genes and the encoded relaxin polypeptides have been
identified in many species including man, pig, rat, sheep and
shark. In all these species only one relaxin gene has been
characterised in mammals, -with the exception of the human and
higher primates where two separate genes have been described. The
separate human genes were identified by the present applicant and
designated H1 (Hudson et al (1983) Nature 301, 628-631) and H2
(Hudson et al (1984) Embo. J. 3, 2333-2339).
[0004] The peptide encoded by the H2 gene is the major stored and
circulating form in the human (Winslow et al (1992) Endrocrinology
130, 2660-2668). Hi relaxin expression is restricted to the
decidua, placenta and prostate (Hansell et al (1991) J. Clin.
Endocrinol. Metab. 72, 899-904), however, the H1 peptide has
similar biological activity to that of H2 relaxin in a rat atrial
bioassay (Tan et al (1998) Br. J. Pharmacol. 123, 762-770).
[0005] The actions of relaxin include an ability to inhibit
myometrial contractions, to stimulate remodelling of connective
tissue and to induce softening of the tissues of the birth canal.
Additionally, relaxin increases growth and differentiation of the
mammary gland and nipple and induces the breakdown of collagen, one
of the main components of connective tissue. Relaxin decreases
collagen synthesis and increases the release of collagenases
(Unemori et al (1990) J. Biol. Chem. 265, 10682-10685). These
findings were recently confirmed by the establishment of the
relaxin gene-knockout mouse (Zhao et al (1999) Endocrinology 140,
445-453), which exhibited a number of phenotypic properties
associated with pregnancy. Female mice lacking a functionally
active relaxin gene failed to relax and elongate the interpubic
ligament of the pubic symphysis and could not suckle their pups,
which in turn, died within 24 hours unless cross-fostered to
relaxin wildtype or relaxin heterozygous foster mothers.
[0006] Evidence has accumulated to suggest that relaxin is more
than a hormone of pregnancy and acts on cells and tissues other
than those of the female reproductive system. Relaxin causes a
widening of blood vessels (vasodilatation) in the kidney,
mesocaecum, lung and peripheral vasculature, which leads to
increased blood flow or perfusion rates in these tissues (Bani et
al (1997) Gen. Pharmacol. 28, 13-22). It also stimulates an
increase in heart rate and coronary blood flow, and increases both
glomerular filtration rate and renal plasma flow (Bani et al (1997)
Gen. Pharmacol. 28, 13-22). The brain is another target tissue for
relaxin where the peptide has been shown to bind to receptors
(Osheroff et al (1991) Proc. Nal. Acad. Sci. U.S.A. 88, 6413-6417;
Tan et al (1999) Br. J. Pharmacol 127, 91-98) in the
circumventricular organs to affect blood pressure and drinking
(Parry et al (1990) J Neuroendocrinol. 2, 53-58; Summerlee et al
(1998) Endocrinology 139, 2322-2328; Sinnahay et al (1999)
Endocrinology 140, 5082-5086).
[0007] Important clinical uses arise for relaxin in various
diseases responding to vasodilation, such as coronary artery
disease, peripheral vascular disease, kidney disease associated
with arteriosclerosis or other narrowing of kidney capillaries, or
other capillaries narrowing in the body, such as in the eyes or in
the peripheral digits, the mesocaecum, lung and peripheral
vasculature.
[0008] The finding of two human relaxin genes, and encoded human
relaxin peptide products nearly 20 years ago was of itself most
surprising.
[0009] Even more surprisingly with the benefit of nearly 20 years
of further research and development in relaxin biology
internationally, the applicant has identified, isolated and
characterised nucleic acid sequences encoding a third human relaxin
gene (H3), the encoded H3 relaxin peptide and the constituent
peptide chains thereof. The production of H3 relaxin and analogues
thereof has been made possible, as have uses and therapeutic
treatment methods.
SUMMARY OF THE INVENTION
[0010] In a first aspect the invention relates to the peptides
human H3 relaxin, H3 prorelaxin and H3 preprorelaxin, to the
individual peptide chains which comprise these sequences and to
analogues thereof, particularly truncated and/or amino acid
substituted modifications. Preferably the peptides are provided as
pharmaceutically acceptable compositions for human or animal
administration, by various therapeutic routes. Peptides are
preferably isolated in purified or homogenous form free of
contaminating peptides and proteins, or in a form of about 90-99%
purity.
[0011] In a second aspect of the invention there is provided a
composition comprising human H3 relaxin or a human H3 relaxin
analogue having an A chain and a B chain,
[0012] the A chain having the amino acid sequence:
1 Asp Val Leu Ala Gly Leu Ser Ser Ser Cys Cys Lys Trp Gly Cys Ser
(SEQ ID NO: 4) 1 5 10 15 Lys Ser Glu Ile Ser Ser Leu Cys 20
[0013] or an amino acid sequence truncated by up to about 9 amino
acids from N-terminus,
[0014] the B chain having the amino sequence:
2 Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly Arg Glu Phe Ile Arg
(SEQ ID NO: 2) 1 5 10 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg
Trp 20 25
[0015] or an amino acid sequence truncated by up to 9 amino acids
from the amino-terminus and/or up to about 5 amino acids from the
carboxyl-terminus,
[0016] the A and B chains being linked by interchain disulphide
bonds at A11-B10, and A24-B22, and wherein the human H3 relaxin or
analogue thereof has relaxin bioactivity.
[0017] In a third aspect of the invention there is provided a
composition comprising a human H3 relaxin analogue having a
modified A chain and/or a modified B chain,
[0018] the H3 relaxin A chain having the amino acid sequence:
3 Asp Val Leu Ala Gly Leu Ser Ser Ser Cys Cys Lys Trp Gly Cys Ser
(SEQ ID NO: 4) 1 5 10 15 Lys Ser Glu Ile Ser Ser Leu Cys 20
[0019] wherein the carboxyl-terminus is an amide derivative and/or
Lys at position 12 is replaced with Glu, and/or Glu at position 19
is replaced with Gln,
[0020] the H3 relaxin B chain having the amino acid sequence:
4 Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly Arg Glu Phe Ile Arg
(SEQ ID NO: 2) 1 5 10 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg
Trp 20 25
[0021] wherein the carboxyl-terminus is an amide derivative,.and/
or Ala at position 2 is replaced with Pro, and/or Arg at position 8
is replaced with Lys,
[0022] the A and B chains being linked by disulphide bonds between
A11-B10 and A24-B22 and wherein the human H3 relaxin analogue has
relaxin bioactivity.
[0023] In accordance with a fourth aspect of the invention there is
provided a composition comprising human H3 preprorelaxin or human
H3 prorelaxin, having a signal, A chain, B chain and C chain in
respect of human H3 preprorelaxin, and an A chain, B chain and C
chain in respect of human H3 prorelaxin, the said amino acid chains
having the amino acid sequences:
[0024] the A chain comprising:
5 Asp Val Leu Ala Gly Leu Ser Ser Ser Cys Cys Lys Trp Gly Cys Ser
(SEQ ID NO: 4) 1 5 10 15 Lys Ser Glu Ile Ser Ser Leu Cys 20
[0025] the B chain comprising:
6 Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly Arg Glu Phe Ile Arg
(SEQ ID NO: 2) 1 5 10 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg
Trp 20 25
[0026] the signal sequence comprising:
7 Met Ala Arg Tyr Met Leu Leu Leu Leu Leu Ala Val Trp Val Leu Thr
(SEQ ID NO: 1) 1 5 10 15 Gly Glu Leu Trp Pro Gly Ala Glu Ala 20
25
[0027] and the C chain comprising:
8 Arg Arg Ser Asp Ile Leu Ala His Glu Ala Met Gly Asp Thr Phe Pro
(SEQ ID NO: 3) 1 5 10 15 Asp Ala Asp Ala Asp Glu Asp Ser Leu Ala
Gly Glu Leu Asp Glu Ala 20 25 30 Met Gly Ser Ser Glu Trp Leu Ala
Leu Thr Lys Ser Pro Gln Ala Phe 35 40 45 Tyr Arg Gly Arg Pro Ser
Trp Gln Gly Thr Pro Gly Val Leu Arg Gly 50 55 60 Ser Arg 65
[0028] In accordance with a fifth aspect of the invention there is
provided a composition comprising the C chain of human H3 relaxin,
the C chain having the amino acid sequence:
9 Arg Arg Ser Asp Ile Leu Ala His Glu Ala Met Gly Asp Thr Phe Pro
(SEQ ID NO: 3) 1 5 10 15 Asp Ala Asp Ala Asp Glu Asp Ser Leu Ala
Gly Glu Leu Asp Glu Ala 20 25 30 Met Gly Ser Ser Glu Trp Leu Ala
Leu Thr Lys Ser Pro Gln Ala Phe 35 40 45 Tyr Arg Gly Arg Pro Ser
Trp Gln Gly Thr Pro Gly Val Leu Arg Gly 50 55 60 Ser Arg 65
[0029] In accordance with a sixth aspect of the invention there is
provided a nucleic acid sequence encoding human prepro-H3 relaxin
comprising the nucleic acid sequence:
10 tataaatggg gggccaagag gcagcagaga cactggccca ctctcacgtt
caaagcgtct 60 (SEQ ID NO: 6) ccgtccagca tggccaggta catgctgctg
ctgctcctgg cggtatgggt gctgaccggg 120 gagctgtggc cgggagctga
ggcccgggca gcgccttacg gggtcaggct ttgcggccga 180 gaattcatcc
gagcagtcat cttcacctgc gggggctccc ggtggagacg atcagacatc 240
ctggcccacg aggctatggg tgaggctggg gagagagtgg atgtagaagg ggaacaggtg
300 gctggatggg tcccaggagc taaggacaga gataagagga ggttgctgga
ggaggagggt 360 ccctgtcctg ccacattcag ccagggacac ctgcccagcc
ttgaaacaag ggctcaggag 420 ttagcagagc tgcagagctg ggatggggtg
ttgcaagcca tccatggggg ctggaagtct 480 gaggacaggt gggggcgggg
agcgtgccat ttgcaaagac aacaccgaag tgttttccaa 540 ccctttccag
caggtaatgt gaagggtgtg gtatacacat agctgggttt gtcacctaat 600
gcatgacctc tccccagcaa gttggttttt cttccgtctc tgagtgtctt ttttttggag
660 atgtggtctc actccattgc ccaggcttga atgcagtggc ccaatcactg
ctcattgcag 720 cctcgacctc ccaggctcaa gtgattctcc tgcctccgcc
tccagagtag ttgagaccac 780 aggcacctga caccatgcct ggctagtttt
aaattttttt tttgtagaaa caggggtctc 840 actatgttgc ctaggctggt
ctcgaactcc tgggctcaag tgatcctccc acctcggcct 900 ccctaagtgc
tgagattaga gtctctgagt gtctttatct tcaaatggga gacacagttc 960
ctgaatcttg caggattaag tggtatgatt aaatcaaaac agattagggc agagtctcag
1020 cagggcagcg gcacaatctg ggatccatca ggagagtcag agggaacaga
agacctagct 1080 tcatgagggg cagggacctg gcaaatagat attcatgatg
gtgagaagga ggataggtat 1140 gagcgtggac atagaagaca caccacttgg
attcagatag tagctctaca atgtaatagt 1200 tgtgtgttca tgtgctacta
tttttttttt ttttgagaca gaatctcatt ctgttgccca 1260 ggctggagtg
cagtggtgca atcttggctc actgtaacct ccatcacctg ggttcaagcg 1320
attctcgtgc ctccagcctc ccaagtagct gggattacag atgtgtgcca ccatacctcg
1380 ctaatctttt tatttttagt agagacagtt tcaccatgtt ggccaggctg
gtctccaact 1440 cctgacctca ggtgatcctc ccacctcagc ctcccaaagt
gctgggatta caggcatgag 1500 ccaccgcgcc cagccatgca aattctttac
tgagtcctgc ctcagtggtc tcctctggaa 1560 aatacgggtg ataactgcac
ccacctcaac tggttatcac tgagaagaat aaagaagtta 1620 acctgctaaa
gcacttaaaa cgttgtttga cacaaagtaa gtgatcaata aattattatt 1680
attattatta ttattattat tattattatt tttgagacag ggtcttgctc tgttgcccag
1740 actggagtgc agtggtgtga tcacagctca ctgcagcttc aacctcttgg
gctcaagcaa 1800 ttctcctgcc tcagcctcct gagtagctgg gactacaggc
ttgtgccaac atgtctaact 1860 ttttattatt tgtagagaca gggtagtgct
gtgttgtcca ggctgttctt gaactcctgg 1920 ttctggtgat cctccagcat
gtgcccctgg aagtgctggg attacaggtg tgagacaccg 1980 tgcccggact
caatagtcat ttttgagtgc tcatcatgtt ccagacattg ttctaagttt 2040
ttttttttaa tgaatattaa ctccttataa aacttgagaa ggttggagta attatttttt
2100 tccactttgc agaaaagaac attgaggctc caagaagtaa atttacttgc
tcacgattag 2160 agaagctgga ttcatgctca gtcagcccag ctcccaaatg
taccaggtcc tcaattaata 2220 aagagtaagg agaaataaat gacagggctg
ggtgcggtgg ctcacgcctg taatcccagc 2280 actttgggtg gctgaggtgg
gcacatcact tgaggtcagg agtttgcgac cagcctgaac 2340 aacatggtga
accccatctc tataacaata caaaaatcag ccaggcctgc tggcagacac 2400
ctgtaatccc acctactctg gcagagccag aatttgaacc caggactggg tggaataaaa
2460 actctgaact atgtctatga ctgttgtcac aagatcagag ctagactggc
caggagccat 2520 gactgtgggt gcagcagcag ctgagccctg atcactaact
ctgttcatct tttgcaggag 2580 ataccttccc ggatgcagat gctgatgaag
acagtctggc aggcgagctg gatgaggcca 2640 tggggtccag cgagtggctg
gccctgacca agtcacccca ggccttttac agggggcgac 2700 ccagctggca
aggaacccct ggggttcttc ggggcagccg agatgtcctg gctggccttt 2760
ccagcagctg ctgcaagtgg gggtgtagca aaagtgaaat cagtagcctt tgctagtttg
2820 agggctgggc agccgtgggc accaggacca atgccccagt cctgccatcc
actcaactag 2880 tgtctggctg ggcacctgtc tttcgagcct cacacattca
ttcattcatc tacaagtcac 2940 agaggcactg tgggctcagg cacagtctcc
cgacaccacc tatccaaccc tgccctttga 3000 ccagcctatc atgaccctgg
cccctaagga agctgtgccc ctgcctggtc aagtggggac 3060 ccccccatcc
tgacccctga cctctcccca gccctaacca tgcgtttgcc tggcctacac 3120
actccactgc cacaactggg tccctactct acctaggctg gccacacaga gacccctgcc
3180 cccttcccag tccaaactgt ggccattgtc ccctgaccag ctaaaatcaa
gcctctgtct 3240 cagtccagcc tttgcacgca cgcttccttt gccctgcttt
ccatcccctc tccctccaac 3300 tcccctgcca gagttccaag gctgtggacc
ccagagaagg tggcaggtgg cccccctagg 3360 agagctctgg gcacattcga
atcttcccaa actccaataa taaaaattcg aagactttgg 3420 cagagagtgt
gtgtgtgtgt gtatggttg 3449
[0030] In accordance with a seventh aspect of the invention there
is provided a nucleic acid sequence encoding human pro-H3 relaxin
including an A chain, B chain and C chain sequence,
[0031] the A chain sequence comprising:
11 gatgtcctgg ctggcctttc cagcagctgc 60 (SEQ ID NO: 7) tgcaagtggg
ggtgtagcaa aagtgaaatc agtagccttt gc 72
[0032] the B chain sequence comprising:
12 cgggcagcgc cttacggggt caggctttgc 60 (SEQ ID NO: 8) ggccgagaat
tcatccgagc agtcatcttc acctgcgggg gctcccggtg g 81
[0033] the C chain sequence comprising:
13 agacgatcag acatcctggc ccacgaggct 60 (SEQ ID NO: 9) atgggagata
ccttcccgga tgcagatgct gatgaagaca gtctggcagg cgagctggat 120
gaggccatgg ggtccagcga gtggctggcc ctgaccaagt caccccaggc cttttacagg
180 gggcgaccca gctggcaagg aacccctggg gttcttcggg gcagccga 198
[0034] In an eighth aspect of the invention there is provided a
nucleic acid sequence encoding human H3 relaxin having an A and B
chain,
[0035] the A chain sequence comprising:
14 gatgtcctgg ctggcctttc cagcagctgc 60 (SEQ ID NO: 7) tgcaagtggg
ggtgtagcaa aagtgaaatc agtagccttt gc 72
[0036] and the B chain sequence comprising:
15 cgggcagcgc cttacggggt caggctttgc 60 (SEQ ID NO: 8) ggccgagaat
tcatccgagc agtcatcttc acctgcgggg gctcccggtg g 81
[0037] In a ninth aspect of the invention there is provided a
nucleic acid sequence encoding the A, B or C peptide chains of
human H3 relaxin, the said chains comprising the nucleic acid
sequences:
[0038] A chain:
16 gatgtcctgg ctggcctttc cagcagctgc 60 (SEQ ID NO: 7) tgcaagtggg
ggtgtagcaa aagtgaaatc agtagccttt gc 72
[0039] B chain:
17 cgggcagcgc cttacggggt caggctttgc 60 (SEQ ID NO: 8) ggccgagaat
tcatccgagc agtcatcttc acctgcgggg gctcccggtg g 81
[0040] and C chain:
18 agacgatcag acatcctggc ccacgaggct 60 (SEQ ID NO: 9) atgggagata
ccttcccgga tgcagatgct gatgaagaca gtctggcagg cgagctggat 120
gaggccatgg ggtccagcga gtggctggcc ctgaccaagt caccccaggc cttttacagg
180 gggcgaccca gctggcaagg aacccctggg gttcttcggg gcagccga 198
[0041] The nucleic acid sequences are isolated and purified nucleic
acids, and may be contained within a vector, such as a plasmid,
bacteriophage or virus DNA or RNA, and may be in single or double
stranded form, and may include promoters or enhancers or other
sequences which confer elevated, enhanced or other effects on
expression in a host system such as a bacterial cell.
[0042] The triplet codons of nucleic acids encode specific amino
acids. More than one codon may encode the same amino acid, as is
well and established in the art. Moreover, methods of modifying or
altering the sequence of nucleic acids are well known in the art.
Insofar as this invention pertains in its various aspects to
nucleic acids encoding human H3 relaxin, pro-H3 relaxin, prepro-H3
relaxin, and constituent peptide chains thereof, the invention
includes nucleic acid variants which encode the same protein
products, or a protein product having relaxin activity.
[0043] Nucleotide sequence aspects of this invention also include
closely related nucleic acid sequences as defined by stringent
hybridization, this being annealing of complimentary sequences
under conditions of 0.25M H.sub.2PO.sub.4, pH 7.2, 1 mmol EDTA, 20%
SDS at 65.degree. C. overnight; followed by 3 washes for 5 min in
2.times.SSC, 0.1% SDS at room temperature; and finally a 30 min
wash at 65.degree. C. in 0.1% SSC; where 6.times.SSC is 0.9M NaCl,
0.3M Na.sub.3CO.sub.2 H.sub.2O at ph 7.0. Such sequences will
encode H3 relaxin polypeptides having biological or immunological
or other activity corresponding to those of H3 relaxin.
[0044] In another aspect of the invention there is provided a
method for the treatment of one or more of: vascular disease
including coronary artery disease, peripheral vascular disease,
vasospasm including Raynaud's phenomenon, microvascular disease
involving the central and peripheral nervous system, kidney, eye
and other organs; treatment of arterial hypertension; diseases
related to uncontrolled or abnormal collagen or fibronectin
formation such as fibrotic disorders (including fibrosis of lung,
heart and cardiovascular system, kidney and genitourinary tract,
gastrointestinal system, cutaneous, rheumatologic and hepatobiliary
systems); kidney disease associated with vascular disease,
interstitial fibrosis, glomerulosclerosis, or other kidney
disorders; psychiatric disorders including anxiety states including
panic attack, agoraphobia, global anxiety, phobic states;
depression or depressive disorders including major depression,
dysthymia, bipolar and unipolar depression; neurologic or
neurodegenerative diseases (including memory loss or other memory
disorders, dementias, Alzheimer's disease); disorders of learning,
attention and motivation (including Attention Deficit Hyperactivity
Disorder, Tourette's disease, impulsivity, antisocial and
personality disorders, negative symptoms of psychoses including
those due to schizophrenia, acquired brain damage and frontal lobe
lesions); addictive disorders (including drug, alcohol and nicotine
addiction); movement and locomotor disorders (including Parkinson's
disease, Huntington's disease, and motor deficits after stoke, head
injury, surgery, tumour or spinal cord injury); immunological
disorders( including immune deficiency states, haematological and
reticuloendothelial malignancy, breast disorders (including
fibrocystic disease, impaired lactation, and cancer); endometrial
disorders including infertility due to impaired implantation;
endocrine disorders (including adrenal, ovarian and testicular
disorders related to steroid or peptide hormone production) ;
delayed onset of labour, impaired cervical ripening, and prevention
of prolonged labour due to fetal dystocia; sinus bradycardia; hair
loss, alopecia; disorders of water balance including impaired or
inappropriate secretion of vasopressin; placental insufficiency;
which comprises administering to a subject in need of any such
treatments a therapeutically effective amount of human H3 relaxin,
or an analogue thereof as herein defined, optionally in association
with one or more pharmaceutically acceptable carriers and/ diluents
and/or excipients.
[0045] In another aspect of the invention there is provided use of
human H3 relaxin or an analogue thereof in the manufacture of
medicaments for the treatment of one or more of: vascular disease
including coronary artery disease, peripheral vascular disease,
vasospasm including Raynaud's phenomenon, microvascular disease
involving the central and peripheral nervous system, kidney, eye
and other organs; treatment of arterial hypertension; diseases
related to uncontrolled or abnormal collagen or fibronectin
formation such as fibrotic disorders (including fibrosis of lung,
heart and cardiovascular system, kidney-and genitourinary tract,
gastrointestinal system, cutaneous, rheumatologic and hepatobiliary
systems); kidney disease associated with vascular disease,
interstitial fibrosis, glomerulosclerosis, or other kidney
disorders; psychiatric disorders including anxiety states including
panic attack, agoraphobia, global anxiety, phobic states;
depression or depressive disorders including major depression,
dysthymia, bipolar and unipolar depression; neurologic or
neurodegenerative diseases (including memory loss or other memory
disorders, dementias, Alzheimer's disease); disorders of learning,
attention and motivation (including Attention Deficit Hyperactivity
Disorder, Tourette's disease, impulsivity, antisocial and
personality disorders, negative symptoms of psychoses including
those due to schizophrenia, acquired brain damage and frontal lobe
lesions); addictive disorders (including drug, alcohol and nicotine
addiction); movement and locomotor disorders (including Parkinson's
disease, Huntington's disease, and motor deficits after stoke, head
injury, surgery, tumour or spinal cord injury); immunological
disorders(including immune deficiency states, haematological and
reticuloendothelial malignancy, breast disorders (including
fibrocystic disease, impaired lactation, and cancer); endometrial
disorders including infertility due to impaired implantation;
endocrine disorders (including adrenal, ovarian and testicular
disorders related to steroid or peptide hormone production);
delayed onset of labour, impaired cervical ripening, and prevention
of prolonged labour due to fetal dystocia; sinus bradycardia; hair
loss, alopecia; disorders of water balance including impaired or
inappropriate secretion of vasopressin; placental insufficiency;
which comprises administering to a subject in need of any such
treatments a therapeutically effective amount of human H3 relaxin,
or an analogue thereof as herein defined, optionally in association
with one or more pharmaceutically acceptable carriers and/ diluents
and/or excipients.
19 Sequence Listing Table SEQ ID NO: 1 Signal peptide sequence SEQ
ID NO: 2 B chain peptide sequence SEQ ID NO: 3 C chain peptide
sequence SEQ ID NO: 4 A chain peptide sequence SEQ ID NO: 6 Genomic
DNA sequence SEQ ID NO: 7 A chain nucleic acid sequence SEQ ID NO:
8 B chain nucleic acid sequence SEQ ID NO: 9 C chain nucleic acid
sequence
DESCRIPTION OF THE FIGURES
[0046] FIG. 1. Assembled DNA sequence of the H3 (A) and M3 (B)
genes.
[0047] Start and Stop codons as well as predicted TATA boxes and
polyadenylation sequences are underlined. The positions of the
putative signal peptide, and B-, C- and A-chain peptide sequences
are indicated by arrows. A- and B-chain sequences are boxed and the
residues implicated in relaxin receptor binding are shaded. The
intron sequence, which is at an identical position in the C-chain
in both the human (A) and mouse (B) sequences, is indicated by
lower case letters and the exact size of the intron is marked.
[0048] FIG. 2. Sequence comparisons of H3 and M3 relaxin with other
relaxin and insulin family members.
[0049] Alignments of A- and B-chain sequences from H3 and M3
relaxin with other human and mouse relaxin sequences (A). The
consensus sequences are boxed; Cons 1,2,3: Consensus sequence
between human relaxins 1, 2 and 3. Cons 3: Consensus sequence
between H3 and M3 relaxin for the B-chain and H3, R3 and M3 relaxin
for the A-chain. Cons Mouse: Consensus sequence between M1 and M3
relaxin. The rat sequence is derived from an EST clone (see results
for details). "+" Denotes a conservative substitution, "." denotes
no homology. Phylogenetic tree of evolution of H3 and M3 relaxin
full-length sequences with human sequences of the
relaxin/insulin/IGF superfamily (B).
[0050] FIG. 3. Bioactivity of H3 compared to H1 and H2 relaxin in a
human relaxin receptor expressing cell line.
[0051] cAMP accumulation in THP-1 cells upon stimulation with
peptides (A). Data are expressed as mean .+-.SEM of the maximum
response (%) to H2 relaxin (n=3). The response to bovine insulin
(bINSL) and human INSL3 (hINSL3) are also shown to highlight the
specificity of the assay. H1, H2, H3; Human 1, 2 and 3 relaxin
respectively. The ability of H1 (n=7), H2 (n=11) and H3 (n=3)
relaxin peptides to compete for .sup.33P-labeled H2 relaxin (B33)
binding to THP-1 cells (B). Data are expressed as mean.+-.SEM of
the specific binding (%).
[0052] FIG. 4. Ability of a well characterized H2 relaxin antibody
to recognize H3 relaxin.
[0053] The H2 relaxin antibody was immobilized onto ELISA plates
and a competition experiment was performed using H1, H2 and H3
relaxin against .sup.125I-labeled H2 relaxin. Results are
mean.+-.SEM of the specific binding (%) of triplicate
determinations from a representative assay.
[0054] Unexpectedly, some 20 years after the identification of
human relaxin, and the surprising identification at that time of
two human relaxin genes, a further relaxin gene has been
identified. This invention in its various aspects provides: the
characterisation of nucleotide sequences encoding human H3 relaxin;
the isolation of purified nucleic acid material; amplification of
nucleotide sequences encoding H3 relaxin (mRNA, cDNA and DNA);
nucleic acid cloning of H3 relaxin sequences; nucleic acid sequence
identification, and peptide sequences encoding human H3
preprorelaxin, H3 prorelaxin and H3 relaxin.
[0055] The human H3 relaxin polypeptide comprises disulphide
bridged A and B chains. The amino acid sequence of human H3 relaxin
is set out in SEQ ID NO: 4. The amino acid sequence of the B chain
of human relaxin is set out in SEQ ID NO: 2.
[0056] The A and B chains of human H3 relaxin are linked through
cysteine residues, A11-B10, A24-B22 disulphide bond formation
taking place between these cysteine linkages.
[0057] Hence, the amino acid sequence of human H3 relaxin A and B
chains are as follows:
20 A Chain (SEQ ID NO: 4) Asp Val Leu Ala Gly Leu Ser Ser 1 5 Ser
Cys Cys Lys Trp Gly Cys Ser 10 15 Lys Ser Glu Ile Ser Ser Leu Cys
20
[0058] B Chain
21 (SEQ ID NO: 2) Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly 1 5
10 Arg Glu Phe Ile Arg 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg
Trp 20 25
[0059] the A and B chains being linked by disulphide bonds between
A11-B10, A24-B22.
[0060] Human H3 relaxin possesses classical relaxin bioactivity.
Human relaxins, H1 and H2 relaxin, bind to cells expressing relaxin
receptors, such as THP-1 cells (Parsell et al (1996) J. Biol. Chem.
271, 27936-27941). H2 relaxin produces a dose dependent increase in
cAMP production from these cells. Synthetic H3 relaxin produced
according to this invention stimulated a dose dependent increase in
cAMP in keeping with human H2 relaxin. The specificity of response
in target cells bearing the human relaxin receptor as exhibited by
H3 relaxin is demonstrated by the inability of bovine insulin
(bINSL) or human insulin (hINSL3) to stimulate cAMP responses at
doses up to 1 um in THP-1 cells.
[0061] The elicitation of a second messager response (cAMP) by
stimulating human relaxin receptors with human H3 relaxin, provides
definitive evidence that human H3 relaxin has classic relaxin
biological activity. Such assays in cells containing relaxin
receptors, for example THP-1 cells as referred to above provides, a
ready way to determine relaxin activity. In addition, the ability
of human H3 relaxin to compete with P.sup.32-labelled H2 relaxin in
binding to relaxin binding sites in cells expressing relaxin
receptors, again provides definitive confirmation of relaxin
activity.
[0062] Other biological activities/assays for determining relaxin
activity are known in the art. For example, bioassays used for the
measurement of active relaxin during pregnancy and non-pregnancy,
such as the guinea pig interpubic ligament assay may be used
(Steinetz et al (1960) Endocrinology 67, 102-115, and Sirosi et al
(1983) American Journal of Obstetrics and Gynaecology 145: 402405)
may be used. Other bioassays include cAMP production in THP-1 cells
(Parsell et al (1996) J. Biol. Chem 271, 27936-27941).
[0063] Applicant has found that H3 relaxin analogues may be
prepared where up to 9 amino acids are truncated from the
N-terminus of the A chain, and up to 9 amino acids are truncated
from the N-terminus of the B chain, and up to 5 amino acids are
truncated from the C-terminus of the B chain.
[0064] The resulting relaxin analogues comprise a H3 relaxin A and
B chain, the A chain having the amno acid sequence
22 Asp Val Leu Ala Gly Leu Ser Ser (SEQ ID NO: 4) 1 5 Ser Cys Cys
Lys Trp Gly Cys Ser 10 15 Lys Ser Glu Ile Ser Ser Leu Cys 20
[0065] truncated by up to about 9 amino acids from
amino-terminus,
[0066] and the B chain having the amino acid sequence:
23 (SEQ ID NO: 2) Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly 1 5
10 Arg Glu Phe Ile Arg 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg
Trp 20 25
[0067] truncated by up to 9 amino acids from the amino-terminus
and/or up to about 5 amino acids from the carboxyl-terminus,
[0068] the A and B chains being linked by disulphide bonds between
A11-B10 and A24-B22, and wherein the human H3 relaxin or analogue
thereof has relaxin bioactivity. The A chain of human H3 relaxin
contains an intrachain disulphide bond between Cys residues 10 and
15.
[0069] In standard assays looking at second messenger elicitation
in cells bearing human relaxin receptors, the H3 relaxin analogues
referred to above all elicited cyclic AMP production in a manner
which was characteristic of full length, non-truncated human H3
relaxin, and indeed human H2 relaxin. Hence, such truncated H3
relaxin analogues possess relaxin bioactivity.
[0070] Another aspect of the present invention relates to
compositions comprising a human H3 relaxin analogue having a
modified A chain and/or a modified B chain. The carboxyl-terminus
of the A chain, and/or the B chain, may be an amide derivative. Lys
at position 12 in the A chain may be replaced with Glu, and/or Glu
at position 19 may be replaced with Gln. In the B chain, the Ala at
position 2 may be replaced with Pro, and/or Arg at position 8 may
be replaced with Lys. The resulting H3 relaxin analogues having
modified amino acids comprise an amino acid sequence which may be
depicted as follows:
[0071] In accordance with another aspect of the invention, there is
provided a human H3 relaxin analogue having a modified A chain
and/or a modified B chain,
[0072] the H3 relaxin A chain having the amino acid sequence:
24 Asp Val Leu Ala Gly Leu Ser Ser Ser (SEQ ID NO: 4) 1 5 Cys Cys
Lys Trp Gly Cys Ser 10 15 Lys Ser Glu Ile Ser Ser Leu Cys 20
[0073] wherein the carboxyl-terminus is an amide derivative and/or
Lys at position 12 is replaced with Glu, and/or Glu at position 19
is replaced with Gln,
[0074] the modified B chain having the amino acid sequence:
25 (SEQ ID NO: 2) Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly 1 5
10 Arg Glu Phe Ile Arg 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg
Trp 20 25
[0075] wherein the carboxyl-terminus is an amide derivative, and/or
Ala at position 2 is replaced with Pro, and/or Arg at position 8 is
replaced with Lys,
[0076] the A and B chains being linked by disulphide bonds between
A11-B10 and A24-B22, and wherein the human H3 relaxin analogue has
relaxin bioactivity.
[0077] The isolation, purification and characterisation of nucleic
acid sequences encoding human H3 relaxin has allowed the
characterisation and production of the signal sequence of human H3
relaxin, and the pro-sequence of human H3 relaxin.
[0078] The identification, purification and characterisation of the
signal sequence and C chain of human H3 relaxin enables the prepro-
and pro-human H3 relaxin to be produced.
[0079] In accordance with another aspect of the invention there is
provided a composition comprising human H3 preprorelaxin or human
H3 prorelaxin, having a signal, A chain, B chain and C chain in
respect of human H3 preprorelaxin, and an A chain, B chain and C
chain in respect of human H3 prorelaxin, the said amino acid chains
having the amino acid sequences:
[0080] the A chain comprising:
26 Asp Val Leu Ala Gly Leu Ser Ser (SEQ ID NO: 4) 1 5 Ser Cys Cys
Lys Trp Gly Cys Ser 10 15 Lys Ser Glu Ile Ser Ser Leu Cys 20
[0081] the B chain comprising:
27 (SEQ ID NO: 2) Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly 1 5
10 Arg Glu Phe Ile Arg 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg
Trp 20 25
[0082] the signal sequence comprising:
28 Met Ala Arg Tyr Met Leu Leu Leu Leu (SEQ ID NO: 1) 1 5 Leu Ala
Val Trp Val Leu Thr 10 15 Gly Glu Leu Trp Pro Gly Ala Glu Ala 20
25
[0083] and the C chain comprising:
29 Arg Arg Ser Asp Ile Leu Ala His Glu Ala Met Gly Asp Thr Phe Pro
(SEQ ID NO: 3) 1 5 10 15 Asp Ala Asp Ala Asp Glu Asp Ser Leu Ala
Gly Glu Leu Asp Glu Ala 20 25 30 Met Gly Ser Ser Glu Trp Leu Ala
Leu Thr Lys Ser Pro Gln Ala Phe 35 40 45 Tyr Arg Gly Arg Pro Ser
Trp Gln Gly Thr Pro Gly Val Leu Arg Gly 50 55 60 Ser Arg 65
[0084] In accordance with a further aspect of the invention there
is provided the C chain of human H3 relaxin, said C chain having
the amino acid sequence:
30 Arg Arg Ser Asp Ile Leu Ala His Glu Ala Met Gly Asp Thr Phe Pro
(SEQ ID NO: 3) 1 5 10 15 Asp Ala Asp Ala Asp Glu Asp Ser Leu Ala
Gly Glu Leu Asp Glu Ala 20 25 30 Met Gly Ser Ser Glu Trp Leu Ala
Leu Thr Lys Ser Pro Gln Ala Phe 35 40 45 Tyr Arg Gly Arg Pro Ser
Trp Gln Gly Thr Pro Gly Val Leu Arg Gly 50 55 60 Ser Arg 65
[0085] Human H3 prorelaxin possesses characteristic relaxin
bioactivity.
[0086] Human H3 relaxin, prorelaxin, preprorelaxin and constitutive
peptide chains may be products using techniques previously
described as useful in the production of relaxin such as U.S. Pat.
No. 5,991,997, U.S. Pat. No. 4,758,516, U.S. Pat. No. 4,871,670,
U.S. Pat. No. 4,835,251, PCT/US90/02085, and PCT/US94/0699.
[0087] Relaxin analogues and derivatives where amino acids are
substituted as indicated above may be produced recombinantly using,
for example, site directed mutagenesis techniques as set forth, for
example, in Tsurushita et al (1988) Gene Tissue: 135-139.
[0088] The disclosed sequence information for human H3 relaxin,
analogues thereof wherein one or more amino acids are truncated
from the N- and/or C-terminus of the A and/or B chains, or human H3
relaxin analogues having amino acid substitutions as referred to
above, may be synthesised according to the methods of Bullesbach
(1991) J. Biol. Chem. 266, 10754-10761, for synthesising relaxin.
Additionally, well known methods of peptide synthesis may be
utilised to produce the various H3 relaxin forms referred to
herein.
[0089] Relaxin has been implicated consequently in the treatment
and diagnosis of various diseases and disorders. For example,
studies provide evidence that relaxin is effective in the treatment
of scleroderma, sinus bradycardia, cardiovascular disease,
neurodegenerative and neurologic disorders, hair loss, depression.
See e.g., U.S. Pat. No. 5,166,191 and International Patent
Application No. PCT/US92/069). Evidence also suggests the use of
relaxin in diseases and disorders related to the abnormal
expression of collagen or fibronectin, such as rheumatoid
arthritis.
[0090] Human H3 relaxin, human H3 relaxin truncated analogues,
amino acid modified H3 relaxin analogues, and human prorelaxin
provided by the instant invention bind to the relaxin receptor and
possess relaxin biological activity. It directly follows that these
human H3 relaxin forms possessing relaxin biological activity may
be used for the treatment of the above-identified diseases and
other diseases.
[0091] In accordance with another aspect of the present invention
there is provided a method for the treatment of one or more of:
vascular disease including coronary artery disease, peripheral
vascular disease, vasospasm including Raynaud's phenomenon,
microvascular disease involving the central and peripheral nervous
system, kidney, eye and other organs; treatment of arterial
hypertension; diseases related to uncontrolled or abnormal collagen
or fibronectin formation such as fibrotic disorders -(including
fibrosis of lung, heart and cardiovascular system, kidney and
genitourinary tract, gastrointestinal system, cutaneous,
rheumatologic and hepatobiliary systems); kidney disease associated
with vascular disease, interstitial fibrosis, glomerulosclerosis,
or other kidney disorders; psychiatric disorders including anxiety
states including panic attack, agoraphobia, global anxiety, phobic
states; depression or depressive disorders including major
depression, dysthymia, bipolar and unipolar depression; neurologic
or neurodegenerative diseases (including memory loss or other
memory disorders, dementias, Alzheimer's disease); disorders of
learning, attention and motivation (including Attention Deficit
Hyperactivity Disorder, Tourette's disease, impulsivity, antisocial
and personality disorders, negative symptoms of psychoses including
those due to schizophrenia, acquired brain damage and frontal lobe
lesions); addictive disorders (including drug, alcohol and nicotine
addiction); movement and locomotor disorders (including Parkinson's
disease, Huntington's disease, and motor deficits after stoke, head
injury, surgery, tumour or spinal cord injury); immunological
disorders( including immune deficiency states, haematological and
reticuloendothelial malignancy; breast disorders (including
fibrocystic disease, impaired lactation, and cancer); endometrial
disorders including infertility due to impaired implantation;
endocrine disorders (including adrenal, ovarian and testicular
disorders related to steroid or peptide hormone production) ;
delayed onset of labour, impaired cervical ripening, and prevention
of prolonged labour due to fetal dystocia; sinus bradycardia; hair
loss, alopecia; disorders of water balance including impaired or
inappropriate secretion of vasopressin; placental insufficiency;
which comprises administering to a subject in need of any such
treatments a therapeutically effective amount of human H3 relaxin,
or an analogue thereof as herein defined, optionally in association
with one or more pharmaceutically acceptable carriers and/ diluents
and/or excipients.
[0092] In accordance with another aspect of the present invention
there is provided use of human H3 relaxin or an analogue thereof in
the manufacture of medicaments for the treatment of one or more of:
vascular disease including coronary artery disease, peripheral
vascular disease, vasospasm including Raynaud's phenomenon,
microvascular disease involving the central and peripheral nervous
system, kidney, eye and other organs; treatment of arterial
hypertension; diseases related to uncontrolled or abnormal collagen
or fibronectin formation such as fibrotic disorders (including
fibrosis of lung, heart and cardiovascular system, kidney and
genitourinary tract, gastrointestinal system, cutaneous,
rheumatologic and hepatobiliary systems); kidney disease associated
with vascular disease, interstitial fibrosis, glomerulosclerosis,
or other kidney disorders; psychiatric disorders including anxiety
states including panic attack, agoraphobia, global anxiety, phobic
states; depression or depressive disorders including major
depression, dysthyria, bipolar and unipolar depression; neurologic
or neurodegenerative diseases (including memory loss or other
memory disorders, dementias, Alzheimer's disease); disorders of
learning, attention and motivation (including Attention Deficit
Hyperactivity Disorder, Tourette's disease, impulsivity, antisocial
and personality disorders, negative symptoms of psychoses including
those due to schizophrenia, acquired brain damage and frontal lobe
lesions); addictive disorders (including drug, alcohol and nicotine
addiction); movement and locomotor disorders (including Parkinson's
disease, Huntington's disease, and motor deficits after stoke, head
injury, surgery, tumour or spinal cord injury); immunological
disorders( including immune deficiency states, haematological and
reticuloendothelial malignancy; breast disorders (including
fibrocystic disease, impaired lactation, and cancer); endometrial
disorders including infertility due to impaired implantation;
endocrine disorders (including adrenal, ovarian and testicular
disorders related to steroid or peptide hormone production) ;
delayed onset of labour, impaired cervical ripening, and prevention
of prolonged labour due to fetal dystocia; sinus bradycardia; hair
loss, alopecia; disorders of water balance including impaired or
inappropriate secretion of vasopressin; placental insufficiency;
which comprises administering to a subject in need of any such
treatments a therapeutically effective amount of human H3 relaxin,
or an analogue thereof as herein defined, optionally in association
with one or more pharmaceutically acceptable carriers and/ diluents
and/or excipients.
[0093] Without wishing to be bound on mechanism of action,
applicant believes that H3 relaxin may act as a neurotransmitter or
neuroregulator in the brain, and other parts of the body including
nerves, for example through inducing eAMP production in cells. H3
relaxin may also allow nutrient uptake by cells, or may be involved
in autoregulatory presynaptic and/or conventional postsynaptic
actions. Applicant further believes that H3 relaxin may also be
axonally transported by nerve projections.
[0094] As defined hereinafter, H3 relaxin has surprisingly been
found to be expressed in neuroanatomical region of the pars
ventromedialis of the dorsal tegmental nucleus (vmDTg), which may
otherwise be referred to as the nucleus incertus (Goto et al (2001)
Journal of Comparative Neurology 438: 86-122). With the extensive
pattern of efferents and afferents to and from key forebrain areas
from the nucleus incertus, this region has been proposed as part of
a brain stem network that may regulate behavioural activation via
influences on attention, motivation, locomotion and learning (Goto
et al) and may give rise to the therapeutic treatment modalities
herein described. This is consistent with the abundent distribution
of relaxin binding sites in cerebral cortex and other relevant
brain areas (Osheroff and Phillips (1991) Proc. Natl. Acad. Sci.
USA 88, 6413-6417; and Tan et al (1999) Br. J. Pharmacol. 127,
91-98).
[0095] H3 relaxin may cross the blood brain barrier, or may be
treated to facilitate crossing of the blood brain barrier, by
methods known in the art incluidng use of one or more sugars or
amino acids, or other substances which open the blood brain barrier
or make it leaky allowing coadministered/timed administration with
H3 relaxin (see for example Naito U.S. Pat. No. 6,294,520), by
intranasal administration according to the methods of Frey (U.S.
Pat. No. 6,313,093), for example using a lipophilic vehicle, and by
methods described in PCT/WO89/10134.
[0096] H3 relaxin and anlogues as herein described may be effective
in the treatment of a wide range of what may broadly be described
as neurologic diseases including psychiatric disorders, disorders
of learning, attention and memory, addictive disorders and movement
and locomotor disorders.
[0097] H3 relaxin binds to the relaxin receptor as described
hereinafter.
[0098] For convenience, human H3 relaxin, analogues of human H3
relaxin where one or more amino acids are truncated from the N--
and/or C-terminus of the A and B chains of human H3 relaxin,
analogues of human H3 relaxin where one or more amino acids are
modified or substituted with another amino acid as described
herein, and human H3 preprorelaxin shall collectively be referred
to as human H3 relaxin, unless otherwise specifically
indicated.
[0099] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
More specifically, a therapeutically effective amount means an
amount effective to prevent development of or to alleviate the
existing symptoms of the subject being treated. Determination of
the effective amounts is well within the capability of those
skilled in the art, especially in light of the detailed disclosure
provided herein.
[0100] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. For example, a dose can be formulated in animal
models to achieve a circulating concentration range that includes
the IC.sub.50 as determined in cell culture. Such information can
be used to more accurately determine useful doses in humans.
[0101] A therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms or a
prolongation of survival in a patient. Toxicity and therapeutic
efficacy of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio between LD.sub.50 and ED.sub.50. Compounds
which exhibit high therapeutic indices are preferred. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition.
(See e.g. Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p1).
[0102] Dosage amount and interval may be adjusted individually to
provide serum levels of the active moiety which are sufficient to
maintain the relaxin activity and effects.
[0103] Administration of H3 relaxin can be via any of the accepted
modes of administration for agents that serve similar utilities,
preferably by systemic administration.
[0104] While human dosage levels for treating many of the
above-identified relaxin related diseases or disorders have yet to
be optimized for H3 relaxin generally, a daily dose is from about
0.05 to 500.0 .mu.g/kg of body weight per day, preferably about 5.0
to 200.0 .mu.g/kg, and most preferably about 10.0 to 100.0
.mu.g/kg. Generally it is sought to obtain a serum concentration of
the H3 relaxin approximating or greater than normal circulating
levels of relaxin in pregnancy, i.e., 1.0 ng/ml, such as 1.0 to 20
ng/ml, preferably 1.0 to 20 ng/ml.
[0105] For administration to a 70 kg person, the dosage range would
be about 7.0 .mu.g to 3.5 mg per day, preferably about 42.0 .mu.g
to 2.1 mg per day, and most preferably about 84.0 to 700.0 .mu.g
per day. The amount of the H3 relaxin administered will, of course,
be dependent on the subject and the severity of the affliction, the
manner and schedule of administration and the judgment of the
prescribing physician and the biological activity of such analog or
derivative. One treatment regimen can employ a higher initial
dosage level (e.g., 100 to 200 .mu.g/kg/day) followed by decreasing
dosages to achieve steady H3 relaxin serum concentration of about
1.0 ng/ml. Another treatment regimen, particularly postpartum
depression, entails administration of an amount of H3 relaxin
sufficient to attain normal pregnancy levels of relaxin (about 1.0
ng/ml) followed by gradual decreasing dosages until H3 relaxin
serum levels are no longer detectable (e.g. less than about 20
picograms/ml), optionally discontinuing treatment upon reaching
that dosage level.
[0106] Any pharmaceutically acceptable mode of administration can
be used. H3 relaxin can be administered either alone or in
combination with other pharmaceutically acceptable excipients,
including solid, semi-solid, liquid or aerosol dosage forms, such
as, for example, tablets, capsules, powders, liquids, gels,
suspensions, suppositories, aerosols or the like. Such proteins can
also be administered in sustained or controlled release dosage
forms (e.g., employing a slow release bioerodable delivery system),
including depot injections, osmotic pumps (such as the Alzet
implant made by Alza), pills, transdermal (including
electrotransport) patches, and the like, for prolonged
administration at a predetermined rate, preferably in unit dosage
forms suitable for single administration of precise dosages. The
compositions will typically include a conventional pharmaceutical
carrier or excipient and/or H3 relaxin, H3 prorelaxin, and H3
preprorelaxin or derivatives thereof. In addition, these
compositions may include other active agents, carriers, adjuvants,
etc.
[0107] In a preferred aspect of the invention, a
sustained/controlled release H3 relaxin formulation was a
selectively permeable outer barrier with a drug dispensing opening,
and an inner H3 relaxin containing portion designed to deliver
dosage of the H3 relaxin progressively diminishing at a
predetermined rate (e.g. containing about 30 mg of H3 relaxin in a
matrix for delivery of initially about 500 .mu.g per day
diminishing as a rate of 10 .mu.g per day.
[0108] In another preferred aspect of the invention, a
sustained/controlled release of H3 relaxin has a selectively
permeable outer barrier with a drug dispensing opening, a first
inner H3 containing portion designed for steady state release of H3
relaxin at a therapeutically effective daily dosage (e.g.
containing about 50 mg of H3 relaxin in a matrix for continuous
delivery of about 500 .mu.g per day), and a second inner H3 relaxin
a portion designed to deliver a dosage of H3 relaxin progressively
diminishing at a predetermined rate (e.g. containing about 3 mg of
H3 relaxin in a matrix for delivery of initially about 500 .mu.g
per day diminishing at a rate of 50 .mu.g per day) commencing upon
exhaustion of the H3 relaxin from the first inner portion.
[0109] Generally, depending on the intended mode of administration,
the pharmaceutically acceptable composition will contain about 0.1%
to 90%, preferably about 0.5% to 50%, by weight of H3 relaxin,
either alone or in combination with H3 relaxin, the remainder being
suitable pharmaceutical excipients, carriers, etc. Actual methods
of preparing such dosage forms are known, or will be apparent, to
those skilled in this art; for example, see Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th
Edition, 1975.
[0110] In cases of local administration or selective uptake, the
effective local concentration of the drug may not be related to
plasma concentration.
[0111] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
[0112] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, or intestinal administration.
Parenteral administration is generally characterized by injection,
either subcutaneously, intradermally, intramuscularly or
intravenously, preferably subcutaneously. Injectable can be
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution or suspension in
liquid prior to injection, or as emulsions. Suitable excipients
are, for example, water, saline, dextrose, glycerol, ethanol or the
like. In addition, if desired, the pharmaceutical compositions to
be administered may also contain minor amounts of non-toxic
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, solubility enhancers, and the like, such as for
example, sodium acetate, sorbitan monolaurate, triethanolamine
oleate, cyclodextrins, and the like.
[0113] A more recently devised approach for parenteral
administration employs the implantation of a slow-release or
sustained-release system, such that a constant level of dosage is
maintained. See, e.g., U.S. Pat. No. 3,710,795.
[0114] Alternately, one may administer the H3 in a local rather
than systemic manner, for example, via injection of the compound
directly into a solid tumor, often in a depot or sustained release
formulation.
[0115] Furthermore, one may administer the drug in a targeted drug
delivery system, for example, in a liposome coated with
tumor-specific antibody. The liposomes will be targeted to and
taken up selectively by the tumor.
[0116] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0117] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0118] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0119] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0120] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0121] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containiing
conventional suppository bases such as cocoa butter or other
glycerides.
[0122] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0123] A pharmaceutical carrier for the hydrophobic compounds of
the invention is a cosolvent system comprising benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. The cosolvent system may be the VPD co-solvent
system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol
300, made up to volume in absolute ethanol. The VPD co-solvent
system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in
water solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon systemic
administration. Naturally, the proportions of a co-solvent system
may be varied considerably without destroying its solubility and
toxicity characteristics. Furthermore, the identity of the
co-solvent components may be varied: for example, other
low-toxicity nonpolar surfactants may be used instead of
polysorbate 80; the fraction size of polyethylene glycol may be
varied; other biocompatible polymers may replace polyethylene
glycol, e.g. polyvinyl pyrrolidone; and other sugars or
polysaccharides may substitute for dextrose.
[0124] Alternatively, other delivery systems for hydrophobic
pharmaceutical compounds may be employed. Liposomes and emulsions
are well known examples of delivery vehicles or carriers for
hydrophobic drugs. Certain organic solvents such as
dimethylsulfoxide also may be employed, although usually at the
cost of greater toxicity. Additionally, the compounds may be
delivered using a sustained-release system, such as semipermeable
matrices of solid hydrophobic polymers containing the therapeutic
agent. Various of sustained-release materials have been established
and are well known by those skilled in the art. Sustained-release
capsules may, depending on their chemical nature, release the
compounds for a few weeks up to over 100 days. Depending on the
chemical nature and the biological stability of the therapeutic
reagent, additional strategies for protein stabilization may be
employed.
[0125] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0126] Formulations comprising human H3 relaxin may also be
administered to the respiratory tract as a nasal or pulmonary
inhalation aerosol or solution for a nebulizer, or as a microfine
powder for insufflation, alone or in combination with an inert
carrier such as lactose, or with other pharmaceutically acceptable
excipients. In such a case, the particles of the formulation may
advantageously have diameters of less than 50 microns, preferably
less than 10 microns. See, e.g., U.S. Pat. No. 5,364,838, which
discloses a method of administration for insulin that can be
adapted for the administration of H3 relaxin.
[0127] H3 relaxin for treatment of such disorders such as alopecia,
may also be administered topically in a formulation adapted for
application to the scalp, such as a shampoo (e.g., as disclosed in
U.S. Pat. No.4,938,953, adapted according to methods known by those
skilled in the art, as necessary for the inclusion of protein
ingredients) or a gel (e.g., as disclosed in allowed U.S. Ser. No.
08/050,745) optionally with increased H3 relaxin concentrations to
facilitate absorption.
[0128] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0129] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0130] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0131] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0132] Various aspects of the invention will be described with
reference to the following non-limiting examples.
[0133] In the examples which follow rats are used as an
experimental model to map H3 relaxin expression at the mRNA and
protein level in the rat brain, this allowing human brain mapping.
In this regard, the rat brain is a standard comparative anatomical
model of the human brain (Goto et al (2001) The Journal of
Comparative Neurology 438: 86-122).
EXAMPLE 1
Nucleotide Sequence Identification, Characterisation, Purification
and Manipulation
[0134] Tissue RNA/DNA Extraction and RT-PCR-Human genomic DNA was
extracted from human CL using standard protocols (Sambrook et al
1989) In Molecular Cloning. A Laboratory Manual, 2nd Ed., Cold
Spring Harbour Laboratory Press, N.Y.). Human CL and mouse tissues
were finely diced in the presence of liquid nitrogen and
immediately homogenized with RNAWiz reagent (Ambion Inc., Austin,
Tex.), and the RNA extracted according to the manufacturer's
instructions. Total RNA (5 .mu.g) from each sample was used for the
reverse transcription (RT) reaction, which was preformed using the
Superscript II RT-PCR kit (Gibco-BRL, Rockville, Md.) in a 20 .mu.l
volume according to the manufacturer's instructions. A 50 .mu.l
reaction containing 100 ng of primers and 150 ng of the cDNA
template was used for all PCR reactions. Mouse tissues were
screened for M3 relaxin expression using specific forward [5'
TGCGGAGGCTCACGATGGCGC 3'] and reverse [5' GACAGCAGCTTGCAGGCACGG 3']
primers, which generated a 319-bp product. Mouse relaxin (M1)
expression was determined using a specific forward [5'
GTGAATATGCCCGTGAATTGATC 3'] and reverse [5' AGCGTCGTATCGAAAGGCTCT
3'] primer based on the published sequence (Evans et al (1993) J.
Mol. Endocrinol. 10, 15-23), generating a 150-bp product.
[0135] Human CL cDNA was used in RT-PCR reactions with specific
primers for H3 relaxin, forward 1 [5' ACGTTCAAAGCGTCTCCGTCC 3'],
forward 2 [5' CGGTGGAGACGATCAGACATC 3'] and reverse [5'
ATGGCAGGACTGGGGCATTGG 3'], generating products of 504- and 310-bp
for forward 1/reverse and forward 2/reverse, respectively. All
primer combinations we subsequently show to cross the single
introns in the mouse and human relaxin sequences, respectively, so
as to control for genomic DNA contamination. In all experiments
GAPDH forward [5' TGATGACATCAAGAAGGTGG 3'] and reverse [5'
TTTCTTACTCCTTGGAGGCC 3'] primers generating a product of 246-bp
were used in separate PCR reactions to control for quality, and
equivalent loading of the cDNA. M3 relaxin expression by RT-PCR was
performed on cDNA samples extracted from at least two animals,
although the results from only one representative experiment are
shown. The mouse PCR reactions were completed in a Perkin Elmer
Gene Amplifier using the following (touch-down) annealing
temperatures: 64.degree. C. (2 cycles), 63.degree. C. (2 cycles),
62.degree. C. (2 cycles), 61.degree. C. (2 cycles), 60.degree. C.
(32 cycles). H3 relaxin expression in human CL cDNA was performed
by RT-PCR at the following annealing temperatures: 60.degree. C. (2
cycles), 59.degree. C. (2 cycles), 58.degree. C. (2 cycles),
57.degree. C. (2 cycles), 56.degree. C. (32 cycles). Aliquots of
the PCR products were electrophoresed on 2% (w/v) agarose gels
stained with ethidium bromide and photographed. Mouse tissue
samples were transfered to Hybond NX membranes (Amersham
International, Aylesbury, UK) for Southern blot analysis.
[0136] An additional PCR reaction was performed using mouse brain
and ovarian cDNA using the reverse M3 primer (above) and a forward
primer from in front of the ATG start codon (5' GGG
TCGCAGGCATCTCAACTG 3'). The resulting product contained the full H3
relaxin coding sequence. PCR was performed as above, but with the
following annealing temperatures: 60.degree. C. (2 cycles),
59.degree. C. (2 cycles), 58.degree. C. (2 cycles), 57.degree. C.
(2 cycles), 56.degree. C. (32 cycles). To generate a specific H3
relaxin cDNA probe for .sup.32P-labeling and to utilize it for
subsequent probing of a human multi tissue array, RT-PCR was
performed on human genomic DNA (50 ng). Specific forward (5'
CGGATGCAGATGCTGATGAAG 3') and reverse (5' GTGCCTGAGCCCACAGTGCCT 3')
primers from the exon II sequence of the H3 relaxin gene were used
at the following annealing temperatures: 60.degree. C. (2 cycles),
59.degree. C. (2 cycles), 58.degree. C. (2 cycles), 56.degree. C.
(2 cycles), 54.degree. C. (32 cycles). These products as well as
the mouse PCR product described above, were separated on 2% agarose
gels. Bands were detected of the expected size under UV light
(mouse 319-bp, 478-bp; human 374-bp), excised and eluted from the
gel using the Ultraclean TM 15 DNA purification kit (Geneworks Pty
Ltd, Adelaide, Australia). The bands were subsequently subcloned
into the pGEM-T vector (Promega, Madison, Wis.) and multiple
subclones were then sequenced on both strands using the ABI PRISM
377 automatic DNA sequencer, according to the manufacturers
instructions (Applied Biosystems, Melbourne, Australia).
[0137] Southern Blot Analysis--PCR products on membranes were
hybridized against specific internal oligonucleotide primers for
the M1 relaxin (5' CAAGCAGAGCTGGCTCCTCCTGGCT CAAAGCCAATCTTC 3') and
M3 relaxin (5' AATTTGGCTCTTGCTACAGCCCCACTCG CAGCAACTGCT 3') cDNA
sequences, which had been labeled using T4 polynucleotide kinase
and [.gamma.-.sup.32P] ATP. Hybridization was performed at
55.degree. C. overnight in 5.times.SSC (1.times.SSC; 0.15 M NaCl,
15 mM sodium citrate, pH 7), 5.times.Denhardts, 1% SDS and 100
.mu.g/ml sonicated herring sperm. Membranes were washed three times
for 5 min in 2.times.SSC, 0.1% SDS at room temperature followed by
a 30 min wash at 55.degree. C. in 0.1.times.SSC, 0.1% SDS, before
being exposed to BioMAX MR film (Eastman Kodak Co., Rochester,
N.Y.) for 24 h at room temperature.
[0138] Northern Blot Analysis--To further examine the expression of
M3 relaxin mRNA, total RNA (5-25 .mu.g) from the heart, brain,
lung, thymus and spleen of male mice, and ovary, endometrium,
myometrium, cervix and vagina of female mice pooled from day 7.5,
10.5 and 18.5 of pregnancy, were run on standard MOPS/formaldehyde
gels. RNA was then transferred to optimized Hybond-NX membranes and
probed for M3 relaxin mRNA with a .sup.32P-labeled probe that
corresponded to the 319-bp PCR product, generated by specific
primers to M3 relaxin (see above). This product was labeled with
[.alpha.-.sup.32P]dCTP using the specific reverse primer (above)
and T7 polymerase as previously described (31). The membrane was
hybridized at 65.degree. C. overnight in 0.25M NaH.sub.2PO.sub.4,
pH 7.2, 1 mM EDTA, 20% SDS, followed by three washes for 5 min in
2.times.SSC, 0.1% SDS at room temperature, and finally a 30 min
wash at 65.degree. C. in 0.1.times.SSC, 0.1% SDS. Membranes were
first exposed to a phosphoimager plate for 48 h at room temperature
before being analysed in a FujiX 2000 Phosphoimager (Fuji Photo
Company, Japan), and then exposed to BioMAX MS film (Integrated
Sciences, Melbourne, Australia) together with a Hyperscreen
(Amersham Pharmacia, Sydney, Australia) at -80.degree. C. In
separate experiments, total RNA (200 .mu.g) from the brain, spleen,
liver and testes was purified to poly-A RNA using an mRNA
purification kit (Amersham Pharmacia), and Northern blotting
performed as described above. A human multiple tissue expression
array (CLONTECH laboratories, Palo Alto, Calif.) was hybridized
with a .sup.32P-labeled H3 relaxin specific probe according to the
manufacturers recommendations. The 374-bp fragment of the H3
relaxin sequence isolated from genomic DNA was labeled with
[.alpha.-.sup.32P]dCTP using the H3 relaxin specific reverse primer
(described above), and T7 polymerase (Bathgate et al (1999) Biol.
Reprod. 61, 1090-1098). The membrane was exposed to a phosphoimager
plate and BioMAX film as described above.
[0139] In Situ Hybridization Histochemistry--Coronal sections (14
.mu.m) were cut on a cryostat at -16.degree. C. and mounted on
silane-coated slides. Sections were delipidated in chloroform for
10 min, rinsed and stored in 100% ethanol at 4.degree. C. Three
oligonucleotides (39 mers) [5' GGTGGTCTGTATTG
GCTTCTCCATCAGCGAAGAAGTCCC 3]'; [5' AATTTGGCTCTTGCTACAGCCCCACTC
GCACGAACTGCT 3'] and [5' TAAGGAGACAGTGGACCCCTTGGTGCCTCGCCTGT AGGA
3'], of the M3 relaxin mRNA sequence, and three oligonucleotides to
[5' GCACATCCGAATGAATCCGTCCATCCACT- CCTCCGAGAC 3'], [5' CAAGCAGAGCT
GGCTCCTCCTGGCTCAAAGCCAATCTTC 3'] and [5' GTTGTAGCTCTGGGAGCGAGGC
CTGAGCCTCAGACAGTA 3'] of the previously known M1 relaxin sequence
(Evans et al (1993) J. Mol. Endocrinol. 10, 15-23) were prepared
commercially (Geneworks Pty Ltd). Probes were labeled with
[.alpha.-.sup.35S]dATP (1200 Ci/mmol; NEN, AMRAD-Biotech,
Melbourne, Australia) to a specific activity of 1.times.10.sup.9
d.p.m./.mu.g using terminal deoxynucleotidyl transferase (Roche
Diagnostics; Wisden et al (1994) In In Situ Hybridization Protocols
for the Brain (Wisden, W. and Morris, B. J. eds), pp 9-34, Academic
Press, London). Screening of the sequences used against gene
sequence databases (Celera, EMBL and Genbank; NCBI/NIH Blast
Service) revealed homology only with the appropriate M1 and M3
relaxin mRNAs.
[0140] Sections were incubated overnight at 42.degree. C. with
multiple .sup.35S-labeled probes (30 fmol each probe/slide) in
hybridization buffer containing 50% formamide, 4.times.SSC, 10%
dextran sulphate and 0.2 M dithiothreitol. Slides were washed in
1.times.SSC at 55.degree. C. for 1 h, rinsed in 0.1.times.SSC, then
dehydrated before being apposed to Kodak BioMAX MR for 10 d.
[0141] The authenticity of the hybridization was confirmed by the
demonstration that the signal could be successfully blocked in all
areas by the addition of a 100-fold excess of unlabeled probes to
the hybridization buffer, except those that corresponded to
non-specific or background hybridization (data not shown). In
addition, three oligonucleotide probes were used that were
complementary to different, non-overlapping regions of the M3
relaxin gene sequence.
[0142] Human Relaxin (H3) Studies:
[0143] Solid Phase Synthesis--A putative peptide sequence encoded
by the H3 gene was assembled by solid phase synthesis procedures
based on the predicted signal peptide and proteolytic enzyme
cleavage sites between the signal peptide and the B-chain, and the
B/C and C/A chain junctions of the H3 relaxin prohormone (see
Results for details). For ease of synthesis we chose to prepare the
A- and B-peptides as their carboxyl-terminal amide derivatives.
Selectively S-protected A- and B-chains were synthesized on a 0.1
mmol scale by the continuous flow Fmoc solid-phase method as
previously described Dawson et al (1999) J. Pept. Res. 53, 542-547.
Selective S-protection was afforded for the following cysteine
residues: trityl (Trt) for A.sup.10,15 and B.sup.22, tert-butyl for
A.sup.24, and acetamidomethyl (Acm) for A.sup.11 and B.sup.10 (see
FIG. 2A for numbering of amino acid residues).
[0144] On completion of the syntheses, the S-protected A- and
B-chains were cleaved from the solid supports and simultaneously
sidechain deprotected by treatment with TFA in the presence of
scavengers. Selective disulfide bond formation was achieved
essentially as described for the synthesis of bombyxin Maruyama et
al (1992) J. Prot. Chem. 11, 1-12.
[0145] Peptide Charactenzation--Peptides were quantitated by
duplicate amino acid analysis of 24 h acid hydrolyzates on a GBC
automatic analyser (Melbourne, Australia). MALDITOF mass
spectrometry (MS) was performed in the linear mode at 19.5 kv on a
Bruker Biflex instrument (Bremen, Germany) equipped with delayed
ion extraction.
[0146] Other Relaxin and Insulin Peptides--Human INSL3 was
synthesized using the same methodology used for ovine INSL3 (Dawson
et al (1999) J. Pept. Res. 53, 542-547), and was characterized by
MS and amino acid analysis as outlined above. H1 relaxin was
synthesized previously (Wade et al (1996) Biomed. Pept. Prot. Nucl.
Acids 2, 27-32), recombinant H2 relaxin was a gift from the
Connetics Corporation (Palo Alto, Calif.) and bovine insulin was
purchased from Roche Diagnostics (Sydney, Australia).
[0147] THP-1 Cell Bioassay--The ability of H3 relaxin to induce
cAMP production in the human monocytic cell line (THP-1) was
compared to H1 and H2 relaxin following the procedure of Parsell
and colleagues (Parsell et al (1996) J. Biol. Chem. 271,
27936-27941), with the following modifications; THP-1 cells which
had been viability tested using Trypan Blue were resuspended in
media, and transferred to a 96 well plate at a density of 60,000
cells/well. Peptides (H1, H2, H3 relaxin, human INSL3 and bovine
insulin) were added to the wells together with 1 .mu.M forskolin
and 50 .mu.M isobutylmethylxanthine (IBMX) in RPMI media, and
incubated at 37.degree. C. for 30 min. The plate was then briefly
centrifuged, the media removed and the cells resuspended in lysis
buffer. cAMP levels were measured in the lysates using the cAMP
Biotrak EIA system (Amersham International, Aylesbury, UK). The
results are expressed as the maximum relaxin response (%/O) in
comparison to the maximum stimulation of cAMP achieved with H2
relaxin. Data represent the mean.+-.SEM of three experiments
performed in quadruplicate, and are plotted using PRISM (Graphpad
Inc., San Diego, Calif.).
[0148] THP-1 Cell Binding Assay--THP-1 cells were spun down and
resuspended in binding buffer (20 mM HEPES, 50 mM NaCl, 1.5 mM
CaCl.sub.2, 1% BSA, 0.1 mg/ml lysine, 0.01% NaN.sub.4, pH 7.5)
(Parsell et al (1996) J. Biol. Chem. 271, 27936-27941) to give
2.times.10.sup.6 cells/well in a 96-well plate. The cells were
incubated in binding buffer with .sup.33P-labeled H2 (B33) relaxin
(100 pM: labeled as previously described (Tan et al (1999) Br. J.
Pharmacol. 127, 91-98) at 25.degree. C. for 90 min in the absence
or presence of increasing concentrations of unlabeled H1, H2 and H3
relaxin (100 pM to 30 nM). Non-specific binding was defined with H2
relaxin (1 .mu.M. Cells were harvested using a Packard 96-well
plate cell harvester and Whatman GF/C glass fibre filters treated
with 0.5% polyethylenimine. The filters were washed three times
with modified binding buffer (20 mM HEPES, 50 mM NaCl, 1.5 mM
CaCl.sub.2), dried in a 37.degree. C. oven, and the radioactivity
counted by liquid scintillation spectrometry (TopCount.TM.,
Canberra Packard, Australia).
[0149] Antibody Crossreactivity--The ability of well characterized
human relaxin antibodies to recognize H3 relaxin was tested in
comparison to H1 and H2 relaxins by radioinmmunoassay. Briefly,
goat anti-H2 relaxin (Lucas et al (1989) J. Endocrinol. 120,
449-57) was coated onto 96 well ELISA plates (Disposable Products,
Adelaide, Australia) at a dilution of 1:1000 with 0.05M sodium
carbonate buffer at 4.degree. C. overnight. After washing twice
with PBS-T (phosphate buffered saline; 0.05% Tween 20, pH 7.4)
dilutions of human relaxin peptides dissolved in 50 .mu.l of assay
buffer (1% BSA in PBS-T) were added together with 50,000 cpm
.sup.125I-labeled relaxin, in 50 .mu.l of assay buffer. H2 relaxin
was .sup.125I-labeled and purified by HPLC (Palejwala et al (1998)
Endocrinology 139, 1208-1212). After an overnight incubation at
4.degree. C. the plates were washed twice with PBS-T. The
antibody-bound-.sup.125I-- labeled H2 relaxin was collected by the
addition of 1M NaOH and decanted into tubes for counting on a
Packard 5010 gamma counter (Canberra Packard). Experiments were
performed at least twice and similar results obtained. Data was
plotted as the mean.+-.SEM from one representative experiment
performed in triplicate and plotted using PRISM.
[0150] Mouse Relaxin (M3) Studies:
[0151] Animals--All male and female mice used in these studies were
age-matched and had the same background (C57BLK6J). Animals were
housed in a controlled environment and maintained on a 14 h light,
10 h dark schedule with access to rodent lab chow (Barastock
Stockfeeds, Melbourne, Australia) and water. Female mice (3.5
months old) were mated and pregnancy timed from the identification
of the vaginal plug. At day 7.5, 10.5 and 18.5 of pregnancy, mice
were sacrificed for tissue collection. Tissues were also collected
from non-pregnant female and male mice (4 months old). These
experiments were approved by the Howard Florey Institute's Animal
Experimental Ethics Committee, which adheres to the Australian Code
of Practice for the care and use of laboratory animals for
scientific purposes.
[0152] Tissue Collection--Animals were killed with an overdose of
Isofluorane (Abbott Australasia Pty Ltd, Sydney, Australia). The
brain, heart, thymus, spleen, lung, liver, kidneys, skin and gut
were collected along with the reproductive organs from female
(ovary, endometrium, myometrium, cervix, vagina; n=2 for each
pregnancy stage) and male (testes, epididymis, prostate; n=3) mice.
From additional animals, male brains (n=3) were dissected into
specific regions including the hypothalamus, cortex, hippocampus,
thalamus, medulla and cerebellum, and immediately placed in liquid
nitrogen and stored at -80.degree. C. until used for RNA
preparation. Female brains (n=3) were collected and immediately
frozen over dry ice for in situ hybridization histochemistry
(Burazin et al (2001) J. Neuroendocrinol. 13, 358-370). Human CL
from women in early pregnancy undergoing surgery for ectopic
pregnancies were utilized with the approval of the Howard Florey
Institute Human Ethics Committee and the written consent of the
patients.
EXAMPLE 2
Human H3 Relaxin Genes in the Human and Mouse
[0153] Both H3 relaxin sequences in the human and mouse contain
features representative of functional genes (FIG. 1A human; 1B
mouse). Each contain a putative TATA box for initiation of
transcription 65, and 59 bp, upstream of putative ATG start codons
for human and mouse, respectively. A polyadenylation signal is
present in the 3' untranslated region of both genes, in a position
582 and 448 bp downstream from an inframe TAG stop codon for the
human, and mouse genes respectively. A single intron interrupts the
coding region in an identical position in the sequence of both
genes, corresponding to a similar position to that of other relaxin
and insulin family members (Hudson et al (1983) Nature 301,
628-631; Evans et al (1993) J. Mol. Endocrinol. 10, 15-23; Ivell, R
(1997) Rev. Reprod. 2, 133-138). The H3 relaxin gene is localized
on chromosome 19 at 19p13.3, whereas the mouse gene is located on
chromosome 8 at 8C2. The derived coding regions of the H3 and M3
relaxin genes were 142, and 141, amino acids, respectively.
[0154] The cysteine residues necessary for disulphide bond
formation are retained in the correct positions, together with
conserved glycine residues necessary for flexibility around the
cysteine linkages (Bullesbach et al (2000) Int. J. Pept. Prot. Res.
46, 238-243). Most importantly, the residues demonstrated to be
essential for relaxin receptor binding in the core of the B-chain
(R--X--X--X--R--X--X--I) (Bullesbach et al (2000) J. Biol. Chem.
275, 35276-35280), have been retained in both the human and mouse
sequences. Therefore, although the human sequence most closely
resembles the hINSL5 peptide sequence on direct amino acid
homology, the presence of this binding motif indicates that the
peptide is more like a relaxin peptide. Interestingly, the M3
relaxin A-chain conforms to the cysteine pattern of family members,
whereas the previously characterized M1 relaxin sequence contains
an extra tyrosine residue before the final cysteine residue (FIG.
2A).
[0155] The H3 (human H3) and M3 (mouse "3" relaxin) sequences share
greater than 70% homology in the coding region at the nucleotide
level. However, the homology is most striking in the derived amino
acid sequence. Both derived pro-hormone sequences contain a typical
signal sequence after the ATG start codon which is likely to be
cleaved at an identical position between alanine and arginine in
both the human and mouse peptides (Nielsen et al (1997) Prot.
Engineer. 10, 1-6). The arginine-arginine pair of basic amino acids
at the B/C junction found with other members of the relaxin family
strongly suggests cleavage between tryptophan and arginine.
Similarly, cleavage at the C/A junction is most likely to occur
between the arginine and aspartic acid as indicated in FIGS. 1A and
1B, as this corresponds to a weak furin (proprotein convertase)
cleavage site (Nakayama, K. (1997) Biochem. J. 327, 625-635.
Therefore, it is believed that both H3 and M3 relaxins comprise a
B-chain of 27 amino acids, a C-peptide of 66 amino acids and an
A-chain of 24 amino acids.
[0156] A comparison of the A- and B-chain sequences of H3 and M3
relaxin with H1, H2 and M1 relaxin is outlined in FIG. 2A. There
are only two amino acid differences in both the A- and B-chains
between the M3 and H3 sequences, of which three of these changes
are conserved. In contrast, the homology between M1 and H2 relaxin
is only 42% and 45% in the A-, and B-, chains respectively.
Furthermore, other than the key core elements in the B-chain and
the key structural elements in the A-chain, there is very little
homology between H2 and H3 relaxin, and between M1 and M3 relaxin.
Interestingly, H3 and M3 relaxin show high homology of the
C-peptide domain (73%), compared with less than 20% homology in
this region of other insulin/relaxin family members. The C-peptide
lengths of H3 and M3 relaxin are 65, and 66 amino acids,
respectively, and are much shorter than that of other relaxins (102
amino acids for H1 and H2 and 99 amino acids for M1 relaxin). The
C-peptide chain length and sequence homology is most similar to
INSL5 (24%).
[0157] The full length amino acid sequences of the two genes were
aligned to other members of the insulin/relaxin family and a
phylogenetic tree generated (FIG. 2B). Additionally, the H3 and M3
relaxin sequences are grouped under a separate branch, indicating
that the evolution of these particular relaxins diverged from other
relaxins early in evolution. This was also the case for INSL5
within this analysis which interestingly shares closest primary
structural similarity to H3 relaxin.
EXAMPLE 3
Peptide Synthesis
[0158] H3 relaxin was prepared by solid phase synthesis in low
overall yield (0.7%). MALDTOF MS showed a single product with an
MH.sup.+of 5,494.7 (theoretical value: 5,497.5). Amino acid
analysis also confirmed its correct composition.
Chemical Synthesis of Human Relaxin H3 [hRlx-3 A(1-24)
amide-B(l-27) amide
[0159] Selectively S-protected A- and B-chains representing the
amino acid sequence of the separate H3 relaxin peptide chains, were
synthesized by the continuous flow 9-fluorenyl methoxycarbonyl
(Fmoc) solid-phase method using the general procedures described in
Atherton, E and Sheppard, R C. (Solid Phase Peptide Synthesis. IRL
Press at Oxford University Press, Oxford, United Kingdom, 1989).
Both peptides were prepared on a 0.1 mmol scale as peptide-carboxyl
terminal amides using Fmoc peptide amide linker polyethylene glycol
polystyrene (Fmoc-PAL-PEG-PS) supports (Applied Biosystems). For
the A-chain assembly, four-fold excesses of Fmoc-amino acids
(Auspep, Melbourne, Australia) were activated by
1,3-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt)
in dimethylformamide (DMF), whereas during the B-chain synthesis
each residue was activated by
2-(1H-benzotrazole-1-yl)-1,1,3,3-tetramethyluron- ium
hexaflurophosphate (HBTU) and diisopropylethylamine (DIEA) in DMF.
N.sup..alpha.--Fmoc deprotection for both chain assemblies was with
20% piperidine in DMF. Couplings were generally of 30 minutes
duration, with the exception of double couplings and extended times
for A-chain residues Ser.sup.7,8,21 and all cysteines, and double
couplings of B-chain residues Arg.sup.1,12,16, Ala.sup.2,3,17 and
Cys.sup.10. Side chain protection was afforded by tert-butyl esters
and ethers for Asp, Glu, Thr and Ser, butoxycarbonyl (Boc) for Lys
and Trp, 2,2,4,6,7-pentamethyldihyd- robenzofurane-5-sulfonyl (Pbf)
for Arg and the amide bond protection
N.sup..alpha.-(2-Fmoc-oxy-4-methoxybenzyl) [FmocHmb] for B-chain
Gly.sup.11. Selective S-protection was afforded for the following
cysteine residues: trityl (Trt) for Cys.sup.10,15 in the A-chain
and Cys.sup.22 in the B-chain, tert-butyl (tBu) for Cys.sup.24 in
the A-chain, and acetamidomethyl (Acm) for A-chain Cys.sup.11 and
B-chain Cys.sup.10.
(i) Synthesis of Human Relaxin H3. A-chain [Cys.sup.11(Acm),
Cys.sup.24(tBu)](1-24) amide [1]
[0160] On completion of the synthesis, the protected A-chain resin
was treated at room temperature for 2.5 hours with 95%
trifluoroacetic acid (TFA)/2.5% ethanedithiol (EDT)/2.5% H.sub.2O
plus 4 drops triethylsilane, to aid the quenching of thiols. TFA
was removed to a minimum volume under a stream of nitrogen and
precipitated twice from chilled diethyl ether. The precipitate was
then dissolved in 0.1% aq. TFA and lyophilized. The crude S-reduced
[thiol-Cys.sup.10,15, Cys.sup.11(Acm), Cys.sup.24(t-Bu)] A-chain
was directly subjected to air oxidation in 0.1M Gly-NaOH, pH 8.3,
for 4 hours at room temperature. Analytical reverse-phase high
performance liquid chromatography (RP-HPLC) monitoring confirmed
the completeness of the intramolecular disulfide bond formation,
after which several drops of neat TFA were added and the crude
oxidized material directly lyophilized.
(ii) Synthesis of Human Relaxin H3, B-chain [Cys.sup.10(Acm)](1-27)
amide [2]
[0161] On completion of the synthesis, the protected B-chain resin
was treated at room temperature for 2.5 hours with 82.5% TFA/5%
phenol/5% H.sub.20/5% thioanisole/2.5% ethanedithiol plus 4 drops
of triethylsilane, to aid the quenching of thiols. TFA was removed
to a mininmum volume under a stream of nitrogen and precipitated
twice from chilled diethyl ether. The precipitate was then
dissolved in 0.1% aq. TFA and lyophilized. The crude B-chain was
then purified by RP-HPLC as described below.
(iii) Synthesis of Human Relaxin H3, A-chain [Cys.sup.11(Acm),
Cys.sup.24(Pyr)](1-24) amide [3]
[0162] 25 mg of peptide 1 (9.65 .mu.mol) and 35 mg (158.86 .mu.mol)
2,2'-dipyridyl disulfide (DPDS) were dissolved together in 4.5 ml
TFA and 0.5 ml thioanisole and the resulting solution then chilled.
To this was added 5 ml trifluoromethanesulfonic acid (TFMSA)/TFA
(1:5 v/v) and the whole mixture allowed to stir at
.ltoreq.0.degree. C. for 30 mins. The [Cys.sup.11(Acm),
Cys.sup.24Pyr)] A-chain amide peptide was precipitated from cold
ether and the pellet obtained on centrifugation then suspended in
6M guanidine hydrochloride (GdHCl), pH 8.0, and purified by
RP-HPLC. (Yield peptide 3: 4%). (Alternative to RP-HPLC
purification, peptide 3 was desalted on a Sephadex G-25 gel
filtration column in 20% aq acetic acid).
(iv) Synthesis of Human Relaxin H3, A[Cys.sup.11(Acm)](1-24)
amide-B[Cys.sup.10(Acm)](1-27) amide [4]
[0163] Purified A-chain peptide 3 (1.0 mg, 0.38 .mu.mol) and
purified B-chain peptide 2 (1.2 mg, 0.38 .mu.mol) were dissolved
separately in 1.0 ml and 0.5 ml 0.1M NH.sub.4HCO.sub.3
respectively. The B-chain solution was then slowly added to A-chain
and the reaction mixture was stirred vigorously at room temperature
for 30 min. The solution was acidified with 0.5 ml glacial acetic
acid and then subjected to RP-HPLC, as detailed below, to isolate
the bis-disulfide bonded chain combined product. (Alternative to
RP-HPLC purification, the resulting A/B product, peptide 4, was
desalted on a Sephadex G-25 gel filtration column in 20% aq acetic
acid).
[0164] (An alternative method for chain combination which improves
B-chain solubility, is as follows: peptide 3 and purified
[Cys.sup.10(Acm)] B-chain were dissolved separately, at a
concentration of 1.0 ml/mg, in 8M GdHCl, pH 4.5 buffer. The B-chain
solution was then slowly added to A-chain and the reaction mixture
was stirred vigorously at 37.degree. C. for 24 hours).
(v) Synthesis of Human Relaxin H3 [hRlx-3A(1-24) amide-B(1-27)
amide
[0165] All of the purified 4 peptide was used to form the third and
final disulfide bond (assuming 100% recovery, estimated at 0.39
.mu.mol). The peptide was dissolved in a solution of 80 mM HCl and
acetic acid. 20 mM iodine in 95% aqueous acetic acid was then added
dropwise (25 equivs of iodine per Acm group). The reaction was
performed for 1 hour in the dark at room temperature after which
excess oxidant was quenched with 20 mM aqueous ascorbic acid.
Purification of the relaxin was by RP-HPLC, with a final yield,
relative to peptide 3 starting material, of 0.74%.
[0166] Purification
[0167] The separate crude chains and intermediate peptides were
purified by RP-HPLC, using a Waters 600 multisolvent delivery
system connected to a model 996 photodiode array detector. A
10.times.250 mm Vydac 218 TP column packed with C.sub.4 silica gel
(330 A pore size, 10 .mu.m particle size) was used. The peptides
were eluted with a solvent system of (A) 0.1% aq. TFA (v/v) and (B)
0.1% TFA in acetonitrile (v/v) in a linear gradient mode (25-50% B
over 30 minutes). The target fractions were collected and
identified by matrix-assisted laser desorption ionization mass
spectrometry (MALDI-TOF MS) and lyophilized.
[0168] Peptide Characterisation
[0169] Peptide quantitation was by duplicate amino acid analysis of
24 hr acid hydrolyzates on a GBC automatic analyser (Melbourne,
Aust). MALDITOF MS was performed in the linear mode at 19.5 kv on a
Bruker Biflex instrument (Bremen, Germany) equipped with delayed
ion extraction.
EXAMPLE 4
Relaxin Biological Activity
[0170] Demonstration of Relaxin Activity of Synthetic H3
Relaxin-Synthetic H3 relaxin C-terminal amide derivatives were
tested for relaxin activity in a relaxin receptor expressing cell
line, THP-1 (Parsell et al (1996) J. Biol. Chem. 271, 27936-27941).
H2 relaxin produces a dose dependent increase in cAMP production
from these cells (FIG. 3A). Synthetic H3 relaxin also stimulated a
dose dependent increase in cAMP (pEC.sub.50=8.68.+-.0.08 [2.11 nM];
n=3), albeit with slightly lower activity than H1
(pEC.sub.50=9.10.+-.0.05 [0.794 nM]; n=3) and H2
(pEC.sub.50=9.67.+-.0.11 [0.214 nM; n=3) relaxin. The specificity
of this response was demonstrated by the inability of bovine
insulin (bINSL), or human insulin 3 (hINSL3), to stimulate cAMP
responses at doses up to 1 .mu.M.
[0171] Synthetic H3 relaxin was also tested for its ability to
compete for .sup.33P-labeled H2 relaxin binding to relaxin binding
sites in THP-1 cells (FIG. 3B), with an affinity
(pK.sub.i=7.5.+-.0.16; n=3) lower than that of H2
(pK.sub.i=8.74+0.11; n=11) and H1 (pK.sub.i=8.9+0.11; n=7) relaxin.
Nevertheless, these data provide definitive evidence that the
synthetic H3 relaxin peptide binds to, and elicits a second
messenger response by stimulating human relaxin receptors.
[0172] Ability of a Well Characterized H2 Relaxin Antibody to
Recognize H3 Relaxin--The ability of a well characterized anti-H2
relaxin antibody to recognize H1 and H3 relaxin was tested by
radioimmunoassay. As shown in FIG. 4, H2 relaxin was able to
displace .sup.125I-labeled H2 relaxin binding to the anti-H2
relaxin antibody with high specificity. In contrast, H1 and H3
relaxin showed poor cross reactivity with the antisera as
determined by their poor ability to displace .sup.125I-labeled H2
relaxin binding. Furthermore, the non-parallellism of the
displacement curves indicates that not all the antibody epitopes
are recognized by the two peptides.
EXAMPLE 5
H3 Relaxin Expression
[0173] Relaxin Gene Expression in the Mouse--The expression of M3
relaxin mRNA was compared to M1 relaxin mRNA expression using
southern blotting of RT-PCR products. Although this technique is
only semi-quantitative, it enabled us to determine the potential
sites of expression of M3 relaxin compared to M1 relaxin. The
results of a representative experiment and duplicate experiments
gave identical results. M3 relaxin mRNA was expressed in a number
of tissues in C57BLK6J mice where M1 relaxin was found, but the
pattern of expression, between the two mouse relaxins was
different. In male non-reproductive tissues, highest levels of M1
relaxin expression were seen in the brain, moderate levels in the
thymus, heart and kidney, lower levels in the lung, spleen and
skin, with no expression seen in the gut. Interestingly, M3 relaxin
expression was detected at highest levels in brain, however, it was
expressed at moderate levels in the thymus, lung and spleen, only
at very low levels in the heart and liver, and not at all in the
kidney, skin and gut. Female mice showed an almost identical
pattern of expression for both genes in these tissues. In male
reproductive tissues M3 relaxin mRNA was significantly expressed
only in the testis whereas, M1 relaxin mRNA was detected in the
testis, epididymis and prostate. Both relaxins were also detected
in female reproductive organs in the mammary gland, ovaries of
non-pregnant, pregnant and lactating mice, and the endometrium and
myometrium of pregnant mice. Significant expression of M3 relaxin
mRNA was observed in all ovarian stages, while M1 relaxin
expression was higher in ovaries of late gestation compared to
ovaries from non-pregnant and lactating mice. High levels of M3
relaxin mRNA were detected in the brain and further analysis of
this tissue revealed that both relaxins were expressed in several
distinct regions. While M1 relaxin mRNA was consistently expressed
in the hypothalamus, hippocampus, cortex, thalamus, pons/medulla
and cerebellum, M3 relaxin mRNA was found to be highly expressed in
the thalamus and pons/medulla, thus suggesting, that the two
relaxins may play distinct roles in the mouse.
[0174] Northern Analysis--Tissues in which M3 relaxin mRNA was
positively identified by RT-PCR and Southern blot analysis, were
further examined by Northern blotting. Total RNA (5-25 .mu.g) from
the heart, brain, lung, thymus, spleen, ovary, endometrium,
myometrium, cervix and vagina were initially probed with a
.sup.32P-labeled M3 relaxin specific probe, but no specific
hybridizing bands were found in any tissue. Poly-A RNA from the
brain (15 .mu.g), spleen (5 .mu.g), liver (5 .mu.g) and testis (25
.mu.g) were then analyzed and a specific .about.1.2-kb hybridizing
band was identified in the brain, consistent with M3 relaxin
expression detected by RT-PCR and Southern blot analysis. The
obtained transcript size was consistent with the predicted size
based on the M3 relaxin transcript sequence (.about.1 kb) plus a
poly-A tail (.about.200-bp).
[0175] Expression of H3 Relaxin in Human Tissues--A Clonetech Multi
Tissue Expression Array was used to examine sites of expression of
H3 relaxin in human tissues. The array contained normalized poly-A
RNA (50-750 ng) from 76 different human tissues including 8
different control RNAs and DNAs, spotted onto a nylon membrane. The
array was probed with a .sup.32P-labeled 374-bp H3 relaxin specific
gene fragment from the 3' end of the H3 relaxin transcript,
generated from genomic DNA. This DNA fragment was sequenced on both
strands. Very weak hybridizing signals were observed in spleen,
thymus, peripheral blood leukocytes, lymph node and testis however,
these signals were barely discernable above background and hence,
the data is not shown. RT-PCR was also performed on human CL from
early pregnancy using two different primer combinations based on
the H3 relaxin sequence. No specific bands were observed in any PCR
reaction even after changing the PCR conditions, whereas
transcripts for H2 relaxin and GAPDH were easily amplified (data
not shown), confirming the integrity of the cDNA.
[0176] Distribution of Relaxin mRNA in the Mouse Brain--Given the
high levels of M3 relaxin mRNA expression detected by RT-PCR and
Northern blotting in the brain, its distribution was further
examined using in situ hybridization histochemistry (Burazin et al
(2001) J. Neuroendocrinol. 13, 358-370. Multiple specific
.sup.35S-labeled oligonucleotide probes were utilized to determine
the cellular distribution of M3 relaxin mRNA throughout the
rostro-caudal extent of the female C57BLK6J mouse brain. M3 relaxin
mRNA was not widely detected throughout brain nuclei, but was most
strongly detected in the pons/medulla (FIG. 7). The strongest level
of M3 relaxin mRNA was present in the pars ventromedialis of the
dorsal tegmental nucleus. In addition, M3 relaxin mRNA was also
detected, albeit at far lower levels, in the hippocampus and
olfactory regions. Brain regions containing low levels of mRNA
encoding M3 relaxin may not have been detected in the current study
due to sensitivity limitations associated with in situ
hybridization histochemistry. The distribution of M3 relaxin mRNA
in the brain differs from that of M1 relaxin mRNA, as no M1 relaxin
mRNA was detected in the pars ventromedialis of the dorsal
tegmental nucleus (data not shown).
EXAMPLE 6
[0177] Prorelaxin H3 cDNA sequences from human, mouse and rat are
expressed in both prokaryotic and eukaryotic cell systems using
appropriate expression transfer vectors.
[0178] These systems include appropriate mammalian host cells,
other higher eukaryotic cells including insect cells, plant cells
and avian cells as well as bacterial and yeast expression systems.
Additionally, fusion protein products of these three sequences are
produced by linking a portion of a prokaryotic or eukaryotic
protein characteristic of the host cell. The fusion products
facilitate the purification of the protein product such that the
fusion product may be subsequently removed. All transfer vectors
may also be modified by codon substitutions/deletions/ad- ditions
with the modifications giving rise to shortened C peptide
prorelaxins with B/C and C/A junction modifications to facilitate
the removal of the modified C peptide sequence.
[0179] Relaxin synthesis using shortened C peptide substitutions
and B/C and C/A junction modifications are described in U.S. Pat.
No. 5,759,807, and such methods may be used for the production of
H3 relaxin.
[0180] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0181] The reference to any prior art in this specification is not,
and should not be taken as an acknowledgment or any form of
suggestion that that prior art forms part of the common general
knowledge in Australia.
Sequence CWU 1
1
51 1 25 PRT Homo sapiens 1 Met Ala Arg Tyr Met Leu Leu Leu Leu Leu
Ala Val Trp Val Leu Thr 1 5 10 15 Gly Glu Leu Trp Pro Gly Ala Glu
Ala 20 25 2 27 PRT Homo sapiens 2 Arg Ala Ala Pro Tyr Gly Val Arg
Leu Cys Gly Arg Glu Phe Ile Arg 1 5 10 15 Ala Val Ile Phe Thr Cys
Gly Gly Ser Arg Trp 20 25 3 66 PRT Homo sapiens 3 Arg Arg Ser Asp
Ile Leu Ala His Glu Ala Met Gly Asp Thr Phe Pro 1 5 10 15 Asp Ala
Asp Ala Asp Glu Asp Ser Leu Ala Gly Glu Leu Asp Glu Ala 20 25 30
Met Gly Ser Ser Glu Trp Leu Ala Leu Thr Lys Ser Pro Gln Ala Phe 35
40 45 Tyr Arg Gly Arg Pro Ser Trp Gln Gly Thr Pro Gly Val Leu Arg
Gly 50 55 60 Ser Arg 65 4 24 PRT Homo sapiens 4 Asp Val Leu Ala Gly
Leu Ser Ser Ser Cys Cys Lys Trp Gly Cys Ser 1 5 10 15 Lys Ser Glu
Ile Ser Ser Leu Cys 20 5 117 PRT Homo sapiens 5 Arg Ala Ala Pro Tyr
Gly Val Arg Leu Cys Gly Arg Glu Phe Ile Arg 1 5 10 15 Ala Val Ile
Phe Thr Cys Gly Gly Ser Arg Trp Arg Arg Ser Asp Ile 20 25 30 Leu
Ala His Glu Ala Met Gly Asp Thr Phe Pro Asp Ala Asp Ala Asp 35 40
45 Glu Asp Ser Leu Ala Gly Glu Leu Asp Glu Ala Met Gly Ser Ser Glu
50 55 60 Trp Leu Ala Leu Thr Lys Ser Pro Gln Ala Phe Tyr Arg Gly
Arg Pro 65 70 75 80 Ser Trp Gln Gly Thr Pro Gly Val Leu Arg Gly Ser
Arg Asp Val Leu 85 90 95 Ala Gly Leu Ser Ser Ser Cys Cys Lys Trp
Gly Cys Ser Lys Ser Glu 100 105 110 Ile Ser Ser Leu Cys 115 6 3449
DNA Homo sapiens 6 tataaatggg gggccaagag gcagcagaga cactggccca
ctctcacgtt caaagcgtct 60 ccgtccagca tggccaggta catgctgctg
ctgctcctgg cggtatgggt gctgaccggg 120 gagctgtggc cgggagctga
ggcccgggca gcgccttacg gggtcaggct ttgcggccga 180 gaattcatcc
gagcagtcat cttcacctgc gggggctccc ggtggagacg atcagacatc 240
ctggcccacg aggctatggg tgaggctggg gagagagtgg atgtagaagg ggaacaggtg
300 gctggatggg tcccaggagc taaggacaga gataagagga ggttgctgga
ggaggagggt 360 ccctgtcctg ccacattcag ccagggacac ctgcccagcc
ttgaaacaag ggctcaggag 420 ttagcagagc tgcagagctg ggatggggtg
ttgcaagcca tccatggggg ctggaagtct 480 gaggacaggt gggggcgggg
agcgtgccat ttgcaaagac aacaccgaag tgttttccaa 540 ccctttccag
caggtaatgt gaagggtgtg gtatacacat agctgggttt gtcacctaat 600
gcatgacctc tccccagcaa gttggttttt cttccgtctc tgagtgtctt ttttttggag
660 atgtggtctc actccattgc ccaggcttga atgcagtggc ccaatcactg
ctcattgcag 720 cctcgacctc ccaggctcaa gtgattctcc tgcctccgcc
tccagagtag ttgagaccac 780 aggcacctga caccatgcct ggctagtttt
aaattttttt tttgtagaaa caggggtctc 840 actatgttgc ctaggctggt
ctcgaactcc tgggctcaag tgatcctccc acctcggcct 900 ccctaagtgc
tgagattaga gtctctgagt gtctttatct tcaaatggga gacacagttc 960
ctgaatcttg caggattaag tggtatgatt aaatcaaaac agattagggc agagtctcag
1020 cagggcagcg gcacaatctg ggatccatca ggagagtcag agggaacaga
agacctagct 1080 tcatgagggg cagggacctg gcaaatagat attcatgatg
gtgagaagga ggataggtat 1140 gagcgtggac atagaagaca caccacttgg
attcagatag tagctctaca atgtaatagt 1200 tgtgtgttca tgtgctacta
tttttttttt ttttgagaca gaatctcatt ctgttgccca 1260 ggctggagtg
cagtggtgca atcttggctc actgtaacct ccatcacctg ggttcaagcg 1320
attctcgtgc ctccagcctc ccaagtagct gggattacag atgtgtgcca ccatacctcg
1380 ctaatctttt tatttttagt agagacagtt tcaccatgtt ggccaggctg
gtctccaact 1440 cctgacctca ggtgatcctc ccacctcagc ctcccaaagt
gctgggatta caggcatgag 1500 ccaccgcgcc cagccatgca aattctttac
tgagtcctgc ctcagtggtc tcctctggaa 1560 aatacgggtg ataactgcac
ccacctcaac tggttatcac tgagaagaat aaagaagtta 1620 acctgctaaa
gcacttaaaa cgttgtttga cacaaagtaa gtgatcaata aattattatt 1680
attattatta ttattattat tattattatt tttgagacag ggtcttgctc tgttgcccag
1740 actggagtgc agtggtgtga tcacagctca ctgcagcttc aacctcttgg
gctcaagcaa 1800 ttctcctgcc tcagcctcct gagtagctgg gactacaggc
ttgtgccaac atgtctaact 1860 ttttattatt tgtagagaca gggtagtgct
gtgttgtcca ggctgttctt gaactcctgg 1920 ttctggtgat cctccagcat
gtgcccctgg aagtgctggg attacaggtg tgagacaccg 1980 tgcccggact
caatagtcat ttttgagtgc tcatcatgtt ccagacattg ttctaagttt 2040
ttttttttaa tgaatattaa ctccttataa aacttgagaa ggttggagta attatttttt
2100 tccactttgc agaaaagaac attgaggctc caagaagtaa atttacttgc
tcacgattag 2160 agaagctgga ttcatgctca gtcagcccag ctcccaaatg
taccaggtcc tcaattaata 2220 aagagtaagg agaaataaat gacagggctg
ggtgcggtgg ctcacgcctg taatcccagc 2280 actttgggtg gctgaggtgg
gcacatcact tgaggtcagg agtttgcgac cagcctgaac 2340 aacatggtga
accccatctc tataacaata caaaaatcag ccaggcctgc tggcagacac 2400
ctgtaatccc acctactctg gcagagccag aatttgaacc caggactggg tggaataaaa
2460 actctgaact atgtctatga ctgttgtcac aagatcagag ctagactggc
caggagccat 2520 gactgtgggt gcagcagcag ctgagccctg atcactaact
ctgttcatct tttgcaggag 2580 ataccttccc ggatgcagat gctgatgaag
acagtctggc aggcgagctg gatgaggcca 2640 tggggtccag cgagtggctg
gccctgacca agtcacccca ggccttttac agggggcgac 2700 ccagctggca
aggaacccct ggggttcttc ggggcagccg agatgtcctg gctggccttt 2760
ccagcagctg ctgcaagtgg gggtgtagca aaagtgaaat cagtagcctt tgctagtttg
2820 agggctgggc agccgtgggc accaggacca atgccccagt cctgccatcc
actcaactag 2880 tgtctggctg ggcacctgtc tttcgagcct cacacattca
ttcattcatc tacaagtcac 2940 agaggcactg tgggctcagg cacagtctcc
cgacaccacc tatccaaccc tgccctttga 3000 ccagcctatc atgaccctgg
cccctaagga agctgtgccc ctgcctggtc aagtggggac 3060 ccccccatcc
tgacccctga cctctcccca gccctaacca tgcgtttgcc tggcctacac 3120
actccactgc cacaactggg tccctactct acctaggctg gccacacaga gacccctgcc
3180 cccttcccag tccaaactgt ggccattgtc ccctgaccag ctaaaatcaa
gcctctgtct 3240 cagtccagcc tttgcacgca cgcttccttt gccctgcttt
ccatcccctc tccctccaac 3300 tcccctgcca gagttccaag gctgtggacc
ccagagaagg tggcaggtgg cccccctagg 3360 agagctctgg gcacattcga
atcttcccaa actccaataa taaaaattcg aagactttgg 3420 cagagagtgt
gtgtgtgtgt gtatggttg 3449 7 72 DNA Homo sapiens 7 gatgtcctgg
ctggcctttc cagcagctgc tgcaagtggg ggtgtagcaa aagtgaaatc 60
agtagccttt gc 72 8 81 DNA Homo sapiens 8 cgggcagcgc cttacggggt
caggctttgc ggccgagaat tcatccgagc agtcatcttc 60 acctgcgggg
gctcccggtg g 81 9 198 DNA Homo sapiens 9 agacgatcag acatcctggc
ccacgaggct atgggagata ccttcccgga tgcagatgct 60 gatgaagaca
gtctggcagg cgagctggat gaggccatgg ggtccagcga gtggctggcc 120
ctgaccaagt caccccaggc cttttacagg gggcgaccca gctggcaagg aacccctggg
180 gttcttcggg gcagccga 198 10 351 DNA Homo sapiens 10 cgggcagcgc
cttacggggt caggctttgc ggccgagaat tcatccgagc agtcatcttc 60
acctgcgggg gctcccggtg gagacgatca gacatcctgg cccacgaggc tatgggagat
120 accttcccgg atgcagatgc tgatgaagac agtctggcag gcgagctgga
tgaggccatg 180 gggtccagcg agtggctggc cctgaccaag tcaccccagg
ccttttacag ggggcgaccc 240 agctggcaag gaacccctgg ggttcttcgg
ggcagccgag atgtcctggc tggcctttcc 300 agcagctgct gcaagtgggg
gtgtagcaaa agtgaaatca gtagcctttg c 351 11 21 DNA Artificial
Sequence M3 relaxin Forward primer 11 tgcggaggct cacgatggcg c 21 12
21 DNA Artificial Sequence M3 relaxin Reverse primer 12 gacagcagct
tgcaggcacg g 21 13 23 DNA Artificial Sequence M1 relaxin forward
primer 13 gtgaatatgc ccgtgaattg atc 23 14 21 DNA Artificial
Sequence M1 relaxin reverse primer 14 agcgtcgtat cgaaaggctc t 21 15
21 DNA Artificial Sequence H3 relaxin forward primer 1 15
acgttcaaag cgtctccgtc c 21 16 21 DNA Artificial Sequence H3 relaxin
forward primer 2 16 cggtggagac gatcagacat c 21 17 21 DNA Artificial
Sequence H3 relaxin reverse primer 17 atggcaggac tggggcattg g 21 18
20 DNA Artificial Sequence GAPDH forward primer 18 tgatgacatc
aagaaggtgg 20 19 20 DNA Artificial Sequence GAPDH reverse primer 19
tttcttactc cttggaggcc 20 20 21 DNA Artificial Sequence M3 forward
primer 2 20 gggtcgcagg catctcaact g 21 21 21 DNA Artificial
Sequence H3 relaxin exon II forward primer 21 cggatgcaga tgctgatgaa
g 21 22 21 DNA Artificial Sequence H3 relaxin exon II reverse
primer 22 gtgcctgagc ccacagtgcc t 21 23 39 DNA Artificial Sequence
M1 relaxin internal probe 23 caagcagagc tggctcctcc tggctcaaag
ccaatcttc 39 24 39 DNA Artificial Sequence M3 relaxin internal
probe 24 aatttggctc ttgctacagc cccactcgca gcaactgct 39 25 78 DNA
Artificial Sequence M3 relaxin mRNA sequence probe 1 25 ggtggtctgt
attggcttct ccatcagcga agaagtcccg gtggtctgta ttggcttctc 60
catcagcgaa gaagtccc 78 26 39 DNA Artificial Sequence M3 relaxin
mRNA sequence probe 2 26 aatttggctc ttgctacagc cccactcgca cgaactgct
39 27 39 DNA Artificial Sequence M3 relaxin mRNA sequence probe 3
27 taaggagaca gtggacccct tggtgcctcg cctgtagga 39 28 39 DNA
Artificial Sequence M1 relaxin mRNA sequence probe 1 28 gcacatccga
atgaatccgt ccatccactc ctccgagac 39 29 39 DNA Artificial Sequence M1
relaxin mRNA sequence probe 2 29 caagcagagc tggctcctcc tggctcaaag
ccaatcttc 39 30 39 DNA Artificial Sequence M1 relaxin mRNA sequence
probe 3 30 gttgtagctc tgggagcgag gcctgagcct cagacagta 39 31 8 PRT
Artificial Sequence relaxin receptor consensus binding domain 31
Arg Xaa Xaa Xaa Arg Xaa Xaa Ile 1 5 32 1194 DNA Homo sapien 32
tataaatggg gggccaagag gcagcagaga cactggccca ctctcacgtt caaagcgtct
60 ccgtccagca tggccaggta catgctgctg ctgctcctgg cggtatgggt
gctgaccggg 120 gagctgtggc cgggagctga ggcccgggca gcgccttacg
gggtcaggct ttgcggccga 180 gaattcatcc gagcagtcat cttcacctgc
gggggctccc ggtggagacg atcagacatc 240 ctggcccacg aggctatggg
tgaggctggg gagagagtgg atgtagaagg ggaacagcac 300 taactctgtt
catcttttgc aggagatacc ttcccggatg cagatgctga tgaagacagt 360
ctggcaggcg agctggatga ggccatgggg tccagcgagt ggctggccct gaccaagtca
420 ccccaggcct tttacagggg gcgacccagc tggcaaggaa cccctggggt
tcttcggggc 480 agccgagatg tcctggctgg cctttccagc agctgctgca
agtgggggtg tagcaaaagt 540 gaaatcagta gcctttgcta gtttgagggc
tgggcagccg tgggcaccag gaccaatgcc 600 ccagtcctgc catccactca
actagtgtct ggctgggcac ctgtctttcg agcctcacac 660 attcattcat
tcatctacaa gtcacagagg cactgtgggc tcaggcacag tctcccgaca 720
ccacctatcc aaccctgccc tttgaccagc ctatcatgac cctggcccct aaggaagctg
780 tgcccctgcc tggtcaagtg gggacccccc catcctgacc cctgacctct
ccccagccct 840 aaccatgcgt ttgcctggcc tacacactcc actgccacaa
ctgggtccct actctaccta 900 ggctggccac acagagaccc ctgccccctt
cccagtccaa actgtggcca ttgtcccctg 960 accagctaaa atcaagcctc
tgtctcagtc cagcctttgc acgcacgctt cctttgccct 1020 gctttccatc
ccctctccct ccaactcccc tgccagagtt ccaaggctgt ggaccccaga 1080
gaaggtggca ggtggccccc ctaggagagc tctgggcaca ttcgaatctt cccaaactcc
1140 aataataaaa attcgaagac tttggcagag agtgtgtgtg tgtgtgtatg gttg
1194 33 1013 DNA Homo sapien 33 tataaatagg ggatcggagg tggtgcagat
agagcacctg ggtcgcaggc atctcaactg 60 atcatggcaa tgctcgggct
gctgctgctg gcttcctggg ctctcctcgg ggctctgggg 120 ctgcaggccg
aggcgaggcc ggcgccctac ggggtgaagc tctgcggtcg ggagttcatc 180
cgcgcggtca tcttcacttg cggaggctca cgatggcgcc gggcggacat cttggcccac
240 gaatctctgg gtgagtgcta ggcaatcaac ctggaacagg tgtcctggta
agcgcaactt 300 ttgcagggga cttcttcgct gatggagaag ccaatacaga
ccacctggcc agcgagctgg 360 atgaagcggt gggctccagc gagtggctgg
ccctaaccaa atccccccag gctttctacg 420 gtggtcgagc cagctggcaa
gggtcacctg gagtggttcg gggcagcaga gatgtgttgg 480 ctggcctttc
cagcagttgc tgcgagtggg gctgtagcaa gagccaaatt agcagcttgt 540
gctaggatca gggttgagca atggagaagc gggccgtgcc tgcaagctgc tgtcagctgt
600 gcgatgttca agagcattcc tacaggcgag gcaccaaggg gtccactgtc
tccttacaga 660 ccctctgcca agatgcacac actacgtgcc aacctttccc
caccttgctg ccggcccctc 720 ctctatccag ccaaacagaa acttgttttt
catgactgag ttcttccgtg ccacaacctc 780 acccccagca gcccagcagc
aaccagatgc ccatcttctt aaactggcta cactagagtc 840 tgccccacct
ccaccctcag tccggcccta attgccgcca ctgtccctgg ctaacctgcc 900
ccccccccaa aaaaaaaaaa acagagcact ctgttgcaga ccccaggact gagggcccct
960 ggtcctcagt actcagactt cctcaccaca taaaataaag gttcagttct gag 1013
34 28 PRT Homo sapien 34 Lys Trp Lys Asp Asp Val Ile Lys Leu Cys
Gly Arg Glu Leu Val Arg 1 5 10 15 Ala Gln Ile Ala Ile Cys Gly Met
Ser Thr Trp Ser 20 25 35 29 PRT Homo sapien 35 Asp Ser Trp Met Glu
Glu Val Ile Lys Leu Cys Gly Arg Glu Leu Val 1 5 10 15 Arg Ala Gln
Ile Ala Ile Cys Gly Met Ser Thr Trp Ser 20 25 36 5 PRT Artificial
Sequence Relaxin consensus sequence 36 Leu Cys Gly Arg Glu 1 5 37
27 PRT Homo sapien 37 Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly
Arg Glu Phe Ile Arg 1 5 10 15 Ala Val Ile Phe Thr Cys Gly Gly Ser
Arg Trp 20 25 38 5 PRT Artificial Sequence Relaxin consensus
sequence 38 Ala Pro Tyr Gly Val 1 5 39 19 PRT Artificial Sequence
Relaxin consensus sequence 39 Leu Cys Gly Arg Glu Phe Ile Arg Ala
Val Ile Phe Thr Cys Gly Gly 1 5 10 15 Ser Arg Trp 40 27 PRT Mus
musculus 40 Arg Pro Ala Pro Tyr Gly Val Lys Leu Cys Gly Arg Glu Phe
Ile Arg 1 5 10 15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg Trp 20 25
41 4 PRT Artificial Sequence Relaxin consensus sequence 41 Cys Gly
Arg Glu 1 42 35 PRT Mus musculus 42 Arg Val Ser Glu Glu Trp Met Asp
Gly Phe Ile Arg Met Cys Gly Arg 1 5 10 15 Glu Tyr Ala Arg Glu Leu
Ile Lys Ile Cys Gly Ala Ser Val Gly Arg 20 25 30 Leu Ala Leu 35 43
24 PRT Homo sapien 43 Arg Pro Tyr Val Ala Leu Phe Glu Lys Cys Cys
Leu Ile Gly Cys Thr 1 5 10 15 Lys Arg Ser Leu Ala Lys Tyr Cys 20 44
24 PRT Homo sapien 44 Gln Leu Tyr Ser Ala Leu Ala Asn Lys Cys Cys
His Val Gly Cys Thr 1 5 10 15 Lys Arg Ser Leu Ala Arg Phe Cys 20 45
24 PRT Homo sapien 45 Asp Val Leu Ala Gly Leu Ser Ser Ser Cys Cys
Lys Trp Gly Cys Ser 1 5 10 15 Lys Ser Glu Ile Ser Ser Leu Cys 20 46
11 PRT Artificial Sequence Relaxin consensus sequence 46 Asp Val
Leu Ala Gly Leu Ser Ser Ser Cys Cys 1 5 10 47 6 PRT Artificial
Sequence Relaxin consensus sequence 47 Trp Gly Cys Ser Lys Ser 1 5
48 5 PRT Artificial Sequence Relaxin consensus sequence 48 Ile Ser
Ser Leu Cys 1 5 49 24 PRT Rattus norvegicus 49 Asp Val Leu Ala Gly
Leu Ser Ser Ser Cys Cys Glu Trp Gly Cys Ser 1 5 10 15 Lys Ser Gln
Ile Ser Ser Leu Cys 20 50 24 PRT Mus musculus 50 Asp Val Leu Ala
Gly Leu Ser Ser Ser Cys Cys Glu Trp Gly Cys Ser 1 5 10 15 Lys Ser
Gln Ile Ser Ser Leu Cys 20 51 25 PRT Mus musculus 51 Glu Ser Gly
Gly Leu Met Ser Gln Gln Cys Cys His Val Gly Cys Ser 1 5 10 15 Arg
Arg Ser Ile Ala Lys Leu Tyr Cys 20 25
* * * * *