U.S. patent application number 14/628586 was filed with the patent office on 2015-10-29 for bioactive peptides and methods of using same.
The applicant listed for this patent is Compugen Ltd.. Invention is credited to Yossi Cohen, Dvir Dahary, Eyal Gofer, Chen Hermesh, Yossef Kliger, Zurit Levine, Ronen Shemesh, Amir Toporik, Assaf Wool.
Application Number | 20150307572 14/628586 |
Document ID | / |
Family ID | 40229171 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150307572 |
Kind Code |
A1 |
Shemesh; Ronen ; et
al. |
October 29, 2015 |
BIOACTIVE PEPTIDES AND METHODS OF USING SAME
Abstract
Disclosed are peptide ligands for G-protein coupled receptors
that are useful for treating disorders associated with G-protein
coupled receptor activation.
Inventors: |
Shemesh; Ronen; (Modiin,
IL) ; Levine; Zurit; (Herzlia, IL) ; Toporik;
Amir; (Holon, IL) ; Hermesh; Chen; (Pardes
Hana, IL) ; Kliger; Yossef; (Rishon Le Zion, IL)
; Gofer; Eyal; (Tel Aviv-Yafo, IL) ; Wool;
Assaf; (Kiriyat Ono, IL) ; Dahary; Dvir; (Tel
Aviv, IL) ; Cohen; Yossi; (Woking, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Compugen Ltd. |
Tel Aviv |
|
IL |
|
|
Family ID: |
40229171 |
Appl. No.: |
14/628586 |
Filed: |
February 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13923690 |
Jun 21, 2013 |
8987191 |
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14628586 |
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12668712 |
Jun 7, 2010 |
8492329 |
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PCT/IB2008/002447 |
Jul 11, 2008 |
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13923690 |
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60959370 |
Jul 12, 2007 |
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Current U.S.
Class: |
514/1.4 ;
514/1.7; 514/13.3; 514/15.1; 514/15.7; 514/16.4; 514/16.7;
514/16.8; 514/16.9; 514/18.6; 514/18.7; 514/19.3; 514/19.4;
514/19.5; 514/19.6; 514/20.6; 530/324; 530/326; 530/328; 530/329;
530/387.9 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
19/08 20180101; A61P 25/28 20180101; A61P 25/00 20180101; A61P
17/00 20180101; A61P 37/02 20180101; C07K 2317/34 20130101; A61P
13/12 20180101; A61P 29/00 20180101; A61P 7/02 20180101; A61P 11/06
20180101; C07K 14/472 20130101; A61P 9/00 20180101; C07K 7/06
20130101; Y02A 50/385 20180101; A61P 3/00 20180101; C07K 14/47
20130101; C07K 16/18 20130101; A61P 13/00 20180101; A61P 9/04
20180101; A61P 19/06 20180101; A61P 9/10 20180101; Y02A 50/30
20180101; A61P 11/00 20180101; A61P 19/00 20180101; A61P 9/12
20180101; A61P 35/00 20180101; C07K 14/78 20130101; A61K 38/00
20130101; A61P 17/02 20180101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; C07K 16/18 20060101 C07K016/18 |
Claims
1-21. (canceled)
22. A purified peptide less than 35 amino acids in length, said
peptide comprising an amino acid sequence of Formula I:
X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-X-
.sup.10-X.sup.11-X.sup.12-X.sup.13-X.sup.14-X.sup.15-X.sup.16-X.sup.17-X.s-
up.18-X.sup.19-X.sup.20-X.sup.21-X.sup.22-X.sup.23-X.sup.24-X.sup.25-X.sup-
.26-X.sup.27-X.sup.28-X.sup.29-X.sup.30-X.sup.31-X.sup.32-X.sup.33
wherein X.sup.1 is absent or G or a small naturally or
non-naturally occurring amino acid; X.sup.2 is absent or Q or a
polar naturally or non-naturally occurring amino acid; X.sup.3 is
absent or K or a basic naturally or non-naturally occurring amino
acid; X.sup.4 is absent or G or a small naturally or non-naturally
occurring amino acid; X.sup.5 is absent or Q or S a polar naturally
or non-naturally occurring amino acid; X.sup.6 is absent or V or A
or P or M or a hydrophobic naturally or non-naturally occurring
amino acid; X.sup.7 is absent or G or a small naturally or
non-naturally occurring amino acid; X.sup.8 is absent or P or L or
A or naturally or non-naturally occurring amino acid; X.sup.9 is
absent or P or Q or a naturally or non-naturally occurring amino
acid; X.sup.10 is absent or G or a small naturally or non-naturally
occurring amino acid; X.sup.11 is absent or A or H or E or D or a
hydrophobic or a small or an acidic naturally or non-naturally
occurring amino acid; X.sup.12 is absent or A or P or Q or S or R
or H or a hydrophobic or a small naturally or non-naturally
occurring amino acid; X.sup.13 is absent or C or V or A or any
amino acid other than C; X.sup.14 is absent or R or K or Q or P or
a basic or a polar naturally or non-naturally occurring amino acid;
X.sup.15 is absent or R or Q or S or a basic or a polar naturally
or non-naturally occurring amino acid; X.sup.16 is absent or A or L
or H or Q or a hydrophobic or a small naturally or non-naturally
occurring amino acid; X.sup.17 is absent or Y or a hydrophobic or
an aromatic naturally or non-naturally occurring amino acid;
X.sup.18 is absent or A or a hydrophobic or small naturally or
non-naturally occurring amino acid; X.sup.19 is absent or A or a
hydrophobic small naturally or non-naturally occurring amino acid;
X.sup.20 is absent or F or a hydrophobic or an aromatic naturally
or non-naturally occurring amino acid; X.sup.21 is absent or S or T
or a polar naturally or non-naturally occurring amino acid;
X.sup.22 is absent or V or a hydrophobic naturally or non-naturally
occurring amino acid; X.sup.23 is absent or G or hydrophobic or
small naturally or non-naturally occurring amino acid or replaced
by an Amide; X.sup.24 is absent or R or a basic naturally or
non-naturally occurring amino acid; X.sup.25 is absent or R or a
basic naturally or non-naturally occurring amino acid; X.sup.26 is
A or a hydrophobic or small naturally or non-naturally occurring
amino acid; X.sup.27 is Y or a hydrophobic or an aromatic naturally
or non-naturally occurring amino acid; X.sup.28 is A or a
hydrophobic or small naturally or non-naturally occurring amino
acid; X.sup.29 is A or a hydrophobic or small naturally or
non-naturally occurring amino acid; X.sup.30 is F or a hydrophobic
naturally or non-naturally occurring amino acid; X.sup.31 is S or T
or a polar naturally or non-naturally occurring amino acid;
X.sup.32 is V or a hydrophobic naturally or non-naturally occurring
amino acid; X.sup.33 is absent or G or hydrophobic or small
naturally or non-naturally occurring amino acid or replaced by an
amide; or a pharmaceutically acceptable salt thereof.
23. The peptide of claim 22, wherein said peptide binds to/and or
activates a GPCR in the LGR family of receptors or the
relaxin-related family of receptors.
24. The peptide of claim 22, wherein said peptide is isolated from
a protein recombinantly produced in a prokaryotic or eukaryotic
cell.
25. The peptide of claim 22, wherein said peptide is chemically
synthesized in vitro.
26. The peptide of claim 22, wherein said peptide includes a
C-terminal amidated amino acid.
27. The peptide of claim 22, wherein peptide is less than 30 amino
acids.
28. The peptide of claim 22, wherein said peptide is less than 20
amino acids.
29. The peptide of claim 22, wherein said peptide is selected from
the group consisting of: TABLE-US-00010 (SEQ ID NO: 1)
AYAAFSV-Amide (SEQ ID NO: 2) AYAAFSV; (SEQ ID NO: 3)
GQKGQVGPPGAACRRAYAAFSV-Amide; (SEQ ID NO: 4)
GQKGQVGPPGAACRRAYAAFSV; (SEQ ID NO: 5)
GQKGQVGPPGAAVRRAYAAFSV-Amide; (SEQ ID NO: 6)
GQKGQVGPPGAAVRRAYAAFSV; (SEQ ID NO: 7)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide; (SEQ ID NO: 8)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV; (SEQ ID NO: 9)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide; (SEQ ID NO: 10)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV; (SEQ ID NO: 11) AYAAFSVG; (SEQ ID
NO: 12) GQKGQVGPPGAACRRAYAAFSVG; (SEQ ID NO: 13)
GQKGQVGPPGAAVRRAYAAFSVG; (SEQ ID NO: 14)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG; (SEQ ID NO: 15)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG; (SEQ ID NO: 20)
GQKGQVGPPGAACQRAYAAFSVG; (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG;
(SEQ ID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; (SEQ ID NO: 23)
GQKGQAGLPGAQCPRAYAAFSVG; (SEQ ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG;
(SEQ ID NO: 25) GQKGSMGAPGERCKSHYAAFSVG; and (SEQ ID NO: 26)
GQKGSMGAPGDHCKSQYAAFSVG.
30. The peptide of claim 22, wherein said peptide is conjugated or
fused to a second peptide or polypeptide.
31. A pharmaceutical composition comprising the peptide of claim 22
and a pharmaceutically acceptable carrier.
32. A method of treating a disorder or a condition selected from
the group consisting of hypertension associated disorder,
cardiovascular disorder, fibrotic condition, endothelial
dysfunction disease, respiratory disease, skin injury, condition
associated with pregnancy, cancer, bone disease,
ischemia-reperfusion injury, inflammatory disorder, autoimmune
disorder, inflammatory condition associated with infection, kidney
disease, and angiogenesis related condition, the method comprising
administering to said subject a therapeutically effective amount of
a peptide of claim 22.
33. The method of claim 32, wherein said peptide is selected from a
group consisting of SEQ ID NOs: 1-15, 20-26.
34. The method of claim 32, wherein the hypertension associated
disorder is selected from a group consisting of hypertensive heart
disease; antihypertension (blood pressure reduction); systemic and
pulmonary high blood pressure; cerebrovascular disease and stroke;
heart failure and stroke; left ventricular hypertrophy (LVH);
congestive heart failure (CHF); hypertension, high blood pressure;
vasodilation; renal hypertension; diuresis; nephritis; natriuresis;
scleroderma renal crisis; angina pectoris (stable and unstable);
myocardial infarction; heart attack; coronary artery disease;
coronary heart disease; cardiac arrhythmias; atrial fibrillation;
portal hypertension; raised intraocular pressure; vascular
restenosis; chronic hypertension; valvular disease; myocardial
ischemia; acute pulmonary edema; acute coronary syndrome;
hypertensive retinopathy; hypertensive pregnancy sickness;
preeclampsia; Raynaud's phenomenon; erectile dysfunction and
glaucoma.
35. The method of claim 32, wherein the cancer is selected from a
group consisting of colon cancer, lung cancer, breast cancer,
prostate cancer, brain cancer, pancreatic cancer, ovarian cancer,
kidney cancer, testicular cancer, bone cancer, osteosarcoma, liver
cancer, melanoma, glioma, sarcoma, leukemia, or lymphoma, and
wherein the cancer is invasive or metastatic.
36. The method of claim 32, wherein the bone disease is selected
from a group consisting of osteoporosis; osteoarthritis;
osteopetrosis; bone inconsistency; osteosarcoma; and cancer
metastasis to the bone.
37. The method of claim 32, wherein the ischemia-reperfusion injury
is associated with ischemic and post-ischemic events in organs and
tissues and is selected from a group consisting of thrombotic
stroke; myocardial infarction; angina pectoris; embolic vascular
occlusions; peripheral vascular insufficiency; splanchnic artery
occlusion; arterial occlusion by thrombi or embolisms, arterial
occlusion by non-occlusive processes such as following low
mesenteric flow or sepsis; mesenteric arterial occlusion;
mesenteric vein occlusion; ischemia-reperfusion injury to the
mesenteric microcirculation; ischemic acute renal failure;
ischemia-reperfusion injury to the cerebral tissue; intestinal
intussusception; hemodynamic shock; tissue dysfunction; organ
failure; restenosis; atherosclerosis; thrombosis; platelet
aggregation, ischemia-reperfusion injury following cardiac surgery;
organ surgery; organ transplantation; angiography; cardiopulmonary
and cerebral resuscitation.
38. The method of claim 32, wherein the inflammatory condition is
associated with an infection, and is selected from the group
consisting of a viral infection caused by human immunodeficiency
virus I (HIV-1) or HIV-2, acquired immune deficiency (AIDS), West
Nile encephalitis virus, coronavirus, rhinovirus, influenza virus,
dengue virus, HCV, HBV, HAV, hemorrhagic fever; an otological
infection; severe acute respiratory syndrome (SARS), sepsis and
sinusitis.
39. The method of claim 32, wherein the inflammatory disorder is
selected from the group consisting of gastritis, gout, gouty
arthritis, arthritis, rheumatoid arthritis, inflammatory bowel
disease, Crohn's disease, ulcerative colitis, ulcers, chronic
bronchitis, asthma, allergy, acute lung injury, pulmonary
inflammation, airway hyper-responsiveness, vasculitis, septic shock
and inflammatory skin disorders, including but not limited to
psoriasis, atopic dermatitis, eczema.
40. An antibody that selectively binds to an epitope in the peptide
of claim 22.
41. The antibody of claim 39, wherein said peptide is selected from
the group consisting of TABLE-US-00011 (SEQ ID NO: 1) AYAAFSV-Amide
(SEQ ID NO: 2) AYAAFSV; (SEQ ID NO: 3)
GQKGQVGPPGAACRRAYAAFSV-Amide; (SEQ ID NO: 4)
GQKGQVGPPGAACRRAYAAFSV; (SEQ ID NO: 5)
GQKGQVGPPGAAVRRAYAAFSV-Amide; (SEQ ID NO: 6)
GQKGQVGPPGAAVRRAYAAFSV; (SEQ ID NO: 7)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide; (SEQ ID NO: 8)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV; (SEQ ID NO: 9)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide; (SEQ ID NO: 10)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV; (SEQ ID NO: 11) AYAAFSVG; (SEQ ID
NO: 12) GQKGQVGPPGAACRRAYAAFSVG; (SEQ ID NO: 13)
GQKGQVGPPGAAVRRAYAAFSVG; (SEQ ID NO: 14)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG; (SEQ ID NO: 15)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG; (SEQ ID NO: 20)
GQKGQVGPPGAACQRAYAAFSVG; (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG;
(SEQ ID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; (SEQ ID NO: 23)
GQKGQAGLPGAQCPRAYAAFSVG; (SEQ ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG;
(SEQ ID NO: 25) GQKGSMGAPGERCKSHYAAFSVG; and (SEQ ID NO: 26)
GQKGSMGAPGDHCKSQYAAFSVG.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/923,690 filed on Jun. 21, 2013 which is a divisional of U.S.
application Ser. No. 12/668,712 filed on Jun. 7, 2010 which is a
National Stage of International Application No. PCT/IB2008/002447
filed on Jul. 11, 2008, which in turn claims priority to U.S.
Provisional Application No. 60/959,370, filed Jul. 12, 2007. The
contents of these applications are hereby incorporated by reference
in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates to bioactive peptides.
BACKGROUND OF THE INVENTION
[0003] Known and uncharacterized GPCRs currently constitute major
targets for drug action and development. There are ongoing efforts
to identify new G protein coupled receptors and to deorphanize
known GPCRs, which can be used to screen for new agonists and
antagonists having potential prophylactic and therapeutical
properties.
SUMMARY OF THE INVENTION
[0004] The invention is based in part on the identification of
novel peptides, variants, homologs, orthologs or derivatives
thereof:
TABLE-US-00001 Peptide P59-S-Amide (Amide): (SEQ ID NO: 1)
AYAAFSV-Amide; Peptide P59-SG (free acid Gly) (SEQ ID NO: 2)
AYAAFSV; Peptide P59-Amide (amide) (SEQ ID NO: 3)
GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid) (SEQ ID NO:
4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide) (SEQ ID
NO: 5) GQKGQVGPPGAAVRRAYAAFSV-Amide Peptide P59C13V (free acid)
(SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV; Peptide P74-Amide (Amide):
(SEQ ID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide Peptide P74
(free acid) (SEQ ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide
P74C13V (amide) (SEQ ID NO: 9)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide Peptide P74C13V (free acid)
(SEQ ID NO: 10) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV Peptide P59SG
(free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acid
Gly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G
(free acid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide
P74-G (free acid Gly) (SEQ ID NO: 14)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG Peptide P74C13V-G (free acid Gly)
(SEQ ID NO: 15) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG Peptide
P59-Chimpanzee (SEQ ID NO: 20) GQKGQVGPPGAACQRAYAAFSVG; Peptide
P59-Orangutan (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG; Peptide
P59-Rhesus (SEQ ID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow
(SEQ ID NO: 23) GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ
ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human)
(SEQ ID NO: 25) GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID
NO: 26) GQKGSMGAPGDHCKSQYAAFSVG;
that are ligands for the relaxin-related GPCRs, selected from the
group consisting of RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 and RXFP4;
and/or of the LGR family GPCRs, selected from a group consisting of
but not limited to LRR (A10) containing GPCRs: FSHR (LGR1), LHCGR
(LGR2), TSHR (LGR3), LGR4, LGR5, LGR6, LGR7 (RXFP1) and LGR8
(RXFP2).
[0005] In one aspect, the invention features peptides less than 100
amino acids in length, wherein the peptide comprises the amino acid
sequence of Formula I:
X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9--
X.sup.10-X.sup.11-X.sup.12-X.sup.13-X.sup.14-X.sup.15-X.sup.16-X.sup.17-X.-
sup.18-X.sup.19-X.sup.20-X.sup.21-X.sup.22-X.sup.23-X.sup.24-X.sup.25-X.su-
p.26-X.sup.27-X.sup.28-X.sup.29-X.sup.30-X.sup.31-X.sup.32-X.sup.33
[0006] Wherein
[0007] X.sup.1 is absent or G or a small naturally or non-naturally
occurring amino acid;
[0008] X.sup.2 is absent or Q or a polar naturally or non-naturally
occurring amino acid;
[0009] X.sup.3 is absent or K or a basic naturally or non-naturally
occurring amino acid;
[0010] X.sup.4 is absent or G or a small naturally or non-naturally
occurring amino acid;
[0011] X.sup.5 is absent or Q or S a polar naturally or
non-naturally occurring amino acid;
[0012] X.sup.6 is absent or V or A or P or M or a hydrophobic
naturally or non-naturally occurring amino acid;
[0013] X.sup.7 is absent or G or a small naturally or non-naturally
occurring amino acid;
[0014] X.sup.8 is absent or P or L or A naturally or non-naturally
occurring amino acid;
[0015] X.sup.9 is absent or P or Q naturally or non-naturally
occurring amino acid;
[0016] X.sup.10 is absent or G or a small naturally or
non-naturally occurring amino acid;
[0017] X.sup.11 is absent or A or H or E or D or a hydrophobic or a
small or an acidic naturally or non-naturally occurring amino
acid;
[0018] X.sup.12 is absent or A or P or Q or S or R or H or a
hydrophobic or a small naturally or non-naturally occurring amino
acid;
[0019] X.sup.13 is absent or C or V or a hydrophobic naturally or
non-naturally occurring amino acid;
[0020] X.sup.14 is absent or R or K or Q or P or a basic or a polar
naturally or non-naturally occurring amino acid;
[0021] X.sup.15 is absent or R or Q or S or a basic or a polar
naturally or non-naturally occurring amino acid;
[0022] X.sup.16 is absent or A or L or H or Q or a hydrophobic or a
small naturally or non-naturally occurring amino acid;
[0023] X.sup.17 is absent or Y or a hydrophobic or an aromatic
naturally or non-naturally occurring amino acid;
[0024] X.sup.18 is absent or A or a hydrophobic or small naturally
or non-naturally occurring amino acid;
[0025] X.sup.19 is absent or A or a hydrophobic small naturally or
non-naturally occurring amino acid;
[0026] X.sup.20 is absent or F or a hydrophobic or an aromatic
naturally or non-naturally occurring amino acid;
[0027] X.sup.21 is absent or S or T or a polar naturally or
non-naturally occurring amino acid;
[0028] X.sup.22 is absent or V or a hydrophobic naturally or
non-naturally occurring amino acid;
[0029] X.sup.23 is absent or G or hydrophobic or small
non-naturally occurring amino acid or replaced by an Amide;
[0030] X.sup.24 is absent or R or a basic naturally or
non-naturally occurring amino acid;
[0031] X.sup.25 is absent or R or a basic naturally or
non-naturally occurring amino acid;
[0032] X.sup.26 is A or a hydrophobic or small naturally or
non-naturally occurring amino acid;
[0033] X.sup.27 is Y or a hydrophobic or an aromatic naturally or
non-naturally occurring amino acid;
[0034] X.sup.28 is A or a hydrophobic or small naturally or
non-naturally occurring amino acid;
[0035] X.sup.29 is A or a hydrophobic or small naturally or
non-naturally occurring amino acid;
[0036] X.sup.30 is F or a hydrophobic naturally or non-naturally
occurring amino acid;
[0037] X.sup.31 is S or T or a polar naturally or non-naturally
occurring amino acid;
[0038] X.sup.32 is V or a hydrophobic naturally or non-naturally
occurring amino acid;
[0039] X.sup.33 is absent or G or hydrophobic or small naturally or
non-naturally occurring amino acid or replaced by an Amide;
or a pharmaceutically acceptable salt thereof.
[0040] In some embodiments, the amino acid sequence of said
peptides differes by at least one, or at least two, or at least
three amino acids as compared to the naturally occurring amino acid
sequence set forth in SEQ ID NOs:1-15, 20-26.
[0041] In some embodiments, the peptide of this invention has at
least one-forth, e.g., one third, one half, or the same activity,
as the activity of a peptide of substantially identical length with
a naturally occurring amino acid sequence. By "substantially
identical length" is meant the same length or a difference in
length of nomore than ten percent.
[0042] In some embodiments, the peptide binds to a G-protein
coupled receptor (GPCR) protein.
[0043] In some embodiments, the GPCR protein belongs to a
relaxin-related family of GPCR proteins selected from the group
consisting of relaxin-related GPCRs, including RXFP1 (LGR7), RXFP2
(LGR8), RXFP3 and RXFP4; and/or of the LGR family of GPCRs,
selected from a group consisting of but not limited to LRR (A10)
containing GPCRs: FSHR (LGR1), LHCGR (LGR2), TSHR (LGR3), LGR4,
LGR5, LGR6, LGR7 and LGR8.
[0044] In some embodiments, the peptide inhibits forskolin-mediated
increases in cAMP levels in an LG7R7 and/or LGR8 expressing CHO-K1
cell.
[0045] In some embodiments, the peptide is a degradation product of
a naturally occurring protein isolated from a cell. In other
embodiments, the peptide is isolated from a protein recombinantly
produced in a cell. The cell can be, e.g., a prokaryotic or
eukaryotic cell.
[0046] In some embodiments, the peptide is chemically synthesized
in vitro.
[0047] If desired, the he peptide can be provided coupled to a
biotin moiety.
[0048] In some embodiments, the peptide includes a disulfide
bond.
[0049] The peptide can be provided as a linear or cyclic peptide.
In some embodiments, the peptide is a cyclic lactam. In some
embodiments, the peptide is a branched peptide.
[0050] In some embodiments, the peptide is phosphorylated, e.g., at
a serine, theronine, or tyrosine residue.
[0051] In some embodiments, a cysteine residue in a Peptide P59 or
Peptide P73 sequence is replaced with a second amino acid, e.g.,
one that prevents dimerization. Examples of suitable amino acids
include leucine, isoleucine, alanine, and valine.
[0052] In some embodiments, the peptide is modified at its amino
terminus. Example of amino terminal modifications include an
N-glycated, N-alkylated, N-acetylated or N-acylated amino acid.
[0053] In some embodiments, the peptide is pegylated.
[0054] In some embodiments, the peptide includes a C-terminal
amidated amino acid. In other embodiments, the peptide does not
include an amidated amino acid at its carboxy terminus.
[0055] In some embodiments, the non-naturally occurring amino acid
is an omega-amino acid, e.g. beta-alanine (beta-Ala), or 3
aminopropionic (3-aP).
[0056] In some embodiment, the peptide includes a small
non-naturally occurring amino acid, e.g., sarcosine (Sar),
.beta.-alanine (.beta.-Ala), 2,3 diaminopropionic (2,3-diaP) or
alpha-aminisobutyric acid (Aib); omega-acid beta-alanine
(beta-Ala), or 3 aminopropionic (3-aP) acid
[0057] In some embodiments, the peptide includes a hydrophobic
non-naturally occurring amino acid, e.g., t butylalanine (t BuA), t
butylglycine (t BuG), N methylisoleucine (N MeIle), norleucine
(Nle), methylvaline (Mvl), cyclohexylalanine (Cha), phenylglycine
(Phg), NaI, .beta.2-thienylalanine (Thi), 2 naphthylalanine (2
Nal), or 1,2,3,4-tetrahydroisoquinoline-3 carboxylic acid
(Tic).
[0058] In some embodiments, the peptide includes a basic
non-naturally occurring amino acid, e.g., ornithine (Orn) or
homoarginine (Har).
[0059] In some embodiments, the peptide includes a neutral and
polar non-naturally occurring amino acid, e.g., citrulline (Cit),
Acetyl Lys, or methionine sulfoxide (MSO).
[0060] In some embodiments, the peptide is less than 225, 200, 175,
150, 125, 100, 75, 50, 30, 25, 20, 15, 10, 9, 8, 7, 6, or 5 amino
acids.
[0061] Peptides can be modified to include one or more
modifications. Thus, in some embodiments, the carboxy terminus is
amidated and an internal cysteine is replaced with a valine at
position 13 (C13V) in Formula I, above. In other embodiments, both
modifications are present:
TABLE-US-00002 Peptide P-59S (amide) (SEQ ID NO: 1) AYAAFSV-Amide;
Peptide P-59S (free acid) (SEQ ID NO: 2) AYAAFSV; Peptide P59-Amide
(amide) (SEQ ID NO: 3) GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59
(free acid) (SEQ ID NO: 4) GQKGQVGPPGAACRRAYAAFSV; Peptide
P59C13V-Amide (amide) (SEQ ID NO: 5) GQKGQVGPPGAAVRRAYAAFSV-Amide
Peptide P59C13V (free acid) (SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV;
Peptide P74-Amide (amide) (SEQ ID NO: 7)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Amide Peptide P74 (free acid) (SEQ
ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide P74C13V-Amide
(amide) (SEQ ID NO: 9) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV Amide
Peptide P74C13V (free acid) (SEQ ID NO: 10)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV. Peptide P59SG (free acid Gly)
(SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acid Gly) (SEQ ID NO:
12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G (free acid Gly) (SEQ
ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide P74-G (free acid Gly)
(SEQ ID NO: 14) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG Peptide P74C13V-G
(free acid Gly) (SEQ ID NO: 15) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG
Peptide P59-Chimpanzee (SEQ ID NO: 20) GQKGQVGPPGAACQRAYAAFSVG;
Peptide P59-Orangutan (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG;
Peptide P59-Rhesus (SEQ ID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; Peptide
P59-Cow (SEQ ID NO: 23) GQKGQAGLPGAQCPRAYAAFSVG; Peptide
P59-Chicken (SEQ ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG; Peptide
P59-C1QTNF1 (Human) (SEQ ID NO: 25) GQKGSMGAPGERCKSHYAAFSVG;
Peptide P59-Rat (SEQ ID NO: 26) GQKGSMGAPGDHCKSQYAAFSVG.
[0062] The invention in another embodiment includes any one of the
foregoing peptides, wherein said peptide is conjugated or fused to
a second peptide or polypeptide, optionally wherein said second
peptide or polypeptide are multiple antigenic peptides (MAP), or
wherein said second peptide or polypeptide comprises a portion of
an immunoglobulin, or wherein said second peptide or polypeptide
comprises albumin or a portion of albumin.
[0063] The invention in another embodiment includes any one of the
foregoing peptides, wherein said second peptide or polypeptide
includes a signal sequence.
[0064] The invention in another embodiment includes any one of the
foregoing peptides, wherein signal sequence comprises:
MAAPALLLLALLLPVGA (SEQ ID NO:11),
TABLE-US-00003 (SEQ ID NO: 12) MAAPALLLLALLLPVGAWP, (SEQ ID NO: 13)
MAAPALLLLALLLPVGAWPGLP.
[0065] The invention in another embodiment includes a
pharmaceutical composition comprising any one of the foregoing
peptides and a pharmaceutically acceptable carrier.
[0066] The invention in another embodiment includes a peptide
comprising a fragment of any one of the foregoing peptides, wherein
said peptide fragment binds or activates a G-protein coupled
receptor (GPCR) protein, optionally wherein said GPCR protein
belongs to the family of proteins, selected from the group
consisting of relaxin-related GPCRs, including RXFP1 (LGR7), RXFP2
(LGR8), RXFP3 and RXFP4; and/or of the LGR family of GPCRs,
selected from a group consisting of but not limited to LRR (A10)
containing GPCRs: FSHR (LGR1), LHCGR (LGR2), TSHR (LGR3), LGR4,
LGR5, LGR6, LGR7 and LGR8.
[0067] The invention in another embodiment includes a purified
nucleic acid sequence encoding any one of the foregoing peptides,
variants, homoplogus, orthologus or derivatives thereof.
[0068] The invention in another embodiment includes a method of
treating a disorder where modulation or activation of a
relaxin-related GPCR and/or LGR family of GPCR receptor or
receptors is efficacious and/or of therapeutic value, the method
comprising administering to the subject a therapeutically effective
amount of a peptide of Formula I. The disorder is selected from but
not limited to hyperplastic disorders, neoplastic disorders,
cancer; fibrotic conditions, disorders of collagen deposition,
fibrotic breakdown, connective tissue remodeling, uncontrolled or
abnormal collagen or fibronectin formation or breakdown; skin
injuries including wound healing and scarring, scleroderma;
urogenital disorders including female reproductive disorders, male
and female infertility, cryptorchidism, disregulation of
spermatogenesis and reproductive development including descent of
the gonads; conditions associated with pregnancy such as
preeclampsia or complication of labor; angiogenesis related
disorders; cardiovascular disorders, vasodilatation,
vasoconstriction or hypertension, endothelial disfunction and
vascular disease, congestive heart failure, coronary artery
disease, ischemia and ischemia-reperfusion, peripheral vascular
disease; kidney disease, renal disease associated with
arteriosclerosis or other narrowing of kidney capillaries;
capillaries narrowing in the body, such as in the eyes or in the
peripheral digits, the mesocaecum, lung and peripheral vasculature;
CNS related disorders, neurological disorders, cognition and memory
related indications, depression, neurological modification;
inflammatory disorders, such as gastritis, gout, gouty arthritis,
arthritis, rheumatoid arthritis, inflammatory bowel disease,
Crohn's disease, ulcerative colitis, ulcers; autoimmune disorders;
inflammatory conditions associated with viral infection and
infection related diseases including fibrosis and cirrhosis;
Raynaud's disease, Raynaud's phenomenon; bone related conditions
including osteoporosis; metabolic disorders including food and
water intake, diabetes, obesity; respiratory or a pulmonary
disorder, including asthma, COPD, bronchial disease, lung diseases,
cystic fibrosis, ARDS, SARS.
[0069] The invention in another embodiment includes a method of
treating a hypertension and its complications, said method
comprising administering to a subject in need thereof a
therapeutically effective amount of any one of the foregoing
peptides, wherein said peptide is Formula I.
[0070] The invention in another embodiment includes the foregoing
method, wherein said hypertension associated disorder is selected
from the group consisting of hypertension and its complications
including but not limited to hypertensive heart disease;
antihypertension (blood pressure reduction); systemic and pulmonary
high blood pressure; cerebrovascular disease and stroke; heart
failure and stroke; left ventricular hypertrophy (LVH); congestive
heart failure (CHF); hypertension, high blood pressure;
vasodilation; renal hypertension; diuresis; nephritis; natriuresis;
scleroderma renal crisis; angina pectoris (stable and unstable);
myocardial infarction; heart attack; coronary artery disease;
coronary heart disease; cardiac arrhythmias; atrial fibrillation;
portal hypertension; raised intraocular pressure; vascular
restenosis; chronic hypertension; valvular disease; myocardial
ischemia; acute pulmonary edema; acute coronary syndrome;
hypertensive retinopathy; hypertensive pregnancy sickness;
preeclampsia; Raynaud's phenomenon; erectile dysfunction, glaucoma.
These peptides are also used as a vasodilator and in antithrombotic
therapy
[0071] The invention in another embodiment includes a method of
treating a cardiovascular diseases and their-complications, said
method comprising administering to a subject in need thereof a
therapeutically effective amount of any one of the foregoing
peptides, wherein said peptide is Formula I.
[0072] The invention in another embodiment includes the foregoing
method, wherein said cardiovascular disorder is selected from a
group consisting of peripheral vascular diseases and coronary
artery diseases, including but not limited to myocardial
infarction; congestive heart failure (CHF); myocardial failure;
myocardial hypertrophy; ischemic cardiomyopathy; systolic heart
failure; diastolic heart failure; stroke; thrombotic stroke;
concentric LV hypertrophy, myocarditis; cardiomyopathy;
hypertrophic cardiomyopathy; myocarditis; decompensated heart
failure; ischemic myocardial disease; congenital heart disease;
angina pectoris; prevention of heart remodeling or ventricular
remodeling after myocardial infarction; ischemia-reperfusion injury
in ischemic and post-ischemic events (e.g. myocardial infarct);
cerebrovascular accident; mitral valve regurgitation; hypertension;
hypotension; restenosis; fibrosis; thrombosis; or platelet
aggregation.
[0073] The invention in another embodiment includes a method of
treating a fibrotic condition in a subject, involving tissue
remodeling following inflammation or ischemia-reperfusion injury,
said method comprising administering to a subject in need thereof a
therapeutically effective amount of anyone of the foregoing
peptides, wherein said peptide is Formula I.
[0074] The invention in another embodiment includes the foregoing
method, wherein said fibrotic conditions is selected from a group
consisting of fibrotic conditions involving tissue remodeling
following inflammation or ischemia-reperfusion injury, including
but not limited to endomyocardial and cardiac fibrosis fibrosis;
mediastinal fibrosis; idiopathy pulmonary fibrosis; pulmonary
fibrosis; retroperitoneal fibrosis; fibrosis of the spleen;
fibrosis of the pancreas; hepatic fibrosis (cirrhosis) alcohol and
non-alcohol related (including viral infection such as HAV, HBV and
HCV); fibromatosis; granulomatous lung disease; glomerulonephritis
myocardial scarring following infarction; endometrial fibrosis and
endometriosis; wound healing whether by injury or surgical
procedures, diabetes related wound fibrosis.
[0075] The invention in another embodiment includes a method of
treating a endothelial dysfunction disease in a subject, said
method comprising administering to a subject in need thereof a
therapeutically effective amount of anyone of the foregoing
peptides, wherein said peptide is Formula I.
The invention in another embodiment includes the foregoing method,
wherein said endothelial dysfunction disease is selected from a
group consisting of cardiovascular diseases, high blood pressure,
atherosclerosis, thrombosis, myocardial infarct, heart failure,
renal diseases, plurimetabolic syndrome, erectile dysfunction;
vasculitis; and diseases of the central nervous system (CNS).
[0076] The invention in another embodiment includes a method of
treating a respiratory disease in a subject, said method comprising
administering to a subject in need thereof a therapeutically
effective amount of anyone of the foregoing peptides, wherein said
peptide is Formula I.
[0077] The invention in another embodiment includes the foregoing
method, wherein said respiratory disease is selected from a group
consisting of including but not limited to asthma, bronchial
disease, lung diseases, chronic obstructive pulmonary disease
(COPD), Acute Respiratory Distress Syndrome (ARDS), severe acute
respiratory syndrome (SARS), Fibrosis related Asthma, cystic
fibrosis.
[0078] The invention in another embodiment includes a method of
preventing or treating a skin injury or tissue repair, said method
comprising administering to a subject in need thereof a
therapeutically effective amount of anyone of the foregoing
peptides, wherein said peptide is Formula I.
[0079] The invention in another embodiment includes the foregoing
method, wherein said skin injury is selected from a group including
but not limited to dermal repair, wound healing; burns, erythemas,
lesions, wound healing following surgical procedures; skin or
tissue lesions including lesions induced by conditions including,
but not limited to Psoriasis, Lupus and Kaposhi Sarcoma;
Scleroderma and collagenous diseases of the skin and skin
tumors.
[0080] The invention in another embodiment includes a method of
treating a urogenital disorder or a genitor-urological disorder in
a subject, said method comprising administering to a subject in
need thereof a therapeutically effective amount of anyone of the
foregoing peptides, wherein said peptide is Formula I.
[0081] The invention in another embodiment includes the foregoing
method, wherein said urogenital disorder or genitor-urological
disorders is selected from group consisting of urogenital disorder
or a genitor-urological disorders including but not limited to
renal disease; a bladder disorder; disorders of the reproductive
system; gynecologic disorders; urinary tract disorder;
incontinence; disorders of the male (spermatogenesis, spermatic
motility), and female reproductive system; sexual dysfunction;
erectile dysfunction; embryogenesis; and conditions associated with
pregnancy. These are also used in pregnancy monitoring. As used
herein, the term "conditions associated with pregnancy" includes,
but is not limited to, conditions of fertilisation, pregnancy,
parturition and lactation. The invention in another embodiment
includes using the peptides of the invention falling within
Formulas I, such as for example, peptides as depicted in SEQ ID
NOs:6-7, 9-16, 18-25, 27-31, wherein said pregnancy related
disorders are selected from a group consisting of Abnormal
endometrial angiogenesis; Placental development defects; Cervical
ripening (softening); Abnormal implantation; Nipple development and
disfunction; Pregnancy related remodeling of the Uterine tissue;
Endometriosis; Preeclampsia; Lactation disorders; Estrogenic and
non-estrogenic related hormonal disorders; Pre-term labor; post
term labor; and Labor complications.
[0082] The invention in another embodiment includes a method of
treating a cancer or inflammation associated with cancer in a
patient, said method comprising administering to a subject in need
thereof a therapeutically effective amount of anyone of the
foregoing peptides, wherein said peptide is Formula I.
[0083] The invention in another embodiment includes the foregoing
method, wherein said cancer is selected from a group consisting of
solid cancer, including but not limited to colon cancer, lung
cancer, breast cancer, prostate cancer, brain cancer, pancreatic
cancer, ovarian cancer, kidney cancer, testicular cancer, bone
cancer, osteosarcoma, or liver cancer (HBV/HCV related or
non-related). The cancer can alternatively be a melanoma, glioma, a
sarcoma, a leukemia, or lymphoma. These peptides are also useful in
the prevention or treatment of invasive and metastatic cancer.
[0084] The invention in another embodiment includes a method of
treating a bone and bone related disorders in a patient, said
method comprising administering to a subject in need thereof a
therapeutically effective amount of anyone of the foregoing
peptides, wherein said peptide is Formula I.
[0085] The invention in another embodiment includes the foregoing
method, wherein said bone disease is selected from a group
including but not limited to Osteoporosis; Osteoarthritis;
Osteopetrosis; Bone inconsistency; Osteosarcoma; and Cancer
matastesis to the bone.
[0086] The invention in another embodiment includes a method of
treating a metabolic and metabolic related disorders in a patient,
said method comprising administering to a subject in need thereof a
therapeutically effective amount of anyone of the foregoing
peptides, wherein said peptide is Formula I.
[0087] The invention in another embodiment includes the foregoing
method, wherein said metabolic disorder is selected from a group
including but not limited to diabetes, diabetes mellitus,
lipodystrophy, hyperthyroidism, glaucoma, hyperlipidaemia,
non-insulin dependent diabetes, Food intake; Water intake; Feeding
and drinking behaviors, Anorexia, Cachexia (cancer and non cancer
related); Fat and lipid metabolism; and Energy control, appetite
control and obesity.
[0088] The invention in another embodiment includes a method of
treating a CNS and cognition disorders in a patient, said method
comprising administering to a subject in need thereof a
therapeutically effective amount of anyone of the foregoing
peptides, wherein said peptide is Formula I.
[0089] The invention in another embodiment includes the foregoing
method, wherein said CNS and cognition disorders are selected from
a group including but not limited to central and peripheral
degenerative neuropathies; neuroprotection; impaired cognition;
anxiety disorders, pain control, food intake, a behavioral
disorder, a learning disorder, a sleep disorder, a memory disorder,
a pathologic response to anesthesia, addiction, depression,
migraine, a menstruation disorder, muscle spasm, opiate dependence,
dementia, Alzheimer's disease, Parkinson's disease, cortical
function, locomotor activity, Alcohol and Drug addiction and abuse;
Impared memory; Feeding and drinking related behaviours; Stress
control, Bipolar disorder; Schyzophrenia; Schyzoaffective; Multiple
Sclerosis (MS); Stroke and stroke damage repair (Ischemia
protection); Vasculature and re-vasculature in the brain; and Brain
tissue regeneration and a peripheral nervous system disorder.
[0090] The invention in another embodiment includes a method of
treating ischemia-reperfusion injury associated with ischemic and
post-ischemic events in organs and tissues in a patient, said
method comprising administering to a subject in need thereof a
therapeutically effective amount of anyone of the foregoing
peptides, wherein said peptide is Formula I.
[0091] The invention in another embodiment includes the foregoing
method, wherein said ischemia-reperfusion injury associated with
ischemic and post-ischemic events in organs and tissues disorders
are selected from a group including but not limited to thrombotic
stroke; myocardial infarction; angina pectoris; embolic vascular
occlusions; peripheral vascular insufficiency; splanchnic artery
occlusion; arterial occlusion by thrombi or embolisms, arterial
occlusion by non-occlusive processes such as following low
mesenteric flow or sepsis; mesenteric arterial occlusion;
mesenteric vein occlusion; ischemia-reperfusion injury to the
mesenteric microcirculation; ischemic acute renal failure;
ischemia-reperfusion injury to the cerebral tissue; intestinal
intussusception; hemodynamic shock; tissue dysfunction; organ
failure; restenosis; atherosclerosis; thrombosis; platelet
aggregation.
[0092] The invention in another embodiment includes a method of
treating ischemia-reperfusion injury following conditions including
but not limited to procedures such as cardiac surgery; organ
surgery; organ transplantation; angiography; cardiopulmonary and
cerebral resuscitation in a patient, said method comprising
administering to a subject in need thereof a therapeutically
effective amount of anyone of the foregoing peptides, wherein said
peptide is Formula I.
[0093] The invention in another embodiment includes a method of
inflammatory conditions associated with an infection, e.g., a
bacterial infection or a viral infection in a patient, said method
comprising administering to a subject in need thereof a
therapeutically effective amount of anyone of the foregoing
peptides, wherein said peptide is Formula I.
[0094] The invention in another embodiment includes the foregoing
method, wherein said inflammatory conditions associated with an
infection, are selected from but not limited to including but not
limited to a viral infection caused by human immunodeficiency virus
I (HIV-1) or HIV-2, acquired immune deficiency (AIDS), West Nile
encephalitis virus, coronavirus, rhinovirus, influenza virus,
dengue virus, HCV, HBV, HAV, hemorrhagic fever; an otological
infection; severe acute respiratory syndrome (SARS), sepsis and
sinusitis.
[0095] The invention in another embodiment includes a method of
kidney diseases in a patient, said method comprising administering
to a subject in need thereof a therapeutically effective amount of
anyone of the foregoing peptides, wherein said peptide is Formula
I.
[0096] The invention in another embodiment includes the foregoing
method, wherein said kidney diseases are selected from but not
limited to diabetic nephropathy; glomerulosclerosis; nephropathies;
renal impairment; scleroderma renal crisis and chronic renal
failure.
[0097] The invention in another embodiment includes a method of
angiogenesis related conditions in a patient, said method
comprising administering to a subject in need thereof a
therapeutically effective amount of anyone of the foregoing
peptides, wherein said peptide is Formula I.
[0098] The invention in another embodiment includes the foregoing
method, wherein said angiogenesis related conditions are selected
from but not limited to retinal angiogenesis in a number of human
ocular diseases such as diabetes mellitus, retinopathy of
prematury, and age-related macular degeneration, or cancer
associated angiogenesis in primary or metastatic cancer, including
but not limited to cancer of the prostate, brain, breast,
colorectal, lung, ovarian, pancreatic, renal, cervical, melanoma,
soft tissue sarcomas, lymphomas, head-and-neck, and
glioblastomas.
[0099] The invention in another embodiment includes a method of
inflammatory disorder in a patient, said method comprising
administering to a subject in need thereof a therapeutically
effective amount of anyone of the foregoing peptides, wherein said
peptide is Formula I.
[0100] The invention in another embodiment includes the foregoing
method, wherein said inflammatory disorder are selected from but
not limited to gastritis, gout, gouty arthritis, arthritis,
rheumatoid arthritis, inflammatory bowel disease, Crohn's disease,
ulcerative colitis, ulcers, chronic bronchitis, asthma, allergy,
acute lung injury, pulmonary inflammation, airway
hyper-responsiveness, vasculitis, septic shock and inflammatory
skin disorders, including but not limited to psoriasis, atopic
dermatitis, eczema.
[0101] The invention in another embodiment includes a method of
autoimmune disorder in a patient, said method comprising
administering to a subject in need thereof a therapeutically
effective amount of anyone of the foregoing peptides, wherein said
peptide is Formula I. The invention in another embodiment includes
the foregoing method, wherein said autoimmune disorder are selected
from but not limited to multiple sclerosis, psoriasis, rheumatoid
arthritis, systemic lupus erythematosus, ulcerative colitis,
Crohn's disease, transplant rejection, immune disorders associated
with graft transplantation rejection, benign lymphocytic angiitis,
lupus erythematosus, Hashimoto's thyroiditis, primary myxedema,
Graves' disease, pernicious anemia, autoimmune atrophic gastritis,
Addison's disease, insulin dependent diabetes mellitis, Good
pasture's syndrome, myasthenia gravis, pemphigus, sympathetic
ophthalmia, autoimmune uveitis, autoimmune hemolytic anemia,
idiopathic thrombocytopenia, primary biliary cirrhosis, chronic
action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic
disease, polymyositis, scleroderma, mixed connective tissue
disease, inflammatory rheumatism, degenerative rheumatism,
extra-articular rheumatism, collagen diseases, chronic
polyarthritis, psoriasis arthropathica, ankylosing spondylitis,
juvenile rheumatoid arthritis, periarthritis humeroscapularis,
panarteriitis nodosa, progressive systemic scleroderma, arthritis
uratica, dermatomyositis, muscular rheumatism, myositis, myogelosis
and chondrocalcinosis.
[0102] The invention in another embodiment includes the cDNA that
encodes the peptide sequences of the invention, which can be used
in gene therapy.
[0103] If desired, gene therapy can be used to deliver to a subject
a peptide according to the invention. A nucleic acid encoding the
peptide can be inserted into vectors, which are then used as gene
therapy vectors. Gene therapy vectors can be delivered to a subject
by, for example, intravenous injection, local administration or by
stereotactic injection. The pharmaceutical preparation of the gene
therapy vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g., retroviral vectors, the pharmaceutical preparation can
include one or more cells that produce the gene delivery
system.
[0104] The invention in another embodiment includes combination
therapy using one or more peptides of the present invention
provided in combination with another therapeutic agent or agents.
As used herein, the term "combination therapy" refers to treatment
of a single condition or disease involving the concomitant use of
more than one therapeutic agent.
[0105] The invention in another embodiment includes an antibody
that selectively binds to an epitope in anyone of the foregoing
peptides.
[0106] In a still further aspect, the invention provides an
antibody that selectively binds to an epitope in the peptide of
Formula I.
[0107] In some embodiments, the antibody binds selectively and/or
is raised to the following sequences:
TABLE-US-00004 Peptide P-59S-Amide (amide) (SEQ ID NO: 1)
AYAAFSV-Amide; Peptide P-59S (free acid) (SEQ ID NO: 2) AYAAFSV;
Peptide P59-Amide (amide) (SEQ ID NO: 3)
GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid) (SEQ ID NO:
4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide) (SEQ ID
NO: 5) GQKGQVGPPGAAVRRAYAAFSV-Amide Peptide P59C13V (free acid)
(SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV; Peptide P74-Amide (amide)
(SEQ ID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Amide Peptide P74
(free acid) (SEQ ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide
P74C13V-Amide (amide) (SEQ ID NO: 9)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV Amide Peptide P74C13V (free acid)
(SEQ ID NO: 10) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV. Peptide P59SG
(free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acid
Gly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G
(free acid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide
P74-G (free acid Gly) (SEQ ID NO: 14)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG Peptide P74C13V-G (free acid Gly)
(SEQ ID NO: 15) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG Peptide
P59-Chimpanzee (SEQ ID NO: 20) GQKGQVGPPGAACQRAYAAFSVG; Peptide
P59-Orangutan (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG; Peptide
P59-Rhesus (SEQ ID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow
(SEQ ID NO: 23) GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ
ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human)
(SEQ ID NO: 25) GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID
NO: 26) GQKGSMGAPGDHCKSQYAAFSVG.
[0108] The invention in another embodiment includes anyone of the
foregoing antibodies, wherein the antibody is a monoclonal
antibody.
[0109] The invention in another embodiment includes anyone of the
foregoing antibodies, wherein the antibody is conjugated or coupled
to a detectable label, a radioactive label, an enzyme, a
fluorescent label, a luminescent label, a bioluminescent label, or
a therapeutic agent.
[0110] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present Specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0111] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0112] FIG. 1 is a graph showing the Gi (cAMP inhibition) effect of
P59C13V (P59) and P74C13V (P74) on forskolin-treated LGR7 and LGR8
transiently transfected CHO-K1 cells.
[0113] FIG. 2 presents a Gi (cAMP inhibition) dose response curve
at concentrations between 1 nM to 10 uM of P59C13V (P59), P74C13V
(P74) and INSL3 on CHO-K1 cells transiently transfected with LGR8
(RXFP2) and pre-treated with 20 uM of Forskolin.
[0114] FIG. 3A is a graph showing a Gs (cAMP increase) assay
following no treatment or treatment with HCG (control), P59C13V
(P59) and P74C13V (P74), respectively on LGR4, LGR5, and LGR6
transfected CHO-K1 cells.
[0115] FIG. 3B is a graph showing a Gi (cAMP inhibition) assay
following pre treatment by 20 uM of Forskolin followed by no
treatment or treatment with HCG (control), P59C13V (P59), and
P74C13V (P74), respectively on LGR5, transfected CHO-K1 cells.
[0116] FIG. 3C is a graph showing a Gi (cAMP inhibition) assay
following pre treatment by 20 uM of Forskolin followed by no
treatment or treatment with HCG (control), P59C13V (P59), and
P74C13V (P74), respectively on LGR4, transfected CHO-K1 cells.
[0117] FIG. 4A is a graph showing cAMP dose response activation
following treatment pre-treated with 20 uM of Forskolin and
treatment with increasing doses (1 nM-10 uM) of P59C13V (P59),
P74C13V (P74) or relaxin, on CHO-K1 cells transiently transfected
with LGR7.
[0118] FIG. 4B is a graph showing Gs (cAMP increase) dose response
activation following treatment with low concentrations (1-100 nM)
of P59C13V (P59) or relaxin on CHO-K1 cells transiently transfected
with LGR7.
[0119] FIG. 5 is a graph showing Gs (cAMP increase) dose respose
activation of Euroscreen recombinant cell lines expressing the
RXFP3 receptor (Cat: D88437, assay cat: ES-656-MG). The cAMP dose
response was compared between Relaxin 3 (positive control) and
P59C13V (P59) at concentrations of 1 pM-1 uM for Relaxin 3 and 1
nM-10 uM for P59C13V (P59).
[0120] FIGS. 6A-B are graphs showing cAMP responsive element
reading (CRE) in response to stimulation of LGR8 expressing HEK293
cells to INSL3 (as a positive control) and the peptides P59-S amide
and P74C13V (P74) (FIG. 6A); or P59C13V (P59) and P59C13V-amide
(P59-amide)) (FIG. 6B). The X axis represents the peptide
concentration (log) and the Y axis represents the CRE activation
relative to INSL3 max activity (in %).
[0121] FIGS. 7A-B are graphs showing cAMP responsive element
reading (CRE) in response to stimulation of LGR7 expressing CHO-K1
cells to pre treatment with 5 uM of Forskolin and H2 relaxin (H2)
as a positive control compared with the peptides P59-S amide and
P74C13V (P74) (FIG. 7A); or P59C13V (P59) and P59C13V-amide
(P59-amide) (FIG. 7B). The X axis represents the peptide
concentration (log) and the Y axis represents the CRE activation
relative to the base line Forskolin activity (in % where 100% is
with no peptide treatment). This graph is the average result of
three independent experiments (N=3).
[0122] FIGS. 8A-B are graphs showing cAMP responsive element
reading (CRE) in response to stimulation of LGR8 expressing CHO-K1
cells to pre treatment with 5 uM of Forskolin and INSL3 as a
positive control compared with the peptides P59-S amide and P74C13V
(P74) (FIG. 8A); or P59C13V (P59) and P59C13V-amide (P59-amide)
(FIG. 8B). The X axis represents the peptide concentration (log)
and the Y axis represents the CRE activation relative to the base
line Forskolin activity (in % where 100% is with no peptide
treatment). This graph is the average result of three independent
experiments (N=3).
[0123] FIG. 9 is a graph showing competitive binding of LGR7
between a Europium (radioactive) conjugated Relaxin (H2) and either
non-radioactive H2 relaxin and each of the peptides: P59-S-amide,
P74C13V (P74), P59C13V (P59) and P59C13V-amide (P59-amide). The X
axis represents the peptide concentration (log) and the Y axis
represents the specific binding represented by radioactivity
(%).
[0124] FIG. 10 is a graph showing competitive binding of LGR8
between a Europium (radioactive) conjugated INSL3 and either
non-radioactive INSL3 and each of the peptides: P59-S-amide,
P74C13V (P74), P59C13V (P59) and P59C13V-amide (P59-amide). The X
axis represents the peptide concentration (log) and the Y axis
represents the specific binding represented by radioactivity
(%).
[0125] FIG. 11 is a graph showing the cell impedance and
cytoskeleton remodeling effect (measured by ACEA RT-CES system) of
H2 Relaxin (1 uM), P74C13V (P74-10 uM), 0.1% BSA (as a negative
control) and 1 uM of Calcitonin (a ligand for CalcR a GPCR
endogenously expressed on CHO-K1 cells, as a positive internal
control) on Untransfected CHO-K1 cells. The X axis represents time
of experiment (challenge time is indicated by a vertical line), and
the Y axis represents the cell index (a measurement of the change
in Cell impedance) normalized to the peptide challenge time. The
code is as follows: circles represent calcitonin 1 uM; short
horizontal lines represents H2 1 uM; squares represent P74 10 uM;
and diamond shapes represent BSA 0.1%.
[0126] FIG. 12 is a graph showing the cell impedance and
cytoskeleton remodeling effect (measured by ACEA RT-CES system) of
H2 Relaxin (1 uM), P74C13V (P74-10 uM), 0.1% BSA (as a negative
control) and 1 uM of Calcitonin (a ligand for CalcR a GPCR
endogenously expressed on CHO-K1 cells, as a positive internal
control) on GPR39 (a non relaxin related GPCR) transfected CHO-K1
cells. The X axis represents time of experiment (challenge time is
indicated by a vertical line), and the Y axis represents the cell
index (a measurement of the change in Cell impedance) normalized to
the peptide challenge time. The code is as follows: circles
represent calcitonin 1 uM; diamond shapes represent P74, 10 uM;
squares represent H2, 1 uM; and short horizontal lines represent
BSA 0.1%.
[0127] FIG. 13 is a graph showing the cell impedance and
cytoskeleton remodeling effect (measured by ACEA RT-CES system) of
H2 Relaxin (1 uM), P74C13V (P74-10 uM), 0.1% BSA (as a negative
control) and 1 uM of Calcitonin (a ligand for CalcR a GPCR
endogenously expressed on CHO-K1 cells, as a positive internal
control) on LGR7 (RXFP1) transfected CHO-K1 cells. The X axis
represents time of experiment (challenge time is indicated by a
vertical line), and the Y axis represents the cell index (a
measurement of the change in Cell impedance) normalized to the
peptide challenge time. The code is as follows: diamond shapes
represent P74, 10 uM; circles represent H2, 1 uM; short horizontal
lines represent calcitonin 1 uM; and squares represent BSA
0.1%.
[0128] FIG. 14 is a graph showing the cell impedance and
cytoskeleton remodeling effect (measured by ACEA RT-CES system) of
H2 Relaxin (1 uM), P74C13V (P74-10 uM), P59C13V (P59), 0.1% BSA (as
a negative control) and 1 uM of Calcitonin (a ligand for CalcR a
GPCR endogenously expressed on CHO-K1 cells, as a positive internal
control) on LGR8 (RXFP2) transfected CHO-K1 cells. The X axis
represents time of experiment (challenge time is indicated by a
vertical line), and the Y axis represents the cell index (a
measurement of the change in Cell impedance) normalized to the
peptide challenge time. The code is as follows: diamond shapes
represent P59, 1 uM; circles represent H2, 1 uM; short horizontal
lines represent calcitonin 1 uM; squares represent P74, 10 uM; and
triangles represent BSA 0.1%.
[0129] FIG. 15 is a graph showing the cell impedance and
cytoskeleton remodeling effect (measured by ACEA RT-CES system) of
H2 Relaxin (1 uM), P74C13V (P74-10 uM), 0.1% BSA (as a negative
control) and 1 uM of Calcitonin (a ligand for CalcR a GPCR
endogenously expressed on CHO-K1 cells, as a positive internal
control) on LGR4 (Orphan) transfected CHO-K1 cells. The X axis
represents time of experiment (challenge time is indicated by a
vertical line), and the Y axis represents the cell index (a
measurement of the change in Cell impedance) normalized to the
peptide challenge time. The code is as follows: circles represent
P74, 10 uM; diamond shapes represent H2, 1 uM; squares represent
calcitonin 1 uM; and short horizontal lines represent BSA 0.1%.
[0130] FIGS. 16A-B is a graph showing the cell impedance and
cytoskeleton remodeling effect (measured by ACEA RT-CES system) as
a dose dependant response of different concentrations of H2 Relaxin
on LGR7 (RXFP1) transfected CHO-K1 cells. FIG. 16A: The X axis
represents time of experiment (challenge time is indicated by a
vertical line), and the Y axis represents the cell index (a
measurement of the change in Cell impedance) normalized to the
peptide challenge time (indicated by the left vertical line). Each
line represents a different concentration (according to legend).
FIG. 16B: a dose response curve as calculated by the ACEA RT-CES
system according to the results in FIG. 16A. The X axis represents
the peptide's concentration in log of M. The Y axis represents the
normalized cell index at the end point of the experiment (indicated
by the right vertical line in FIG. 16A). The code for FIG. 16A is
as follows: 0.16 nM; 0.8 nM; 4 nM; 20 nM; 100 nM; and 500 nM are
represented by black circles; triangles; squares; diamond-shapes;
white circles; and horizontal lines, respectively.
[0131] FIGS. 17A-B is a graph showing the cell impedance and
cytoskeleton remodeling effect (measured by ACEA RT-CES system) as
a dose dependant response of different concentrations of P74C13V
(P74) on LGR7 (RXFP1) transfected CHO-K1 cells. FIG. 17A: The X
axis represents time of experiment (challenge time is indicated by
a vertical line), and the Y axis represents the cell index (a
measurement of the change in Cell impedance) normalized to the
peptide challenge time (indicated by the left vertical line). Each
line represents a different concentration (according to legend).
FIG. 17B: a dose response curve as calculated by the ACEA RT-CES
system according to the results in FIG. 17A. The X axis represents
the peptide's concentration in log of M. The Y axis represents the
normalized cell index at the end point of the experiment (indicated
by the right vertical line in FIG. 17A). The code for FIG. 17A is
as follows: 41 nM; 123 nM; 370 nM; 1.1 uM; 3.3 uM; and 10 uM are
represented by black circles; triangles; squares; diamond-shapes;
white circles; and horizontal lines, respectively.
[0132] FIG. 18A-B is a graph showing the cell impedance and
cytoskeleton remodeling effect (measured by ACEA RT-CES system) as
a dose dependant response of different concentrations of P59C13V
(P59) on LGR7 (RXFP1) transfected CHO-K1 cells. FIG. 18A: The X
axis represents time of experiment (challenge time is indicated by
a vertical line), and the Y axis represents the cell index (a
measurement of the change in Cell impedance) normalized to the
peptide challenge time (indicated by the left vertical line). Each
line represents a different concentration (according to legend).
FIG. 18B: a dose response curve as calculated by the ACEA RT-CES
system according to the results in FIG. 18A. The X axis represents
the peptide's concentration in log of M. The Y axis represents the
normalized cell index at the end point of the experiment (indicated
by the right vertical line in FIG. 18A). The code for FIG. 18A is
as follows: 41 nM; 123 nM; 370 nM; 1.1 uM; 3.3 uM; and 10 uM are
represented by black circles; triangles; squares; diamond-shapes;
white circles; and horizontal lines, respectively.
[0133] FIG. 19A-B is a graph showing the cell impedance and
cytoskeleton remodeling effect (measured by ACEA RT-CES system) as
a dose dependant response of different concentrations of P59S-Amide
on LGR7 (RXFP1) transfected CHO-K1 cells. FIG. 18A: The X axis
represents time of experiment (challenge time is indicated by a
vertical line), and the Y axis represents the cell index (a
measurement of the change in Cell impedance) normalized to the
peptide challenge time (indicated by the left vertical line). Each
line represents a different concentration (according to legend).
FIG. 19B: a dose response curve as calculated by the ACEA RT-CES
system according to the results in FIG. 19A. The X axis represents
the peptide's concentration in log of M. The Y axis represents the
normalized cell index at the end point of the experiment (indicated
by the right vertical line in FIG. 19A). The code for FIG. 19A is
as follows: 41 nM; 123 nM; 370 nM; 1.1 uM; 3.3 uM; and 10 uM are
represented by black circles; triangles; squares; diamond-shapes;
white circles; and horizontal lines, respectively.
[0134] FIG. 20: This figure shows a multiple alignment of the
sequence of P59-G (SEQ ID No. 12) as a part of the native precursor
(C1QTNF8--SEQ. ID. No. 19), including the flanking dibasic (K or R)
cleavage sites, with homologous sequences derived from different
organisms, including Chimpanzee (SEQ ID No. 20), Orangutan (SEQ ID
No. 21), Rhesus (SEQ ID No. 22), Cow (SEQ ID No. 23), Chicken (SEQ
ID No. 24) and Rat (SEQ ID No. 26), and the corresponding peptide
sequence from the human paralogue C1QTNF1 (SEQ ID No. 25) (all
sequences include the flanking dibasic cleavage sites). As can be
seen both the N-terminal end (4 Amino acids) and C-terminal end (7
Amino acids) are identical to all species. The cleavage sites at
both ends are also highly conserved (with occasional replacement of
K for an R). The middle Cysteine residue (C13) that was replaced
with Valine for dimerization purposes also is highly conserved.
DETAILED DESCRIPTION OF THE INVENTION
[0135] The invention provides bioactive peptides. These peptides
are useful, inter alia, for treating a variety of indications and
disorders, which are discussed in detail below. In some
embodiments, the peptides are ligands for GPCR receptors.
[0136] The peptides disclosed herein are related to GPCR ligand of
the LGR and Relaxin family: [0137] 1. The LGR family: consists of
all LRR containing GPCRs including LGR1 (FSHR), LGR2 (LHCGR), LGR3
(TSHR), KGR4, LGR5, LGR6, LGR7 (RXFP1) and LGR8 (RXFP2). [0138] 2.
The Relaxin related family: consists of all Relaxin activated
receptors including RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135) and
RXFP4 (GPCR142).
[0139] LGR8 (RXFP2) (SwissProt accession number: RXFP2_HUMAN) is a
lower affinity receptor for relaxin (H2 and H1). The activity of
this receptor is mostly but not exclusively mediated by G proteins
leading to stimulation of adenylate cyclase and an increase of
cAMP. Binding of the ligand activates a TRK pathway. LGR8 is also a
high affinity receptor for the Leydig insulin-like 3 peptide
(INSL3). LGR8 belongs to the G-protein coupled receptor 1 family.
It contains 1 LDL-receptor class A domain and 10 LRR (leucine-rich)
repeats. LGR8 is expressed in the brain, kidney, muscle, thyroid,
testis, placenta, uterus, ovary, adrenal, prostate, skin,
peripheral blood cells, bone marrow, osteoblasts and heart. Defects
in LGR8 are a cause of cryptorchidism, also known as impaired
testicular descent, one of the most frequent congenital
abnormalities in humans, involving 2-5% of male births.
Cryptorchidism is associated with increased risk of infertility and
testicular cancer. LGR8 is involved in CNS related diseases like
Alzheimer (Shen P J., et al., Ann N Y Acad Sci. 2005; 1041:510-5).
Stimulation with relaxin increases cell proliferation,
invasiveness, and adhesion in vitro. The suppression of relaxin
decreased cell invasiveness by 90% and growth by 25% and increased
cell apoptosis up by 2.2 times (Feng S, et al. Clin. Cancer Res.
(2007). LGR8 is also involved in Osteoporosis, Female infertility
and non cryptorchidism related male infertility.
[0140] LGR7 (RXFP1) (SwissProt Accession Number: RXFP1_HUMAN) is a
receptor for relaxin. LGR7 binds all human relaxin peptides with
high affinity, but has very low affinity for INSL3. The activity of
this receptor is mediated by G proteins leading both to stimulation
of adenylate cyclase and an increase of cAMP, and inhibition of
adenylate cyclase and a decrease of cAMP depending on the tissue
and cell type. Binding of the ligand activates a TRK pathway that
inhibits the activity of a phosphodiesterase that degrades cAMP.
LGR7 belongs to the G-protein coupled receptor 1 family. It
contains 1 LDL-receptor class A domain and 10 LRR (leucine-rich)
repeats. LGR7 is expressed in the brain, liver, kidney, testis,
breast placenta, uterus, uterus endometrium, cervix, vagina, ovary,
adrenal, prostate, skin, osteoclasts and heart. The LGR7 receptor
is regulated by Estrogen and the activity of Estrogen receptors.
LGR7 is involved in many processes including Collagen deposition
and scaring, Pregnancy and labor processes including implantation
and cervical ripening, Vasodilatation, Tumor progression and
invasiveness, food intake and Osteoporosis.
[0141] LGR4 (SwissProt Accession Number: LGR4_HUMAN; G-protein
coupled receptor 48 (GPR48)) is an orphan seven trans-membrane
receptor that belongs to the G-protein coupled receptor 1 family.
It contains 15 LRR (leucine-rich) repeats. LGR4 is expressed in
multiple steroidogenic tissues: placenta, ovary, testis and
adrenal. It is also expressed in spinal cord, thyroid, stomach,
trachea, heart, pancreas, kidney, prostate and spleen.
[0142] LGR5 (SwissProt Accession Number: LGR5_HUMAN; G-protein
coupled receptor 49; G-protein coupled receptor 67 (GPR49, GPR67))
is an orphan seven trans-membrane receptor that belongs to the
G-protein coupled receptor 1 family. It contains 17 LRR
(leucine-rich) repeats. LGR5 is prediocted to be an important
receptor for signals controlling growth and differentiation of
specific embryonic tissues [Morita H, et al. Mol Cell Biol. 2004
November; 24(22):9736-43].
[0143] LGR6 (SwissProt Accession Number: LGR6_HUMAN) is an orphan
seven trans-membrane receptor that belongs to the G-protein coupled
receptor 1 family. It contains 16 LRR (leucine-rich) repeats. LGR6
is expressed in multiple steroidogenic tissues: placenta, ovary,
testis, and adrenal. It is also expressed in spinal cord, thyroid,
stomach trachea, heart, pancreas, kidney, prostate and spleen.
[0144] GPCR135 (RXFP3) (SwissProt Accession Number: RL3R1_HUMAN;
Relaxin-3 receptor 1, RLN3 receptor 1, Relaxin family peptide
receptor 3, Somatostatin- and angiotensin-like peptide receptor, G
protein-coupled receptor SALPR, GPCR135), is the receptor for
relaxin-3 (H3 Relaxin). The RXFP3 receptor also has a lower
affinity for Relaxin 2 (H2 Relaxin). Binding of the ligand inhibits
cAMP accumulation by coupling with Gi proteins. This receptor is
Expressed predominantly in brain regions. Highest expression in
substantia nigra and pituitary, followed by hippocampus, spinal
cord, amygdala, caudate nucleus and corpus callosum, quite low
level in cerebellum. In peripheral tissues, relatively high levels
in adrenal glands, low levels in pancreas, salivary gland,
placenta, mammary gland and testis. Defects in this receptor's
activity, as well as reduction in its primary ligand Relaxin 3
cause a decrease in body weight and feeding behaviour in mice.
[0145] GPCR142 (RXFP4) (SwiisProt Accession Number: RL3R2_HUMAN;
Relaxin-3 receptor 2, Relaxin family peptide receptor 4, G-protein
coupled receptor 100, GPCR142), is the receptor for INSL5. This
receptor is also activated by Relaxin 3, Relaxin 2 as well as
bradykinin and kallidin. Binding of the ligand inhibits cAMP
accumulation by coupling with Gi proteins. This receptor is
expressed in a broader range of tissues including brain, kidney,
testis, thymus, placenta, prostate, salivary gland, thyroid and
colon.
[0146] The relaxin ligands superfamily currently comprises 10
members with a relatively high degree of sequence homology. These
family members include insulin, insulin-like grown factors I and
II, relaxin 1, 2 and 3, and the insulin-like hormones INSL3, 4, 5
and 6. The relaxin superfamily members have a wide range of
biological activities which are well described in the art.
[0147] The actions of relaxin (mostly 1 and 2) include an ability
to inhibit myometrial contractions, stimulation of remodelling of
connective tissue and induction of softening of the tissues of the
cervix and birth canal. Additionally, relaxin (1, 2) increases
growth and differentiation of the mammary gland and nipple and
induces the breakdown of collagen (mostly by induction of MMP
proteins that breakdown ECM components such as collagen as well as
inhibition of TIMP proteins which induce ECM, such as collagen,
synthesis), one of the main components of connective tissue as well
as fibrotic tissues. Relaxin decreases collagen synthesis and
increases the release of collagenases. 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 wild type or relaxin heterozygous foster
mothers.
[0148] Relaxin (1 and 2) has additionally been reported to cause 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. It also
stimulates an increase in heart rate and coronary blood flow, and
increases both glomerular filtration rate and renal plasma flow.
Relaxin (1,2) has also been found to inhibit histamine release and
the accumulation of calcium, as well as promote nitric oxide
synthesis, during cardiac anaphylaxis.
[0149] The brain is another target tissue for relaxin where the
peptides have been shown to bind to receptors in the
circumventricular organs to affect blood pressure, food intake and
drinking
[0150] Relaxin (1 and 2) has also been implicated in depression of
platelet aggregation and their release by megakaryocytes, and may
thus be associated with clotting disorders.
[0151] The INSL peptides, such as INSL3, has been shown to be
involved in maturation and descent of the testes, as well as the
survival of sperm cells, development of ovarian follicles and
maturation of the oocyte. Therefore, potential clinical
applications of INSL3 agonists and antagonists include the
treatment of fertility disorders, or the control of fertility
levels.
[0152] INSL3 has been implicated in regulation of relaxin activity
in the heart. Thus, INL3 agoinsits of the invention can are useful
for treating heart disease. Furthermore, INSL3 polymorphisms have
been hyperplastic and neoplastic disorders of the thyroid gland,
suggesting a role for this relaxin superfamily member in the
etiology of these pathologies.
[0153] Provided by the invention are bioactive peptides falling
within Formula I.
[0154] Formula I includes compounds falling within the following
formula:
[0155] Wherein
[0156] X.sup.1 is absent or G or a small naturally or non-naturally
occurring amino acid;
[0157] X.sup.2 is absent or Q or a polar naturally or non-naturally
occurring amino acid;
[0158] X.sup.3 is absent or K or a basic naturally or non-naturally
occurring amino acid;
[0159] X.sup.4 is absent or G or a small naturally or non-naturally
occurring amino acid;
[0160] X.sup.5 is absent or Q or S a polar naturally or
non-naturally occurring amino acid;
[0161] X.sup.6 is absent or V or A or P or M or a hydrophobic
naturally or non-naturally occurring amino acid;
[0162] X.sup.7 is absent or G or a small naturally or non-naturally
occurring amino acid;
[0163] X.sup.8 is absent or P or L or A naturally or non-naturally
occurring amino acid;
[0164] X.sup.9 is absent or P or Q naturally or non-naturally
occurring amino acid;
[0165] X.sup.10 is absent or G or a small naturally or
non-naturally occurring amino acid;
[0166] X.sup.11 is absent or A or H or E or D or a hydrophobic or a
small or an acidic naturally or non-naturally occurring amino
acid;
[0167] X.sup.12 is absent or A or P or Q or S or R or H or a
hydrophobic or a small naturally or non-naturally occurring amino
acid;
[0168] X.sup.13 is absent or C or V or a hydrophobic naturally or
non-naturally occurring amino acid;
[0169] X.sup.14 is absent or R or K or Q or P or a basic or a polar
naturally or non-naturally occurring amino acid;
[0170] X.sup.15 is absent or R or Q or S or a basic or a polar
naturally or non-naturally occurring amino acid;
[0171] X.sup.16 is absent or A or L or H or Q or a hydrophobic or a
small naturally or non-naturally occurring amino acid;
[0172] X.sup.17 is absent or Y or a hydrophobic or an aromatic
naturally or non-naturally occurring amino acid;
[0173] X.sup.18 is absent or A or a hydrophobic or small naturally
or non-naturally occurring amino acid;
[0174] X.sup.19 is absent or A or a hydrophobic small naturally or
non-naturally occurring amino acid;
[0175] X.sup.20 is absent or F or a hydrophobic or an aromatic
naturally or non-naturally occurring amino acid;
[0176] X.sup.21 is absent or S or T or a polar naturally or
non-naturally occurring amino acid;
[0177] X.sup.22 is absent or V or a hydrophobic naturally or
non-naturally occurring amino acid;
[0178] X.sup.23 is absent or G or hydrophobic or small naturally or
non-naturally occurring amino acid or replaced by an Amide;
[0179] X.sup.24 is absent or R or a basic naturally or
non-naturally occurring amino acid;
[0180] X.sup.25 is absent or R or a basic naturally or
non-naturally occurring amino acid;
[0181] X.sup.26 is A or a hydrophobic or small naturally or
non-naturally occurring amino acid;
[0182] X.sup.27 is Y or a hydrophobic or an aromatic naturally or
non-naturally occurring amino acid;
[0183] X.sup.28 is A or a hydrophobic or small naturally or
non-naturally occurring amino acid;
[0184] X.sup.29 is A or a hydrophobic or small naturally or
non-naturally occurring amino acid;
[0185] X.sup.30 is F or a hydrophobic naturally or non-naturally
occurring amino acid;
[0186] X.sup.31 is S or T or a polar naturally or non-naturally
occurring amino acid;
[0187] X.sup.32 is V or a hydrophobic naturally or non-naturally
occurring amino acid;
[0188] X.sup.33 is absent or G or hydrophobic or small naturally or
non-naturally occurring amino acid or replaced by an Amide;
or a pharmaceutically acceptable salt thereof.
[0189] In some embodiments, a peptide of Formula I includes the
amino acid sequence of one of the following:
TABLE-US-00005 Peptide P59-S-Amide (Amide): (SEQ ID NO: 1)
AYAAFSV-Amide; Peptide P59-SG (free acid Gly) (SEQ ID NO: 2)
AYAAFSV; Peptide P59-Amide (amide) (SEQ ID NO: 3)
GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid) (SEQ ID NO:
4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide) (SEQ ID
NO: 5) GQKGQVGPPGAAVRRAYAAFSV-Amide Peptide P59C13V (free acid)
(SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV; Peptide P74-Amide (Amide):
(SEQ ID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide Peptide P74
(free acid) (SEQ ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide
P74C13V (amide) (SEQ ID NO: 9)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide Peptide P74C13V (free acid)
(SEQ ID NO: 10) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV Peptide P59SG
(free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acid
Gly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G
(free acid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide
P74-G (free acid Gly) (SEQ ID NO: 14)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG Peptide P74C13V-G (free acid Gly)
(SEQ ID NO: 15) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG Peptide
P59-Chimpanzee (SEQ ID NO: 20) GQKGQVGPPGAACQRAYAAFSVG; Peptide
P59-Orangutan (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG; Peptide
P59-Rhesus (SEQ ID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow
(SEQ ID NO: 23) GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ
ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human)
(SEQ ID NO: 25) GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID
NO: 26) GQKGSMGAPGDHCKSQYAAFSVG,
[0190] that are ligands for the relaxin-related GPCRs selected from
the group consisting of RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135)
and RXFP4 (GPCR142); and/or of the LGR family of GPCRs, selected
from a group consisting of but not limited to LRR containing GPCRs:
FSHR (LGR1), LHCGR (LGR2), TSHR (LGR3), LGR4, LGR5, LGR6, LGR7
(RXFP1) and LGR8 (RXFP2).
[0191] The present invention also encompasses polypeptides encoded
by the polynucleotide sequences of the present invention, as well
as polypeptides according to the amino acid sequences described
herein.
[0192] The present invention also encompasses homologues of these
polypeptides, such homologues can be at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 85%, at least 90%, at least 95% or more say
100% homologoues to the amino acid sequence set forth below, as can
be determined using BlastP software of the National Center of
Biotechnology Information (NCBI) using default parameters,
optionally and preferably including the following: filtering on
(thisoption filters repetitive or low-complexity sequences from the
query using the Seg (protein) program), scoring matrix is BLOSUM62
for proteins, word size is 3, E value is 10, gap costs are 11, 1
(initialization and extension). Optionally and preferably, nucleic
acid sequence identity/homology is determined with BlastN software
of the National Center of Biotechnology Information (NCBI) using
default parameters, which preferably include using the DUST filter
program, and also preferably include having an E value of 10,
filtering low complexity sequences and a word size of 11. Finally
the present invention also encompasses fragments of the above
described polypeptides and polypeptides having mutations, such as
deletions, insertions or substitutions of one or more amino acids,
either naturally occurring or artificially induced, either randomly
or in a targeted fashion.
[0193] The term "homolog" relating to a peptide of the invention as
used herein should be understood to encompass a peptide which has
substantially the same amino acid sequence and substantially the
same biological activity as peptides depicted in SEQ ID NOs: 1-15,
20-26. Thus, a homolog may differ from the peptides depicted in SEQ
ID NOs: 1-15, 20-26 by the addition, deletion or substitution of
one or more amino acid residues, provided that the resulting
peptide retains the biological activity of peptides depicted in SEQ
ID NOs: 1-15, 20-26, respectively. Persons skilled in the art can
readily determine which amino acid residues may be added, deleted
or substituted (including with which amino acids such substitutions
may be made) using established well known procedures. Examples of
homologs of peptides depicted in SEQ ID NOs: 1-15, 20-26 are
deletion homologs containing less than all the amino acid residues
of peptides depicted in SEQ ID NOs: 1-15, 20-26, respectively,
substitution homologs wherein one or more amino acid residues
specified are replaced by other amino acid residues (eg. amino acid
with similar properties or by D-amino acids, or by non-natural
amino acids) and addition homologs wherein one or more amino acid
residues are added to a terminal or medial portion of peptides
depicted in SEQ ID NOs: 1-15, 20-26, respectively.
[0194] The term "derivative" relating to a peptide of the invention
should be understood to encompass a peptide which has substantially
the same amino acid sequence and substantially the same biological
activity as peptides depicted in SEQ ID NOs: 1-15, 20-26,
respectively. Thus, a derivative may differ from the peptides
depicted in SEQ ID NOs: 1-15, 20-26 by a modification, such as but
not limited to glycosylation, amidation, acetylation, alkylation,
alkenylation, alkynylation, phosphorylation, sulphorization,
hydroxylation, hydrogenation and so forth. Thus, a derivative of a
peptide of the invention may differ from the peptides depicted in
SEQ ID NOs: 1-15, 20-26 by a modification on one or more amino acid
residues, provided that the resulting peptide retains the
biological activity of peptides depicted in SEQ ID NOs: 1-15,
20-26, respectively. Persons skilled in the art can readily
determine which amino acid residues may be modified using
established well known procedures. In one embodiment, a peptide of
the invention is amidated at its C-terminus and acetylated at its
N-terminus.
[0195] By "variant" is meant a polypeptide that differs from a
reference polypeptide, but retains essential properties. Generally,
differences are limited so that the sequences of the reference
polypeptide and the variant are closely similar overall and, in
many regions, identical. A variant and reference polypeptide may
differ in amino acid sequence by one or more substitutions,
additions, and/or deletions, in any combination. A substituted or
inserted amino acid residue may or may not be one encoded by the
genetic code. A variant of a polypeptide may be a naturally
occurring such as an allelic variant, or it may be a variant that
is not known to occur naturally. Non-naturally occurring variants
of polypeptides may be made by mutagenesis techniques or by direct
synthesis.
[0196] Generally, the variant differs from the reference
polypeptide by conservative amino acid substitutions, whereby a
residue is substituted by another with like characteristics (e.g.
acidic, basic, aromatic, etc.). Typical substitutions are among
Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues
Asp and Glu; among Asn and Gln; and among the basic residues Lys
and Arg; or aromatic residues Phe and Tyr.
[0197] "A peptide with substantially the same biological activity"
as used herein should be understood to encompass a peptide which
has at least has at least one-forth, e.g., one third, one half, or
the same activity, as the activity of a peptide of substantially
identical length with a naturally occurring amino acid sequence. By
"substantially identical length" is meant the same length or a
difference in length of no more than ten percent.
[0198] A peptide within Formula I can be provided as part of a
longer peptide that includes the specified amino acid sequence. For
example, the peptide can be provided on a peptide that is less than
200, 150, 125, 100, 75, 50, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,
15, 14, 13, 13, 11, 10, 9, 8 or 7 amino acids.
[0199] In preferred embodiments, the peptide fragment retains one
or more of the activities associated with the full-length peptide,
e.g., binding to and/or activation of a GPCR receptor, or activity
against a condition described herein. In some embodiments, a
peptide within Formula I binds a G-protein coupled receptor (GPCR)
protein, which is preferably relaxin-related GPCR or LGR family
GPCR. In a further preferred embodiments the relaxin-related GPCR
is selected from the group consisting of but not limited to RXFP1,
RXFP2, RXFP3 or RXFP4. or from the LGR related family group
consisting of but not limited to FSHR (LGR1), LHCGR (LGR2), TSHR
(LGR3), LGR4, LGR5, LGR6 LGR7 (RXFP1) and/or LGR8 (RXFP2)
proteins.
[0200] In some embodiments, a peptide within Formula I activates a
GPCR protein. Activation of a GPCR protein can be measured using
methods known in the art.
[0201] A peptide within Formula I can be provided conjugated to a
second peptide or polypeptide. Examples of second peptides or
polypeptides are multiple antigenic peptides (MAP) and a signal
sequence. Suitable signal sequences include, e.g.
TABLE-US-00006 (SEQ ID NO: 16) MAAPALLLLALLLPVGA, (SEQ ID NO: 17)
MAAPALLLLALLLPVGAWP, (SEQ ID NO: 18) MAAPALLLLALLLPVGAWPGLP.
[0202] In some embodiments, the second peptide or polypeptide is an
immunoglobulin sequence (e.g., an IgG sequence). Immunoreactive
ligands for use as a targeting moiety in the invention include an
antigen-recognizing immunoglobulin (also referred to as
"antibody"), or antigen-recognizing fragment thereof, e.g.,
immunoglobulins that can recognize a tumor-associated antigen. As
used herein, "immunoglobulin" refers to any recognized class or
subclass of immunoglobulins such as IgG, IgA, IgM, IgD, or IgE.
[0203] Preferred are those immunoglobulins which fall within the
IgG class of immunoglobulins. The immunoglobulin can be derived
from any species. Preferably, however, the immunoglobulin is of
human, murine, or rabbit origin. In addition, the immunoglobulin
may be polyclonal or monoclonal, but is preferably monoclonal.
[0204] Conjugates of the invention may include an
antigen-recognizing immunoglobulin fragment. Such immunoglobulin
fragments may include, for example, the Fab', F(ab') 2, Fv or Fab
fragments, or other antigen-recognizing immunoglobulin fragments.
Such immunoglobulin fragments can be prepared, for example, by
proteolytic enzyme digestion, for example, by pepsin or papain
digestion, reductive alkylation, or recombinant techniques. The
materials and methods for preparing such immunoglobulin fragments
are well-known to those skilled in the art. See Parham, J.
Immunology, 131, 2895, 1983; Lamoyi et al., J. Immunological
Methods, 56, 235, 1983.
[0205] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an analog or mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. The terms encompass any peptide (including
cyclic peptides) or protein comprising two or more amino acids
joined to each other by peptide bonds or modified peptide bonds.
"Polypeptide" refers to both short chains, commonly referred to as
peptides, oligopeptides or oligomers, and to longer chains,
generally referred to as proteins.
[0206] "Polypeptides" include amino acid sequences modified either
by natural processes, or by chemical modification techniques which
are well known in the art. Modifications may occur anywhere in a
polypeptide, including the peptide backbone, the amino acid
side-chains, and the amino or carboxyl termini. Polypeptides can be
modified, e.g., by the addition of carbohydrate residues to form
glycoproteins. The terms "polypeptide," "peptide" and "protein"
include glycoproteins, as well as non-glycoproteins.
[0207] Peptides within the invention can be produced using methods
known in the art, e.g., by purifying the peptide sequence from a
naturally occurring protein or peptide. Purification can be
performed along with a cleavage or degradation (either enzymatic or
non-enzymatic) to produce the desired peptide using methods known
in the art.
[0208] Alternatively, products can be biochemically synthesized
using, e.g., solid phase synthesis, partial solid phase synthesis
methods, fragment condensation, classical solution synthesis. These
methods are preferably used when the peptide is relatively short
(i.e., 10 kDa) and/or when it cannot be produced by recombinant
techniques (i.e., not encoded by a nucleic acid sequence).
[0209] Solid phase polypeptide synthesis procedures are well known
in the art and further described by John Morrow Stewart and Janis
Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce
Chemical Company, 1984).
[0210] Synthetic polypeptides can be purified by preparative high
performance liquid chromatography [Creighton T. (1983) Proteins,
structures and molecular principles. WH Freeman and Co. N.Y.] and
the composition of which can be confirmed via amino acid
sequencing.
[0211] Polypeptides or peptides can alternatively be synthesized
using recombinant techniques such as those described by Bitter et
al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990)
Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature
310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311, Coruzzi et
al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science
224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and
Weissbach & Weissbach, 1988, Methods for Plant Molecular
Biology, Academic Press, NY, Section VIII, pp 421-463.
[0212] A peptide within the invention may include one or more
modifications. For example, it may be provided phosphorylated
(typically at a serine, threonine, or tyrosine residue), pegylated,
coupled to a biotin moiety, or include a disulfide bond to another
peptide, polypeptide or amino acid. The peptide may be provided in
a cyclic form, e.g., as a cyclic peptide or as a lactam.
Alternatively, or in addition, the peptide may be provided as a
branched peptide.
[0213] The peptide may be additionally modified (when linear) at
its amino terminus or carboxy terminus. Examples of amino terminal
modifications include, e.g., N-glycated, N-alkylated, N-acetylated
or N-acylated amino acid. A terminal modification can include a
pegylation. An example of a carboxy terminal modification is a
c-terminal amidated amino acid.
[0214] A peptide of the invention may contain amino acids other
than the 20 gene-encoded amino acids. When amino acids are not
designated as either D- or L-amino acids, the amino acid is either
an L-amino acid or could be either a D- or L-amino acid, unless the
context requires a particular isomer.
[0215] The notations used herein for the polypeptide amino acid
residues are those abbreviations commonly used in the art. The less
common abbreviations Abu, Cpa, Nle, Pal, Tle, Dip, 4-Fpa, and Nal
stand for 2-amino-butyric acid, p-chloroPhenylalanine, norleucine,
3-pyridyl-2-alanine, tert-leucine, 2,2-diphenylalanine,
4-fluoro-phenylalanine, and 3-(2-naphthyl)-alanine or
3-(1-naphthyl)-alanine, respectively.
[0216] One example of a non-naturally occurring amino acid is an
omega-amino acid, e.g., beta-alanine (beta-Ala), or 3
aminopropionic (3-aP). Other examples are non-naturally occurring
amino acids, e.g., sarcosine (Sar), .beta.-alanine (.beta.-Ala),
2,3 diaminopropionic (2,3-diaP) or alpha-aminisobutyric acid (Aib);
omega-acid is beta-alanine (beta-Ala), or 3 aminopropionic (3-aP);
a hydrophobic non-naturally occurring amino acid, such as
t-butylalanine (t BuA), t butylglycine (t BuG), N methylisoleucine
(N MeIle), norleucine (Nle), methylvaline (Mvl), cyclohexylalanine
(Cha), phenylglycine (Phg), NaI, .beta.2-thienylalanine (Thi), 2
naphthylalanine (2 Nal), or 1,2,3,4-tetrahydroisoquinoline-3
carboxylic acid (Tic); a basic amino acid, such as ornithine (Orn)
or homoarginine (Har); and a neutral/polar non-naturally occurring
amino acid is citrulline (Cit), Acetyl Lys, or methionine sulfoxide
(MSO).
[0217] Other non-conventional amino acids are listed in Table
6.
TABLE-US-00007 TABLE 6 Non-conventional amino acid Code
Non-conventional amino acid Code .alpha.-aminobutyric acid Abu
L-N-methylalanine Nmala .alpha.-amino-.alpha.-methylbutyrate Mgabu
L-N-methylarginine Nmarg aminocyclopropane- Cpro
L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid
Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys
aminonorbornyl- Norb L-N-methylglutamine Nmgin carboxylate
L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa
L-N-methylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyrate Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha.-methylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcyclopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methylbutyrate
Nmaabu D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cyclododeclglycine Ncdod
D-.alpha.-methylalnine Dnmala N-cyclooctylglycine Ncoct
D-.alpha.-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-.alpha.-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-.alpha.-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-.alpha.-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nva
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomo phenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methylvaline Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylleucine Mval L-N-methylhomophenylalanine Nmhphe
Nnbhm N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl)
carbamylmethyl-glycine Nnbhm carbamylmethyl(1)glycine Nnbhe
1-carboxy-1-(2,2-diphenylethylamino)cyclopropane Nmbc
Modifications
Fusion Proteins
[0218] A fusion protein may be prepared from a peptide according to
the present invention by fusion with a portion of an immunoglobulin
comprising a constant region of an immunoglobulin.
[0219] More preferably, the portion of the immunoglobulin comprises
a heavy chain constant region which is optionally and more
preferably a human heavy chain constant region. The heavy chain
constant region is most preferably an IgG heavy chain constant
region, and optionally and most preferably is an Fc chain, most
preferably an IgG Fc fragment that comprises CH2 and CH3 domains.
Although any IgG subtype may optionally be used, the IgG1 subtype
is preferred. The Fc chain may optionally be a known or "wild type"
Fc chain, or alternatively may be mutated. Non-limiting,
illustrative, exemplary types of mutations are described in US
Patent Application No. 20060034852, published on Feb. 16 2006,
hereby incorporated by reference as if fully set forth herein. The
term "Fc chain" also optionally comprises any type of Fc
fragment.
[0220] Several of the specific amino acid residues that are
important for antibody constant region-mediated activity in the IgG
subclass have been identified. Inclusion, substitution or exclusion
of these specific amino acids therefore allows for inclusion or
exclusion of specific immunoglobulin constant region-mediated
activity. Furthermore, specific changes may result in
aglycosylation for example and/or other desired changes to the Fc
chain. At least some changes may optionally be made to block a
function of Fc which is considered to be undesirable, such as an
undesirable immune system effect, as described in greater detail
below.
[0221] Non-limiting, illustrative examples of mutations to Fc which
may be made to modulate the activity of the fusion protein include
the following changes (given with regard to the Fc sequence
nomenclature as given by Kabat, from Kabat E A et al: Sequences of
Proteins of
[0222] Immunological Interest. US Department of Health and Human
Services, NIH, 1991): 220C->S; 233-238 ELLGGP->EAEGAP;
265D->A, preferably in combination with 434N->A; 297N->A
(for example to block N-glycosylation); 318-322 EYKCK->AYACA;
330-331AP->SS; or a combination thereof (see for example M.
Clark, "Chemical Immunol and Antibody Engineering", pp 1-31 for a
description of these mutations and their effect). The construct for
the Fc chain which features the above changes optionally and
preferably comprises a combination of the hinge region with the CH2
and CH3 domains.
[0223] The above mutations may optionally be implemented to enhance
desired properties or alternatively to block non-desired
properties. For example, aglycosylation of antibodies was shown to
maintain the desired binding functionality while blocking depletion
of T-cells or triggering cytokine release, which may optionally be
undesired functions (see M. Clark, "Chemical Immunol and Antibody
Engineering", pp 1-31). Substitution of 331 proline for serine may
block the ability to activate complement, which may optionally be
considered an undesired function (see M. Clark, "Chemical Immunol
and Antibody Engineering", pp 1-31). Changing 330 alanine to serine
in combination with this change may also enhance the desired effect
of blocking the ability to activate complement.
[0224] Residues 235 and 237 were shown to be involved in
antibody-dependent cell-mediated cytotoxicity (ADCC), such that
changing the block of residues from 233-238 as described may also
block such activity if ADCC is considered to be an undesirable
function.
[0225] Residue 220 is normally a cysteine for Fc from IgG1, which
is the site at which the heavy chain forms a covalent linkage with
the light chain. Optionally, this residue may be changed to a
serine, to avoid any type of covalent linkage (see M. Clark,
"Chemical Immunol and Antibody Engineering", pp 1-31).
[0226] The above changes to residues 265 and 434 may optionally be
implemented to reduce or block binding to the Fc receptor, which
may optionally block undesired functionality of Fc related to its
immune system functions (see "Binding site on Human IgG1 for Fc
Receptors", Shields et al. vol 276, pp 6591-6604, 2001).
[0227] The above changes are intended as illustrations only of
optional changes and are not meant to be limiting in any way.
Furthermore, the above explanation is provided for descriptive
purposes only, without wishing to be bound by a single
hypothesis.
Addition of Groups
[0228] If a peptide according to the present invention is a linear
molecule, it is possible to place various functional groups at
various points on the linear molecule which are susceptible to or
suitable for chemical modification. Functional groups can be added
to the termini of linear forms of the peptide. In some embodiments,
the functional groups improve the activity of the peptide with
regard to one or more characteristics, including but not limited
to, improvement in stability, penetration (through cellular
membranes and/or tissue barriers), tissue localization, efficacy,
decreased clearance, decreased toxicity, improved selectivity,
improved resistance to expulsion by cellular pumps, and the like.
For convenience sake and without wishing to be limiting, the free
N-terminus of one of the sequences contained in the compositions of
the invention will be termed as the N-terminus of the composition,
and the free C-terminal of the sequence will be considered as the
C-terminus of the composition. Either the C-terminus or the
N-terminus of the sequences, or both, can be linked to a carboxylic
acid functional groups or an amine functional group,
respectively.
[0229] Non-limiting examples of suitable functional groups are
described in Green and Wuts, "Protecting Groups in Organic
Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991, the
teachings of which are incorporated herein by reference. Preferred
protecting groups are those that facilitate transport of the active
ingredient attached thereto into a cell, for example, by reducing
the hydrophilicity and increasing the lipophilicity of the active
ingredient, these being an example for "a moiety for transport
across cellular membranes".
[0230] These moieties can optionally and preferably be cleaved in
vivo, either by hydrolysis or enzymatically, inside the cell.
(Ditter et al., J. Pharm. Sci. 57:783 (1968); Ditter et al., J.
Pharm. Sci. 57:828 (1968); Ditter et al., J. Pharm. Sci. 58:557
(1969); King et al., Biochemistry 26:2294 (1987); Lindberg et al.,
Drug Metabolism and Disposition 17:311 (1989); and Tunek et al.,
Biochem. Pharm. 37:3867 (1988), Anderson et al., Arch. Biochem.
Biophys. 239:538 (1985) and Singhal et al., FASEB J. 1:220 (1987)).
Hydroxyl protecting groups include esters, carbonates and carbamate
protecting groups. Amine protecting groups include alkoxy and
aryloxy carbonyl groups, as described above for N-terminal
protecting groups. Carboxylic acid protecting groups include
aliphatic, benzylic and aryl esters, as described above for
C-terminal protecting groups. In one embodiment, the carboxylic
acid group in the side chain of one or more glutamic acid or
aspartic acid residue in a composition of the present invention is
protected, preferably with a methyl, ethyl, benzyl or substituted
benzyl ester, more preferably as a benzyl ester.
[0231] Non-limiting, illustrative examples of N-terminal protecting
groups include acyl groups (--CO--R1) and alkoxy carbonyl or
aryloxy carbonyl groups (--CO--O--R1), wherein R1 is an aliphatic,
substituted aliphatic, benzyl, substituted benzyl, aromatic or a
substituted aromatic group. Specific examples of acyl groups
include but are not limited to acetyl, (ethyl)-CO--, n-propyl-CO--,
iso-propyl-CO--, n-butyl-CO--, sec-butyl-CO--, t-butyl-CO--, hexyl,
lauroyl, palmitoyl, myristoyl, stearyl, oleoyl phenyl-CO--,
substituted phenyl-CO--, benzyl-CO-- and (substituted benzyl)-CO--.
Examples of alkoxy carbonyl and aryloxy carbonyl groups include
CH3-O--CO--, (ethyl)-O--CO--, n-propyl-O--CO--, iso-propyl-O--CO--,
n-butyl-O--CO--, sec-butyl-O--CO--, t-butyl-O--CO--,
phenyl-O--CO--, substituted phenyl-O--CO-- and benzyl-O--CO--,
(substituted benzyl)-O--OC--, Adamantan, naphtalen, myristoleyl,
toluen, biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl,
cyclohexane, norbornane, or Z-caproic. In order to facilitate the
N-acylation, one to four glycine residues can be present in the
N-terminus of the molecule.
[0232] The carboxyl group at the C-terminus of the compound can be
protected, for example, by a group including but not limited to an
amide (i.e., the hydroxyl group at the C-terminus is replaced with
--NH.sub.2, --NHR.sub.2 and --NR.sub.2R.sub.3) or ester (i.e. the
hydroxyl group at the C-terminus is replaced with --OR.sub.2).
R.sub.2 and R.sub.3 are optionally independently an aliphatic,
substituted aliphatic, benzyl, substituted benzyl, aryl or a
substituted aryl group. In addition, taken together with the
nitrogen atom, R.sub.2 and R.sub.3 can optionally form a C4 to C8
heterocyclic ring with from about 0-2 additional heteroatoms such
as nitrogen, oxygen or sulfur. Non-limiting suitable examples of
suitable heterocyclic rings include piperidinyl, pyrrolidinyl,
morpholino, thiomorpholino or piperazinyl. Examples of C-terminal
protecting groups include but are not limited to --NH.sub.2,
--NHCH.sub.3, --N(CH.sub.3).sub.2, --NH(ethyl), --N(ethyl).sub.2,
--N(methyl) (ethyl), --NH(benzyl), --N(C1-C4 alkyl)(benzyl),
--NH(phenyl), --N(C1-C4 alkyl) (phenyl), --OCH.sub.3, --O-(ethyl),
--O-(n-propyl), --O-(n-butyl), --O-(iso-propyl), --O-(sec-butyl),
--O-(t-butyl), --O-benzyl and --O-phenyl.
Substitution by Peptidomimetic Moieties
[0233] A "peptidomimetic organic moiety" can optionally be
substituted for amino acid residues in the composition of this
invention both as conservative and as non-conservative
substitutions. These moieties are also termed "non-natural amino
acids" and may optionally replace amino acid residues, amino acids
or act as spacer groups within the peptides in lieu of deleted
amino acids. The peptidomimetic organic moieties optionally and
preferably have steric, electronic or configurational properties
similar to the replaced amino acid and such peptidomimetics are
used to replace amino acids in the essential positions, and are
considered conservative substitutions. However such similarities
are not necessarily required. According to preferred embodiments of
the present invention, one or more peptidomimetics are selected
such that the composition at least substantially retains its
physiological activity as compared to the native peptide protein
according to the present invention.
[0234] Peptidomimetics may optionally be used to inhibit
degradation of the peptides by enzymatic or other degradative
processes. The peptidomimetics can optionally and preferably be
produced by organic synthetic techniques. Non-limiting examples of
suitable peptidomimetics include D amino acids of the corresponding
L amino acids, tetrazol (Zabrocki et al., J. Am. Chem. Soc.
110:5875-5880 (1988)); isosteres of amide bonds (Jones et al.,
Tetrahedron Lett. 29: 3853-3856 (1988));
LL-3-amino-2-propenidone-6-carboxylic acid (LL-Acp) (Kemp et al.,
J. Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in
Kemp et al., Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp
et al., Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al.,
Tetrahedron Lett. 29:4935-4938 (1988) and Kemp et al., J. Org.
Chem. 54:109-115 (1987). Other suitable but exemplary
peptidomimetics are shown in Nagai and Sato, Tetrahedron Lett.
26:647-650 (1985); Di Maio et al., J. Chem. Soc. Perkin Trans.,
1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317 (1989); Olson
et al., J. Am. Chem. Soc. 112:323-333 (1990); Garvey et al., J.
Org. Chem. 56:436 (1990). Further suitable exemplary
peptidomimetics include
hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et
al., J. Takeda Res. Labs 43:53-76 (1989));
1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (Kazmierski et al.,
J. Am. Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolone
carboxylic acid (HIC) (Zechel et al., Int. J. Pep. Protein Res. 43
(1991)); (2S,3S)-methyl-phenylalanine,
(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and
(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, Tetrahedron
Lett. (1991)).
[0235] Exemplary, illustrative but non-limiting non-natural amino
acids include beta-amino acids (beta3 and beta2), homo-amino acids,
cyclic amino acids, aromatic amino acids, Pro and Pyr derivatives,
3-substituted Alanine derivatives, Glycine derivatives,
ring-substituted Phe and Tyr Derivatives, linear core amino acids
or diamino acids. They are available from a variety of suppliers,
such as Sigma-Aldrich (USA) for example.
Chemical Modifications
[0236] In the present invention any part of a peptide may
optionally be chemically modified, i.e. changed by addition of
functional groups. For example the side amino acid residues
appearing in the native sequence may optionally be modified,
although as described below alternatively other part(s) of the
protein may optionally be modified, in addition to or in place of
the side amino acid residues. The modification may optionally be
performed during synthesis of the molecule if a chemical synthetic
process is followed, for example by adding a chemically modified
amino acid. However, chemical modification of an amino acid when it
is already present in the molecule ("in situ" modification) is also
possible.
[0237] The amino acid of any of the sequence regions of the
molecule can optionally be modified according to any one of the
following exemplary types of modification (in the peptide
conceptually viewed as "chemically modified"). Non-limiting
exemplary types of modification include carboxymethylation,
acylation, phosphorylation, glycosylation or fatty acylation. Ether
bonds can optionally be used to join the serine or threonine
hydroxyl to the hydroxyl of a sugar. Amide bonds can optionally be
used to join the glutamate or aspartate carboxyl groups to an amino
group on a sugar (Garg and Jeanloz, Advances in Carbohydrate
Chemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz,
Ang. Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal
bonds can also optionally be formed between amino acids and
carbohydrates. Fatty acid acyl derivatives can optionally be made,
for example, by acylation of a free amino group (e.g., lysine)
(Toth et al., Peptides: Chemistry, Structure and Biology, Rivier
and Marshal, eds., ESCOM Publ., Leiden, 1078-1079 (1990)).
[0238] As used herein the term "chemical modification", when
referring to a protein or peptide according to the present
invention, refers to a protein or peptide where at least one of its
amino acid residues is modified either by natural processes, such
as processing or other post-translational modifications, or by
chemical modification techniques which are well known in the art.
Examples of the numerous known modifications typically include, but
are not limited to:
[0239] acetylation, acylation, amidation, ADP-ribosylation,
glycosylation, GPI anchor formation, covalent attachment of a lipid
or lipid derivative, methylation, myristylation, pegylation,
prenylation, phosphorylation, ubiquitination, or any similar
process.
[0240] Other types of modifications optionally include the addition
of a cycloalkane moiety to a biological molecule, such as a
protein, as described in PCT Application No. WO 2006/050262, hereby
incorporated by reference as if fully set forth herein. These
moieties are designed for use with biomolecules and may optionally
be used to impart various properties to proteins.
[0241] Furthermore, optionally any point on a protein may be
modified. For example, pegylation of a glycosylation moiety on a
protein may optionally be performed, as described in PCT
Application No. WO 2006/050247, hereby incorporated by reference as
if fully set forth herein. One or more polyethylene glycol (PEG)
groups may optionally be added to O-linked and/or N-linked
glycosylation. The PEG group may optionally be branched or linear.
Optionally any type of water-soluble polymer may be attached to a
glycosylation site on a protein through a glycosyl linker.
[0242] Covalent modifications of the peptides of the present
invention are included within the scope of this invention. Other
types of covalent modifications of the peptides are introduced into
the molecule by reacting targeted amino acid residues with an
organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues.
[0243] Cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
[0244] Histidyl residues are derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively
specific for the histidyl side chain. Para-bromophenacyl bromide
also is useful; the reaction is preferably performed in 0.1M sodium
cacodylate at pH 6.0.
[0245] Lysinyl and amino-terminal residues are reacted with
succinic or other carboxylic acid anhydrides. Derivatization with
these agents has the effect of reversing the charge of the lysinyl
residues. Other suitable reagents for derivatizing
.alpha.-amino-containing residues include imidoesters such as
methyl picolinimidate, pyridoxal phosphate, pyridoxal,
chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea,
2,4-pentanedione, and transaminase-catalyzed reaction with
glyoxylate.
[0246] Arginyl residues are modified by reaction with one or
several conventional reagents, among them phenylglyoxal,
2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues requires that the reaction be
performed in alkaline conditions because of the high pKa of the
guanidine functional group. Furthermore, these reagents may react
with the groups of lysine as well as the arginine epsilon-amino
group.
[0247] The specific modification of tyrosyl residues may be made,
with particular interest in introducing spectral labels into
tyrosyl residues by reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizole and
tetranitromethane are used to form 0-acetyl tyrosyl species and
3-nitro derivatives, respectively. Tyrosyl residues are iodinated
using 125 I or 131 I to prepare labeled proteins for use in
radioimmunoassay, the chloramine T method described above being
suitable.
[0248] Carboxyl side groups (aspartyl or glutamyl) are selectively
modified by reaction with carbodiimides (R--N.dbd.C.dbd.N--R'),
where R and R' are different alkyl groups, such as
1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0249] Derivatization with bifunctional agents is useful for
crosslinking CHF to a water-insoluble support matrix or surface for
use in the method for purifying anti-CHF antibodies, and
vice-versa. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and
4,330,440 are employed for protein immobilization.
[0250] Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl residues,
respectively. These residues are deamidated under neutral or basic
conditions. The deamidated form of these residues falls within the
scope of this invention.
[0251] Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, methylation of the .alpha.-amino groups of lysine,
arginine, and histidine side chains (T. E. Creighton, Proteins:
Structure and Molecular Properties, W. H. Freeman & Co., San
Francisco, pp. 79-86 [1983]), acetylation of the N-terminal amine,
and amidation of any C-terminal carboxyl group.
Altered Glycosylation
[0252] Peptides of the invention may be modified to have an altered
glycosylation pattern (i.e., altered from the original or native
glycosylation pattern). As used herein, "altered" means having one
or more carbohydrate moieties deleted, and/or having at least one
glycosylation site added to the original protein.
[0253] Glycosylation of proteins is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences, asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-acetylgalactosamine, galactose, or xylose to a
hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
[0254] Addition of glycosylation sites to peptides of the invention
is conveniently accomplished by altering the amino acid sequence of
the protein such that it contains one or more of the
above-described tripeptide sequences (for N-linked glycosylation
sites). The alteration may also be made by the addition of, or
substitution by, one or more serine or threonine residues in the
sequence of the original protein (for O-linked glycosylation
sites). The protein's amino acid sequence may also be altered by
introducing changes at the DNA level.
[0255] Another means of increasing the number of carbohydrate
moieties on proteins is by chemical or enzymatic coupling of
glycosides to the amino acid residues of the protein. Depending on
the coupling mode used, the sugars may be attached to (a) arginine
and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups
such as those of cysteine, (d) free hydroxyl groups such as those
of serine, threonine, or hydroxyproline, (e) aromatic residues such
as those of phenylalanine, tyrosine, or tryptophan, or (f) the
amide group of glutamine. These methods are described in WO
87/05330, and in Aplin and Wriston, CRC Crit. Rev. Biochem., 22:
259-306 (1981).
[0256] Removal of any carbohydrate moieties present on peptides of
the invention may be accomplished chemically or enzymatically.
Chemical deglycosylation requires exposure of the protein to
trifluoromethanesulfonic acid, or an equivalent compound. This
treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine),
leaving the amino acid sequence intact.
[0257] Chemical deglycosylation is described by Hakimuddin et al.,
Arch. Biochem. Biophys., 259: 52 (1987); and Edge et al., Anal.
Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate
moieties on proteins can be achieved by the use of a variety of
endo- and exo-glycosidases as described by Thotakura et al., Meth.
Enzymol., 138: 350 (1987).
Antibodies to Bioactive Peptides
[0258] The invention also includes an antibody to a bioactive
peptide disclosed herein, or a fragment of the bioactive peptide.
In some embodiments, the bioactive peptide is a GPCR ligand.
[0259] "Antibody" refers to a polypeptide ligand substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof, which specifically binds and recognizes an
epitope (e.g., an antigen). The antibody can be provided as, e.g.,
an intact immunoglobulin or as fragment, e.g., a fragment produced
by digestion with various peptidases. This includes, e.g., Fab' and
F(ab)'.sub.2 Fv (defined as a genetically engineered fragment
containing the variable region of the light chain and the variable
region of the heavy chain expressed as two chains); and (5) Single
chain antibody ("SCA"), a genetically engineered molecule
containing the variable region of the light chain and the variable
region of the heavy chain, linked by a suitable polypeptide linker
as a genetically fused single chain molecule. The term "antibody,"
as used herein, also includes antibody fragments either produced by
the modification of whole antibodies or those synthesized de novo
using recombinant DNA methodologies. It includes polyclonal
antibodies, monoclonal antibodies, chimeric antibodies, humanized
antibodies, or single chain antibodies. "Fc" portion of an antibody
refers to that portion of an immunoglobulin heavy chain that
comprises one or more heavy chain constant region domains, CH1, CH2
and CH3, but does not include the heavy chain variable region.
[0260] Antibodies are raised against, e.g., an epitope in a peptide
of Formula I. In some embodiments, anti-GPCR peptide ligand
antibodies are raised against
TABLE-US-00008 Peptide P59-S-Amide (Amide): (SEQ ID NO: 1)
AYAAFSV-Amide; Peptide P59-SG (free acid Gly) (SEQ ID NO: 2)
AYAAFSV; Peptide P59-Amide (amide) (SEQ ID NO: 3)
GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid) (SEQ ID NO:
4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide) (SEQ ID
NO: 5) GQKGQVGPPGAAVRRAYAAFSV-Amide Peptide P59C13V (free acid)
(SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV; Peptide P74-Amide (Amide):
(SEQ ID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide Peptide P74
(free acid) (SEQ ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide
P74C13V (amide) (SEQ ID NO: 9)
GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide Peptide P74C13V (free acid)
(SEQ ID NO: 10) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV Peptide P59SG
(free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acid
Gly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G
(free acid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide
P74-G (free acid Gly) (SEQ ID NO: 14)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG Peptide P74C13V-G (free acid Gly)
(SEQ ID NO: 15) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG Peptide
P59-Chimpanzee (SEQ ID NO: 20) GQKGQVGPPGAACQRAYAAFSVG; Peptide
P59-Orangutan (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG; Peptide
P59-Rhesus (SEQ ID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow
(SEQ ID NO: 23) GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ
ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human)
(SEQ ID NO: 25) GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID
NO: 26) GQKGSMGAPGDHCKSQYAAFSVG.
[0261] Methods of producing polyclonal and monoclonal antibodies as
well as fragments thereof are well known in the art (See for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference).
[0262] Antibody fragments can be prepared by proteolytic hydrolysis
of the antibody or by expression in E. coli or mammalian cells
(e.g. Chinese hamster ovary cell culture or other protein
expression systems) of DNA encoding the fragment. Antibody
fragments can be obtained by pepsin or papain digestion of whole
antibodies by conventional methods.
[0263] The bioactive peptide antibody can additionally be provided
as a peptide coding corresponding a single
complementarity-determining region (CDR). CDR peptides ("minimal
recognition units") can be obtained by constructing genes encoding
the CDR of an antibody of interest. Such genes are prepared, for
example, by using the polymerase chain reaction to synthesize the
variable region from RNA of antibody-producing cells. See, for
example, Larrick and Fry [Methods, 2: 106-10 (1991)].
[0264] Humanized forms of non-human (e.g., murine) antibodies are
chimeric molecules of immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab') or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0265] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0266] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introduction of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10: 779-783 (1992);
Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg
and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
[0267] The antibody preferably binds specifically (or selectively)
to a GPCR peptide ligand. The phrase "specifically (or selectively)
binds" to an antibody or "specifically (or selectively)
immunoreactive with," when referring to a protein or peptide,
refers to a binding reaction that is determinative of the presence
of the protein in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times the background and do not substantially bind in a significant
amount to other proteins present in the sample. Specific binding to
an antibody under such conditions may require an antibody that is
selected for its specificity for a particular protein. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies
specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Antibodies, A Laboratory Manual (1988), for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity). Typically a specific or selective
reaction will be at least twice background signal or noise and more
typically more than 10 to 100 times background.
[0268] If desired, the antibody can be provided conjugated or
coupled to a detectable label, a radioactive label, an enzyme, a
fluorescent label, a luminescent label, a bioluminescent label, or
a therapeutic agent.
Methods of Treatment
[0269] According to an additional aspect of the present invention
there is provided a method of treating disease, disorder or
condition, as described hereinabove, in a subject.
[0270] The subject according to the present invention is a mammal,
preferably a human which is diagnosed with one of the disease,
disorder or conditions described hereinabove, or alternatively is
predisposed to at least one type of disease, disorder or conditions
described hereinabove.
[0271] As used herein the term "treating" refers to preventing,
curing, reversing, attenuating, alleviating, minimizing,
suppressing or halting the deleterious effects of the
above-described diseases, disorders or conditions.
[0272] Treating, according to the present invention, can be
effected by specifically upregulating the expression of at least
one of the polypeptides of the present invention in the
subject.
[0273] Optionally, upregulation may be effected by administering to
the subject at least one of the polypeptides of the present
invention (e.g., recombinant or synthetic) or an active portion
thereof, as described herein. The polypeptide or peptide may
optionally be administered in as part of a pharmaceutical
composition, described in more detail below.
[0274] It will be appreciated that treatment of the above-described
diseases according to the present invention may be combined with
other treatment methods known in the art (i.e., combination
therapy). Thus, treatment of malignancies using the agents of the
present invention may be combined with, for example, radiation
therapy, antibody therapy and/or chemotherapy, surgery or in
combination therapy with other biological agents, conventional
drugs and/or with any other anti-cancer agent, such as
immunosuppressants or cytotoxic drugs for cancer, and or in
combination with therapeutic agents targeting complement regulatory
proteins (CRPs). Each one of the above mentioned compounds or
pharmaceutical composition of the invention may also be
administered in conjunction with other compounds. For example, the
combination therapy can include the compound of the present
invention combined with at least one other therapeutic or immune
modulatory agent, including, but not limited to, chemotherapeutic
agents such as cytotoxic and cytostatic agents, immunological
modifiers such as interferons and interleukins, growth hormones or
other cytokines, folic acid, vitamins, minerals, aromatase
inhibitors, RNAi, Histone Deacetylase Inhibitors, proteasome
inhibitors, and so forth.
[0275] Alternatively or additionally, an upregulating method may
optionally be effected by specifically upregulating the amount
(optionally expression) in the subject of at least one of the
polypeptides of the present invention or active portions
thereof.
[0276] Upregulating expression of the therapeutic peptides of the
present invention may be effected via the administration of at
least one of the exogenous polynucleotide sequences of the present
invention, ligated into a nucleic acid expression construct
designed for expression of coding sequences in eukaryotic cells
(e.g., mammalian cells). Accordingly, the exogenous polynucleotide
sequence may be a DNA or RNA sequence encoding the peptides of the
present invention or active portions thereof.
[0277] It will be appreciated that the nucleic acid construct can
be administered to the individual employing any suitable mode of
administration including in vivo gene therapy (e.g., using viral
transformation as described hereinabove). Alternatively, the
nucleic acid construct is introduced into a suitable cell via an
appropriate gene delivery vehicle/method (transfection,
transduction, homologous recombination, etc.) and an expression
system as needed and then the modified cells are expanded in
culture and returned to the individual (i.e., ex-vivo gene
therapy).
[0278] Such cells (i.e., which are transfected with the nucleic
acid construct of the present invention) can be any suitable cells,
such as kidney, bone marrow, keratinocyte, lymphocyte, adult stem
cells, cord blood cells, embryonic stem cells which are derived
from the individual and are transfected ex vivo with an expression
vector containing the polynucleotide designed to express the
polypeptide of the present invention as described hereinabove.
[0279] Administration of the ex vivo transfected cells of the
present invention can be effected using any suitable route such as
intravenous, intra peritoneal, intra kidney, intra gastrointestinal
track, subcutaneous, transcutaneous, intramuscular, intracutaneous,
intrathecal, epidural and rectal. According to presently preferred
embodiments, the ex vivo transfected cells of the present invention
are introduced to the individual using intravenous, intra kidney,
intra gastrointestinal track and/or intra peritoneal
administrations.
[0280] The ex vivo transfected cells of the present invention can
be derived from either autologous sources such as self bone marrow
cells or from allogeneic sources such as bone marrow or other cells
derived from non-autologous sources. Since non-autologous cells are
likely to induce an immune reaction when administered to the body
several approaches have been developed to reduce the likelihood of
rejection of non-autologous cells. These include either suppressing
the recipient immune system or encapsulating the non-autologous
cells or tissues in immunoisolating, semipermeable membranes before
transplantation.
[0281] Encapsulation techniques are generally classified as
microencapsulation, involving small spherical vehicles and
macroencapsulation, involving larger flat-sheet and hollow-fiber
membranes (Uludag, H. et al. Technology of mammalian cell
encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).
[0282] Methods of preparing microcapsules are known in the arts and
include for example those disclosed by Lu M Z, et al., Cell
encapsulation with alginate and
alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol
Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Procedures for
microencapsulation of enzymes, cells and genetically engineered
microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et
al., A novel cell encapsulation method using photosensitive poly
(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul.
2000, 17: 245-51.
[0283] For example, microcapsules are prepared by complexing
modified collagen with a ter-polymer shell of 2-hydroxyethyl
methylacrylate (HEMA), methacrylic acid (MAA) and methyl
methacrylate (MMA), resulting in a capsule thickness of 2-5 .mu.m.
Such microcapsules can be further encapsulated with additional 2-5
.mu.m ter-polymer shells in order to impart a negatively charged
smooth surface and to minimize plasma protein absorption (Chia, S.
M. et al. Multi-layered microcapsules for cell encapsulation
Biomaterials. 2002 23: 849-56).
[0284] Other microcapsules are based on alginate, a marine
polysaccharide (Sambanis, A. Encapsulated islets in diabetes
treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its
derivatives. For example, microcapsules can be prepared by the
polyelectrolyte complexation between the polyanions sodium alginate
and sodium cellulose sulphate with the polycation
poly(methylene-co-guanidine) hydrochloride in the presence of
calcium chloride.
Pharmaceutical Compositions and Delivery Thereof
[0285] The bioactive peptide ligand is typically provided in a
pharmaceutically acceptable carrier suitable for administering the
pharmaceutical composition to a human patient. As would be
appreciated by one of skill in this art, the carriers may be chosen
based on the route of administration as described below, the
location of the target issue, the drug being delivered, the time
course of delivery of the drug, etc.
[0286] The term "pharmaceutically acceptable carrier" means a
non-toxic, inert solid, semi-solid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. One
exemplary pharmaceutically acceptable carrier is physiological
saline. Other physiologically acceptable carriers and their
formulations are known to one skilled in the art and described, for
example, in Remington's Pharmaceutical Sciences, (18.sup.th
edition), A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.
Some examples of materials which can serve as pharmaceutically
acceptable carriers include, but are not limited to, sugars such as
lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil; sesame oil; olive oil; corn oil and soybean
oil; glycols such as propylene glycol; esters such as ethyl oleate
and ethyl laurate; agar; detergents such as TWEEN.TM. 80; buffering
agents such as magnesium hydroxide and aluminum hydroxide; alginic
acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol; and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator.
[0287] By "pharmaceutically acceptable salt" is meant non-toxic
acid addition salts or metal complexes which are commonly used in
the pharmaceutical industry. Examples of acid addition salts
include organic acids such as acetic, lactic, pamoic, maleic,
citric, malic, ascorbic, succinic, benzoic, palmitic, suberic,
salicylic, tartaric, methanesulfonic, toluenesulfonic, or
trifluoroacetic acids or the like; polymeric acids such as tannic
acid, carboxymethyl cellulose, or the like; and inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid
phosphoric acid, or the like. Metal complexes include zinc, iron,
and the like.
[0288] The pharmaceutical compositions can be administered to a
patient by any means known in the art including oral and parenteral
routes. The term "patient", as used herein, refers to humans as
well as non-humans, including, for example, mammals, birds,
reptiles, amphibians and fish. Preferably, the non-humans are
mammals (e.g., a rodent (including a mouse or rat), a rabbit, a
monkey, a dog, a cat, sheep, cow, pig, horse). The non-human animal
could alternatively be a bird, e.g., a chicken or turkey.
[0289] In certain embodiments parenteral routes are preferred since
they avoid contact with the digestive enzymes that are found in the
alimentary canal. According to such embodiments, inventive
compositions including a therapeutic agent may be administered by
injection (e.g., intravenous, subcutaneous or intramuscular,
intraperitoneal injection), rectally, vaginally, topically (as by
powders, creams, ointments, or drops), or by inhalation (as by
sprays), intranasal, pulmonary, or intrabuccal.
[0290] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions, may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. In a
particularly preferred embodiment, a therapeutic agent is suspended
in a carrier fluid comprising 1% (w/v) sodium carboxymethyl
cellulose and 0.1% (v/v) TWEEN80.TM.. The injectable formulations
can be sterilized, for example, by filtration through a
bacteria-retaining filter, or by incorporating sterilizing agents
in the form of sterile solid compositions which can be dissolved or
dispersed in sterile water or other sterile injectable medium prior
to use.
[0291] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
therapeutic agent with suitable non-irritating excipients or
carriers such as cocoa butter, polyethylene glycol, or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the therapeutic agent.
[0292] Dosage forms for topical or transdermal administration of a
pharmaceutical composition including a therapeutic agent include
ointments, pastes, creams, lotions, gels, powders, solutions,
sprays, inhalants, or patches. The therapeutic agent is admixed
under sterile conditions with a pharmaceutically acceptable carrier
and any needed preservatives or buffers as may be required.
Ophthalmic formulations, ear drops and eye drops are also
contemplated as being within the scope of this invention. The
ointments, pastes, creams and gels may contain, in addition to the
therapeutic agents of this invention, excipients such as animal and
vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc and zinc oxide, or mixtures thereof. Transdermal
patches have the added advantage of providing controlled delivery
of a compound to the body. Such dosage forms can be made by
dissolving or dispensing the therapeutic agents in a proper medium.
Absorption enhancers can also be used to increase the flux of the
compound across the skin. The rate can be controlled by either
providing a rate controlling membrane or by dispersing the
therapeutic agents in a polymer matrix or gel.
[0293] Powders and sprays can also contain excipients such as
lactose, talc, silicic acid, aluminum hydroxide, calcium silicates
and polyamide powder, or mixtures of these drugs. Sprays can
additionally contain customary propellants such as
chlorofluorohydrocarbons.
[0294] When administered orally, the therapeutic agent is
optionally encapsulated. A variety of suitable encapsulation
systems are known in the art ("Microcapsules and Nanoparticles in
Medicine and Pharmacy," Edited by Doubrow, M., CRC Press, Boca
Raton, 1992; Mathiowitz and Langer J. Control. Release 5:13, 1987;
Mathiowitz et al., Reactive Polymers 6:275, 1987; Mathiowitz et
al., J. Appl. Polymer Sci. 35:755, 1988; Langer Acc. Chem. Res.
33:94, 2000; Langer J. Control. Release 62:7, 1999; Uhrich et al.,
Chem. Rev. 99:3181, 1999; Zhou et al., J. Control. Release 75:27,
2001; and Hanes et al., Pharm. Biotechnol. 6:389, 1995). For
example, the therapeutic agent can be encapsulated within
biodegradable polymeric microspheres or liposomes. Examples of
natural and synthetic polymers useful in the preparation of
biodegradable microspheres include carbohydrates such as alginate,
cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes,
polypropylfumarates, polyethers, polyacetals, polycyanoacrylates,
biodegradable polyurethanes, polycarbonates, polyanhydrides,
polyhydroxyacids, poly(ortho esters) and other biodegradable
polyesters. Examples of lipids useful in liposome production
include phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides and gangliosides.
[0295] Pharmaceutical compositions for oral administration can be
liquid or solid. Liquid dosage forms suitable for oral
administration of inventive compositions include pharmaceutically
acceptable emulsions, microemulsions, solutions, suspensions,
syrups and elixirs. In addition to an encapsulated or
unencapsulated therapeutic agent, the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants, wetting agents, emulsifying and suspending agents,
sweetening, flavoring and perfuming agents. As used herein, the
term "adjuvant" refers to any compound which is a nonspecific
modulator of the immune response. In certain preferred embodiments,
the adjuvant stimulates the immune response. Any adjuvant may be
used in accordance with the present invention. A large number of
adjuvant compounds are known in the art (Allison, Dev. Biol. Stand.
92:3, 1998; Unkeless et al., Annu. Rev. Immunol. 6:251, 1998; and
Phillips et al., Vaccine 10: 151, 1992).
[0296] Solid dosage forms for oral administration include capsules,
tablets, pills, powders and granules. In such solid dosage forms,
the encapsulated or unencapsulated therapeutic agent is mixed with
at least one inert, pharmaceutically acceptable excipient or
carrier such as sodium citrate or dicalcium phosphate and/or (a)
fillers or extenders such as starches, lactose, sucrose, glucose,
mannitol and silicic acid, (b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose and acacia, (c) humectants such as glycerol, (d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates and sodium
carbonate, (e) solution retarding agents such as paraffin, (f)
absorption accelerators such as quaternary ammonium compounds, (g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, (h) absorbents such as kaolin and bentonite clay and
(i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate and mixtures
thereof. In the case of capsules, tablets and pills, the dosage
form may also comprise buffering agents.
[0297] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills and granules can be prepared with
coatings and shells such as enteric coatings and other coatings
well known in the pharmaceutical formulating art.
[0298] The exact dosage of the therapeutic agent is chosen by the
individual physician in view of the patient to be treated. In
general, dosage and administration are adjusted to provide an
effective amount of the therapeutic agent to the patient being
treated. As used herein, the "effective amount" of an therapeutic
agent refers to the amount necessary to elicit the desired
biological response. As will be appreciated by those of ordinary
skill in this art, the effective amount of therapeutic agent may
vary depending on such factors as the desired biological endpoint,
the drug to be delivered, the target tissue, the route of
administration, etc. For example, the effective amount of
therapeutic agent containing an anti-cancer drug might be the
amount that results in a reduction in tumor size by a desired
amount over a desired period of time. Additional factors which may
be taken into account include the severity of the disease state;
age, weight and gender of the patient being treated; diet, time and
frequency of administration; drug combinations; reaction
sensitivities; and tolerance/response to therapy. Long acting
pharmaceutical compositions might be administered every 3 to 4
days, every week, or once every two weeks depending on half-life
and clearance rate of the particular composition.
[0299] The therapeutic agents of the invention are preferably
formulated in dosage unit form for ease of administration and
uniformity of dosage. The expression "dosage unit form" as used
herein refers to a physically discrete unit of therapeutic agent
appropriate for the patient to be treated. It will be understood,
however, that the total daily usage of the compositions of the
present invention will be decided by the attending physician within
the scope of sound medical judgment. For any therapeutic agent, the
therapeutically effective dose can be estimated initially either in
cell culture assays or in animal models, usually mice, rabbits,
dogs, or pigs. The animal model is also used to achieve a desirable
concentration range and route of administration. Such information
can then be used to determine useful doses and routes for
administration in humans. Therapeutic efficacy and toxicity of
therapeutic agents can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., ED50
(the dose is therapeutically effective in 50% of the population)
and LD50 (the dose is lethal to 50% of the population). The dose
ratio of toxic to therapeutic effects is the therapeutic index and
it can be expressed as the ratio, LD50/ED50. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies is
used in formulating a range of dosage for human use.
[0300] If several different therapeutic modalities (e.g., with
different therapeutic agents) are to be administered simultaneously
then they may be combined into a single pharmaceutical composition.
Alternatively, they may be prepared as separate compositions that
are then mixed or simply administered one after the other. If
several different therapeutic agents (e.g., with different
therapeutic agents) are to be administered at different times then
they are preferably prepared as separate compositions. If
additional drugs are going to be included in a combination therapy
they can be added to one or more of these therapeutic agents or
prepared as separate compositions.
[0301] A peptide could be chemically modified in order to alter its
properties such as biodistribution, pharmacokinetics and
solubility. Various methods have been used to increase the
solubility and stability of drugs, among them the use of organic
solvents, their incorporation within emulsions or liposomes, the
adjustment of pH, their chemical modifications and their
complexation with the cyclodextrins. The cyclodextrins are
oligosacharides cyclic family, which include six, seven or eight
units of glucopyranose. Due to sterics interactions, the
cyclodextrins form a cycle structure in the shape of a cone with an
internal cavity. Those are compounds chemically stable that can be
modified. The cyclodextrins hosts form complexes with various
hydrophobic guests in their cavity. The cyclodextrins are used for
the solubilization and encapsulation of drugs.
[0302] Liposomes and Controlled Release:
[0303] In order to design a drug delivery system, various kinds of
high performance carrier materials are being developed to deliver
the necessary amount of drug to the targeted site for a necessary
period of time, both efficiently and precisely.
Cyclodextrins, biodegradable or non biodegradable polymers,
liposomes, emulsions. Multiple emulsions are potential candidates
for such a role, because of their ability to alter physical,
chemical and biological properties of guest molecules. There are
number of drug delivery systems including but not limited to
polymer microcapsules, microparticles, nanoparticles, liposomes and
emulsion. Many of these are prepared from synthetic biodegradable
polymers such as polyanhydrides and poly hydroxy acids. In these
systems the drugs incorporate in polymeric microspheres, which
release the drug inside the organism in small and controlled daily
doses during days months or until years.
[0304] Several polymers already were tested in controlled release
systems. Such as: polyuretans for its elasticity, polysiloxans or
silicons for being a good one insulating, polymethyl-metacrilate
for its physical form; polyvinilalcohol for its hydrofobicity and
resistance, polyethilene for its hardness and impermeability
(Gilding, D. K. Biodegradable polymers. Biocompat. Clin. Impl.
Mater. 2:209-232, 1981). Biodegradable polymers and biocompatible
polymers, have been extensively investigated as vehicle for
controlled release systems due to their ability to undergo surface
degradation. These kind of polymers can be chose from:
poly(2-hidroxi-ethylmetacrilate), polyacrilamide, polymer from
lactic acid (PLA), from glicolic acid (PGA), and the respective
ones co-polymers, (PLGA) and the poly(anidrides), as described by
Tamada and Langer, J. Biomater. Sci. Polym. Edn, 3(4):315-353.
[0305] Suitable controlled release vehicles include, but are not
limited to, biocompatible polymers, other polymeric matrices,
capsules, microcapsules, nanocapsules, microparticles,
nanoparticles, bolus preparations, osmotic pumps, diffusion
devices, liposomes, lipospheres and transdermal delivery systems,
implantable or not.
[0306] Satisfactory systems of controlled release include, but are
not limited to, the ciclodextrines, biocompatible polymers,
biodegradable polymers, other polymeric matrixes, capsules,
micro-capsules, microparticles, bolus preparations, osmotic pumps,
diffusion devices, lipossomes, lipoesferes, and systems of
transdermic administration. Other compositions of controlled
release include liquids that, when submitted the temperature
changes, form a solid or a gel in situ.
[0307] Liposomes are lipid vesicles that include aqueous internal
compartments in which molecules, for example drugs, are
encapsulated with the objective of reaching a controlled release of
the drug after administration in individuals. Many different
techniques have been proposed for the preparation of liposomes
[U.S. Pat. No. 4,552,803, Lenk; U.S. Pat. No. 4,310,506,
Baldeschwieler; U.S. Pat. No. 4,235,871, Papahadjopoulos; U.S. Pat.
No. 4,224,179, Schneider; U.S. Pat. No. 4,078,052, Papahadjopoulos;
U.S. Pat. No. 4,394,372, Tailor; U.S. Pat. No. 4,308,166,
Marchetti; U.S. Pat. No. 4,485,054, Mezei; and U.S. Pat. No.
4,508,703, Redziniak; Woodle and Papahadjopoulos, Methods Enzymol.
171:193-215 (1989]; Unilamellar vesicles display a single membrane
[Huang, Biochemistry 8:334-352 (1969] while muitilamellar vesicles
(MLVs) have numerous concentric membranes [Bangham et al., J. Mol.
Biol. 13:238-252 (1965]. The procedure of Bangham [J. Mol. Biol.
13:238-252 (1965] produces "ordinary MLVs", that present unequal
solute distributions among the aqueous compartments and,
consequently, differences of osmotic pressure. Lenk et al. (U.S.
Pat. No. 4,522,803; U.S. Pat. No. 5,030,453 and U.S. Pat. No.
5,169,637), Fountain et al. (U.S. Pat. No. 4,588,578), Cullis et
al. (U.S. Pat. No. 4,975,282) and Gregoriadis et al. (Pat. W.O.
99/65465) introduced methods for the preparation of MLVs that
present substantially equal solute distributions among the
compartments. Similar solute distributions among the different
compartments mean a larger drug encapsulation efficiency as well as
smaller differences of osmotic pressure that turns these MLVs more
stable than ordinary MLVs. Unilamellar vesicles can be produced by
sonication of MLVs [Papahadjopoulos et al. (1968)] or by extrusion
through polycarbonate membranes [Cullis et al. (U.S. Pat. No.
5,008,050) and Loughrey et al. (U.S. Pat. No. 5,059,421)].
[0308] Satisfactory lipids include for example,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
cardiolipin, cholesterol, phosphatidic acid, sphingolipids,
glycolipids, fatty acids, sterols, phosphatidylethanolamine,
polymerizable lipids in their polymerized or non-polymerized form,
mixture of these lipids.
[0309] The composition of the liposomes can be manipulated such as
to turn them specific for an organ or a cell type. The targeting of
liposomes has been classified either on the basis of anatomical
factors or on the basis of the mechanism of their interaction with
the environment. The anatomical classification is based on their
level of selectivity, for example, organ-specific or cell-specific.
From the point of view of the mechanisms, the--targeting can be
considered as passive or active.
[0310] The passive targeting exploits the natural tendency of
conventional liposomes to be captured by the cells of the
reticulo-endotheliai system, i.e. mainly the fixed macrophages in
the liver, spleen and bone marrow.
[0311] Sterically stabilized liposomes (also well-known as
"PEG-liposomes") are characterized by a reduced rate of elimination
from the blood circulation [Lasic and Martin, Stealth Liposomes,
CRC Press, Inc., Boca Raton, Fla. (1995)].
[0312] PEG-liposomes present a polyethylene glycol polymer
conjugated to the head group of some phospholipid that reduces
their interaction with plasma proteins, such as opsonins, and
reduces the rate of their uptake by cells. The resulting steric
barrier allows these liposomes to remain for a longer period of
time within the circulation than conventional liposomes [Lasic and
Martin, Stealth Liposomes, CRC Press, Inc., Boca Raton, Fla.
(1995); Woodle et al., Biochim. Biophys. Acta 1105:193-200 (1992);
Litzinger et al., Biochim. Biophys. Acta 1190:99-107 (1994); Bedu
Addo, et al., Pharm. Res. 13:718-724 (1996]. The drug encapsulation
within PEG-liposomes has resulted in the improvement of the
effectiveness of many chemotherapeutic agents [Lasic and Martin,
Stealth liposomes, CRC Press, Inc., Boca Raton, Fla. (1995)] and
bioactive peptides [Allen T. M. In: Liposomes, New Systems, New
Trends in their Applications (F. Puisieux, P. Couvreur, J.
Delattre, J.-P. Devissaguet Ed.), Editions de la Sante, France,
1995, pp. 125].
[0313] Studies in this area demonstrated that different factors
affect the effectiveness of PEG-liposomes. Ideally, the diameter of
the vesicles should be below 200 nm, the number of units in PEG of
approximately 2.000 and the proportion of Pegylated lipid from 3 to
5 mol % [Lasic and Martin, Stealth Liposomes, CRC Press, Inc., Boca
Raton, Fla. (1995); Woodle et al., Biochim. Biophys. Acta
1105:193-200 (1992); Litzinger et al., Biochim. Biophys. Acta
1190:99-107(1994); Bedu Addo et al., Pharm. Res.
13:718-724(1996)].
[0314] The active targeting involves alteration of liposomes
through their association with a liguand, such as a monoclonal
antibody, a sugar, a glycolipid, protein, a polymer or by changing
the lipid composition or the liposome size to target them to organs
and cells different from those which accumulate conventional
liposomes.
Mucosal Delivery Enhancing Agents
[0315] "Mucosal delivery enhancing agents" are defined as chemicals
and other excipients that, when added to a formulation comprising
water, salts and/or common buffers and peptide within the present
invention (the control formulation) produce a formulation that
produces a significant increase in transport of peptide across a
mucosa as measured by the maximum blood, serum, or cerebral spinal
fluid concentration (Cmax) or by the area under the curve, AUC, in
a plot of concentration versus time. A mucosa includes the nasal,
oral, intestional, buccal, bronchopulmonary, vaginal, and rectal
mucosal surfaces and in fact includes all mucus-secreting membranes
lining all body cavities or passages that communicate with the
exterior. Mucosal delivery enhancing agents are sometimes called
carriers.
Compositions and Methods of Sustained Release
[0316] The present invention provides improved mucosal (e.g.,
nasal) delivery of a formulation comprising the peptide within the
present invention in combination with one or more mucosal
delivery-enhancing agents and an optional sustained
release-enhancing agent or agents. Mucosal delivery-enhancing
agents of the present invention yield an effective increase in
delivery, e.g., an increase in the maximal plasma concentration
(Cmax) to enhance the therapeutic activity of
mucosally-administered peptide. A second factor affecting
therapeutic activity of the peptide in the blood plasma and CNS is
residence time (RT). Sustained release-enhancing agents, in
combination with intranasal delivery-enhancing agents, increase
Cmax and increase residence time (RT) of the peptide. Polymeric
delivery vehicles and other agents and methods of the present
invention that yield sustained release-enhancing formulations, for
example, polyethylene glycol (PEG), are disclosed herein. Within
the mucosal delivery formulations and methods of the invention, the
peptide is frequently combined or coordinately administered with a
suitable carrier or vehicle for mucosal delivery. As used herein,
the term "carrier" means a pharmaceutically acceptable solid or
liquid filler, diluent or encapsulating material. A
water-containing liquid carrier can contain pharmaceutically
acceptable additives such as acidifying agents, alkalizing agents,
antimicrobial preservatives, antioxidants, buffering agents,
chelating agents, complexing agents, solubilizing agents,
humectants, solvents, suspending and/or viscosity-increasing
agents, tonicity agents, wetting agents or other biocompatible
materials. A tabulation of ingredients listed by the above
categories, can be found in the U.S. Pharmacopeia National
Formulary, 1857-1859, (1990). As used herein, "mucosal
delivery-enhancing agents" include agents which enhance the release
or solubility (e.g., from a formulation delivery vehicle),
diffusion rate, penetration capacity and timing, uptake, residence
time, stability, effective half-life, peak or sustained
concentration levels, clearance and other desired mucosal delivery
characteristics (e.g., as measured at the site of delivery, or at a
selected target site of activity such as the bloodstream or central
nervous system) of the peptide or other biologically active
compound(s). Within certain aspects of the invention,
absorption-promoting agents for coordinate administration or
combinatorial formulation with the peptide of the invention are
selected from small hydrophilic molecules, including but not
limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethanol,
propylene glycol, and the 2-pyrrolidones. Alternatively, long-chain
amphipathic molecules, for example, deacylmethyl sulfoxide, azone,
sodium laurylsulfate, oleic acid, and the bile salts, may be
employed to enhance mucosal penetration of the peptide. In
additional aspects, surfactants (e.g., polysorbates) are employed
as adjunct compounds, processing agents, or formulation additives
to enhance intranasal delivery of the peptide. Agents such as DMSO,
polyethylene glycol, and ethanol can, if present in sufficiently
high concentrations in delivery environment (e.g., by
pre-administration or incorporation in a therapeutic formulation),
enter the aqueous phase of the mucosa and alter its solubilizing
properties, thereby enhancing the partitioning of the peptide from
the vehicle into the mucosa. The mucosal therapeutic and
prophylactic compositions of the present invention may be
supplemented with any suitable penetration-promoting agent that
facilitates absorption, diffusion, or penetration of the peptide
across mucosal barriers. The penetration promoter may be any
promoter that is pharmaceutically acceptable.
[0317] Charge Modifying and pH Control Agents and Methods
[0318] To improve the transport characteristics of biologically
active agents (including the peptide within the present invention),
for enhanced delivery across hydrophobic mucosal membrane barriers,
the invention also provides techniques and reagents for charge
modification of selected biologically active agents or
delivery-enhancing agents described herein. In this regard, the
relative permeabilities of macromolecules is generally be related
to their partition coefficients. The degree of ionization of
molecules, which is dependent on the pKa of the molecule and the pH
at the mucosal membrane surface, also affects permeability of the
molecules. Permeation and partitioning of biologically active
agents, including the peptide within the present invention, for
mucosal delivery may be facilitated by charge alteration or charge
spreading of the active agent or permeabilizing agent, which is
achieved, for example, by alteration of charged functional groups,
by modifying the pH of the delivery vehicle or solution in which
the active agent is delivered, or by coordinate administration of a
charge- or pH-altering reagent with the active agent. Consistent
with these general teachings, mucosal delivery of charged
macromolecular species, including the peptide within the present
invention is substantially improved when the active agent is
delivered to the mucosal surface in a substantially un-ionized, or
neutral, electrical charge state.
[0319] Certain peptide and protein components of mucosal
formulations for use within the invention will be charge modified
to yield an increase in the positive charge density of the peptide
or protein. These modifications extend also to cationization of
peptide and protein conjugates, carriers and other delivery forms
disclosed herein.
Degradative Enzyme Inhibitory Agents and Methods
[0320] Another excipient that may be included in a trans-mucosal
preparation is a degradative enzyme inhibitor. Any inhibitor that
inhibits the activity of an enzyme to protect the biologically
active agent(s) may be usefully employed in the compositions and
methods of the invention. Useful enzyme inhibitors for the
protection of biologically active proteins and peptides include,
for example, soybean trypsin inhibitor, pancreatic trypsin
inhibitor, chymotrypsin inhibitor and trypsin and chrymotrypsin
inhibitor isolated from potato (solanum tuberosum L.) tubers. A
combination or mixtures of inhibitors may be employed. The
inhibitor(s) may be incorporated in or bound to a carrier, e.g., a
hydrophilic polymer, coated on the surface of the dosage form which
is to contact the nasal mucosa, or incorporated in the superficial
phase of the surface, in combination with the biologically active
agent or in a separately administered (e.g., pre-administered)
formulation. Additional enzyme inhibitors for use within the
invention are selected from a wide range of non-protein inhibitors
that vary in their degree of potency and toxicity. As described in
further detail below, immobilization of these adjunct agents to
matrices or other delivery vehicles, or development of chemically
modified analogues, may be readily implemented to reduce or even
eliminate toxic effects, when they are encountered. Among this
broad group of candidate enzyme inhibitors for use within the
invention are organophosphorous inhibitors, such as
diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride
(PMSF), which are potent, irreversible inhibitors of serine
proteases (e.g., trypsin and chymotrypsin). Yet another type of
enzyme inhibitory agent for use within the methods and compositions
of the invention are amino acids and modified amino acids that
interfere with enzymatic degradation of specific therapeutic
compounds.
[0321] The therapeutic agents of the invention can be used to treat
disorders for which modulation of GPCR-related signal transduction
pathways is efficacious. For example, the peptides of the invention
falling within Formula I are used to treat disorders for which
modulation or activation of relaxin-related GPCR receptors and/or
LGR family of GPCRs responses in a cell or in a subject is
efficacious. Examples of such peptides are depicted in SEQ ID NOs:
1-15, 20-26. In another example, the peptide of the invention
falling within Formula I are further used to treat any disease or
condition that involves agonist associated activation and/or
modulation of relaxin or relaxin related peptides activity on
relaxin-related family of GPCRs and/or LGR family of GPCRs, either
by itself or in combination with relaxin or any relaxin related
peptide or another compound, wherein the activation and/or
modulation of the receptor can be either by direct activation of
downstream pathways directly related to the receptors or to
G-proteins activated by the receptors or any other related pathway,
or by indirect activation by either relaxin or any relaxin related
peptide or another compound. The disorder is selected from but not
limited to hyperplastic disorders, neoplastic disorders, cancer;
fibrotic conditions, disorders of collagen deposition, fibrotic
breakdown, connective tissue remodeling, uncontrolled or abnormal
collagen or fibronectin formation or breakdown; skin injuries
including wound healing and scarring, scleroderma; urogenital
disorders including female reproductive disorders, male and female
infertility, cryptorchidism, disregulation of spermatogenesis and
reproductive development including descent of the gonads;
conditions associated with pregnancy such as preeclampsia or
complication of labor; angiogenesis related disorders;
cardiovascular disorders, vasodilatation, vasoconstriction or
hypertension, endothelial disfunction and vascular disease,
congestive heart failure, coronary artery disease, ischemia and
ischemia-reperfusion, peripheral vascular disease; kidney disease,
renal disease associated with arteriosclerosis or other narrowing
of kidney capillaries; capillaries narrowing in the body, such as
in the eyes or in the peripheral digits, the mesocaecum, lung and
peripheral vasculature; CNS related disorders, neurological
disorders, cognition and memory related indications, depression,
neurological modification; inflammatory disorders, such as
gastritis, gout, gouty arthritis, arthritis, rheumatoid arthritis,
inflammatory bowel disease, Crohn's disease, ulcerative colitis,
ulcers; autoimmune disorders; inflammatory conditions associated
with viral infection and infection related diseases including
fibrosis and cirrhosis; Raynaud's disease, Raynaud's phenomenon;
bone related conditions including osteoporosis; metabolic disorders
including food and water intake, diabetes, obesity; respiratory or
a pulmonary disorder, including asthma, COPD, bronchial disease,
lung diseases, cystic fibrosis, ARDS, SARS.
[0322] In another aspect, the invention provides a method of
treating, preventing or ameliorating the symptoms of a
relaxin-related GPCR family or LGR family of GPCRs-mediated or
related disorders or conditions in a patient, the disorder or
condition selected from the group consisting of but not limited to
hyperplastic disorders, neoplastic disorders, cancer; fibrotic
conditions, disorders of collagen deposition, fibrotic breakdown,
connective tissue remodeling, uncontrolled or abnormal collagen or
fibronectin formation or breakdown; skin injuries including wound
healing and scarring, scleroderma; urogenital disorders including
female reproductive disorders, male and female infertility,
cryptorchidism, disregulation of spermatogenesis and reproductive
development including descent of the gonads; conditions associated
with pregnancy such as preeclampsia or complication of labor;
angiogenesis related disorders; cardiovascular disorders,
vasodilatation, vasoconstriction or hypertension, endothelial
disfunction and vascular disease, congestive heart failure,
coronary artery disease, ischemia and ischemia-reperfusion,
peripheral vascular disease; kidney disease, renal disease
associated with arteriosclerosis or other narrowing of kidney
capillaries; capillaries narrowing in the body, such as in the eyes
or in the peripheral digits, the mesocaecum, lung and peripheral
vasculature; CNS related disorders, neurological disorders,
cognition and memory related indications, depression, neurological
modification; inflammatory disorders, such as gastritis, gout,
gouty arthritis, arthritis, rheumatoid arthritis, inflammatory
bowel disease, Crohn's disease, ulcerative colitis, ulcers;
autoimmune disorders; inflammatory conditions associated with viral
infection and infection related diseases including fibrosis and
cirrhosis; Raynaud's disease, Raynaud's phenomenon; bone related
conditions including osteoporosis; metabolic disorders including
food and water intake, diabetes, obesity; respiratory or a
pulmonary disorder, including asthma, COPD, bronchial disease, lung
diseases, cystic fibrosis, ARDS, SARS,
[0323] the method comprising administering to the patient an
effective amount of a peptide of the invention.
[0324] In a further aspect, the invention contemplates the use of
the analogues and/or pharmaceutical compositions of the present
invention in the manufacture of a medicament for the treatment of
any disease or condition that involves relaxin-related family of
GPCRs or LGR GPCRs family related disorder or condition in a
patient, the disorder selected from the group consisting of but not
limited to: hyperplastic disorders, neoplastic disorders, cancer;
fibrotic conditions, disorders of collagen deposition, fibrotic
breakdown, connective tissue remodeling, uncontrolled or abnormal
collagen or fibronectin formation or breakdown; skin injuries
including wound healing and scarring, scleroderma; urogenital
disorders including female reproductive disorders, male and female
infertility, cryptorchidism, disregulation of spermatogenesis and
reproductive development including descent of the gonads;
conditions associated with pregnancy such as preeclampsia or
complication of labor; angiogenesis related disorders;
cardiovascular disorders, vasodilatation, vasoconstriction or
hypertension, endothelial disfunction and vascular disease,
congestive heart failure, coronary artery disease, ischemia and
ischemia-reperfusion, peripheral vascular disease; kidney disease,
renal disease associated with arteriosclerosis or other narrowing
of kidney capillaries; capillaries narrowing in the body, such as
in the eyes or in the peripheral digits, the mesocaecum, lung and
peripheral vasculature; CNS related disorders, neurological
disorders, cognition and memory related indications, depression,
neurological modification; inflammatory disorders, such as
gastritis, gout, gouty arthritis, arthritis, rheumatoid arthritis,
inflammatory bowel disease, Crohn's disease, ulcerative colitis,
ulcers; autoimmune disorders; inflammatory conditions associated
with viral infection and infection related diseases including
fibrosis and cirrhosis; Raynaud's disease, Raynaud's phenomenon;
bone related conditions including osteoporosis; metabolic disorders
including food and water intake, diabetes, obesity; respiratory or
a pulmonary disorder, including asthma, COPD, bronchial disease,
lung diseases, cystic fibrosis, ARDS, SARS.
[0325] The term "relaxin-related family of GPCRs or LGR GPCRs
family related disorder or condition" as used herein refers to
conditions where relaxin-related family of GPCRs or LGR GPCRs
family are involved or implicated in the development, maintenance
or progression of the disease or condition.
[0326] The peptides of the invention falling within Formula I such
as for example, peptides as depicted in SEQ ID NOs:1-15, 20-26 are
also useful for the treatment of fibrotic conditions involving
tissue remodeling following inflammation or ischemia-reperfusion
injury, including but not limited to endomyocardial and cardiac
fibrosis fibrosis; mediastinal fibrosis; idiopathy pulmonary
fibrosis; pulmonary fibrosis; retroperitoneal fibrosis; fibrosis of
the spleen; fibrosis of the pancreas; hepatic fibrosis (cirrhosis)
alcohol and non-alcohol related (including viral infection such as
HAV, HBV and HCV); fibromatosis; granulomatous lung disease;
glomerulonephritis, myocardial scarring following infarction;
endometrial fibrosis and endometriosis; wound healing whether by
injury or surgical procedures, diabetes related wound fibrosis.
[0327] The peptides of the invention falling within Formula I, such
as for example, peptides as depicted in SEQ ID NOs: 1-15, 20-26 are
also useful in treating cardiovascular diseases and
their-complications, peripheral vascular diseases and coronary
artery diseases, including but not limited to myocardial
infarction; congestive heart failure (CHF); myocardial failure;
myocardial hypertrophy; ischemic cardiomyopathy; systolic heart
failure; diastolic heart failure; stroke; thrombotic stroke;
concentric LV hypertrophy, myocarditis; cardiomyopathy;
hypertrophic cardiomyopathy; myocarditis; decompensated heart
failure; ischemic myocardial disease; congenital heart disease;
angina pectoris; prevention of heart remodeling or ventricular
remodeling after myocardial infarction; ischemia-reperfusion injury
in ischemic and post-ischemic events (e.g. myocardial infarct);
cerebrovascular accident; mitral valve regurgitation; hypertension;
hypotension; restenosis; fibrosis; thrombosis; or platelet
aggregation.
[0328] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26 are useful in treating ischemia-reperfusion injury associated
with ischemic and post-ischemic events in organs and tissues,
including but not limited to thrombotic stroke; myocardial
infarction; angina pectoris; embolic vascular occlusions;
peripheral vascular insufficiency; splanchnic artery occlusion;
arterial occlusion by thrombi or embolisms, arterial occlusion by
non-occlusive processes such as following low mesenteric flow or
sepsis; mesenteric arterial occlusion; mesenteric vein occlusion;
ischemia-reperfusion injury to the mesenteric microcirculation;
ischemic acute renal failure; ischemia-reperfusion injury to the
cerebral tissue; intestinal intussusception; hemodynamic shock;
tissue dysfunction; organ failure; restenosis; atherosclerosis;
thrombosis; platelet aggregation.
[0329] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are useful in treating ischemia-reperfusion injury following
conditions including but not limited to procedures such as cardiac
surgery; organ surgery; organ transplantation; angiography;
cardiopulmonary and cerebral resuscitation.
[0330] In another aspect, the peptides of the invention falling
within Formulas I, such as for example, peptides as depicted in SEQ
ID NOs:1-15, 20-26, are used for prevention and treatment of
hypertension and its complications including but not limited to
hypertensive heart disease; antihypertension (blood pressure
reduction); systemic and pulmonary high blood pressure;
cerebrovascular disease and stroke; heart failure and stroke; left
ventricular hypertrophy (LVH); congestive heart failure (CHF);
hypertension, high blood pressure; vasodilation; renal
hypertension; diuresis; nephritis; natriuresis; scleroderma renal
crisis; angina pectoris (stable and unstable); myocardial
infarction; heart attack; coronary artery disease; coronary heart
disease; cardiac arrhythmias; atrial fibrillation; portal
hypertension; raised intraocular pressure; vascular restenosis;
chronic hypertension; valvular disease; myocardial ischemia; acute
pulmonary edema; acute coronary syndrome; hypertensive retinopathy;
hypertensive pregnancy sickness; preeclampsia; Raynaud's
phenomenon; erectile dysfunction and glaucoma. These peptides are
also used as a vasodilator and in antithrombotic therapy.
[0331] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are also useful in treating inflammatory conditions
associated with an infection, e.g., a bacterial infection or a
viral infection, including but not limited to a viral infection
caused by human immunodeficiency virus I (HIV-1) or HIV-2, acquired
immune deficiency (AIDS), West Nile encephalitis virus,
coronavirus, rhinovirus, influenza virus, dengue virus, HCV, HBV,
HAV, hemorrhagic fever; an otological infection; severe acute
respiratory syndrome (SARS), sepsis and sinusitis.
[0332] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are also useful in the prevention or treatment of cancer, or
inflammation associated with cancer such as solid cancer, including
but not limited to colon cancer, lung cancer, breast cancer,
prostate cancer, brain cancer, pancreatic cancer, ovarian cancer,
kidney cancer, testicular cancer, bone cancer, osteosarcoma, or
liver cancer (HBV/HCV related or non-related). The cancer can
alternatively be a melanoma, glioma, a sarcoma, a leukemia, or
lymphoma. These peptides are also useful in the prevention or
treatment of invasive and metastatic cancer.
[0333] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are used for the prevention and treatment in skin injuries,
including but not limited to dermal repair, wound healing; burns,
erythemas, lesions, wound healing following surgical procedures;
skin or tissue lesions including lesions induced by conditions
including, but not limited to Psoriasis, Lupus and Kaposhi Sarcoma;
Scleroderma and collagenous diseases of the skin and skin
tumors.
[0334] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are used in the prevention or treatment of a urogenital
disorder or a genitor-urological disorders including but not
limited to renal disease; a bladder disorder; disorders of the
reproductive system; gynecologic disorders; urinary tract disorder;
incontinence; disorders of the male (spermatogenesis, spermatic
motility), and female reproductive system; sexual dysfunction;
erectile dysfunction; embryogenesis; and conditions associated with
pregnancy. These are also used in pregnancy monitoring. As used
herein, the term "conditions associated with pregnancy" includes,
but is not limited to, conditions of fertilisation, pregnancy,
parturition and lactation. The invention in another embodiment
includes using the peptides of the invention falling within
Formulas I, such as for example, peptides as depicted in SEQ ID
NOs: 1-15, 20-26, wherein said pregnancy related disorders are
selected from a group consisting of Abnormal endometrial
angiogenesis; Placental development defects; Cervical ripening
(softening); Abnormal implantation; Nipple development and
disfunction; Pregnancy related remodeling of the Uterine tissue;
Endometriosis; Preeclampsia; Lactation disorders; Estrogenic and
non-estrogenic related hormonal disorders; Pre-term labor; post
term labor; and Labor complications.
[0335] The invention also provides peptides falling within Formulas
I, such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, that are used in the prevention or treatment of respiratory
diseases, including but not limited to asthma, bronchial disease,
lung diseases, chronic obstructive pulmonary disease (COPD), Acute
Respiratory Distress Syndrome (ARDS), severe acute respiratory
syndrome (SARS), Fibrosis related Asthma, cystic fibrosis.
[0336] The invention also provides peptides falling within Formulas
I, such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, that are used in the prevention or treatment of metabolic
disorders including but not limited to diabetes, diabetes mellitus,
lipodystrophy, hyperthyroidism, glaucoma, hyperlipidaemia,
non-insulin dependent diabetes, Food intake; Water intake; Feeding
and drinking behaviors, Anorexia, Cachexia (cancer and non cancer
related); Fat and lipid metabolism; and Energy control, appetite
control and obesity.
[0337] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are also used in the prevention and treatment of kidney
diseases including but not limited to diabetic nephropathy;
glomerulosclerosis; nephropathies; renal impairment; scleroderma
renal crisis and chronic renal failure. These peptides can also be
used as antidiuretics.
[0338] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are also used in the prevention and treatment of
angiogenesis related conditions including but not limited to
retinal angiogenesis in a number of human ocular diseases such as
diabetes mellitus, retinopathy of prematury, and age-related
macular degeneration, or cancer associated angiogenesis in primary
or metastatic cancer, including but not limited to cancer of the
prostate, brain, breast, colorectal, lung, ovarian, pancreatic,
renal, cervical, melanoma, soft tissue sarcomas, lymphomas,
head-and-neck, and glioblastomas.
[0339] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are also used to treat a central nervous system (CNS)
disorder, including but not limited to central and peripheral
degenerative neuropathies; neuroprotection; impaired cognition;
anxiety disorders, pain control, food intake, a behavioral
disorder, a learning disorder, a sleep disorder, a memory disorder,
a pathologic response to anesthesia, addiction, depression,
migraine, a menstruation disorder, muscle spasm, opiate dependence,
dementia, Alzheimer's disease, Parkinson's disease, cortical
function, locomotor activity, Alcohol and Drug addiction and abuse;
Impared memory; Feeding and drinking related behaviours; Stress
control, Bipolar disorder; Schyzophrenia; Schyzoaffective; Multiple
Sclerosis (MS); Stroke and stroke damage repair (Ischemia
protection); Vasculature and re-vasculature in the brain; and Brain
tissue regeneration and a peripheral nervous system disorder.
[0340] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are also used to treat a bone and bone related disorders in
a patient, including but not limited to Osteoporosis;
Osteoarthritis; Osteopetrosis; Bone inconsistency; Osteosarcoma;
and Cancer matastesis to the bone.
[0341] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are also used to treat endothelial dysfunction disease,
selected from a group consisting of cardiovascular diseases, high
blood pressure, atherosclerosis, thrombosis, myocardial infarct,
heart failure, renal diseases, plurimetabolic syndrome, erectile
dysfunction; vasculitis; and diseases of the central nervous system
(CNS).
[0342] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are also used to treat inflammatory disorder in a subject.
The inflammatory disorder can be gastritis, gout, gouty arthritis,
arthritis, rheumatoid arthritis, inflammatory bowel disease,
Crohn's disease, ulcerative colitis, ulcers, chronic bronchitis,
asthma, allergy, acute lung injury, pulmonary inflammation, airway
hyper-responsiveness, vasculitis, septic shock and inflammatory
skin disorders, including but not limited to psoriasis, atopic
dermatitis, eczema.
[0343] The peptides of the invention falling within Formulas I,
such as for example, peptides as depicted in SEQ ID NOs: 1-15,
20-26, are also used to treat autoimmune disease or disorder in a
subject. The autoimmune disease can be multiple sclerosis,
psoriasis, rheumatoid arthritis, systemic lupus erythematosus,
ulcerative colitis, Crohn's disease, transplant rejection, immune
disorders associated with graft transplantation rejection, benign
lymphocytic angiitis, lupus erythematosus, Hashimoto's thyroiditis,
primary myxedema, Graves' disease, pernicious anemia, autoimmune
atrophic gastritis, Addison's disease, insulin dependent diabetes
mellitis, Good pasture's syndrome, myasthenia gravis, pemphigus,
sympathetic ophthalmia, autoimmune uveitis, autoimmune hemolytic
anemia, idiopathic thrombocytopenia, primary biliary cirrhosis,
chronic action hepatitis, ulceratis colitis, Sjogren's syndrome,
rheumatic disease, polymyositis, scleroderma, mixed connective
tissue disease, inflammatory rheumatism, degenerative rheumatism,
extra-articular rheumatism, collagen diseases, chronic
polyarthritis, psoriasis arthropathica, ankylosing spondylitis,
juvenile rheumatoid arthritis, periarthritis humeroscapularis,
panarteriitis nodosa, progressive systemic scleroderma, arthritis
uratica, dermatomyositis, muscular rheumatism, myositis, myogelosis
and chondrocalcinosis.
[0344] Compounds of Formula I can be used to treat disorders,
diseases and/or conditions as described herein, by administering to
a subject in need thereof a therapeutically effective amount of a
peptide falling within Formula I.
[0345] Also provided by the invention is a method of treating
disorders for which modulation of GPCR-related signal transduction
pathways is efficacious. For example, provided by the invention is
a method of treating disorders for which modulation of
relaxin-related family of GPCR and/or LGR family of GPCRs is
efficacious in a subject by administering to a subject in need
thereof a therapeutically effective amount of a compound falling
within Formula I, such as for example, peptides as depicted in SEQ
ID NOs: 1-15, 20-26. Such diseases disorders and/or condition are
selected from but not limited to hyperplastic disorders, neoplastic
disorders, cancer; fibrotic conditions, disorders of collagen
deposition, fibrotic breakdown, connective tissue remodeling,
uncontrolled or abnormal collagen or fibronectin formation or
breakdown; skin injuries including wound healing and scarring,
scleroderma; urogenital disorders including female reproductive
disorders, male and female infertility, cryptorchidism,
disregulation of spermatogenesis and reproductive development
including descent of the gonads; conditions associated with
pregnancy such as preeclampsia or complication of labor;
angiogenesis related disorders; cardiovascular disorders,
vasodilatation, vasoconstriction or hypertension, endothelial
disfunction and vascular disease, congestive heart failure,
coronary artery disease, ischemia and ischemia-reperfusion,
peripheral vascular disease; kidney disease, renal disease
associated with arteriosclerosis or other narrowing of kidney
capillaries; capillaries narrowing in the body, such as in the eyes
or in the peripheral digits, the mesocaecum, lung and peripheral
vasculature; CNS related disorders, neurological disorders,
cognition and memory related indications, depression, neurological
modification; inflammatory disorders, such as gastritis, gout,
gouty arthritis, arthritis, rheumatoid arthritis, inflammatory
bowel disease, Crohn's disease, ulcerative colitis, ulcers;
autoimmune disorders; inflammatory conditions associated with viral
infection and infection related diseases including fibrosis and
cirrhosis; Raynaud's disease, Raynaud's phenomenon; bone related
conditions including osteoporosis; metabolic disorders including
food and water intake, diabetes, obesity; respiratory or a
pulmonary disorder, including asthma, COPD, bronchial disease, lung
diseases, cystic fibrosis, ARDS, SARS.
[0346] Also provided by the invention is a method of treating an
inflammatory disorder in a subject by administering to a subject in
need thereof a therapeutically effective amount of a compound
falling within Formula I, such as for example, peptides as depicted
in SEQ ID NOs: 1-15, 20-26. The inflammatory disorder can be
gastritis, gout, gouty arthritis, arthritis, rheumatoid arthritis,
inflammatory bowel disease, Crohn's disease, ulcerative colitis,
ulcers, chronic bronchitis, asthma, allergy, acute lung injury,
pulmonary inflammation, airway hyper-responsiveness, vasculitis,
septic shock and inflammatory skin disorders, including but not
limited to psoriasis, atopic dermatitis, eczema.
[0347] Also provided by the invention is a method of treating
fibrotic conditions involving tissue remodeling in a subject by
administering to a subject in need thereof a therapeutically
effective amount of a compound falling within Formula I such as for
example, peptides as depicted in SEQ ID NOs: 1-15, 20-26. These
fibrotic conditions can be endomyocardial and cardiac fibrosis
fibrosis; mediastinal fibrosis; idiopathy pulmonary fibrosis;
pulmonary fibrosis; retroperitoneal fibrosis; fibrosis of the
spleen; fibrosis of the pancreas; hepatic fibrosis (cirrhosis)
alcohol and non-alcohol related (including viral infection such as
HAV, HBV and HCV); fibromatosis; granulomatous lung disease;
glomerulonephritis, myocardial scarring following infarction;
endometrial fibrosis and endometriosis; wound healing whether by
injury or surgical procedures, diabetes related wound fibrosis.
[0348] Also provided by the invention is a method of treating an
autoimmune disease or disorder in a subject by administering to a
subject in need thereof a therapeutically effective amount of a
compound falling within Formula I, such as for example, peptides as
depicted in SEQ ID NOs: 1-15, 20-26. The autoimmune disease can be
multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus
erythematosus, ulcerative colitis, Crohn's disease, transplant
rejection, immune disorders associated with graft transplantation
rejection, benign lymphocytic angiitis, lupus erythematosus,
Hashimoto's thyroiditis, primary myxedema, Graves' disease,
pernicious anemia, autoimmune atrophic gastritis, Addison's
disease, insulin dependent diabetes mellitis, Good pasture's
syndrome, myasthenia gravis, pemphigus, sympathetic ophthalmia,
autoimmune uveitis, autoimmune hemolytic anemia, idiopathic
thrombocytopenia, primary biliary cirrhosis, chronic action
hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic
disease, polymyositis, scleroderma, mixed connective tissue
disease, inflammatory rheumatism, degenerative rheumatism,
extra-articular rheumatism, collagen diseases, chronic
polyarthritis, psoriasis arthropathica, ankylosing spondylitis,
juvenile rheumatoid arthritis, periarthritis humeroscapularis,
panarteriitis nodosa, progressive systemic scleroderma, arthritis
uratica, dermatomyositis, muscular rheumatism, myositis, myogelosis
and chondrocalcinosis.
[0349] Also provided by the invention is a method of treating
cardiovascular diseases and their complications in a subject by
administering to a subject in need thereof a therapeutically
effective amount of a compound falling within Formula I, such as
for example, peptides as depicted in SEQ ID NOs: 1-15, 20-26. The
cardiovascular diseases can be peripheral vascular diseases and
coronary artery diseases, including but not limited to myocardial
infarction; coronary heart disease; congestive heart failure (CHF);
myocardial failure; myocardial hypertrophy; ischemic
cardiomyopathy; systolic heart failure; diastolic heart failure;
stroke; thrombotic stroke; concentric LV hypertrophy, myocarditis;
cardiomyopathy; hypertrophic cardiomyopathy; myocarditis;
decompensated heart failure; ischemic myocardial disease;
congenital heart disease; angina pectoris; prevention of heart
remodeling or ventricular remodeling after myocardial infarction;
ischemia-reperfusion injury in ischemic and post-ischemic events
(e.g. myocardial infarct); cerebrovascular accident; mitral valve
regurgitation; hypertension; hypotension; restenosis; fibrosis;
thrombosis; or platelet aggregation.
[0350] Also provided by the invention is a method of treating an
ischemia-reperfusion injury in a subject by administering to a
subject in need thereof a therapeutically effective amount of a
compound falling within Formula I, such as for example, peptides as
depicted in SEQ ID NOs: 1-15, 20-26. The ischemia-reperfusion
injury can be associated with ischemic and post-ischemic events in
organs and tissues, including but not limited to thrombotic stroke;
myocardial infarction; angina pectoris; embolic vascular
occlusions; peripheral vascular insufficiency; splanchnic artery
occlusion; arterial occlusion by thrombi or embolisms, arterial
occlusion by non-occlusive processes such as following low
mesenteric flow or sepsis; mesenteric arterial occlusion;
mesenteric vein occlusion; ischemia-reperfusion injury to the
mesenteric microcirculation; ischemic acute renal failure;
ischemia-reperfusion injury to the cerebral tissue; intestinal
intussusception; hemodynamic shock; tissue dysfunction; organ
failure; restenosis; atherosclerosis; thrombosis; platelet
aggregation. The ischemia-reperfusion injury can be alternatively
following conditions including but not limited to procedures such
as cardiac surgery; organ surgery; organ transplantation;
angiography; cardiopulmonary and cerebral resuscitation.
[0351] Also provided by the invention is a method of preventing and
treating an hypertension and its complications in a subject by
administering to a subject in need thereof a therapeutically
effective amount of a compound falling within Formula I, such as
for example, peptides as depicted in SEQ ID NOs: 1-15, 20-26. The
hypertension and its complications can be hypertensive heart
disease; antihypertension (blood pressure reduction); systemic and
pulmonary high blood pressure; cerebrovascular disease and stroke;
heart failure and stroke; left ventricular hypertrophy (LVH);
congestive heart failure (CHF); hypertension, high blood pressure;
vasodilation; renal hypertension; diuresis; nephritis; natriuresis;
scleroderma renal crisis; angina pectoris (stable and unstable);
myocardial infarction; heart attack; coronary artery disease;
coronary heart disease; cardiac arrhythmias; atrial fibrillation;
portal hypertension; raised intraocular pressure; vascular
restenosis; chronic hypertension; valvular disease; myocardial
ischemia; acute pulmonary edema; acute coronary syndrome;
hypertensive retinopathy; hypertensive pregnancy sickness;
preeclampsia; Raynaud's phenomenon; erectile dysfunction and
glaucoma. These peptides are also used as a vasodilator and in
antithrombotic therapy.
[0352] Also provided by the invention is a method of treating an
inflammatory disorder and/or conditions associated with an
infection in a subject by administering to a subject in need
thereof a therapeutically effective amount of a compound falling
within Formulas I, such as for example, peptides as depicted in SEQ
ID NOs: 1-15, 20-26. The inflammatory conditions associated with an
infection, can be a bacterial infection or a viral infection,
including but not limited to a viral infection caused by human
immunodeficiency virus I (HIV-1) or HIV-2, acquired immune
deficiency (AIDS), HAV, HCV, HBV, West Nile encephalitis virus,
coronavirus, rhinovirus, influenza virus, dengue virus, hemorrhagic
fever; an otological infection; severe acute respiratory syndrome
(SARS), sepsis and sinusitis.
[0353] Also provided by the invention is a method of treating
cancer, or inflammation associated with cancer in a subject by
administering to a subject in need thereof a therapeutically
effective amount of a compound falling within Formulas I, such as
for example, peptides as depicted in SEQ ID NOs: 1-15, 20-26. The
cancer, or inflammation associated with cancer can be solid cancer,
including but not limited to colon cancer, lung cancer, breast
cancer, prostate cancer, brain cancer, pancreatic cancer, ovarian
cancer, testicular cancer, bone cancer, osteosarcoma, liver cancer
(HBV/HCV related or non-related), or kidney cancer. The cancer can
alternatively be a melanoma, glioma, a sarcoma, a leukemia, or
lymphoma. These peptides are also useful in the prevention or
treatment of invasive and metastatic cancer.
[0354] Also provided by the invention is a method of treating skin
injury diseases, disorders and/or conditions in a subject by
administering to a subject in need thereof a therapeutically
effective amount of a compound falling within Formulas I, such as
for example, peptides as depicted in SEQ ID NOs: 1-15, 20-26. The
skin injury diseases, disorders and/or conditions can be dermal
repair, wound healing; burns, erythemas, lesions, wound healing
following surgical procedures; skin or tissue lesions including
lesions induced by conditions including, but not limited to
Psoriasis, Lupus and Kaposhi Sarcoma; Scleroderma and collagenous
diseases of the skin and skin tumors.
[0355] Also provided by the invention is a method of prevention or
treatment of a urogenital disorder or a genitor-urological
disorders in a subject by administering to a subject in need
thereof a therapeutically effective amount of a compound falling
within Formulas I such as for example, peptides as depicted in SEQ
ID NOs: 1-15, 20-26. The a urogenital disorder or a
genitor-urological disorders can be renal disease; a bladder
disorder; disorders of the reproductive system; gynecologic
disorders; urinary tract disorder; incontinence; disorders of the
male (spermatogenesis, spermatic motility), and female reproductive
system; sexual dysfunction; erectile dysfunction; embryogenesis;
and conditions associated with pregnancy. These are also used in
pregnancy monitoring. As used herein, the term "conditions
associated with pregnancy" includes, but is not limited to,
conditions of fertilisation, pregnancy, parturition and lactation.
The invention in another embodiment includes the foregoing method,
wherein said pregnancy related disorders are selected from a group
consisting of Abnormal endometrial angiogenesis; Placental
development defects; Cervical ripening (softening); Abnormal
implantation; Nipple development and disfunction; Pregnancy related
remodeling of the Uterine tissue; Endometriosis; Preeclampsia;
Lactation disorders; Estrogenic and non-estrogenic related hormonal
disorders; Pre-term labor; post term labor; and Labor
complications.
[0356] Also provided by the invention is a method of prevention or
treatment of respiratory diseases in a subject by administering to
a subject in need thereof a therapeutically effective amount of a
compound falling within Formulas I, II, IV and VI, such as for
example, peptides as depicted in SEQ ID NOs: 1-15, 20-26. The
respiratory diseases can be asthma, bronchial disease, lung
diseases, chronic obstructive pulmonary disease (COPD), Acute
Respiratory Distress Syndrome (ARDS), severe acute respiratory
syndrome (SARS), Fibrosis related Asthma, cystic fibrosis.
[0357] Also provided by the invention is a method of prevention or
treatment of metabolic disorders in a subject by administering to a
subject in need thereof a therapeutically effective amount of a
compound falling within Formulas I, such as for example, peptides
as depicted in SEQ ID NOs: 1-15, 20-26. The metabolic disorders can
be diabetes, diabetes mellitus, lipodystrophy, hyperthyroidism,
glaucoma, hyperlipidaemia, non-insulin dependent diabetes, Food
intake; Water intake; Feeding and drinking behaviors, Anorexia,
Cachexia (cancer and non cancer related); Fat and lipid metabolism;
and Energy control, appetite control and obesity.
[0358] Also provided by the invention is a method of prevention and
treatment of kidney diseases in a subject by administering to a
subject in need thereof a therapeutically effective amount of a
compound falling within I, such as for example, peptides as
depicted in SEQ ID NOs: 1-15, 20-26. The kidney diseases can be
diabetic nephropathy; glomerulosclerosis; nephropathies; renal
impairment; scleroderma renal crisis and chronic renal failure.
[0359] Also provided by the invention is a method of prevention and
treatment of angiogenesis related conditions in a subject by
administering to a subject in need thereof a therapeutically
effective amount of a compound falling within Formulas I such as
for example, peptides as depicted in SEQ ID NOs: 1-15, 20-26. The
angiogenesis related conditions including but not limited to
retinal angiogenesis in a number of human ocular diseases such as
diabetes mellitus, retinopathy of prematury, and age-related
macular degeneration, or cancer associated angiogenesis in primary
or metastatic cancer, including but not limited to cancer of the
prostate, brain, breast, colorectal, lung, ovarian, pancreatic,
renal, cervical, melanoma, soft tissue sarcomas, lymphomas,
head-and-neck, and glioblastomas.
[0360] Also provided by the invention is a method of treating
central nervous system (CNS) disorder, in a subject by
administering to a subject in need thereof a therapeutically
effective amount of a compound falling within Formulas I, such as
for example, peptides as depicted in SEQ ID NOs: 1-15, 20-26. The
central nervous system (CNS) disorder, including but not limited to
central and peripheral degenerative neuropathies; neuroprotection;
impaired cognition; anxiety disorders, pain control, food intake, a
behavioral disorder, a learning disorder, a sleep disorder, a
memory disorder, a pathologic response to anesthesia, addiction,
depression, migraine, a menstruation disorder, muscle spasm, opiate
dependence, dementia, Alzheimer's disease, Parkinson's disease,
cortical function, locomotor activity, Alcohol and Drug addiction
and abuse; Impared memory; Feeding and drinking related behaviours;
Stress control, Bipolar disorder; Schyzophrenia; Schyzoaffective;
Multiple Sclerosis (MS); Stroke and stroke damage repair (Ischemia
protection); Vasculature and re-vasculature in the brain; and Brain
tissue regeneration and a peripheral nervous system disorder.
[0361] Also provided by the invention is a method of treating bone
and bone related disorders in a subject by administering to a
subject in need thereof a therapeutically effective amount of a
compound falling within Formulas I, such as for example, peptides
as depicted in SEQ ID NOs: 1-15, 20-26. The bone and bone related
disorders including but not limited to Osteoporosis;
Osteoarthritis; Osteopetrosis; Bone inconsistency; Osteosarcoma;
and Cancer matastesis to the bone.
[0362] Also provided by the invention is a method of treating
endothelial dysfunction disease, in a subject by administering to a
subject in need thereof a therapeutically effective amount of a
compound falling within Formulas I, such as for example, peptides
as depicted in SEQ ID NOs: 1-15, 20-26. The endothelial dysfunction
disease is selected from a group consisting of cardiovascular
diseases, high blood pressure, atherosclerosis, thrombosis,
myocardial infarct, heart failure, renal diseases, plurimetabolic
syndrome, erectile dysfunction; vasculitis; and diseases of the
central nervous system (CNS).
[0363] Optionally, the cDNA that encodes the peptide sequences of
the invention are used in gene therapy to treat the respective
diseases, disorders and/or conditions, as detailed hereinabove.
[0364] The invention will be further illustrated in the following
examples.
Example 1
Peptides Syntheses
[0365] All P74, P59-S and P59 related peptides derive from C1QT8, a
Collagen repeat containing Hypothetical protein (SEQ ID NO:19) or
its orthologous or homologous sequences:
>P60827 C1QT8_HUMAN Complement C1q tumor necrosis factor-related
protein 8-Homo sapiens (Human).
TABLE-US-00009 MAAPALLLLALLLPVGAWPGLPRRPCVHCCRPAWPPGPYARVSDRDLW
RGDLWRGLPRVRPTIDIEILKGEKGEAGVRGRAGRSGKEGPPGARGLQG
RRGQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVGRREGLHSSDHFQA
VPFDTELVNLDGAFDLAAGRFLCTVPGVYFLSLNVHTWNYKETYLHIML
NRRPAAVLYAQPSERSVMQAQSLMLLLAAGDAVWVRMFQRDRDNAIY
GEHGDLYITFSGHLVKPAAEL Peptide P59-S-Amide (Amide): (SEQ ID NO: 1)
AYAAFSV-Amide; Peptide P59-SG (free acid Gly) (SEQ ID NO: 2)
AYAAFSV; Peptide P59-Amide (amide) (SEQ ID NO: 3)
GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid) (SEQ ID NO:
4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide) (SEQ ID
NO: 5) GQKGQVGPPGAAV*RRAYAAFSV-Amide Peptide P59C13V (free acid)
(SEQ ID NO: 6) GQKGQVGPPGAAV*RRAYAAFSV; Peptide P74-Amide (Amide):
(SEQ ID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide Peptide P74
(free acid) (SEQ ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide
P74C13V (amide) (SEQ ID NO: 9)
GQKGQVGPPGAAV*RRAYAAFSVGRRAYAAFSV-Amide Peptide P74C13V (free acid)
(SEQ ID NO: 10) GQKGQVGPPGAAV*RRAYAAFSVGRRAYAAFSV Peptide P59SG
(free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acid
Gly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G
(free acid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAV*RRAYAAFSVG; Peptide
P74-G (free acid Gly) (SEQ ID NO: 14)
GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG Peptide P74C13V-G (free acid Gly)
(SEQ ID NO: 15) GQKGQVGPPGAAV*RRAYAAFSVGRRAYAAFSVG Peptide
P59-Chimpanzee (SEQ ID NO: 20) GQKGQVGPPGAACQRAYAAFSVG; Peptide
P59-Orangutan (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG; Peptide
P59-Rhesus (SEQ ID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow
(SEQ ID NO: 23) GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ
ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human)
(SEQ ID NO: 25) GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID
NO: 26) GQKGSMGAPGDHCKSQYAAFSVG. *The Cys residue within the
peptides was replaced by V in the synthesized peptides made to
prevent dimerization at the cysteine residue in the peptides.
[0366] Peptides were synthesized by the solid phase peptide
synthesis (SPPS) method, cleaved from the resin, and purified by
RP-HPLC unless stated otherwise. The peptide's identity was
verified by mass spectrometry. Final purity of peptide was >90%
as measured by RP-HPLC. Peptides were diluted in PBS containing
0.1% BSA. All plates were stored at -80C until use.
Example 2
Effect of Peptides P59C13V (P59) (SEQ ID NO:6) and P74C13V (P74)
(SEQ ID NO:10) on Cyclic Amp Accumulation in Cho-K1 Cells
Expressing LGR7 and LGR8
[0367] The ability of Peptides to affect cAMP concentration was
examined in CHO-K1 cells transiently transfected with LGR7 (RXFP1)
or LGR8 (RXFP2). The experiment measured cAMP concentration in
response to the addition of the peptides.
[0368] LGR7 (RXFP1) or LGR8 (RXFP2) were transiently transfected
into CHO-K1 cells as follows: Cells (12 million) were plated into
T75 flasks on the day preceding transfection. Cells were
transfected with a GPCR DNA and G.sub..alpha.,16 using a lipid
technique according to the manufacturer's recommendation. Cells
were transfected for 5 hours, then re-plated into 96-well dishes
(60,000 cells per well) and grown overnight. Transfected cells were
plated into 24 wells of a 96-well plate. Cells were pre-treated
with 0.5 mM IBMX (stimulation buffer) for 10 min at 37.degree. C.,
then stimulated with either a positive control (Relaxin 2) or a
candidate peptide (for Gs functional examination), followed by
stimulation or a preincubation with 10 .mu.M forskolin (for Gi
functional examination). Following a 20 minutes stimulation by
incubation with 1 uM of either positive control or the tested
peptides, either with or without forskolin, intracellular cAMP was
assayed using the Hit Hunter cAMP kit (DiscoveRx Corporation)
according to the manufacturer's recommended protocol. Data was
converted to nmol of cAMP by running a standard cAMP curve.
Relaxin was used as a positive control. cAMP level was elevated by
pre-treatment with forskolin.
[0369] The transfected CHO-K1 cells were treated with 10 uM of
Forskolin (Applied cell sciences (ACS)) for 10 minutes and then
challenged with 1 uM of H2 Relaxin (ACS) (as a positive control), 1
uM of Peptide P59C13V (P59) (SEQ ID NO: 6), and Peptide P74C13V
(P74) (SEQ ID NO: 10).
[0370] The results are shown in FIG. 1. As can be seen from FIG. 1,
both peptides P59C13V (P59) (SEQ ID NO: 6) and P74C13V (SEQ ID NO:
10) showed a cAMP inhibitory effect with respect to stimulation by
Forskolin, and in relation to the effect H2 Relaxin positive
control had on the cells. A clear cAMP inhibition (Gi) effect was
demonstrated by P59C13V (P59) (SEQ ID NO: 6) and P74C13V (P74) (SEQ
ID NO: 10) on both receptors at 1 uM. Relaxin affected only the
LGR7 (RXFP1) receptor. P59C13V (P59) (SEQ ID NO: 6) showed a
stronger effect, than P74C13V (P74) (SEQ ID NO: 10), however, both
peptides showed stronger effect when compared to the reduction in
cAMP with the positive control (Relaxin 2).
[0371] A dose response effect of P59C13V (P59) and P74C13V (P74) on
CHO-K1 cells transiently transfected with LGR8 (RXFP2) was further
examined (Method as above). CHO-K1 cells transiently transfected
with LGR8 (RXFP2) were treated with 20 uM of Forskolin for 10
minutes and then challenged with either INSL3 (as a positive
control), P59C13V (SEQ ID NO: 6) or P74C13V (P74) (SEQ ID NO: 10)
at increasing concentrations from 1 nM to 10 uM. The results are
shown in FIG. 2. As can be seen from FIG. 2, P59C13V (P59) (SEQ ID
NO: 10) showed a slight decrease in cAMP at concentrations lower
than 100 nM; (Gi effect) and a slight increase (Gs effect) at
concentrations higher than 100 nM, as compared to INSL3. INSL3
showed a slight dose dependent increase in lower concentrations
(<100 nM; Gs effect), and a sharp decrease (Gi effect) in higher
concentrations (>100 nM). P74C13V (P74) (SEQ ID NO: 10) showed a
pattern similar to INSL3 at lower concentrations (slight increase
in cAMP) but no effect at higher concentrations.
Example 3
Effect of Peptides P59C13V (P59) (SEQ ID NO: 6) and P74C13V (P74)
(SEQ ID NO: 10) on cAMP Accumulation in CHO-K1 Cells Expressing
LGR4, LGR5 or LGR6
[0372] The ability of Peptides to affect cAMP concentration was
examined in CHO-K1 cells transiently transfected the GPCR's LGR4,
LGR5, or LGR6 and treated with 1 uM of P59C13V (P59) (SEQ ID NO:
6), P74C13V (P74) ((SEQ ID NO: 10), or HCG (ACS) which was used as
a positive control. cAMP accumulation in the cells was then
measured as described above with (Gi) or without (Gs) pre-treatment
with Forskolin.
[0373] The results are shown in FIG. 3. According to FIG. 3A, a
significant increase in cAMP accumulation was demonstrated for LGR6
transfected cells when challenged with P74C13V (P74) (SEQ ID NO:
10). No significant Gs activation was found for any of the peptides
on any of the other LGR transfected cells.
[0374] According to FIG. 3B, a significant Gi effect (decrease in
cAMP accumulation) was demonstrated for LGR5 transfected cells when
challenged with P59C13V (P59) (SEQ ID NO: 6) or P74C13V (P74) (SEQ
ID NO: 10). No significant Gi activation was found for the positive
control (HCG).
[0375] According to FIG. 3C, a slight yet statistically significant
Gi effect (decrease in cAMP accumulation) was demonstrated for LGR4
transfected cells when challenged with P59C13V (P59) (SEQ ID NO: 6)
or P74C13V (P74) (SEQ ID NO: 10). A slight but significant Gi
activation was found for the positive control (HCG).
[0376] FIG. 3 (A-C) indicate a slight Gs activation of LGR6 by
P74C13V (P74), a slight Gi activation of LGR5 by both P59C13V (P59)
(SEQ ID NO: 6) and P74C13V (P74) (SEQ ID NO: 10) and a slight Gi
activation of LGR4 by both P59C13V (P59) (SEQ ID NO: 6) and P74C13V
(P74) (SEQ ID NO: 10) as well as HCG.
Example 4
Dose Response Stimulation of Camp Mediated by Peptides P59 (SEQ ID
NO: 6) and P74 (SEQ ID NO: 10) in CHO-K1 Cells Expressing LGR7
[0377] CHO-K1 cells transiently transfected with LGR7 (RXFP1) were
treated with 10 uM of Forskolin for 10 minutes and then challenged
with either H2 Relaxin (as a positive control), P59C13V (P59) (SEQ
ID NO: 6) or P74C13V (P74) (SEQ ID NO: 10) at increasing
concentrations, from 1 nm to 10 uM (FIG. 4A) and from 3 nM to 100
nM (FIG. 4B). cAMP concentrations were determined at each point, as
described above with (FIG. 4A) or without (FIG. 4B) the presence of
Forskolin.
[0378] The results are presented in FIG. 4. As can be seen from
FIG. 4A, P59C13V (SEQ ID NO: 6) showed a bell-shaped curve as in
lower concentrations there was an increase in cAMP (Gs effect), in
the 30 and 100 nM doses, as compared to H2 Relaxin, whereas Relaxin
showed a dose dependent decrease (Gi) effect in higher
concentrations (>100 nM). P74C13V (SEQ ID NO: 10) showed no
significant effect in this assay.
[0379] FIG. 4B describes a Gs (cAMP stimulation and increase in
cAMP accumulation) dose response was measured for LGR7
(RXFP1)-transfected CHO-K1 cells using low concentrations (3-100
nM) of P59C13V (P59) (SEQ ID NO: 6) or H2 Relaxin. Both P59C13V
(P59) and H2 Relaxin showed a dose dependent increase in cAMP. As
shown in FIG. 4B, P59C13V (P59) (SEQ ID NO: 6) was more effective
than Relaxin in increasing cAMP levels in these studies.
[0380] The ability of P59C13V (SEQ ID NO: 6) and H3 Relaxin to
activate CHO-K1 cells transfected by GPR135 (RXFP3), which is a
non-LGR, Relaxin related receptor, was tested. The assay was
performed by Euroscreen. For cAMP concentration analysis,
Euroscreen cat. ES-656-A-cells (RXFP3, Euroscreen cat. ES-656-A)
grown to mid-log phase in culture media without antibiotics were
detached with PBS-EDTA, centrifuged and resuspended in assay buffer
at a concentration of 7.5.times.105 cells/ml. The test was
performed in 96 well plates. For agonist testing, 12 .mu.l of cells
(5.times.103 cells/well) were mixed with 12 .mu.l of agonist Either
H3-Relaxin or P59C13V (P59) (SEQ ID NO: 6) at increasing
concentrations. The plates were then incubated for 30 min at room
temperature. After addition of the lysis buffer, cAMP
concentrations were estimated, according to the manufacturer
specification, with the HTRF kit from Cis-Bio International (cat
n.degree. 62AM2PEB).
[0381] Relaxin 3 Fit (Eeuroscreen) was used as a positive control.
RXFP3 (GPCR135) transfected cells were challenged with increasing
concentrations of Relaxin 3 (H3 Relaxin--as a positive control--1
pM-1 uM) or P59C13V (P59) (SEQ ID NO: 6) (1 nM-3 uM). The results
are shown in FIG. 5. As can be seen from FIG. 5, P59C13V (P59) (SEQ
ID NO: 6) didn't show any cAMP activation (Gs) of RXFP3, while a
strong cAMP stimulation was demonstrated by H3 Relaxin.
[0382] In this experiment the P59C13V (P59) (SEQ ID NO: 6) did not
activate a cAMP stimulation effect on RXFP3 transfected CHO-K1
cells. However, without wishing to be bound by theory, the P59C13V
(P59) (SEQ ID NO: 6) peptide can either bind or activate a
different pathway when applied on RXFP3 transfected cells. This
experiment does not rule out the possibility that the P59C13V (P59)
(SEQ ID NO: 6) might activate a Gi or a different G-protein
mediated effect on RXFP3 receptor, nor determine the effect of and
of the other peptides on this receptor (i.e. SEQ. ID. No. 1-5 or
7-15).
Example 5
Activity of P74 (SEQ ID NO: 10) and P59 (SEQ ID NO: 6) in Cells
Expressing LGR7 or LGR8 Together with pCRE-.beta.-GAL
[0383] In order to further test the activity of P59C13V (P59) (SEQ
ID NO: 6), P59S-Amide (P59S) (SEQ ID NO: 1), P59C13V-Amide
(P59Amide) (SEQ ID NO: 5) and P74C13V (P74) (SEQ ID NO: 10), the
following assay was performed. The assay was done to assess the
ability of P59C13V (P59) (SEQ ID NO: 6), P59S-Amide (P59S) (SEQ ID
NO: 1), P59C13V-Amide (P59Amide) (SEQ ID NO: 5) and P74C13V (P74)
(SEQ ID NO: 10) peptides to replace H2 Relaxin or INSL3 activity in
stable HEK-293T cell lines expressing the LGR7 (RXFP1) or the LGR8
(RXFP2) receptors, respectively, by assessment of the ability of
these peptides to increase cAMP activity in cell lines expressing
LGR7 (RXFP1) or LGR8 (RXFP2) respectively, together with
pCRE-.beta.-gal. The pCRE-.beta.-gal assay is a standard and well
established assay at the Howard Florey Institute (Melbourne,
Australia) (Halls M L, Bathgate R A, Summers R J.--Comparison of
signaling pathways activated by the relaxin family peptide
receptors, RXFP1 and RXFP2, using reporter genes. J Pharmacol Exp
Ther. 2007 January; 320(1):281-90.)
[0384] The ability of P59C13V (P59) (SEQ ID NO: 6), P59S-Amide
(P59S) (SEQ ID NO: 1), P59C13V-Amide (P59Amide) (SEQ ID NO: 5) and
P74C13V (P74) (SEQ ID NO: 10) to influence cAMP activity in
HEK-293T LGR7/pCRE-.beta.-gal stable or LGR8/pCRE-.beta.-gal
transfected cells was tested in parallel to H2 relaxin and INSL3
respectively, according to the protocol described in Halls M L, et
al., J Pharmacol Exp Ther. 2007 January; 320(1):281-90.
[0385] The experiment was repeated at least three times. The
following concentrations of each peptide were tested: 0.01 nM, 0.1
nM, 1 nM, 10 nM, 100 nM, 1 .mu.M.
[0386] No activation was shown for any of the peptides on
LGR7/pCRE-.beta.-gal transfected HEK-293T cells (data not
shown).
[0387] The results demonstrating activation of LGR8 by P74C13V (SEQ
ID NO: 10), P59C13V with (P59-Amide) (SEQ ID NO: 5) and without
(P59) amide (SEQ ID NO: 6) and P59S-Amide (SEQ ID NO: 1), are shown
in FIGS. 6A and 6B. The results are presented as a percentage of
INSL3 activity in LGR8/pCRE-.beta.-gal transfected HEK-293T cells
as a function of the concentration (Represented in Log [M]).
[0388] FIG. 6A demonstrates a bell shaped response of
LGR8/pCRE-.beta.-gal transfected HEK-293T cells to P74C13V (SEQ ID
NO: 10) in all three of the assays. The P59S-Amide (SEQ ID NO: 1)
effect was less pronounced. FIG. 6B shows that P59C13V (P59) (SEQ
ID NO: 6) demonstrates a weak activation effect (.about.25% of
INSL3) on LGR8/pCRE-.beta.-gal transfected HEK-293T cells, while
P59C13V-amide (P59-Amide) (SEQ ID NO: 5) had a very weak
effect.
[0389] The ability of P74C13V (P74) (SEQ ID NO: 10) and P59C13V
(P59) and P59C13V-Amide (P59-Amide) (SEQ ID NO: 6 and 5
respectively) to activate LGR7 (RXFP1) and LGR8 (RXFP2) was further
tested by testing the ability of these peptides to influence cAMP
activity induced by 5 .mu.M Forskolin in CHO-K1 cells transiently
transfected with LGR7/pCRE-.beta.-gal or LGR8/pCRE-.beta.-gal as
compared to H2 relaxin and INSL3 respectively.
[0390] LGR7 transfected CHO-K1 cells were treated with 5 uM of
Forskilin to stimulate cAMP. The cells were then challenged with
increasing doses of H2 Relaxin (as positive control), P74C13V (P74)
(SEQ ID NO: 10), P59C13V with (P59-Amide) and without (P59) amide
and P59S-Amide (P59S) (SEQ ID NO: 5, 6 and 1, respectively). The
results demonstrating the effect of P74C13V (P74) (SEQ ID NO: 10),
P59C13V with (P59-Amide) and without (P59) amide (SEQ ID NO: 5 and
6, respectively) and P59S-Amide (SEQ ID NO: 1) on
LGR7/pCRE-.beta.-gal transiently transfected CHO-K1 cells is
presented in FIGS. 7A and 7B. The results are presented as a
percentage of Forskolin activity at increasing concentrations of
each peptide (represented in log [M]). The baseline for the
experiment was the activity of Forskolin alone (100%). Each point
represents three unrelated repeats for each experiment together
with their standard error bars.
[0391] FIG. 7A presents a bell-shaped activation pattern shown by
P74C13V (P74) (SEQ ID NO: 10) on LGR7/pCRE-.beta.-gal transiently
transfected CHO-K1 cells. The activation of P74C13V (P74) (SEQ ID
NO: 10) seems to be stronger than that of H2 Relaxin at the lower
concentrations (as was already demonstrated in FIGS. 4A and 4B),
whereas H2 Relaxin showed an increased activation at higher
concentrations. P59S-Amide (SEQ ID NO: 1) showed a mild activation
(of only .about.X2 or Forskolin) at higher concentrations only (100
nM and 1-10 uM)
[0392] FIG. 7B presents a dual activation pattern effect,
demonstrating an activation peak at lower concentrations and second
peak in cAMP activation at higher concentrations, shown by P59C13V
(P59) (SEQ ID NO: 6); and a slight activation pattern shown by
P59C13V-amide (P59-Amide) (SEQ ID NO: 5) on LGR7/pCRE-.beta.-gal
transiently transfected CHO-K1 cells.
[0393] LGR8 transfected CHO-K1 cells were treated with 5 uM of
Forskilin to stimulate cAMP. The cells were then challenged with
increasing doses of INSL3 (as positive control), P74C13V (P74) (SEQ
ID NO: 10), P59C13V with (P59-Amide) and without (P59) amide (SEQ
ID NO: 5 and 6, respectively) and P59S-Amide (P59-S) (SEQ ID NO:
1). The results demonstrating the effect of P74C13V (P74) (SEQ ID
NO: 10), P59C13V with (P59-Amide) and without (P59) amide (SEQ ID
NO: 5 and 6, respectively) and P59S-Amide (P59S-Amide) (SEQ ID NO:
1) on LGR8/pCRE-.beta.-gal transiently transfected CHO-K1 cells is
presented in FIGS. 8A and 8B. The results are presented as a
percentage of Forskolin activity at various concentrations of the
peptide (logarithmic scale). Each point represents three unrelated
repeats for each experiment.
[0394] FIG. 8A presents a bell-shaped activation pattern, shown by
P74C13V (P74) (SEQ ID NO: 10) similar to that of INSL3 at lower
concentrations on LGR8/pCRE-.beta.-gal transiently transfected
CHO-K1 cells. The activation of P74C13V (P74) (SEQ ID NO: 10) seems
to be similar yet slightly weaker than that or INSL3 at the lower
concentrations (as was already demonstrated in FIG. 7A for LGR7),
whereas INSL3 showed an increased activation at higher
concentrations. P59S-Amide (SEQ ID NO: 1) showed a mild activation
(of only .about.X2 or Forskolin) at higher concentrations only (100
nM and 1-10 uM).
[0395] FIG. 8B presents a dual activation pattern effect,
demonstrating a peak in activation at lower concentrations and
second increase in cAMP activation at higher concentrations shown
by P59C13V (P59) (SEQ ID NO: 6); and a slight activation pattern
shown by P59C13V-amide (P59-Amide) (SEQ ID NO: 5) on
LGR8/pCRE-.beta.-gal transiently transfected CHO-K1 cells.
Example 6
Competition Assays of P74C13V (SEQ ID NO: 10) and P59C13V (SEQ ID
NO: 6) with Europium Labelled H2 Relaxin or INSL3 in Cells
Expressing LGR7 or LGR8
[0396] The ability of Peptides P74C13V (SEQ ID NO: 10) and P59C13V
(SEQ ID NO: 6) to compete with the binding of known ligands to LGR7
or LGR8 was tested by testing the ability of P74C13V (SEQ ID NO:
10) and P59C13V (SEQ ID NO: 6) to compete with the binding of
Europium labelled H2 Relaxin or INSL3 (manufactured by the H.
Florey institute) to stable HEK-293T cell lines expressing LGR7 or
LGR8 respectively.
[0397] HEK-293T cell lines stabled expressing LGR7 (RXFP1) were
treated with Europium labelled H2 Relaxin (manufactured by the H.
Florey institute) The cells were then treated with decimal
increasing concentrations (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1
.mu.M, 10 .mu.M) of un-labeled H2 Relaxin (as a positive control)
and the tested peptides P59C13V (P59) (SEQ ID NO: 6), P59C13V-Amide
(P59-Amide) (SEQ ID NO: 5), P59-S-Amide (P59S) (SEQ ID NO: 1) and
P74C13V (P74) (SEQ ID NO: 10). The competition results of three
independent experiments are shown in FIG. 9. The binding results
are shown as a percentage of specific binding of the radioactive
test H2 Relaxin (where 100% is the baseline radiation level). As
can be seen from FIG. 9, no competition was shown for the binding
of Europium-H2 Relaxin by any of the tested peptides. Without
wishing to be bound by a single theory, the activity of peptides
P59C13V (SEQ ID NO: 6), P59C13V-Amide (SEQ ID NO: 5), P59S-Amide
(SEQ ID NO: 1) and P74C13V (SEQ ID NO: 10) on LGR7 (RXFP1), might
be mediated by binding of the peptides to a different binding site,
since the binding of the above peptides does not interfere, compete
or affect the binding of the Europium H2 Relaxin to LGR7.
[0398] HEK-293T cell lines expressing LGR8 (RXFP2) were treated
with Europium labeled INSL3 (manufactured by the H. Florey
institute). The cells were then treated with decimal increasing
concentrations (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 .mu.M, 10
.mu.M) of un-labeled INSL3 (as a positive control) and the tested
peptides P59C13V (P59) (SEQ ID NO: 6), P59C13V-Amide (P59-Amide)
(SEQ ID NO: 5), P59-S-Amide (P59S) (SEQ ID NO: 1) and P74C13V (P74)
(SEQ ID NO: 10). The competition results of three independent
experiments are shown in FIG. 10. The binding results are shown as
a percentage of specific binding of the radioactive test INSL3
(where 100% is the baseline radiation level). As can be seen from
FIG. 10, P59C13V (P59) (SEQ ID NO: 6) demonstrates a positive
cooperative or allosteric effect. showing .about.125% of specific
binding at 1 nM and higher concentrations. This means that the
binding affinity of the Eur-INSL3 was increased by binding/activity
of the P59C13V (P59) (SEQ ID NO: 6) peptide on the LGR8 receptor.
The results with the other peptides (P59C13V-amide (P59-Amide) (SEQ
ID NO: 5), P59-S-Amide (P59S) (SEQ ID NO: 1) and P74C13V (P74) (SEQ
ID NO: 10)) were less apparent but all four peptides showed
alteration and competition/alosteric effect on the binding of
Europium-INSL3 on LGR8 (RXFP2). Without wishing to be bound by a
single theory, the binding of the peptides to the LGR8 receptor,
probably through a different binding site than that of INSL3, there
is an allosteric effect that might alter the affinity of the
receptor to the INSL3 ligand, might suggesting a synergistic effect
of the above peptides and INSL3 on the activity of the LGR8 (RXFP2)
receptor.
Example 7
Real Time Activation Assay Tested by ACEA Cell Impedance System
Method for Analysis of Peptides' Ability to Activate
Relaxin-Related Family of Receptors Using ACEA RT-CES Screen
ACEA RT-CES Screen Background:
[0399] ACEA RT-CES screen is a noninvasive and label-free assay for
GPCRs that can be used with both engineered and nonengineered cell
lines. The assay is based on using cell-electrode impedance to
measure minute changes in cellular morphology as a result of
ligand-dependent GPCR activation and is described in Naichen Yu, et
al., Anal. Chem., 78 (1), 35-43, 2006. 10.1021/ac051695v
S0003-2700(05)01695-1. The Rho family of small GTPases, which
include Rho, Rac, and CDC42, are well-characterized effectors of
oncogenes, growth factor and adhesion-mediated signaling pathways
and are not classically thought of as being key effectors for
GPCRs. Rho family GTPases participate in a number of cellular
processes, the main one being regulation and maintenance of
specific structures within the actin cytoskeleton framework. GPCRs
have been shown to modulate the actin cytoskeleton and hence cell
morphology in a very specific manner depending on the Rho family
GTPase being activated. The current view of the actin cytoskeleton
is that of a dynamic and plastic system that is a reflection and
manifestation of the intracellular signaling and not simply a
static structure designed to maintain cellular architecture. Since
GPCRs couple to the actin cytoskeletal network and induce very
defined morphological changes, it is possible to harness this
information as a functional and biologically relevant readout for
GPCRs.
[0400] ACEA biosciences, has designed electronic cell sensor arrays
embedded in the bottom of the well of microtiter plates that are
capable of measuring minute changes in cell morphology. The
electronic sensors measure changes in cell-substrate impedance as a
result of the disruption of the ionic environment due to the
presence of cell and cell morphology dynamics. The main advantages
offered by using cell-substrate impedance and cell morphology as a
readout are that both exogenously expressed and endogenous
receptors can be assayed without the need for engineering the cell
with promiscuous G proteins and reporters or labeling the cells
with dyes. In addition, since the readout is noninvasive, multiple
stimulations with the same ligand or different ligands can be
performed to assess events such as desensitization and receptor
cross-talk. Finally, another major aspect of using cell-substrate
impedance and cell morphology as a readout is that potentially all
GPCRs, regardless of the signaling pathways, can be functionally
monitored.
ACEA RT-CES Screen Experimental Procedure & Protocol
[0401] Peptides were synthesized by the solid phase peptide
synthesis (SPPS) method, cleaved from the resin, and purified by
RP-HPLC unless stated otherwise. The peptide's identity was
verified by mass spectrometry. Final purity of peptide was >90%
as measured by RP-HPLC. Peptides were diluted in Water (DDW--high
purity) containing 0.1% BSA. In some cases peptides were dissolved
in Tris-HCl buffer (pH8.2). In other cases either the addition of
1% DMSO or s brief sonication was required. All plates were stored
at -80C until use.
[0402] Cells: CHOk1 cell-line (ATCC CCL-61). Cells were maintained
and propagated in F-12 HAM nutrient mix (Gibco. Cat#21765-029)
supplemented with 10% HI-FBS (Biological Ind. Cat#04-121-1), 2.5 mL
of 200 mm L-Glutamine in Saline Solution (Biological Ind.
Cat#03-020-1), 5 mL of Penicillin-Streptomycin Solution
10000/mLPenicillin G & 10 mg/mL Streptomycin Sulfate
(Biological Ind. Cat#03-031-1).
Subculturing Cells:
[0403] Cells were freshly thawed from liquid N2 and were
sub-cultured 1:10 twice a week according to the following
protocol:
[0404] Remove and discard culture medium.
[0405] Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53
mM EDTA solution to remove all traces of serum which contains
trypsin inhibitor.
[0406] Incubate for 5 minutes at 37.degree. C. in CO2 humidified
incubator.
[0407] Examine under the microscope until cell layer is
dispersed.
[0408] Add 10 to 12 mL of complete growth medium and aspirate cells
by gently pipetting.
[0409] Take a 300 .mu.l sample from the cell suspension and count
concentration, viability and aggregate rate using the CEDEX
conuter.
[0410] Centrifuge cells for 6 minutes at 1200 rpm.
[0411] Re-suspend cells in complete growth medium to the desirable
cell concentration.
[0412] DNA Transfection:
[0413] All transfections were performed using the FUGENE6 reagent
(Roche. Cat#11-814-443-001, Expiry date: May 2008).
Transfection Protocol:
[0414] One day before the transfection 2.times.106 CHO-k1 cells
were plated in T75 flask containing 10 mL of complete growth
medium. On the transfection date, FUGENE6 transfection reagent was
warmed to ambient temperature by 15 minutes incubation at room
temperature and mixed prior to use by vortex.
[0415] The following was diluted in a sterile microfuge tube: 50
.mu.l of FUGENE6 into 650 .mu.l of serum free F12-HAM medium and
tubes were incubated for 10 min at RT.
[0416] The transfected DNA mix was prepared in another sterile
microfuge tube; Total of 20 .mu.g plasmid DNA mixture which is
composed of 18 .mu.g of the target GPCR plasmid and of 2 .mu.g of
pIRES plasmid carrying eGFP reporter gene.
[0417] The plasmids DNA mixture was then added to the transfection
reagent tube and incubated 35 min at RT with gently tapping once
every 5 minutes to mix the contents. The DNA-FuGene complex was
added drop wise to flask and spread by gentle swirling.
[0418] The transfected cells were kept for 24 hours at 37.degree.
C. in CO2 humidified incubator and monitored under fluorescent
microscope for the detection of green light omitted from GFP
transfected cells.
ACEA RT-CES Protocol; Screening for GPCR Activating Peptides:
[0419] In this protocol the RCD96 E-plate device (ACEA Biosciences
Inc.) were used for seeding, monitoring and activating the CHO-k1
transfected cells.
Pre-treatment E-Plates: The E-plate wells were coated with 100 uL
of 1 mg/ml Gelatine (Fluka. Cat#48720). The gelatine was dissolved
at 8 mg/mL in sterile DDW and was diluted 1:8 in DDW before added
to the E-plate wells. The plates covered with gelatine were
incubated for 40 minutes at 37.degree. C. and 5% CO2 in a
humidified atmosphere.
[0420] 100 .mu.l of sterile water were added and all liquids were
removed. The plates were washed twice more, now with 200 .mu.l of
sterile water and plate was taken for cells seeding
Cells Seeding:
[0421] Before the transfected cells were seeded, 80 ml SFM F12-HAM
medium were added to each well of the E-plates and background
levels were recorded. 24-26 hours past transfection, medium was
aspirated and cells were trypsinized with 0.25% (w/v) Trypsin/EDTA
solution and counted on the CEDEX counter.
Total cell number was calculated and cells were re-suspended in
complete growth medium to cell concentration ranging from
0.3125.times.106/mL up to 0.4375.times.106/mL. From that dilution,
80 .mu.l of cell suspension were seeded, resulting in 25,000 up to
35000 total cells in 5% FCS--complete growth medium per each
E-plate well.
Peptide Challenging:
[0422] 22-26 hours past seeding (46-52 hours past transfection) and
with concordance to the recorded Cell Index (CI) the cells were
prepared for the addition of the activating peptides. Prior to
adding the peptides, the complete medium was aspirated and replaced
with 120 ml 37.degree. C. pre-warmed SFM F12-HAM nutrient
mixture.
[0423] 1 hour later or when the CI reads are stabilized, the
E-plate was removed from the 96X E-Plate Station for peptide
challenge. Challenging with the peptides was performed in
duplicates by adding 5 .mu.l of peptide solution from an already
made 250 .mu.M daughter stock plates, resulting in final peptide
concentration of 10 .mu.M. The peptide solution contains the
peptide dissolved in DDW supplemented with 0.1% Albumin
(Sigma-aldrich Cat# A3059, Fraction V, .about.99%, Essentially
.gamma.-globulin free).
The screening was done on transiently transfected CHO-K1 cells and
was done in two stages: [0424] 1. Screening phase--Challenging the
relaxin-related family of receptors transfected CHO-K1 cells with
10 uM of each screened peptide, using Calcitonin (1 uM) as an assay
internal positive control (the receptor for Calcitonin is
endogenously expressed by CHO-K1 cells), 0.1% BSA as a negative
control. 1 uM of H2 Relaxin was used as a positive control for the
transfected receptor. [0425] 2. Dose response phase--Challenging
the LGR7 transfected CHO-K1 with 0.03 uM, 0.1 uM, 0.3 uM, 1 uM, 3
uM and 10 uM of each screened peptide, using H2 Relaxin as a
positive control for the transfected receptor.
Example 7-1
P74 (SEQ ID NO: 10) and P59 (SEQ ID NO: 6) Mediated Activation of
the LGR-Related Family of Receptors in ACEA Cell Impedance
Device-Screening Phase
[0426] The ability of Peptides P74C13V (P74) (SEQ ID NO: 10) and
P59C13V (P59) (SEQ ID NO: 6) to activate LGR7 (RXFP1) and LGR8
(RXFP2) receptors was checked as described above in ACEA cell
impedance device. The following results were obtained:
[0427] First the ability of Peptide P74C13V (SEQ ID NO: 10) (at 10
uM) and H2 Relaxin (at 1 uM) to cause changes in cell impedence was
measured in untransfected CHO-K1 cells. The results are presented
in FIG. 11. Cell index was normalized to time point T1 (after
peptide administration), marked in FIG. 11 by a vertical solid line
(The dashed Vertical line indicates the 20.sup.th hour from the
beginning of the experiment). Calcitonin was used as an internal
positive control to evaluate the validity of the assay, as the
calcitonin receptor is endogenously expressed on CHO-K1 cells. BSA
0.1% was used as a negative control. As can be seen from FIG. 11,
both H2 Relaxin and peptide P74C13V (P74) demonstrated a moderate
effect on untransfected CHO-K1 cells. Without wishing to be bound
by a single theory, the conclusion from this result is that even
though not detected at standard procedures, either the LGR7 or LGR8
receptors are expressed, even if in a residual amount, by the
CHO-K1 cell line.
[0428] Next, the ability of Peptide P74C13V (P74) (SEQ ID NO: 10)
(at 10 uM) and H2 Relaxin (at 1 uM) to cause changes in cell
impedence was measured in CHO-K1 cells transfected with a
non-relaxin related GPCR: GPR39 (GPR39Human in SwissProt accession:
043194). The results are presented in FIG. 12. Cell index was
normalized to time point T1 (after peptide administration), marked
in FIG. 12 by a vertical solid line (the dashed vertical line
indicates the 24.sup.th hour from the beginning of the experiment).
Calcitonin was used as a positive control and BSA 0.1% was used as
a negative control. As can be seen from FIG. 12, both H2 Relaxin
and peptide P74C13V (P74) (SEQ ID NO: 10) demonstrated a moderate
yet visible effect on GPR39 transfected CHO-K1 cells.
[0429] From these experiments, it can be seen that the transfection
process itself was not the cause for the response presented above
(in FIG. 11).
[0430] Next, the ability of Peptide P74C13V (P74) (SEQ ID NO: 10)
(at 10 uM) and H2 Relaxin (at 1 uM) to cause changes in cell
impedence was measured in CHO-K1 cells transfected with a LGR7
(RXFP1). The results are presented in FIG. 13. Cell index was
normalized to time point T1 (after peptide administration), marked
in FIG. 13 by a vertical solid line. Calcitonin was used as a
positive control and BSA 0.1% was used as a negative control. As
can be seen from FIG. 13, both H2 Relaxin and peptide P74C13V (SEQ
ID NO10) demonstrated a cellular cell impedance (as shown by the
ACEA measurement) effect on LGR7 transfected CHO-K1 cells. The
effect of P74C13V (SEQ ID NO: 10) was stronger than the effect of
H2 Relaxin, however, both peptides (H2 Relaxin and P74C13V (SEQ ID
NO10)) were found to be capable of activating a cellular response
in LGR7 transfected CHO-K1 cells.
[0431] The ability of Peptides P74C13V (P74) (SEQ ID NO: 10) (at 10
uM), P59C13V (P59) (SEQ ID NO: 6) (at 10 uM) and H2 Relaxin (at 1
uM) to cause changes in cell impedence was further measured in
CHO-K1 cells transfected with a LGR8 (RXFP2). The results are
presented in FIG. 14. As above, cell index was normalized to time
point T1 (after peptide administration), marked in FIG. 14 by a
vertical solid line (the dashed Vertical line indicates the
23.sup.rd hour from the beginning of the experiment). As above,
Calcitonin was used as a positive control and BSA 0.1% was used as
a negative control. As can be seen from FIG. 14, both H2 Relaxin
and peptide P74C13V (SEQ ID NO: 10) demonstrated a strong cellular
effect on LGR8 transfected CHO-K1 cells. P59C13V (P59) (SEQ ID NO:
6) showed no significant effect as compared to BSA. From these
results it seems that both peptides (H2 Relaxin and P74C13V (SEQ ID
NO: 10)) are capable of activating a cellular response in LGR8
transfected CHO-K1 cells, however, no effect of P59C13V (P59) (SEQ
ID NO: 6) on LGR8 transfected CHO-K1 cells was demonstrated in this
experiment. This could be explained by technical problem, such as
degradation of the P59 peptide in this experiment.
[0432] The ability of Peptide P74C13V (P74) (SEQ ID NO: 10) (10 uM)
and H2 Relaxin (1 uM) to cause changes in cell impedance was
further measured in CHO-K1 cells transfected with a LGR4. The
results are presented in FIG. 15. As above, cell index was
normalized to time point T1 (after peptide administration), marked
in FIG. 15 by a vertical solid line. As above, Calcitonin was used
as a positive control to and BSA 0.1% was used as a negative
control. As can be seen from FIG. 15, peptide P74C13V (SEQ ID NO:
10) demonstrated a moderate effect on LGR4 transfected CHO-K1
cells. H2 Relaxin demonstrated a much stronger effect in this
assay. Without wishing to be bound by a single theory, this result
may suggest that H2 Relaxin is capable of binding and activating a
cellular response though activation of the LGR4 receptor in LGR4
transfected CHO-K1 cells, probably mediated by a non cAMP related
pathway.
Example 7-1
P74 (SEQ ID NO: 10) and P59 (SEQ ID NO: 6) Mediated Activation of
Relaxin-Related Family of Receptors in ACEA Cell Impedance
Device-Dose Response
[0433] Dose response activation testing of both (H2) Relaxin and
P74C13V (P74) (SEQ ID NO: 10) in untransfected CHO-K1 cells showed
no dose dependent activation but only a moderate activation of the
highest concentrations (i.e. 10 uM for P74C13V and 1 uM for H2
Relaxin) (data not shown).
[0434] The experiment was done similarly to the ACEA protocol
above. Each peptide was added in triplicates at increasing
concentrations (presented in Log [M] on FIGS. 16B, 17B, 18B and
19B). Cell impedance was tested as described and cell index was
normalized to the time of peptide addition (T1--left vertical line
in FIGS. 16A, 17A, 18A and 19A). Dose response was examined at end
point of the experiment (one time point--indicated by the right
vertical line on FIGS. 16A-19A).
[0435] FIG. 16A presents a normalized cell index and a dose
response curve for LGR7 (RXFP1) transfected CHO-K1 cells challenged
by increasing concentrations of H2 Relaxin (the dashed Vertical
line indicates the 25.sup.th hour from the beginning of the
experiment). The H2 Relaxin concentrations tested were 0.16 nM; 0.8
nM; 4 nM; 20 nM; 100 nM; 500 nM. As shown in FIG. 16A, there is a
clear dose response for the higher concentrations, with an
estimated EC50 of .about.100 nM.
[0436] FIG. 16B presents a normalized cell index and a dose
response curve for LGR7 transfected CHO-K1 cells challenged by
increasing concentrations of H2 Relaxin at time point 25.5 hours
(vertical right line in FIG. 16A). A dose response is presented as
a normalized cell index per Log of concentration (M). As
demonstrated in FIG. 16B, a clear dose dependent activation of LGR7
by H2 relaxin was found.
[0437] FIG. 17A presents a normalized cell index and a dose
response curve for LGR7 (RXFP1) transfected CHO-K1 cells challenged
by increasing concentrations of peptide P74C13V (P74) (SEQ ID
NO:10) (the dashed Vertical lines indicate the 25.sup.th and
26.sup.th hours from the beginning of the experiment). The P74C13V
(P74) (SEQ ID NO: 10) concentrations tested were 41 nM; 123 nM; 370
nM; 1.1 uM; 3.3 uM; 10 uM. As shown in FIG. 17A, there is a clear
dose response for the high concentrations, with an estimated EC50
of .about.300 nM.
[0438] FIG. 17B presents a normalized cell index and a dose
response curve for LGR7 transfected CHO-K1 cells challenged by
increasing concentrations of peptide P74C13V (SEQ ID NO:10) at time
point 26.5 hours (vertical right line in FIG. 17A). A dose response
is presented as a normalized cell index per Log of concentration
(M). As can be seen from FIG. 17B a clear dose dependent activation
of LGR7 by P74C13V (SEQ ID NO:10) was found. The affinity found for
P74C13V (SEQ ID NO:10) to LGR7 was lower than that of H2
Relaxin.
[0439] FIG. 18A presents a normalized cell index and a dose
response curve for LGR7 (RXFP1) transfected CHO-K1 cells challenged
by increasing concentrations of peptide P59C13V (P59) (SEQ ID NO:
6) (the dashed Vertical lines indicate the 25.sup.th and 26.sup.th
hours from the beginning of the experiment). The P59C13V (P59) (SEQ
ID NO: 6) concentrations tested were 41 nM; 123 nM; 370 nM; 1.1 uM;
3.3 uM; 10 uM. As shown in FIG. 18A, there is a moderate dose
response for the high concentrations.
[0440] FIG. 18B presents a normalized cell index and a dose
response curve for LGR7 transfected CHO-K1 cells challenged by
increasing concentrations of P59 (SEQ ID NO:6) at time point 26.5
hours (vertical right line in FIG. 18A). A dose response is
presented as a normalized cell index per Log of concentration (M).
As can be seen from FIG. 18B a mild dose dependent activation of
LGR7 by P59C13V (SEQ ID NO:6) was found. Only the highest
concentration (10 uM) showed a significant higher cell index rate
as compared to the other concentrations. Since the same peptide
batch was used for this experiment and the experiment presented in
FIG. 14 supports the assumption that this particular batch of
P59C13V peptide was of bad quality. The affinity found for P59C13V
(SEQ ID NO:6) to LGR7 was significantly lower than that of H2
Relaxin or P74C13V (SEQ ID NO:10). Therefore, even though P59C13V
(SEQ ID NO:6) peptide is capable of activating the receptor at high
concentration, no real dose response was demonstrated.
[0441] FIG. 19A presents a normalized cell index and a dose
response curve for LGR7 transfected CHO-K1 cells challenged by
increasing concentrations of peptide P59S-Amide (P59S) (SEQ ID NO:
1) (the dashed Vertical lines indicates the 26.sup.th hour from the
beginning of the experiment). The P59S (SEQ ID NO: 1)
concentrations tested were 41 nM; 123 nM; 370 nM; 1.1 uM; 3.3 uM;
10 uM. As shown in FIG. 19A, there is a moderate dose response for
the high concentrations.
[0442] FIG. 19B presents a normalized cell index and a dose
response curve for LGR7 transfected CHO-K1 cells challenged by
increasing concentrations of P59S-Amide (P59S) (SEQ ID NO: 1) at
time point 26.5 hours (vertical right line in FIG. 18A). A dose
response is presented as a normalized cell index per Log of
concentration (M). As can be seen from FIG. 19B, a mild dose
dependent activation of LGR7 by P59C13V (SEQ ID NO:6) was found.
Only the highest concentration (10 uM) showed a significant higher
cell index rate as compared to the other concentrations. Therefore,
even though this peptide is capable of activating the receptor at
high concentration, no dose response was demonstrated.
Example 8
The P59 Sequence in the C1QTNF8 Protein is Highly Conserved
Throughout Other Species and Orthologs, as Well as at Least One
Human Paralog (C1QTNF1)
[0443] FIG. 20 shows a multiple alignment comparison of the
sequence of P59-G (SEQ ID No. 12), representing a fragment of the
native human precursor (C1QTNF8--SEQ. ID. No. 19), and homologous
sequences derived from various organisms, including Chimpanzee (SEQ
ID No. 20), Orangutan (SEQ ID No. 21), Rhesus (SEQ ID No. 22), Cow
(SEQ ID No. 23), Chicken (SEQ ID No. 24) and Rat (SEQ ID No. 26).
The multiple sequence alignment comparison includes the
corresponding peptide sequence derived from the human paralogue
C1QTNF1 (SEQ ID No. 25). As can be seen both the N-terminal end (4
Amino acids) and C-terminal end (7 Amino acids) are identical to
all species. The cleavage sites at both ends are also highly
conserved (with occasional replacement of K for an R). The middle
Cysteine residue (C13) that was replaced with Valine for
dimerization purposes is highly conserved as well.
[0444] The descriptions given are intended to exemplify, but not
limit, the scope of the invention. Additional embodiments are
within the claims.
Sequence CWU 1
1
3517PRTArtificial sequenceChemically synthesized peptide 1Ala Tyr
Ala Ala Phe Ser Val 1 5 27PRTArtificial sequenceChemically
synthesized peptide 2Ala Tyr Ala Ala Phe Ser Val 1 5
322PRTArtificial sequenceChemically synthesized peptide 3Gly Gln
Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Cys Arg Arg Ala 1 5 10 15
Tyr Ala Ala Phe Ser Val 20 422PRTArtificial sequenceChemically
synthesized peptide 4Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala
Ala Cys Arg Arg Ala 1 5 10 15 Tyr Ala Ala Phe Ser Val 20
522PRTArtificial sequenceChemically synthesized peptide 5Gly Gln
Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Val Arg Arg Ala 1 5 10 15
Tyr Ala Ala Phe Ser Val 20 622PRTArtificial sequenceChemically
synthesized peptide 6Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala
Ala Val Arg Arg Ala 1 5 10 15 Tyr Ala Ala Phe Ser Val 20
732PRTArtificial sequenceChemically synthesized peptide 7Gly Gln
Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Cys Arg Arg Ala 1 5 10 15
Tyr Ala Ala Phe Ser Val Gly Arg Arg Ala Tyr Ala Ala Phe Ser Val 20
25 30 832PRTArtificial sequenceChemically synthesized peptide 8Gly
Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Cys Arg Arg Ala 1 5 10
15 Tyr Ala Ala Phe Ser Val Gly Arg Arg Ala Tyr Ala Ala Phe Ser Val
20 25 30 932PRTArtificial sequenceChemically synthesized peptide
9Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Val Arg Arg Ala 1
5 10 15 Tyr Ala Ala Phe Ser Val Gly Arg Arg Ala Tyr Ala Ala Phe Ser
Val 20 25 30 1032PRTArtificial sequenceChemically synthesized
peptide 10Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Val Arg
Arg Ala 1 5 10 15 Tyr Ala Ala Phe Ser Val Gly Arg Arg Ala Tyr Ala
Ala Phe Ser Val 20 25 30 118PRTArtificial sequenceChemically
synthesized peptide 11Ala Tyr Ala Ala Phe Ser Val Gly 1 5
1223PRTArtificial sequenceChemically synthesized peptide 12Gly Gln
Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Cys Arg Arg Ala 1 5 10 15
Tyr Ala Ala Phe Ser Val Gly 20 1323PRTArtificial sequenceChemically
synthesized peptide 13Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala
Ala Val Arg Arg Ala 1 5 10 15 Tyr Ala Ala Phe Ser Val Gly 20
1433PRTArtificial sequenceChemically synthesized peptide 14Gly Gln
Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Cys Arg Arg Ala 1 5 10 15
Tyr Ala Ala Phe Ser Val Gly Arg Arg Ala Tyr Ala Ala Phe Ser Val 20
25 30 Gly 1533PRTArtificial sequenceChemically synthesized peptide
15Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Val Arg Arg Ala 1
5 10 15 Tyr Ala Ala Phe Ser Val Gly Arg Arg Ala Tyr Ala Ala Phe Ser
Val 20 25 30 Gly 1617PRTArtificial sequenceChemically synthesized
peptide 16Met Ala Ala Pro Ala Leu Leu Leu Leu Ala Leu Leu Leu Pro
Val Gly 1 5 10 15 Ala 1719PRTArtificial sequenceChemically
synthesized peptide 17Met Ala Ala Pro Ala Leu Leu Leu Leu Ala Leu
Leu Leu Pro Val Gly 1 5 10 15 Ala Trp Pro 1822PRTArtificial
sequenceChemically synthesized peptide 18Met Ala Ala Pro Ala Leu
Leu Leu Leu Ala Leu Leu Leu Pro Val Gly 1 5 10 15 Ala Trp Pro Gly
Leu Pro 20 19262PRTHomo sapiens 19Met Ala Ala Pro Ala Leu Leu Leu
Leu Ala Leu Leu Leu Pro Val Gly 1 5 10 15 Ala Trp Pro Gly Leu Pro
Arg Arg Pro Cys Val His Cys Cys Arg Pro 20 25 30 Ala Trp Pro Pro
Gly Pro Tyr Ala Arg Val Ser Asp Arg Asp Leu Trp 35 40 45 Arg Gly
Asp Leu Trp Arg Gly Leu Pro Arg Val Arg Pro Thr Ile Asp 50 55 60
Ile Glu Ile Leu Lys Gly Glu Lys Gly Glu Ala Gly Val Arg Gly Arg 65
70 75 80 Ala Gly Arg Ser Gly Lys Glu Gly Pro Pro Gly Ala Arg Gly
Leu Gln 85 90 95 Gly Arg Arg Gly Gln Lys Gly Gln Val Gly Pro Pro
Gly Ala Ala Cys 100 105 110 Arg Arg Ala Tyr Ala Ala Phe Ser Val Gly
Arg Arg Ala Tyr Ala Ala 115 120 125 Phe Ser Val Gly Arg Arg Glu Gly
Leu His Ser Ser Asp His Phe Gln 130 135 140 Ala Val Pro Phe Asp Thr
Glu Leu Val Asn Leu Asp Gly Ala Phe Asp 145 150 155 160 Leu Ala Ala
Gly Arg Phe Leu Cys Thr Val Pro Gly Val Tyr Phe Leu 165 170 175 Ser
Leu Asn Val His Thr Trp Asn Tyr Lys Glu Thr Tyr Leu His Ile 180 185
190 Met Leu Asn Arg Arg Pro Ala Ala Val Leu Tyr Ala Gln Pro Ser Glu
195 200 205 Arg Ser Val Met Gln Ala Gln Ser Leu Met Leu Leu Leu Ala
Ala Gly 210 215 220 Asp Ala Val Trp Val Arg Met Phe Gln Arg Asp Arg
Asp Asn Ala Ile 225 230 235 240 Tyr Gly Glu His Gly Asp Leu Tyr Ile
Thr Phe Ser Gly His Leu Val 245 250 255 Lys Pro Ala Ala Glu Leu 260
2023PRTPan sp. 20Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Ala
Cys Gln Arg Ala 1 5 10 15 Tyr Ala Ala Phe Ser Val Gly 20
2123PRTPongo sp. 21Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Pro
Cys Gln Arg Ala 1 5 10 15 Tyr Ala Ala Phe Ser Val Gly 20
2223PRTMacaca sp. 22Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Pro
Cys Gln Arg Ala 1 5 10 15 Tyr Ala Ala Phe Ser Val Gly 20 2323PRTBos
sp. 23Gly Gln Lys Gly Gln Ala Gly Leu Pro Gly Ala Gln Cys Pro Arg
Ala 1 5 10 15 Tyr Ala Ala Phe Ser Val Gly 20 2423PRTGallus sp.
24Gly Gln Lys Gly Gln Pro Gly Pro Gln Gly His Ser Cys Lys Gln Leu 1
5 10 15 Tyr Ala Ala Phe Ser Val Gly 20 2523PRTHomo sapiens 25Gly
Gln Lys Gly Ser Met Gly Ala Pro Gly Glu Arg Cys Lys Ser His 1 5 10
15 Tyr Ala Ala Phe Ser Val Gly 20 2623PRTRattus sp. 26Gly Gln Lys
Gly Ser Met Gly Ala Pro Gly Asp His Cys Lys Ser Gln 1 5 10 15 Tyr
Ala Ala Phe Ser Val Gly 20 2727PRTHomo sapiens 27Arg Arg Gly Gln
Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Cys Arg 1 5 10 15 Arg Ala
Tyr Ala Ala Phe Ser Val Gly Arg Arg 20 25 2827PRTPan sp. 28Arg Lys
Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Ala Cys Gln 1 5 10 15
Arg Ala Tyr Ala Ala Phe Ser Val Gly Arg Arg 20 25 2927PRTPongo sp.
29Arg Lys Gly Gln Lys Gly Gln Val Gly Pro Pro Gly Ala Pro Cys Gln 1
5 10 15 Arg Ala Tyr Ala Ala Phe Ser Val Gly Arg Arg 20 25
3027PRTMacaca sp. 30Arg Lys Gly Gln Lys Gly Gln Val Gly Pro Pro Gly
Ala Pro Cys Gln 1 5 10 15 Arg Ala Tyr Ala Ala Phe Ser Val Gly Arg
Arg 20 25 3127PRTBos sp. 31Arg Lys Gly Gln Lys Gly Gln Ala Gly Leu
Pro Gly Ala Gln Cys Pro 1 5 10 15 Arg Ala Tyr Ala Ala Phe Ser Val
Gly Arg Arg 20 25 3227PRTGallus sp. 32Arg Lys Gly Gln Lys Gly Gln
Pro Gly Pro Gln Gly His Ser Cys Lys 1 5 10 15 Gln Leu Tyr Ala Ala
Phe Ser Val Gly Arg Arg 20 25 3327PRTHomo sapiens 33Pro Lys Gly Gln
Lys Gly Ser Met Gly Ala Pro Gly Glu Arg Cys Lys 1 5 10 15 Ser His
Tyr Ala Ala Phe Ser Val Gly Arg Lys 20 25 3427PRTRattus sp. 34Pro
Lys Gly Gln Lys Gly Ser Met Gly Ala Pro Gly Asp His Cys Lys 1 5 10
15 Ser Gln Tyr Ala Ala Phe Ser Val Gly Arg Arg 20 25
3527PRTArtificial sequenceConsensus sequence 35Arg Lys Gly Gln Lys
Gly Gln Xaa Gly Pro Pro Gly Xaa Xaa Cys Lys 1 5 10 15 Xaa Xaa Tyr
Ala Ala Phe Ser Val Gly Arg Arg 20 25
* * * * *