U.S. patent application number 13/693372 was filed with the patent office on 2013-04-18 for method of treating skeletal dysplasias using vessel dilator.
This patent application is currently assigned to UNITED STATES DEPARTMENT OF VETERANS AFFAIRS. The applicant listed for this patent is United States Department of Veterans Affairs, University of South Florida. Invention is credited to David Lynn Vesely.
Application Number | 20130096061 13/693372 |
Document ID | / |
Family ID | 45067104 |
Filed Date | 2013-04-18 |
United States Patent
Application |
20130096061 |
Kind Code |
A1 |
Vesely; David Lynn |
April 18, 2013 |
METHOD OF TREATING SKELETAL DYSPLASIAS USING VESSEL DILATOR
Abstract
C-natriuretic peptide (CNP) has been shown to regulate
proliferation of mouse and rat osteoblasts. Genetic deletion of CNP
results in dwarfism. CNP effects on bone growth involve inhibition
of MEK 1 and ERK 1/2 kinases mediated via the intracellular
messenger cyclic GMP. Vessel dilator is another natriuretic peptide
synthesized by the atrial natriuretic peptide gene whose biologic
half-life is 12 times longer than CNP. Vessel dilator's biologic
effects on proliferating cells are mediated via inhibiting MEK 1/2
and ERK 1/2 kinases via cyclic GMP. Vessel dilator was not studied
previously on osteoblasts. CNP and vessel dilator were tested in
dose-response studies enhanced human osteoblasts' proliferation,
showing that vessel dilator has identical mechanisms of action to
CNP but much longer biologic half-life and effects at lower
concentrations. Vessel dilator exhibited therapeutic effect for use
in human achondroplasia, short stature and osteoporosis by
stimulating osteoblast proliferation.
Inventors: |
Vesely; David Lynn; (Tampa,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of South Florida;
United States Department of Veterans Affairs; |
Tampa
Washington |
FL
DC |
US
US |
|
|
Assignee: |
UNITED STATES DEPARTMENT OF
VETERANS AFFAIRS
Washington
DC
UNIVERSITY OF SOUTH FLORIDA
Tampa
FL
|
Family ID: |
45067104 |
Appl. No.: |
13/693372 |
Filed: |
December 4, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/US2011/039277 |
Jun 6, 2011 |
|
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13693372 |
|
|
|
|
61351534 |
Jun 4, 2010 |
|
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Current U.S.
Class: |
514/12.4 |
Current CPC
Class: |
C07K 14/58 20130101;
A61P 19/08 20180101; A61K 38/00 20130101; A61P 19/10 20180101; A61K
38/22 20130101; A61K 38/2242 20130101 |
Class at
Publication: |
514/12.4 |
International
Class: |
A61K 38/22 20060101
A61K038/22 |
Claims
1. A method of treating skeletal disorders, comprising
administering a therapeutically effective amount of vessel dilator
to a patient in need thereof; wherein the skeletal disorder is
achondroplasia skeletal dysplasia, short stature, osetopenia,
osteoporosis, osteomalacia, hypoparathyroidism, tumor associated
osteomalacia, rickets, osteogenesis imperfecta, osteitis fibrosa
cystic secondary to hyperparathyroidism, Paget's Disease, or
osteitis deformans.
2. The method of claim 1, wherein the vessel dilator is
administered at a concentration of between 10 pM and 10 nM.
3. The method of claim 2, wherein the vessel dilator is
administered at 1 nM.
4. The method of claim 2, wherein the vessel dilator is
administered at 100 pM.
5. The method of claim 2, wherein the vessel dilator is
administered at 10 pM.
6. The method of claim 1, wherein the vessel dilator is
administered at a concentration of between 0.000258 ng/kg to 0.0028
pg/kg of body weight.
7. The method of claim 1, wherein the vessel dilator is
administered at a concentration of between 10 pM and 1 nM.
8. The method of claim 1, wherein the vessel dilator is
administered 4 times per day.
9. The method of claim 8, wherein the vessel dilator is
administered about every 6 hours.
10. The method of claim 9, wherein the vessel dilator is
administered every 6 hours.
11. A method of treating osteoporosis, comprising administering a
therapeutically effective amount of vessel dilator to a patient in
need thereof.
12. The method of claim 11, wherein the vessel dilator is
administered at a concentration of between 10 pM and 10 nM.
13. The method of claim 12, wherein the vessel dilator is
administered at 1 nM.
14. The method of claim 12, wherein the vessel dilator is
administered at 100 pM.
15. The method of claim 12, wherein the vessel dilator is
administered at 10 pM.
16. The method of claim 11, wherein the vessel dilator is
administered at a concentration of between 0.000258 ng/kg to 0.0028
pg/kg of body weight.
17. The method of claim 11, wherein the vessel dilator is
administered 4 times per day.
18. The method of claim 17, wherein the vessel dilator is
administered about every 6 hours.
19. The method of claim 18, wherein the vessel dilator is
administered every 6 hours.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of and claims priority to
International Patent Application No. PCT/US2011/039277, entitled
"Method of Treating Skeletal Dysplasias using Vessel Dilator",
filed on Jun. 6, 2011 which is a non-provisional of and claims
priority to U.S. Provisional Patent Application 61/351,534, with
the same title, filed Jun. 4, 2010; which is fully incorporated
herein by reference.
FIELD OF INVENTION
[0002] This invention relates to treatment of skeletal and
osteopathic disorders. Specifically, the invention provides for the
stimulation of bone growth using vessel dilator.
BACKGROUND OF INVENTION
[0003] One in 26,000 births results in achondroplasia, a common
cause of dwarfism caused by an autosomal dominant genetic disorder.
Bone formation and longitudinal bone growth in long bones, ribs and
vertebrae occurs via endochondral ossification in the cartilaginous
growth plate, which is located at both ends of the growth plate
(Karsenty G, Wagner E F 2002 Reaching a genetic and molecular
understanding of skeletal development. Dev Cell 2:389-406; Olsen B
R, et al. 2000 Bone development. Annu Rev Cell Dev Biol
16:191-220). One autocrine regulator of bone growth is
C-natriuretic peptide (CNP) (Hagiwara H, et al. 1994 Autocrine
regulation of rat chondrocyte proliferation by natriuretic peptide
C and its receptor, natriuretic peptide receptor-B. Biol Chem
269:10729-10733; Hagiwara H, et al. 1996 cGMP produced in response
to ANP and CNP regulates proliferation and differentiation of
osteoblastic cells. Am J Physiol 270:C1311-C1318; Suda M, et al.
1996 C-type natriuretic peptide as an autocrine/paracrine regulator
of osteoblasts. Biochem Biophys Res Commun 223:1-6; Yasoda A, et
al. 1998 Natriuretic peptide regulation of endochondral
ossification. Evidence for possible roles of the C-type natriuretic
peptide/guanylyl cyclase-B pathway. J Biol Chem 273:11695-11700;
Mericq V, et al. 2000 Regulation of fetal rat bone growth by C-type
natriuretic peptide and cGMP. Pediatr Res 47:189-193), a member of
the natriuretic peptide hormone family which circulates at a very
low level, suggesting that it has very little systemic activity on
bone (Kalra P R, et al. 2001 The role of C-natriuretic peptide in
cardiovascular medicine. Eur Heart J 22:997-1007; Daggubati Set al.
1997 Adrenomedullin, endothelin, neuropeptide Y, atrial, brain, and
C-natriuretic prohormone peptides compared as early heart failure
indicators. Cardiovasc Res 36:246-255).
[0004] Studies using primary cultures of osteoblast-like cells and
chondrocytes have revealed that natriuretic peptides with short
half-lives such as CNP and atrial natriuretic peptide (ANP) can
regulate proliferation and differentiation of osteoblasts and
chondrocytes (Hagiwara H, et al. 1994 Autocrine regulation of rat
chondrocyte proliferation by natriuretic peptide C and its
receptor, natriuretic peptide receptor-B. J Biol Chem
269:10729-10733; Hagiwara H, et al. 1996 cGMP produced in response
to ANP and CNP regulates proliferation and differentiation of
osteoblastic cells. Am J Physiol 270:C1311-C1318; Suda M, et al.
1996 C-type natriuretic peptide as an autocrine/paracrine regulator
of osteoblasts. Biochem Biophys Res Commun 223:1-6; Yasoda A, et
al. 1998 Natriuretic peptide regulation of endochondral
ossification. Evidence for possible roles of the C-type natriuretic
peptide/guanylyl cyclase-B pathway. J Biol Chem 273:11695-11700;
Mericq V, et al. 2000 Regulation of fetal rat bone growth by C-type
natriuretic peptide and cGMP. Pediatr Res 47:189-193). CNP
stimulates the intracellular messenger cyclic GMP (cGMP) 10-fold
more in chondrocytes than ANP (Hagiwara H, et al. 1994 Autocrine
regulation of rat chondrocyte proliferation by natriuretic peptide
C and its receptor, natriuretic peptide receptor-B. J Biol Chem
269:10729-10733). cGMP itself is important for bone development and
plays a role in regulating growth and differentiation of
osteoblasts (Hagiwara H, et al. 1996 cGMP produced in response to
ANP and CNP regulates proliferation and differentiation of
osteoblastic cells. Am J Physiol 270:C1311-C1318; Suda M, et al.
1996 C-type natriuretic peptide as an autocrine/paracrine regulator
of osteoblasts. Biochem Biophys Res Commun 223:1-6; Yasoda A, et
al. 1998 Natriuretic peptide regulation of endochondral
ossification. Evidence for possible roles of the C-type natriuretic
peptide/guanylyl cyclase-B pathway. J Biol Chem 273:11695-11700;
Mericq V, et al. 2000 Regulation of fetal rat bone growth by C-type
natriuretic peptide and cGMP. Pediatr Res 47:189-193, Pfeifer A, et
al. 1996 Intestinal secretory defects and dwarfism in mice lacking
cGMP-dependent protein kinase II. Science 274:2082-2086; Yasoda A,
et al. 2004 Overexpression of CNP in chondrocytes rescues
achondroplasia through a MAPK-dependent pathway. Nat Med
10:80-86).
[0005] Genetic deletion of CNP or its signaling results in severe
skeletal dysplasias caused by reduced chondrocyte proliferation and
differentiation (Chusho H, et al. 2001 Dwarfism and early death in
mice lacking C-type natriuretic peptide. Proc Natl Acad Sci USA
98:4016-4021; Yoder A R, et al. 2008 Reduced ability to C-type
natriuretic peptide (CNP) to activate natriuretic peptide receptor
B (NPR-B) causes dwarfism in 1bab -/- mice. Peptides 29:1575-1581).
In mice lacking CNP, dwarfism and early death occur (Chusho H, et
al. 2001 Dwarfism and early death in mice lacking C-type
natriuretic peptide. Proc Natl Acad Sci USA 98:4016-4021). At
birth, these mice have a 10% reduction in bone length, but the
growth retardation becomes more severe postnatally and 70% of the
mice die in the first 100 days after birth (Chusho H, et al. 2001
Dwarfism and early death in mice lacking C-type natriuretic
peptide. Proc Natl Acad Sci USA 98:4016-4021). Cartilage-specific
overexpression of CNP partially rescues the achondroplasia dwarfism
of the CNP-deficient mice, suggesting that CNP stimulates bone
growth through direct effects on chondrocytes (Yasoda A, et al.
2004 Overexpression of CNP in chondrocytes rescues achondroplasia
through a MAPK-dependent pathway. Nat Med 10:80-86). Contrarily,
mice with overexpression of CNP in cartilage have prominent
skeletal overgrowth (Yasoda A, et al. 2004 Overexpression of CNP in
chondrocytes rescues achondroplasia through a MAPK-dependent
pathway. Nat Med 10:80-86). Overexpression of CNP has also been
associated with overgrowth and bone abnormalities in a 14-year-old
girl (Bocciardi R, et al. 2007 Overexpression of the C-type
natriuretic peptide (CNP) is associated with overgrowth and bone
anomalies in an individual with balanced t(2;7) translocation. Hum
Mutat 28:724-731). Functional inactivation of the natriuretic
peptide (NPR)-B receptor that binds CNP (Tamura N, et al. 2004
Critical roles of the guanylyl cyclase B receptor in endochondral
ossification and development of female reproductive organs. Proc
Natl Acad Sci USA 101:17300-17305; Tsuji T, Kunieda T 2005 A
loss-of-function mutation in natriuretic peptide receptor 2 (Npr2)
gene is responsible for disproportionate dwarfism in cn/cn mouse. J
Biol Chem 280:14288-14292) or gene encoding for cGMP protein kinase
II through which cGMP effects are mediated also produces dwarfism
(Pfeifer A, et al. 1996 Intestinal secretory defects and dwarfism
in mice lacking cGMP-dependent protein kinase II. Science
274:2082-2086; Miyazawa T, et al. 2002 Cyclic GMP-dependent protein
kinase II plays a critical role in C-type natriuretic
peptide-mediated endochondral ossification. Endocrinology
143:3604-3610; Teixeira C C, et al. 2008 Nitric oxide, C-type
natriuretic peptide and cGMP as regulators of endochondral
ossification. Dev Biol 319:171-178).
[0006] CNP and ANP are ring-structured natriuretic peptides with
very short half-lives of <3 min in the circulation (Kalra P R,
et al. 2001 The role of C-natriuretic peptide in cardiovascular
medicine. Eur Heart J 22:997-1007, Teixeira C C, et al. 2008 Nitric
oxide, C-type natriuretic peptide and cGMP as regulators of
endochondral ossification. Dev Biol 319:171-178; Nakao K, et al.
1986 The pharmacokinetics of .alpha.-human natriuretic polypeptide
in healthy subjects. Eur J Clin Pharmacol 31:101-103; Yandle T G,
et al. 1986 Metabolic clearance rate and plasma half life of
alpha-human atrial natriuretic peptide in man. Life Sci
38:1827-1833). Their biologic effects last for <30 min. Vessel
dilator is a linear natriuretic peptide synthesized by the ANP gene
(Brenner B M, et al. 1990 Diverse biological action of atrial
natriuretic peptide. Physiol Rev 70:665-699; Vesely D L 2003
Natriuretic peptides and acute renal failure. Am J Physiol Renal
Physiol 285:F167-F177; Vesely D L 2007 Natruiretic hormones. In:
Alpern R J, Herbert S C (eds.) Seldin and Giebisch's The Kidney:
Physiology and Pathophysiology. 4th ed. Elsevier, Inc., Amsterdam,
The Netherlands, pp 947-977) that has a circulatory half-life of
107 min (Ackerman B H, et al. 1997 Disposition of vessel dilator
and long-acting natriuretic peptide in healthy humans after a
one-hour infusion. J Pharmacol Exp Ther 282:603-608) and its
biologic effects last >6 h (Vesely D L, et al. 1994 Three
peptides from the atrial natriuretic factor prohormone amino
terminus lower blood pressure and produce diuresis, natriuresis,
and/or kaliuresis in humans. Circulation 90:1129-1140).
[0007] The compositions currently used for treatment of skeletal
disorders have a short-lived in vivo residence. It would therefore
be beneficial to develop longer-lived compounds, facilitating fewer
treatments with improved effect.
SUMMARY OF THE INVENTION
[0008] Vessel dilator has biologic effects that last 12-times
longer than CNP, ANP or BNP as above which makes it unique and
preferable for therapy as with its longer half-life it can be given
less frequently for treatment. Because vessel dilator is a
natriuretic peptide hormones with similar cGMP mechanism of action
but much longer biologic effects than CNP or ANP (Kalra P R, et al.
2001 The role of C-natriuretic peptide in cardiovascular medicine.
Eur Heart J 22:997-1007; Teixeira C C, et al. 2008 Nitric oxide,
C-type natriuretic peptide and cGMP as regulators of endochondral
ossification. Dev Biol 319:171-178; Nakao K, et al. 1986 The
pharmacokinetics of a-human natriuretic polypeptide in healthy
subjects. Eur J Clin Pharmacol 31:101-103; Yandle T G, et al. 1986
Metabolic clearance rate and plasma half life of alpha-human atrial
natriuretic peptide in man. Life Sci 38:1827-1833; Vesely D L, et
al. 1994 Three peptides from the atrial natriuretic factor
prohormone amino terminus lower blood pressure and produce
diuresis, natriuresis, and/or kaliuresis in humans. Circulation
90:1129-1140), it was determined that a natriuretic peptide with at
least 12-fold longer biologic effects (Vesely D L, et al. 1994
Three peptides from the atrial natriuretic factor prohormone amino
terminus lower blood pressure and produce diuresis, natriuresis,
and/or kaliuresis in humans. Circulation 90:1129-1140) increased
osteoblasts' proliferation such as CNP. Vessel dilator and CNP were
compared directly against each other in dose response curves to
determine their comparative ability to enhance osteoblast
proliferation.
[0009] Surprisingly, it was discovered that the cardiac hormone
vessel dilator stimulated the proliferation of osteoblasts, which
results in the formation of new bone. Vessel dilator exhibited
biologic effects 12 times longer than CNP, ANP, or BNP. As such,
vessel dilator was used to treat skeletal disorders in patients.
Optionally, vessel dilator is administered at a concentration of
between 10 pM and 10 nM, including 1 nM, 100 pM, and 10 pM.
Appropriate concentrations of vessel dilator for administration may
be be calculated in pg and/or ng/kg body weight for infusion by
dividing the desired concentration in molarity by vessel dilator's
known molecular weight of 3878.31. For example, dividing 100 .mu.M
by the molecular weight provides an administration amount of 0.026
pg/kg.
[0010] Vessel dilator and C-natriuretic peptide (CNP) were compared
directly against each other in dose-response curves to determine
their comparative ability to enhance osteoblast proliferation, with
vessel dilator exhibiting better results than CNP. Vessel dilator
was found to stimulate osteoblasts at 1000-lower concentrations
than CNP, and possess biologic effects that last longer than 6
hours compared to less than 30 minutes for CNP, ANP and BNP. This
permits vessel dilator to be administered 4 times per day, such as
about every 6 hours or at every 6 hours.
[0011] The unique findings for cardiac hormone vessel dilator are
useful for the treatment of achondroplastic dwarfs and other
skeletal dysplasias. Examples of skeletal disorders that are
treatable with the present invention include achondroplasia
skeletal dysplasias and other dysplasias, short stature,
osetopenia, osteoporosis, osteomalacia, hypoparathyroidism, tumor
associated osteomalacia, rickets, osteogenesis imperfecta, osteitis
fibrosa cystic secondary to hyperparathyroidism, Paget's Disease,
and osteitis deformans, short stature, and osteoporosis. For
example, osteoporosis is a common disease in adults with current
treatments such as biphosphonates, parathyroid hormone, calcitonin
and 1,25-dihydroxy vitamin D all working via inhibiting
osteoclasts. Current treatment for osteoporosis inhibits the
activity of osteoclasts, preventing break-down of old bone.
Conversely, the invention stimulates osteoblasts to form new bone.
There is no information on this novel use of vessel dilator
stimulating osteoblasts. Stimulating osteoblasts to form new
healthy bone is a beneficial advance in the treatment of
osteoporosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] For a fuller understanding of the invention, reference
should be made to the following detailed description, taken in
connection with the accompanying drawings, in which:
[0013] FIG. 1 is a graph showing C-natriuretic peptide (CNP)
enhances human osteoblast proliferation at its 10 nM concentration
by 27% (p=0.02) when evaluated by the Mann-Whitley (Wilcoxon
rank-sum test). CNP did not significantly enhance human osteoblast
proliferation at its 1 nM, 100 pM, and 10 pM concentrations when
evaluated by Mann-Whitley test.
[0014] FIG. 2 is a graph showing vessel dilator enhanced the
proliferation of human osteoblasts over a concentration of range of
10 nM to 10 pM (p<0.01 or less) when evaluated by Mann-Whitley
test. The 100 pM and 10 pM concentrations in this graph are in the
circulating physiologic range of vessel dilator (Daggubati, et al.,
1997 Adrenomedullin, endothelin, neuropeptide Y, atrial, brain, and
C-natriuretic prohormone peptides compared as early heart failure
indicators. Cardiovascular Res. 36:246-255).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0015] Unless otherwise noted, the terms used herein are to be
understood according to conventional usage by those of ordinary
skill in the relevant art. In addition to the definitions of terms
provided below, definitions of common terms in molecular biology
may also be found in Rieger et al., 1991 Glossary of genetics:
classical and molecular, 5th Ed., Berlin: Springer-Verlag; and in
Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds.,
Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (1998
Supplement). It is to be understood that as used in the
specification and in the claims, "a" or "an" can mean one or more,
depending upon the context in which it is used. Thus, for example,
reference to "a cell" can mean that at least one cell can be
utilized.
[0016] As used herein, "about" means approximately or nearly and in
the context of a numerical value or range set forth means.+-.15% of
the numerical.
[0017] As used herein, "atrial natriuretic peptide" (ANP), also
known as atrial natriuretic factor (ANF), atrial natriuretic
hormone (ANH), or atriopeptin, is a vasodilator, and hormone
encoded by C-terminal residues 99-126 of the natriuretic peptide
precursor C gene (NPPC; GenBank Accession Number NM 024409.1). ANP
is a 28-amino acid peptide with a 17-amino acid ring in the middle
of the molecule. It is secreted by heart muscle cells to reduce
blood pressure by lowering water, sodium and adipose loads on the
circulatory system.
[0018] As used herein, "C-type natriuretic peptide" (CNP) is a 22
amino acid peptide having a 17-amino acid ring, as described by
Brevic (U.S. application Ser. No. 12/677,304, filed Sep. 9, 2008),
and is generated from the natriuretic peptide precursor C gene
(NPPC; GenBank Accession Number NM.sub.--024409.1).
[0019] As used herein, "extracellular signal-regulated kinase 1/2"
(ERK 1/2) are 44-kDa(ERK1) and 42-kDa (ERK2) serine-threonine
protein kinases that regulate cardiac hypertrophy and myocyte
survival, cell proliferation, and cell differentiation.
[0020] As used herein, "mitogen-activated protein kinase kinase"
(MAP kinase kinase 1/2, MEK1/2) are dual specificity kinases that
activate MAPKs (ERK-1/2) having a size of about 45 kDa (MEK 1) and
44 kDa (MEK 2). MEK 1/2 are highly specific, phosphorylating and
activating the 44 kDa and 42 kDa MAP kinases, and responsible for
promoting cell cycle progression. MEK 1/2 also play an important
role in modulating the survival of hematopoietic cells, and the
differentiation of certain cell types, such as neuronal cells,
maturation of thymocytes from CD4.sup.-CD8.sup.- to
CD4.sup.+CD8.sup.+ cells, and development of the visual cortex.
[0021] As used herein, "mitogen-activated protein kinase" (MAPK),
is a serine/threonine-specific protein kinase that responds to
extracellular stimuli to regulate various cellular activities, such
as gene expression, mitosis, differentiation, proliferation, and
cell survival/apoptosis.
[0022] The term "patient" is used herein to describe an animal,
preferably a human, to whom treatment, including prophylactic
treatment, with the compounds according to the present invention,
is provided. For treatment of the conditions or disease states
which are specific for a specific animal such as a human patient,
the term patient refers to that specific animal.
[0023] The term "effective amount" is used herein to describe
concentrations or amounts of compounds, such as vessel dilator,
that are effective for producing an intended result including
regulating growth and differentiation of osteoblasts, to address
skeletal disorders or other pathologic conditions including
achondroplasia skeletal dysplasias and other dysplasias, short
stature, osetopenia, osteoporosis, osteomalacia,
hypoparathyroidism, tumor associated osteomalacia, rickets,
osteogenesis imperfecta, osteitis fibrosa cystic secondary to
hyperparathyroidism, Paget's Disease, and osteitis deformans.
Compositions according to the present invention may be used to
effect proliferation and differentiation of osteoblastic cells to
produce a favorable change in the bone or skeletal tissue, or in
the disease or condition treated, whether that change is an
improvement such as stopping or reversing the degeneration of a
disease or condition, reducing a bone density deficit, or a
complete cure of the disease or condition treated.
[0024] The term "administration" or "administering" is used
throughout the specification to describe the process by which
compounds of the subject invention, such as vessel dilator, are
delivered to a patient for therapeutic purposes. Compounds of the
subject invention can be administered a number of ways including,
but not limited to, parenteral (such term referring to intravenous
and intra-arterial as well as other appropriate parenteral routes),
subcutaneous, intraperitoneal, intraventricular, among others which
term allows compounds of the subject invention to diffuse to the
ultimate target site where needed. The compounds can be
administered systemically or to a target anatomical site,
permitting the compounds to contact target cells, causing the
target cells to proliferate and/or differentiate in response to the
compounds (e.g., site-specific differentiation).
[0025] Administration will often depend upon the disease or
condition treated and may preferably be via a parenteral route, for
example, intravenously, or by direct administration into the
affected bone. For example, vessel dilator may be administered via
direct injection into the bone, or may be administered
systemically. In a preferred embodiment of the present invention,
the route of administration for treating an individual is systemic,
via intravenous, intra-arterial administration, subcutaneous, or
intraperitoneal administration.
[0026] The pharmaceutical compositions may further comprise a
pharmaceutically acceptable carrier. The compositions used in the
present methods can also be in a variety of forms. These include,
for example, solid, semi-solid, and liquid dosage forms, such as
tablets, pills, powders, liquid solutions or suspension,
suppositories, injectable and infusible solutions, and sprays. The
preferred form depends on the intended mode of administration and
therapeutic application. The compositions also preferably include
conventional pharmaceutically acceptable carriers and diluents
which are known to those skilled in the art. Examples of carriers
or diluents for use with the subject compounds include, but are not
limited to, water, saline, ethanol, dimethyl sulfoxide, gelatin,
cyclodextrans, magnesium stearate, dextrose, cellulose, sugars,
calcium carbonate, glycerol, alumina, starch, and equivalent
carriers and diluents, or mixtures of any of these. For example,
vessel dilator can be diluted to give a concentration in either
0.9% saline (ie normal saline) or D5W (dextrose 5% in water) for
infusion.
[0027] The present invention may be understood more readily by
reference to the following detailed description of the preferred
embodiments of the invention and the Examples included herein.
However, before the present compounds, compositions, and methods
are disclosed and described, it is to be understood that this
invention is not limited to specific nucleic acids, specific
polypeptides, specific cell types, specific host cells, specific
conditions, or specific methods, etc., as such may, of course,
vary, and the numerous modifications and variations therein will be
apparent to those skilled in the art. It is also to be understood
that the terminology used herein is for the purpose of describing
specific embodiments only and is not intended to be limiting.
[0028] In Vitro Testing of Vessel Dilator.
[0029] A cell line (ATCC number CRL-11372) of human osteoblast
cells was purchased from the American Type Culture Association
(ATCC, Manassas, Va.). Propagation of the human osteoblast cells
was in a 1:1 mixture of Ham's F12 Medium and Dulbecco's Modified
Eagles Medium (DMEM) with 2.5 mM L-glutamine without phenol red.
Base medium was supplemented with 0.3 mg/mL of Geneticin (G418)
antibiotic and 10% fetal bovine serum (Harris S A, et al. 1995
Developmental and characterization of a conditionally immortalized
human fetal osteoblastic cell line. J Bone Miner Res 10:178-186).
Cells were incubated at a temperature of 34.degree. C. in 5%
CO.sub.2 at which they have rapid cell division, doubling every 36
hours (Harris S A, et al. 1995 Developmental and characterization
of a conditionally immortalized human fetal osteoblastic cell line.
J Bone Miner Res 10:178-186). Immunostaining of these
post-confluent differentiated human osteoblasts showed that high
levels of osteopontin, osteonectin, bone sialoprotein and type 1
collagen were expressed (Harris S A, et al. 1995 Developmental and
characterization of a conditionally immortalized human fetal
osteoblastic cell line. J Bone Miner Res 10:178-186). Cells were
dispensed into new flasks with subculturing every 6-8 days. The
medium was changed every 3 days.
[0030] After the osteoblast cells were subcultured for 24 h,
.about.5000 cells in 200 .mu.L of the above media were then seeded
(day 1) into 96-well plates (Nuclon, Roskilde, Denmark). After
overnight incubation at 34.degree. C. in 5% CO.sub.2, the media was
removed (day 2), and 50 .mu.L of fresh media was added to control
wells, blank wells (with no cells inside), and 50 .mu.L of media
with 10 picomolar (pM), 100 pM, 1 nanomolar (nM), or 10 nM of CNP
or vessel dilator. At day 5, in these experiments, 50 .mu.L of
fresh media was added to the controls, blank wells, and 50 .mu.L of
media with 1 nM, 10 nM, 10 pM, and 100 pM of the respective
natriuretic hormones for a total volume of 100 .mu.L of media in
each well. At day 7, 20 .mu.L of Cell Titer 96.RTM. Aqueous One
Solution (Promega Corporation, Madison, Wis.) was added to each
well containing 100 .mu.L of medium and allowed to incubate for 4 h
in 5% CO.sub.2 atmosphere before recording absorbance at 490 nm
with a 96-well plate reader (Cory A H, et al. 1991 Use of aqueous
soluble tetrazolium/formazan assay for growth assays in culture.
Cancer Commun 3:207-212). There were 15 observations of vessel
dilator at each concentration and 16 observations of CNP at each
concentration. The peptide hormones used in this investigation were
from Phoenix Pharmaceuticals, Inc., Burlingame, Calif.
[0031] Cell Proliferation.
[0032] Cell proliferation of human osteoblasts was examined with
the Cell Titer 96 Aqueous One Solution cell proliferation assay
(Promega Corp.). This colorimetric method determines the viable
cells' proliferation by recording the absorption at 490 nm with a
96-well plate reader (Cory A H, et al. 1991 Use of aqueous soluble
tetrazolium/formazan assay for growth assays in culture. Cancer
Commun 3:207-212) after incubating the respective cells at
37.degree. C. for 4 h in a 5% CO.sub.2 atmosphere.
[0033] Approximately 5000 human osteoblast cells were in each well.
The proliferation assay detects the number of viable cells in
proliferation using a tetrazolium compound
(3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl-
]-2H-tetrazolium, inner salt; MTS) and an electron coupling reagent
[phenazine ethosulfate (PES)]. PES has enhanced chemical stability,
which allows it to be combined with MTS to form a stable solution
(Cory A H, et al. 1991 Use of aqueous soluble tetrazolium/formazan
assay for growth assays in culture. Cancer Commun 3:207-212). The
MTS tetrazolium compound (Owen's reagent) is bioreduced by living
cells into a colored formazan product that is measurable at 490 nM
in a spectrophotometer, thereby eliminating any nonviable (i.e.
dead) cells that would not be proliferating (Cory A H, et al. 1991
Use of aqueous soluble tetrazolium/formazan assay for growth assays
in culture. Cancer Commun 3:207-212). This method measure only
viable cells' proliferation as dead cells are unable to reduce the
MTS tetrazolium compound to a colored formazan product.
[0034] All data are expressed as mean.+-.SEM. Statistical
significance was determined by the Mann-Whitney test (also called
Wilcoxon rank-sum test) for different sample sizes. For the CNP
group, there were 16 data points for each concentration and eight
controls. For the vessel dilator group, there were 15 data points
for each concentration and 24 controls.
[0035] CNP at its 10 nM concentration enhanced human osteoblast
proliferation 27% (n=16) compared with controls, seen in FIG. 1
(n=8; p=0.02). There was no significant enhancement of osteoblast
proliferation at CNP concentrations of 1 nM, 100 pM, and 10 pM,
seen in FIG. 1. Thus, at 1 nM, there was a minus 1% enhancement,
and at 100 pM, there was a minus 16% enhancement of osteoblast
proliferation with CNP, seen in FIG. 1.
[0036] Vessel dilator at its 10 nM concentration (n=15) enhanced
the proliferation of human osteoblasts 8% compared with controls,
seen in FIG. 2 (n=24; p=0.0018). Decreasing the concentration of
vessel dilator 10-fold to 1 nM resulted in a 6% enhancement of the
proliferation of human osteoblasts (p<0.01). With a 100-fold
decrease in the concentration of vessel dilator to 100 pM, there
was still a 7% enhancement of the proliferation of human
osteoblasts, as seen in FIG. 2 (p=0.0073). Vessel dilator at 10 pM
stimulated human osteoblast proliferation 8% (p=0.01).
[0037] Comparing the effects of CNP and vessel dilator on human
osteoblast proliferation, as seen in FIG. 1 versus FIG. 2, revealed
that CNP-stimulated osteoblast proliferation to a greater extent at
its 10 nM concentration versus 10 nM concentration of vessel
dilator (p=0.048). However, at their respective 1 nM and 100 pM
concentrations vessel dilator caused a more significant (p<0.05)
enhancement of human osteoblast proliferation.
[0038] CNP is expressed in fetal bones and accelerates longitudinal
growth of fetal rat metatarsal bones in organ culture (Mericq V, et
al. 2000 Regulation of fetal rat bone growth by C-type natriuretic
peptide and cGMP. Pediatr Res 47:189-193). CNP in the present
investigation was found to stimulate human osteoblast proliferation
for the first time, extending previous findings that CNP can
enhance osteoblast proliferation in rat (Hagiwara H, et al. 1996
cGMP produced in response to ANP and CNP regulates proliferation
and differentiation of osteoblastic cells. Am J Physiol
270:C1311-C1318) and mouse (Suda M, et al. 1996 C-type natriuretic
peptide as an autocrine/paracrine regulator of osteoblasts. Biochem
Biophys Res Commun 223:1-6) osteoblasts. CNP dose-response studies
on human osteoblasts revealed that at 10 pM, which is CNP's
physiological circulating concentration (Daggubati S, et al. 1997
Adrenomedullin, endothelin, neuropeptide Y, atrial, brain, and
C-natriuretic prohormone peptides compared as early heart failure
indicators. Cardiovasc Res 36:246-255), CNP could not enhance human
osteoblast proliferation suggesting that CNP may not be a systemic
physiologic regulator of osteoblast function. This would confirm
previous studies of CNP on osteoblast function of mice osteoblasts
(Suda M, et al. 1996 C-type natriuretic peptide as an
autocrine/paracrine regulator of osteoblasts. Biochem Biophys Res
Commun 223:1-6), rat osteoblasts (Hagiwara H, e al. 1996 cGMP
produced in response to ANP and CNP regulates proliferation and
differentiation of osteoblastic cells. Am J Physiol
270:C1311-C1318), and rat chondrocytes (Hagiwara H, et al. S 1994
Autocrine regulation of rat chondrocyte proliferation by
natriuretic peptide C and its receptor, natriuretic peptide
receptor-B. J Biol Chem 269:10729-10733; Mericq V, et al. 2000
Regulation of fetal rat bone growth by C-type natriuretic peptide
and cGMP. Pediatr Res 47:189-193) where CNP did not have any
effects on osteoblasts in the pM range. However, the importance of
CNP in bone growth is illustrated by genetic deletion of CNP
resulting in skeletal dysplasia (Chusho H, et al. 2001 Dwarfism and
early death in mice lacking C-type natriuretic peptide. Proc Natl
Acad Sci USA 98:4016-4021; Yoder A R, Kruse A C, Earhart C A,
Ohlendorf D H, Potter L R 2008 Reduced ability to C-type
natriuretic peptide (CNP) to activate natriuretic peptide receptor
B (NPR-B) causes dwarfism in 1bab -/- mice. Peptides 29:1575-1581)
with mice lacking CNP having dwarfism (Chusho H, et al. 2001
Dwarfism and early death in mice lacking C-type natriuretic
peptide. Proc Natl Acad Sci USA 98:4016-4021). Further evidence of
CNP importance for bone growth is that mice overexpressing CNP in
cartilage have skeletal overgrowth (Yasoda A, et al. 2004
Overexpression of CNP in chondrocytes rescues achondroplasia
through a MAPK-dependent pathway. Nat Med 10:80-86), and a 14-y-old
girl with overexpression of CNP, with a doubling of CNP in plasma,
had bone overgrowth and who was >97 percentile in length at
birth and had arachnodactyly of hands and feet with a very long
hallux bilaterally at 14 years old (Bocciardi Re al. 2007
Overexpression of the C-type natriuretic peptide (CNP) is
associated with overgrowth and bone anomalies in an individual with
balanced t(2;7) translocation. Hum Mutat 28:724-731). These studies
would suggest that because CNP does not stimulate human, rat, or
mouse osteoblasts at its circulating physiologic concentrations,
its effects on bone are via an autocrine/paracrine process.
[0039] The gene for CNP is expressed in bone (Mericq V, et al. 2000
Regulation of fetal rat bone growth by C-type natriuretic peptide
and cGMP. Pediatr Res 47:189-193) to allow it to be an
autocrine/paracrine regulator of bone. This is the first
investigation demonstrating that vessel dilator, a linear
structured peptide hormone as opposed to a ring-structured CNP
(Brenner B M, et al. 1990 Diverse biological action of atrial
natriuretic peptide. Physiol Rev 70:665-699; Vesely D L 2003
Natriuretic peptides and acute renal failure. Am J Physiol Renal
Physiol 285:F167-F177; Vesely D L 2007 Natruiretic hormones. In:
Alpern R J, Herbert S C (eds) Seldin and Giebisch's The Kidney:
Physiology and Pathophysiology. 4th ed. Elsevier, Inc., Amsterdam,
The Netherlands, pp 947-977), can stimulate osteoblast
proliferation. That vessel dilator can enhance human osteoblast
proliferation is important because its circulating half-life is
36-fold longer than CNP, at 107 min for vessel dilator versus <3
min for CNP; (Kalra P R, et al. 2001 The role of C-natriuretic
peptide in cardiovascular medicine. Eur Heart J 22:997-1007;
Teixeira C C, et al. 2008 Nitric oxide, C-type natriuretic peptide
and cGMP as regulators of endochondral ossification. Dev Biol
319:171-178; Nakao K, et al. 1986 The pharmacokinetics of
.alpha.-human natriuretic polypeptide in healthy subjects. Eur J
Clin Pharmacol 31:101-103; Yandle T G, et al. 1986 Metabolic
clearance rate and plasma half life of alpha-human atrial
natriuretic peptide in man. Life Sci 38:1827-1833; Ackerman B H, et
al. 1997 Disposition of vessel dilator and long-acting natriuretic
peptide in healthy humans after a one-hour infusion. J Pharmacol
Exp Ther 282:603-608); and its biologic effects last for >6 h
compared with <30 min for ring-structured natriuretic peptides
such as CNP and ANP (Vesely D L, et al. 1994 Three peptides from
the atrial natriuretic factor prohormone amino terminus lower blood
pressure and produce diuresis, natriuresis, and/or kaliuresis in
humans. Circulation 90:1129-1140), which also has enhancing effects
in bone growth (Hagiwara H, et al. 1996 cGMP produced in response
to ANP and CNP regulates proliferation and differentiation of
osteoblastic cells. Am J Physiol 270:C1311-C1318). Vessel dilator,
but not CNP, was found to enhance human osteoblast proliferation at
its physiologic concentrations in the circulation (Vesely D L, e
al. 1994 Three peptides from the atrial natriuretic factor
prohormone amino terminus lower blood pressure and produce
diuresis, natriuresis, and/or kaliuresis in humans. Circulation
90:1129-1140), further suggesting that vessel dilator may be
important for physiologic regulation of bone growth by stimulating
osteoblasts. Increasing the concentration of vessel dilator above
the physiologic range to pharmacological concentrations did not
cause a further increase in its ability to enhance osteoblast
proliferation. This information would suggest that bone proteases
may be proteolytically degrading this peptide hormone at its higher
concentrations. With more vessel dilator present in bone, the bone
proteases may become more active in a negative feedback manner,
cleaving this peptide hormone resulting in loss of any enhanced
biologic activity beyond that observed with physiologic
concentrations of vessel dilator.
[0040] With respect to the mechanisms of vessel dilator and CNP's
enhancement of osteoblast proliferation, cGMP would seem to be an
important mediator of their effects because CNP can increase this
intracellular mediator in chondrocytes (Hagiwara H, et al. 1994
Autocrine regulation of rat chondrocyte proliferation by
natriuretic peptide C and its receptor, natriuretic peptide
receptor-B. J Biol Chem 269:10729-10733) and the majority of vessel
dilator's effects are mediated via cGMP (Brenner B M, et al. 1990
Diverse biological action of atrial natriuretic peptide. Physiol
Rev 70:665-699; Vesely D L 2003 Natriuretic peptides and acute
renal failure. Am J Physiol Renal Physiol 285:F167-F177; Vesely D L
2007 Natruiretic hormones. In: Alpern R J, Herbert S C (eds) Seldin
and Giebisch's The Kidney: Physiology and Pathophysiology. 4th ed.
Elsevier, Inc., Amsterdam, The Netherlands, pp 947-977; Sun Y, et
al. 2007 Vessel dilator and kaliuretic peptide inhibit MEK 1/2
activation in human prostate cancer cells. Anticancer Res
27:1387-1392). cGMP itself is important for bone development, which
have been shown to regulate proliferation and differentiation of
osteoblasts and chondrocytes (Hagiwara H, et al. 1996 cGMP produced
in response to ANP and CNP regulates proliferation and
differentiation of osteoblastic cells. Am J Physiol
270:C1311-C1318; Suda M, et al. 1996 C-type natriuretic peptide as
an autocrine/paracrine regulator of osteoblasts. Biochem Biophys
Res Commun 223:1-6; Yasoda A, et al. 1998 Natriuretic peptide
regulation of endochondral ossification. Evidence for possible
roles of the C-type natriuretic peptide/guanylyl cyclase-B pathway.
J Biol Chem 273:11695-11700; Mericq V, et al. 2000 Regulation of
fetal rat bone growth by C-type natriuretic peptide and cGMP.
Pediatr Res 47:189-193; Pfeifer A, et al. 1996 Intestinal secretory
defects and dwarfism in mice lacking cGMP-dependent protein kinase
II. Science 274:2082-2086; Yasoda A, et al. 2004 Overexpression of
CNP in chondrocytes rescues achondroplasia through a MAPK-dependent
pathway. Nat Med 10:80-86).
[0041] Inactivation of the gene encoding for cGMP protein kinase
II, through which cGMP effects are mediated in bone, also produces
achondroplastic dwarfism (Pfeifer A, et al. 1996 Intestinal
secretory defects and dwarfism in mice lacking cGMP-dependent
protein kinase II. Science 274:2082-2086; Miyazawa T, et al. 2002
Cyclic GMP-dependent protein kinase II plays a critical role in
C-type natriuretic peptide-mediated endochondral ossification.
Endocrinology 143:3604-3610; Teixeira C C, et al. 2008 Nitric
oxide, C-type natriuretic peptide and cGMP as regulators of
endochondral ossification. Dev Biol 319:171-178). Overexpression of
CNP in chondrocytes rescues achondroplasia through inhibition of
MEK 1 kinase in the mitogen-activated protein kinase (MAPK) pathway
(Yasoda A, et al. 2004 Overexpression of CNP in chondrocytes
rescues achondroplasia through a MAPK-dependent pathway. Nat Med
10:80-86). Constitutive activation of MEK 1 kinase in chondrocytes
causes achondroplasia-like dwarfism in mice (Murakami S, et al.
2004 Constitutive activation of MEK1 in chondrocytes causes
Stat1-independent achondroplasia-like dwarfism and rescues the
Fgfr3-deficient mouse phenotype. Genes Dev 18:290-305). Vessel
dilator inhibits the activation, i.e. phosphorylation of MEK 1/2
kinases by 98% in proliferating prostate cancer cells (Sun Y, et
al. 2007 Vessel dilator and kaliuretic peptide inhibit MEK 1/2
activation in human prostate cancer cells. Anticancer Res
27:1387-1392). Vessel dilator appears to inhibit MEK 1/2 kinases in
proliferating cells through cGMP. For example, contacting cells
with a cGMP antibody blocks vessel dilator effects on MEK 1/2
kinases. Further, cGMP itself can inhibit MEK 1/2 kinases in
proliferating cells (Sun Y, et al. 2007 Vessel dilator and
kaliuretic peptide inhibit MEK 1/2 activation in human prostate
cancer cells. Anticancer Res 27:1387-1392). CNP and 8-bromo cGMP
also inhibit mitogen-(fibroblast growth factor) stimulated ERK 1/2
kinases' phosphorylation in ATDC5 cells, a mouse chondrogenic cell
line (Ozasa A, et al. 2005 Complementary antagonistic actions
between C-type natriuretic peptide and the MAPK pathway through
FGFR-3 in ATDC5 cells. Bone 36:1056-1064). Vessel dilator inhibits
96% of the phosphorylation of basal activity of ERK 1/2 kinases in
proliferating cells (Sun Y, et al. 2006 Vessel dilator and
kaliuretic peptide inhibit activation of ERK 1/2 in human prostate
cancer cells. Anticancer Res 26:3217-3222) and completely blocks
mitogen; epidermal growth factor, (EGF); and stimulation of ERK 1/2
kinases (Sun Y, et al. 2007 Insulin and epidermal growth factor
activation of ERK 1/2 and DNA synthesis is inhibited by four
cardiac hormones. J. Cancer Mol 3:113-120). Thus, both vessel
dilator and CNP seem to have identical molecular mechanisms of
action of stimulating osteoblasts and bone growth via inhibiting
MAP kinases MEK 1/2 and ERK 1/2, mediated at least in part by cGMP
(Yasoda A, et al. 2004 Overexpression of CNP in chondrocytes
rescues achondroplasia through a MAPK-dependent pathway. Nat Med
10:80-86, 29-32; Murakami, et al., 2004 Constitutive activation of
MEK1 in chondrocytes causes Stat1-independent achondroplasia-like
dwarfism and rescues the Fgfr3-deficient mouse phenotype. Gene
& Dev 18:290-305; Ozasa, et al., 2005 Complementary
antagonistic actions between C-type natriuretic peptide and the
MAPK pathway through FGFR-3 in ATDC5 cells. Bone 36:1056-1064; Sun,
et al., 2006 Vessel dilator and kaliuretic peptide inhibit
activation of ERK 1/2 in human prostate cancer cells. Anticancer
Res. 26:3217-3222; Sun, et al., 2007 Insulin and epidermal growth
factor activation of ERK 1/2 and DNA synthesis is inhibited by four
cardiac hormones. J. Cancer Mol. 3:113-120).
[0042] With respect to potential treatment of bone diseases, CNP
has been suggested to be a new treatment strategy for
achondroplasia (Ozasa A, et al. 2005 Complementary antagonistic
actions between C-type natriuretic peptide and the MAPK pathway
through FGFR-3 in ATDC5 cells. Bone 36:1056-1064). Vessel dilator,
with its 36-fold longer half-life and significantly longer biologic
effects than CNP, i.e. >12 times longer (Vesely D L, et al. 1994
Three peptides from the atrial natriuretic factor prohormone amino
terminus lower blood pressure and produce diuresis, natriuresis,
and/or kaliuresis in humans. Circulation 90:1129-1140), would seem
to be a better choice for treatment of bone disease such as
dwarfism because it can be given less frequently with similar
therapeutic results. Furthermore, vessel dilator stimulates
osteoblastic proliferation over a concentration range of 10 nM
through 10 pM, whereas CNP at concentration <10 nM did not
significantly enhance human osteoblast proliferation. CNP's
half-life is very short, at about 3 min, in vivo whereas vessel
dilator's half-life of >6 h (Vesely D L, et al. 1994 Three
peptides from the atrial natriuretic factor prohormone amino
terminus lower blood pressure and produce diuresis, natriuresis,
and/or kaliuresis in humans. Circulation 90:1129-1140) would
suggest it could be given four times per day to affect bone growth.
As vessel dilator can be given on a reasonable schedule of four
times per day, it may have a role in the treatment of short stature
in children by enhancing their osteoblast proliferation, indicating
that vessel dilator can be utilized in lower concentrations to
obtain the same effects as CNP on bone.
[0043] In addition to growth disorders in children, CNP and vessel
dilator may have a therapeutic role in treating a common bone
disease in adults, i.e. osteoporosis. Current therapeutic agents
for osteoporosis concentrate on inhibiting osteoclasts (Rubin J E,
Rubin C T 2009 Biology, physiology, and morphology of bone. In:
Firestein G S, et al. (eds) Kelly's Textbook of Rheumatology. 8th
ed. Elsevier, Philadelphia, Pa., pp 71-91). Bisphosphonates such as
alendronate, parathyroid hormone (PTH), calcitonin, and
1,25-dihydroxy vitamin D, all work via inhibiting osteoclasts
(Rubin J E, Rubin C T 2009 Biology, physiology, and morphology of
bone. In: Firestein G S, et al. (eds) Kelly's Textbook of
Rheumatology. 8th ed. Elsevier, Philadelphia, Pa., pp 71-91). Sex
steroids such as estrogens and testosterone do stimulate
osteoblasts (Rubin J E, Rubin C T 2009 Biology, physiology, and
morphology of bone. In: Firestein G S, et al. (eds) Kelly's
Textbook of Rheumatology. 8th ed. Elsevier, Philadelphia, Pa., pp
71-91) but are usually given only in cases of documented low
testosterone and/or estrogens because of their side effects.
Estrogens, for example, are not currently given for osteoporosis
even when the person is post-menopausal with low estrogen levels by
some physicians because of their potential cardiovascular risk
(Rubin J E, Rubin C T 2009 Biology, physiology, and morphology of
bone. In: Firestein G S, et al. (eds) Kelly's Textbook of
Rheumatology. 8th ed. Elsevier, Philadelphia, Pa., pp 71-91).
Sodium fluoride stimulates osteoblasts and has been used for
vertebral fractures but even though bone mass increased secondary
to sodium fluoride, it does not decrease the incidence of
fractures. An agent that stimulates osteoblasts without the side
effects of sodium fluoride or sex steroids and that will cause bone
formation via osteoblasts rather than inhibiting old bone in place
(via osteoclasts) has been sought for decades. As seen herein,
vessel dilator was demonstrated to stimulate human osteoblasts,
suggesting that it may provide a new therapeutic option for bone
disease. Vessel dilator would be a preferred option over CNP
because of its much longer biologic activity for >6 h compared
with <30 min for CNP (Vesely D L, et al. 1994 Three peptides
from the atrial natriuretic factor prohormone amino terminus lower
blood pressure and produce diuresis, natriuresis, and/or kaliuresis
in humans. Circulation 90:1129-1140), and that treatment every 30
min with CNP would be very impractical.
[0044] In the preceding specification, all documents, acts, or
information disclosed does not constitute an admission that the
document, act, or information of any combination thereof was
publicly available, known to the public, part of the general
knowledge in the art, or was known to be relevant to solve any
problem at the time of priority.
[0045] The disclosures of all publications cited above are
expressly incorporated herein by reference, each in its entirety,
to the same extent as if each were incorporated by reference
individually.
[0046] While there has been described and illustrated specific
embodiments of a method of treatment of skeletal and osteopathic
disorders using vessel dilator, it will be apparent to those
skilled in the art that variations and modifications are possible
without deviating from the broad spirit and principle of the
present invention. It is also to be understood that the following
claims are intended to cover all of the generic and specific
features of the invention herein described, and all statements of
the scope of the invention which, as a matter of language, might be
said to fall therebetween.
* * * * *