U.S. patent application number 15/311689 was filed with the patent office on 2017-04-20 for compositions and methods for treating and preventing pancreatitis, renal injury and cancer.
The applicant listed for this patent is Yale University. Invention is credited to Gary Desir.
Application Number | 20170107499 15/311689 |
Document ID | / |
Family ID | 54480934 |
Filed Date | 2017-04-20 |
United States Patent
Application |
20170107499 |
Kind Code |
A1 |
Desir; Gary |
April 20, 2017 |
Compositions and Methods for Treating and Preventing Pancreatitis,
Renal Injury and Cancer
Abstract
The present invention includes compositions and methods for
detecting, treating and preventing renal and pancreatic diseases
and disorders.
Inventors: |
Desir; Gary; (Woodbridge,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yale University |
New Haven |
CT |
US |
|
|
Family ID: |
54480934 |
Appl. No.: |
15/311689 |
Filed: |
May 15, 2015 |
PCT Filed: |
May 15, 2015 |
PCT NO: |
PCT/US15/30980 |
371 Date: |
November 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61994279 |
May 16, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/18 20180101; A61P
13/12 20180101; A61P 37/04 20180101; C12N 9/0036 20130101; C12N
2310/14 20130101; C12Q 1/26 20130101; G01N 2333/90209 20130101;
A61P 35/04 20180101; G01N 2800/06 20130101; A61P 35/00 20180101;
C12N 15/1137 20130101; C12Y 306/03008 20130101; A61P 9/12 20180101;
A61P 35/02 20180101; A61K 38/00 20130101; C12Y 106/03 20130101 |
International
Class: |
C12N 9/02 20060101
C12N009/02; C12Q 1/26 20060101 C12Q001/26 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grants
RC1DK086465, RC1DK086402 and R01DK081037, awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of treating or preventing a renal disease or disorder
in a subject in need thereof, the method comprising administering
to the subject a therapeutically effective amount of a composition
comprising at least one PMCA4b activator.
2. (canceled)
3. The method of claim 1, wherein the PMCA4b activator is a
renalase polypeptide.
4. The method of claim 3, wherein the renalase polypeptide
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO: 8 and SEQ ID NO: 9.
5. (canceled)
6. The method of claim 1, wherein the PMCA4b activator is a
renalase polypeptide fragment.
7. The method of claim 6, wherein the renalase polypeptide fragment
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.
8. (canceled)
9. (canceled)
10. The method of claim 1, wherein the at least one PMCA4b
activator is administered one time or repeatedly.
11. (canceled)
12. The method of claim 1, wherein the at least one PMCA4b
activator is administered locally, regionally or systemically.
13. The method of claim 1, wherein the PMCA4b activator is an
activator of PMCA4b expression, an activator of PMCA4b activity, or
a combination thereof.
14. The method of claim 1, wherein the renal disease or disorder is
selected from the group consisting of acute kidney injury (AKI),
chronic kidney disease (CKD), renal ischemic injury, renal
reperfusion injury, renal ischemic-reperfusion injury, toxic renal
injury, renal tubular necrosis, renal tubular inflammation, renal
tubular apoptosis, hypertension, and any combination thereof.
15. (canceled)
16. A method of treating or preventing cancer in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of a composition comprising at
least one PMCA4b inhibitor.
17. The method of claim 16, wherein the PMCA4b inhibitor is at
least one selected from the group consisting of a chemical
compound, a protein, a peptide, a peptidomemetic, an antibody, a
ribozyme, a small molecule chemical compound, and an antisense
nucleic acid molecule.
18. The method of claim 16, wherein the PMCA4b inhibitor is one
selected from the group consisting of caloxin 1b and cisplatin.
19. (canceled)
20. The method of claim 16, wherein the cancer is selected from the
group consisting of brain cancer, bladder cancer, breast cancer,
cervical cancer, colorectal cancer, liver cancer, kidney cancer,
lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma,
mesothelioma, neuroblastoma, ovarian cancer, prostate cancer,
pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma,
non-Hodgkin's lymphoma, Hodgkin's lymphoma, uterine cancer, and any
combination thereof.
21. (canceled)
22. A method of treating or preventing a pancreatic disease or
disorder in a subject in need thereof, the method comprising
administering to the subject a therapeutically effective amount of
a composition comprising at least one agent, wherein the at least
one agent is at least one selected from the group consisting of a
renalase polypeptide, a renalase polypeptide fragment, and an
activator of renalase.
23. The method of claim 22, wherein the renalase polypeptide is a
recombinant renalase polypeptide.
24. The method of claim 22, wherein the renalase polypeptide
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO: 8 and SEQ ID NO: 9.
25. (canceled)
26. The method of claim 22, wherein the renalase polypeptide
fragment comprises an amino acid sequence selected from the group
consisting of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. The method of claim 22, wherein the activator of renalase is an
activator of renalase expression, an activator of renalase
activity, or a combination thereof.
33. (canceled)
34. The method of claim 22, wherein the pancreatic disease or
disorder is at least one selected from the group consisting of
acute pancreatitis and chronic pancreatitis.
35. A method of diagnosing a pancreatic disease or disorder in a
subject in need thereof, the method comprising: a. determining the
level of renalase in a biological sample of the subject, b.
comparing the level of renalase in the biological sample of the
subject with a comparator control, and c. diagnosing the subject
with a pancreatic disease or disorder when the level of renalase in
the biological sample of subject is reduced when compared with the
level of renalase of the comparator control.
36. The method of claim 35, comprising the further step of
administering a treatment to the subject that was diagnosed as
having a pancreatic disease or disorder.
37. The method of claim 35, wherein the level of renalase in the
biological sample is determined by measuring at least one of the
group consisting of the level of renalase mRNA in the biological
sample, the level of renalase polypeptide in the biological sample,
and an enzymatic activity of renalase polypeptide in the biological
sample.
38. (canceled)
39. (canceled)
40. The method of claim 35, wherein the comparator control is at
least one selected from the group consisting of: a positive
control, a negative control, a historical control, a historical
norm, or the level of a reference molecule in the biological
sample.
41. The method of claim 35, wherein the pancreatic disease or
disorder is at least one selected from the group consisting of
acute pancreatitis and chronic pancreatitis.
42. (canceled)
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Application No. 61/994,279, filed May 16, 2014, which is hereby
incorporated by reference in its entirety herein.
BACKGROUND OF THE INVENTION
[0003] Renalase (also designated RNLS and gene C10orf59) is a novel
secretory flavoprotein oxidase (Farzaneh-Far et al., 2010, PLoS One
5:e13496; Desir et al., 2012, J. Am. Heart Assoc. 1:e002634; Desir
et al., 2012, 6:417-426; J. Am. Soc. Hypertension; Xu et al., 2005,
J. Clin. Invest. 115:1275-1280; Li et al., 2008, Circulation
117:1277-1282). Single nucleotide polymorphisms present in the gene
are associated with hypertension, cardiac disease and diabetes
(Farzaneh-Far et al., 2010, PLoS One 5:e13496; Barrett et al.,
2009, Nat. Genet. 41:703-707; Buraczynska et al., 2011,
Neuromolecular Med. 13:321-327; Malyszko et al., 2012, Ren. Fail.
34:727-731; Zhao et al., 2007, J. Mol. Med. (Berl). 85:877-885).
Renalase's crystal structure has been solved (Milani et al., 2011,
J. Mol. Biol. 411:463-473), and the protein binds epinephrine (Xu
et al., 2005, J. Clin. Invest. 115:1275-1280) and functions as an
oxidase/anomerase, using molecular oxygen to convert
.alpha.-NAD(P)H to .beta.-NAD+, with hydrogen peroxide as reaction
byproduct (Beaupre et al., 2013, J. Am. Chem. Soc.
135:13980-13987).
[0004] The administration of renalase in wild type (WT) mice lowers
plasma catecholamines and systemic blood pressure. Renalase
deletion in mice (renalase KO) raises catecholamine levels and
blood pressure (Wu et al., 2011, Kidney Int. 79:853-860). Gene
deletion also aggravates acute ischemic kidney (AKI) (Lee et al.,
2013, J. Am. Soc. Nephrol. 24:445-455), and cardiac injury (Wu et
al., 2011, Kidney Int. 79:853-860). Recombinant renalase prevents
ischemic injury in wild type mice (Lee et al., 2013, J. Am. Soc.
Nephrol. 24:445-455). Two single nucleotide polymorphisms (SNPs) in
the renalase gene (rs2576178 GG genotype and rs2296545 CC) are
associated with essential hypertension (Zhao et al., 2007, J. Mol.
Med. (Berl.) 85:877-885). Moreover, rs2296545 CC results in a
conservative amino acid change (glutamic to aspartic acid at amino
acid 37), and is associated with cardiac hypertrophy, ventricular
dysfunction, poor exercise capacity, and inducible ischemia in
persons with stable coronary artery disease (Farzaneh-Far et al.,
2010, PLoS One 5:e13496). A significant reduction in cardiac
renalase levels was observed in a rat model of chronic kidney
disease (5/6 Nx) (Li et al., 2008, Circulation 117:1277-1282).
Administration of recombinant renalase decreases blood pressure in
rodents (Xu et al., 2005, J. Clin. Invest 115:1275-1280; Milani et
al., 2011, J. Mol. Biol. 411:463-473), and protects mice against
ischemic AKI (Lee et al., 2013, J. Am. Soc. Nephrol. 24:445-455).
Renalase also acts as an oxidase/anomerase to convert
.alpha.-NAD(P)H to .beta.-NAD+, with hydrogen peroxide as reaction
byproduct. Independent of its intrinsic enzymatic activities,
extracellular renalase activates MAPK signaling and prevents acute
kidney injury (AKI) in wild type (WT) mice (Wang et al., 2014, J.
Am. Soc. Nephrol. DOI:10.1681/asn.2013060665).
[0005] Renalase has cytoprotective effects that are initiated in
renal injury via a pattern of MAPK signaling, and that this
signaling is mediated by an interaction of renalase with a plasma
membrane Ca ATPase. Renal injury can lead to chronic kidney disease
(CKD), which is associated with increased morbidity and mortality,
is largely due to cardiovascular complications. Approximately 30%
of veterans suffer from chronic kidney disease (CKD), which is
associated with increased morbidity and mortality, largely due to
cardiovascular complications. Acute kidney injury (AKI) is a
clinical condition commonly associated with sepsis, surgery, and
certain drugs, and which affects up to 20% of hospitalized
veterans. Epidemiologic data indicate that severity of AKI is
associated with in-hospital and long-term mortality. Increased
sympathetic tone has been shown to be pathogenic in CKD and
AKI.
[0006] Although renalase's crystal structure has been solved, its
mechanism of action remains uncertain. It has been recently
reported that renalase promotes cell and organ survival through a
receptor-mediated process that is independent of its intrinsic
enzymatic activities (Wang L, Velazquez H, Moeckel G et al.
Renalase Prevents AKI Independent of Amine Oxidase Activity. J Am
Soc Nephrol 2014). Renalase was also discovered to activate B-cell
lymphoma 2 (Bcl-2), and inhibition of c-Jun N-terminal kinase
(JNK).
[0007] Acute pancreatitis is a potentially life-threatening disease
that affects more than 250,000 people each year in the United
States, making it the leading cause of hospitalization for
gastrointestinal disorders (Wu and Banks, 2013, Gastroenterology
144, 1272-1281; Whitcomb, 2006, N Engl J Med 354, 2142-2150).
Because the pathogenesis of acute pancreatitis is incompletely
understood, the mainstay of treatment continues to be supportive
care. As a result, there remains a 10-30% mortality rate among
patients with severe acute pancreatitis (Petrov et al., 2010,
Gastroenterology 139, 813-820; McKay and Imrie, 2004, The British
journal of surgery 91, 1243-1244).
[0008] This acute inflammatory disease of the pancreas is believed
to most often begin by injury to the pancreatic acinar cell. Though
abnormalities in acinar cell signaling mediated by protein kinase C
isoforms, AMPK, and NF.kappa.B have been found early in disease,
most studies suggest that abnormalities in cytosolic Ca.sup.2+
signaling may be the most important for disease initiation. A
series of sequential events involving vascular damage with enhanced
permeability, inflammation, and reduced blood flow then follow and
culminate in pancreatic cell death. In individuals with severe
disease, there is multi-organ damage that typically involves the
lungs and kidneys. That pancreatitis represents a series of
distinct temporal events means that interventions aimed at
preventing disease or decreasing its severity should be appropriate
disease-stage specific mechanisms to be effective.
[0009] Acute pancreatitis has a highly variable clinical course,
with possible outcomes ranging from complete recovery to death.
Therefore, predicting which patients will develop severe disease is
essential to appropriately triage patients (Mounzer et al., 2012,
Gastroenterology 142, 1476-1482). At initial patient presentation,
measures of kidney function, including blood urea nitrogen (BUN)
and creatinine, are among the most reliable predictors of a severe
disease course and mortality (Wu et al., 2011, Archives of internal
medicine 171, 669-676; Muddana et al., 2009, The American journal
of gastroenterology 104, 164-170). Thus, AKI in the setting of
acute pancreatitis is associated with a 10-fold increase in
mortality (Kes et al., 1996, Ren Fail 18, 621-628; Tran et al.,
1993, Nephrol Dial Transplant 8, 1079-1084).
[0010] The presence of pre-existing CKD and end stage renal disease
(ESRD) also predispose to acute pancreatitis and worsen its
severity. Patients with CKD and ESRD are up to 50-times more likely
to develop acute pancreatitis than matched controls (Lankisch et
al., 2008, Nephrol Dial Transplant 23, 1401-1405; Rutsky et al.,
1986, Archives of internal medicine 146, 1741-1745; Quraishi et
al., 2005, The American journal of gastroenterology 100,
2288-2293). Additionally, CKD patients with acute pancreatitis have
a higher likelihood of serious complications and death (Pitchumoni
et al., 1996, The American journal of gastroenterology 91,
2477-2482; Golay and Roychowdhary, 2012, Ren Fail 34, 1338-1340).
The etiology of acute pancreatitis in patients with CKD is unknown
in the majority of cases, suggesting that CKD might sensitize to
the development of acute pancreatitis (Pitchumoni et al., 1996, The
American journal of gastroenterology 91, 2477-2482). One study
demonstrated that acute renal failure in rats causes a distinct
pattern of pancreatic injury (Lerch et al., 1994, Gut 35, 401-407),
but whether it sensitizes to the development or severity of
experimental pancreatitis is unknown. An understanding of the
mechanisms responsible for the sensitizing effects of AKI and CKD
on acute pancreatitis could lead to targeted therapies.
[0011] There is thus a need in the art for compositions and methods
for the treatment and prevention of renal injury, such as AKI and
CKD, and pancreatitis. The present invention addresses this unmet
need in the art.
SUMMARY OF THE INVENTION
[0012] In one embodiment, the invention is a method of treating or
preventing a renal disease or disorder in a subject in need
thereof, by administering to the subject a therapeutically
effective amount of a composition comprising at least one PMCA4b
activator. In various embodiments, the PMCA4b activator is a
chemical compound, a protein, a peptide, a peptidomemetic, an
antibody, a small molecule chemical compound, or a combination
thereof. In one embodiment, the PMCA4b activator is a renalase
polypeptide, or a fragment or conjugate or analogue or homolog
thereof. In one embodiment, the renalase polypeptide comprises the
amino acid sequence of SEQ ID NO: 8, or a fragment or conjugate or
analogue or homolog thereof. In another embodiment, the renalase
polypeptide comprises the amino acid sequence of SEQ ID
[0013] NO: 9, or a fragment or conjugate or analogue or homolog
thereof. In another embodiment, the PMCA4b activator is a renalase
polypeptide fragment. In one embodiment, the renalase polypeptide
fragment comprises the amino acid sequence of SEQ ID NO: 3, or a
fragment or conjugate or analogue or homolog thereof. In another
embodiment, the renalase polypeptide fragment comprises the amino
acid sequence of SEQ ID NO: 4, or a fragment or conjugate or
analogue or homolog thereof. In one embodiment, the renalase
polypeptide fragment comprises the amino acid sequence of SEQ ID
NO: 5, or a fragment or conjugate or analogue or homolog thereof.
In some embodiments, the at least one PMCA4b activator is
administered one time. In some embodiments, the at least one PMCA4b
activator is administered repeatedly. In some embodiments, the at
least one PMCA4b activator is administered locally, regionally or
systemically. In various embodiments, the PMCA4b activator is an
activator of PMCA4b expression, an activator of PMCA4b activity, or
a combination thereof. In various embodiments, the renal disease or
disorder that is treated or prevented is selected from the group
consisting of acute kidney injury (AKI), chronic kidney disease
(CKD), renal ischemic injury, renal reperfusion injury, renal
ischemic-reperfusion injury, toxic renal injury, renal tubular
necrosis, renal tubular inflammation, renal tubular apoptosis,
hypertension, and any combination thereof. In one embodiment, the
subject is human.
[0014] In another embodiment, the invention is a method of treating
or preventing cancer in a subject in need thereof, by administering
to the subject a therapeutically effective amount of a composition
comprising at least one PMCA4b inhibitor. In various embodiments,
the PMCA4b inhibitor is a chemical compound, a protein, a peptide,
a peptidomemetic, an antibody, a ribozyme, a small molecule
chemical compound, an antisense nucleic acid molecule, or any
combination thereof. In one embodiment, the PMCA4b inhibitor is
caloxin 1b, or analogue or homolog thereof. In another embodiment,
the PMCA4b inhibitor is cisplatin, or analogue or homolog thereof.
In various embodiments, the cancer that is treated or prevented is
selected from the group consisting of brain cancer, bladder cancer,
breast cancer, cervical cancer, colorectal cancer, liver cancer,
kidney cancer, lymphoma, leukemia, lung cancer, melanoma,
metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer,
prostate cancer, pancreatic cancer, renal cancer, skin cancer,
thymoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma,
uterine cancer, or any combination thereof. In one embodiment, the
subject is human.
[0015] In one embodiment, the invention is a method of treating or
preventing a pancreatic disease or disorder in a subject in need
thereof, by administering to the subject a therapeutically
effective amount of a composition comprising at least one agent,
wherein the at least one agent is at least one selected from the
group consisting of a renalase polypeptide, a renalase polypeptide
fragment, and an activator of renalase, or a fragment or conjugate
or analogue or homolog thereof. In one embodiment, the renalase
polypeptide is a recombinant renalase polypeptide, or a fragment or
conjugate or analogue or homolog thereof. In another embodiment,
the renalase polypeptide comprises the amino acid sequence of SEQ
ID NO: 8, or a fragment or conjugate or analogue or homolog
thereof. In one embodiment, the renalase polypeptide comprises the
amino acid sequence of SEQ ID NO: 9, or a fragment or conjugate or
analogue or homolog thereof. In another embodiment, the renalase
polypeptide fragment comprises the amino acid sequence of SEQ ID
NO: 3, or a fragment or conjugate or analogue or homolog thereof.
In one embodiment, the renalase polypeptide fragment comprises the
amino acid sequence of SEQ ID NO: 4, or a fragment or conjugate or
analogue or homolog thereof. In another embodiment, the renalase
polypeptide fragment comprises the amino acid sequence of SEQ ID
NO: 5, or a fragment or conjugate or analogue or homolog thereof.
In some embodiments, the at least one agent is administered one
time. In some embodiments, the at least one agent is administered
repeatedly. In various embodiments, the at least one agent is
administered locally, regionally or systemically. In one
embodiment, the activator of renalase is an activator of renalase
expression, an activator of renalase activity, or a combination
thereof. In various embodiments, the activator of renalase is a
chemical compound, a protein, a peptide, a peptidomemetic, a small
molecule chemical compound, or a combination thereof. In various
embodiments, the pancreatic disease or disorder is at least one
selected from the group consisting of acute pancreatitis and
chronic pancreatitis.
[0016] In one embodiment, the invention is a method of diagnosing a
pancreatic disease or disorder in a subject in need thereof,
including the steps of determining the level of renalase in a
biological sample of the subject, comparing the level of renalase
in the biological sample of the subject with a comparator control,
and diagnosing the subject with a pancreatic disease or disorder
when the level of renalase in the biological sample of subject is
reduced when compared with the level of renalase of the comparator
control. In one embodiment, the method also includes the step of
administering a treatment to the subject that was diagnosed as
having a pancreatic disease or disorder. In some embodiments, the
level of renalase in the biological sample is determined by
measuring the level of renalase mRNA in the biological sample. In
some embodiments, the level of renalase in the biological sample is
determined by measuring the level of renalase polypeptide in the
biological sample. In some embodiments, the level of renalase in
the biological sample is determined by measuring an enzymatic
activity of renalase polypeptide in the biological sample. In
various embodiments, the comparator control is at least one
selected from the group consisting of: a positive control, a
negative control, a historical control, a historical norm, or the
level of a reference molecule in the biological sample. In various
embodiments, the pancreatic disease or disorder acute pancreatitis
or chronic pancreatitis. In one embodiment, the subject is
human.
[0017] In another embodiment, the invention is a method of
identifying a test compound as a modulator of the activity of a
renalase receptor, including the steps of determining the level of
activity of a renalase receptor in the presence of a test compound,
determining the level of activity of a renalase receptor in the
absence of a test compound, comparing the level of activity of a
renalase receptor in the presence of the test compound with the
level of activity of a renalase receptor in the absence of the test
compound, identifying the test compound as a modulator of the
activity of a renalase receptor when the level of activity of a
renalase receptor in the presence of the test compound is different
than the level of activity of a renalase receptor in the absence of
the test compound. In some embodiments, when the level of activity
of a renalase receptor is increased in the presence of the test
compound, the test compound is identified as an activator. In some
embodiments, when the level of activity of a renalase receptor is
reduced in the presence of the test compound, the test compound is
identified as an inhibitor. In one embodiment, the level of
activity of a renalase receptor is determined by measuring the
level of mitogen-activated protein kinase (MAPK) signaling. In one
embodiment, the level of activity of a renalase receptor is
determined by measuring ATPase activity. In one embodiment, the
renalase receptor is PMCA4b. In various embodiments, the test
compound is a chemical compound, a protein, a peptide, a
peptidomemetic, an antibody, a nucleic acid, an antisense nucleic
acid, an siRNA, a miRNA, a shRNA, a ribozyme, an allosteric
modulator, a small molecule chemical compound, or a combination
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The following detailed description of preferred embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, there are shown in the drawings embodiments which are
presently preferred. It should be understood, however, that the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0019] FIG. 1, comprising FIGS. 1A-1C, depicts how renalase
peptides exhibit protective effects. FIG. 1A is an illustration
depicting the amino acid sequence of renalase peptides (SEQ ID NOs
1-7): RP-Scr220 (SEQ ID NO: 7): scrambled RP-220. FIG. 1B is a
graph depicting the effect of renalase peptides on survival of HK-2
cells exposed to 20 .mu.M cisplatin for 24 hrs; cell survival is
depicted as % change in survival compared to that in
cisplatin-treated HK-2 cells without renalase peptides; cell
survival was measured by the WST-1 method; RP-A220: mutated RP-220,
and RP-19 and RP-128: control peptides; peptide concentration
(.mu.g/ml) indicated in top line; n=4, *=p<0.05. FIG. 1C is a
graph depicting a comparison of protective effect of recombinant
renalase; RP-224, RP-220, and RP-H220 on survival of HK-2 cells
exposed to 20 .mu.M cisplatin for 24 hrs; cell survival is depicted
as % change in survival compared to that in cisplatin-treated HK-2
cells without renalase peptides; n=4, *=p<0.05. FIG. 1D is a
graph depicting a dose response curve for RP-220 and RP-H220; HK-2
cells were exposed to 20 .mu.M cisplatin for 24 hrs; cell survival
is depicted as % change in survival compared to that in
cisplatin-treated HK-2 cells without renalase peptides; n=4,
*=p<0.05
[0020] FIG. 2, comprising FIGS. 2A-2C, depicts how RP-220 alters
the pattern of MAPK activation in cisplatin treated cells. FIG. 2A
is an image of a gel depicting MAPK phosphorylation with cisplatin
alone (CP) and with CP and RP-220; representative study: p-p38:
phosphorylated p38 MAPK; p-ERK: phosphorylated ERK. FIG. 2B is a
graph depicting quantification of ERK activation. Signals were
normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
loading control; n=3, *=P<0.05. FIG. 2C is a graph depicting
quantification of p38 activation, signals normalized to GAPDH
loading control; n=3, *=P<0.05.
[0021] FIG. 3, comprising FIGS. 3A-3B, depicts how the inhibition
of p38 abrogates the protective effect of RP-220. FIG. 3A is a
graph depicting how the inhibition of either p38 (SB203580) or ERK
(U0126) did not adversely affect the survival of HK-2 cells
measured using the WST-1 method; cell survival is depicted as %
change in survival compared to that of untreated HK-2 cells; n=4,
*=P<0.05. FIG. 3B is a graph depicting how the inhibition of p38
(10 .mu.M SB203580) abrogated the protective action of RP-220 for
HK-2 cells exposed to 20 .mu.M cisplatin (Cis) for 24 hrs; n=4,
*=P<0.05.
[0022] FIG. 4, comprising FIGS. 4A-4D, depicts the identification
of plasma membrane calcium ATPase isoform PMCA4b as a renalase
binding protein. FIG. 4A is an image of a gel depicting HK-2 cells
incubated with either labeled RP-Scr220 or RP-220, biotin-labeled
proteins purified using streptavidin column, separated by SDS-PAGE
and visualized by western blot using streptavidin-HRP; *=regions
evaluated by mass spectrometry in samples labeled with either
RP-Scr220 or RP-220; #=RP-220 band containing the plasma membrane
calcium ATPase isoform PMCA4b. FIG. 4B is an image of a gel
depicting the endogenous expression of PMCA4b in HK-2 cells,
western immunoblot using isoform specific monoclonal; CCL-119:
human leukemic cell line; thyroid tumor=human thyroid tumor cell
line (ATCC, CRL-1803) 10 .mu.g protein loaded in each lane. FIG. 4C
is an image of co-immunolocalization of PMCA4b and renalase in HK-2
cells. Images were acquired using a Zeiss laser scanning confocal
microscope; scale bar=9 .mu.m; arrow=plasma membrane; FIG. 4D is an
image of gel depicting co-immunoprecipitation of PMCA4b and
renalase from HK-2 cell lysates; renalase-Ab-beads=renalase
antibody coated beads; PMCA4b-Ab-beads=PMCA4b antibody coated
beads.
[0023] FIG. 5, comprising FIGS. 5A-5E, depicts how PMCA4b
inhibition abrogates renalase peptide mediated MAPK signaling and
cytoprotection. FIG. 1A is a series of panels depicting how PMCA4b
inhibition abrogates RP-220 mediated ERK and p38 signaling in HK-2
cells; caloxin1b=peptide inhibitor of PMCA4; left panel: RP-220
mediated ERK and p38 activation, phospho=phosphorylated,
representative blot; middle panel: inhibition of RP-220 mediated
ERK and p38 activation by caloxin1b (100 .mu.M); right panel:
quantification of phosphorylated p38 (P-p38) and phosphorylated ERK
(p-ERK); signals were normalized to glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) loading control; n=3, *=P<0.05. FIG. 5B is
a series of panels depicting how siRNA mediated inhibition of
PMCA4b expression downregulates RP-220 mediated MAPK signaling;
left panel: RP-220 mediated ERK and p38 activation in HK-2 cells
transfected with non-targeting siRNA, p=phosphorylated,
representative immunoblot; middle panel: inhibition of RP-220
mediated ERK and p38 activation in HK-2 cells transfected with
PMCA4b siRNA, representative blot; right panel: quantification of
phosphorylated ERK (p-ERK), signals normalized to glyceraldehyde
3-phosphate dehydrogenase (GAPDH) loading control; n=3,
*=P<0.05. FIG. 5 is a series of panels depicting the lack of
effect of siRNA mediated inhibition of PMCA4b expression on
epidermal growth factor (EGF)-mediated MAPK signaling; left panel:
EGF (100 ng/ml) mediated ERK, p38 activation and c-Jun N-Terminal
Kinase (JNK) in HK-2 cells transfected with non-targeting siRNA,
p=phosphorylated, representative blot; right panel: EGF-mediated
ERK, p38 and JNK activation in HK-2 cells unaffected by
transfection with PMCA4b siRNA and downregulation of PMCA4b
expression; representative blot (n=3). FIG. 5D is a graph depicting
how the inhibition of PMCA4b expression abrogates protective effect
of renalase peptides for HK-2 cells exposed to cisplatin: HK-2
cells exposed to 20 .mu.M cisplatin for 24 hrs; cell survival is
depicted as % change in survival compared to that in
cisplatin-treated HK-2 cells without renalase peptides; cell
survival was measured by the WST-1 method, peptide concentration 15
.mu.g/ml, indicated in top line; n=4, *=p<0.05. FIG. 5E is an
image of a gel depicting endogenous expression of PMCA4b in WT and
renalase KO mice; western immunoblot using an anti-renalase
monoclonal antibody; 10 protein loaded in each lane.
[0024] FIG. 6, comprising FIGS. 6A-6D, depicts the results of
experiments showing a plasma membrane calcium ATPase is a receptor
the secretory protein renalase and appears to mediate the
cytoprotective effects of renalase. (FIG. 6A) HK-2 cells incubated
with either labeled RP-Scr220 or RP-220, biotin-labeled proteins
purified using streptavidin column, separated by SDS-PAGE and
visualized by western blot using streptavidin-HRP; *=regions
evaluated by mass spectrometry in samples labeled with either
RP-Scr220 or RP-220; #RP-220 band containing the plasma membrane
calcium ATPase isoform PMCA4b. (FIG. 6B) Co-immunolocalization of
PMCA4b and renalase in HK-2 cells, images acquired using a Zeiss
laser scanning confocal microscope; arrow points to plasma
membrane. (FIG. 6C) Co-immunoprecipitation of PMCA4b and renalase
from HK-2 cell lysates; renalase-Ab-beads=renalase antibody coated
beads; PMCA4b-Ab-beads=PMCA4b antibody coated beads. (FIG. 6D)
Inhibition of PMCA4b expression abrogates protective effect of
renalase peptides for HK-2 cells exposed to cisplatin: HK-2 cells
exposed to 20 .mu.M cisplatin for 24 hrs; cell survival is depicted
as % change in survival compared to that in cisplatin-treated HK-2
cells without renalase peptides; cell survival measured by the
WST-1 method, peptide concentration 15 .mu.g/ml, indicated in top
line; n=4, *=p<0.05.
[0025] FIG. 7, comprising FIGS. 7A-7C, depicts the results of
experiments showing that renalase is present in mouse acinar cells
and its levels are reduced in the serum after inducing acute
experimental pancreatitis. FIGS. 7A and 7B show that renalase is
expressed in the pancreatic acinar cell, but at lower levels than
the kidney. Since renalase is present in the serum (FIG. 7C),
renalase could affect the acinar cells through autocrine/paracrine
pathways and also as a hormone. Serum renalase levels decrease in a
time-dependent manner in mice after initiating cerulein-induced
pancreatitis, falling by about 70% after 7 hrs of cerulein
pancreatitis (FIG. 7C). This finding has several important
implications: i) the protective effects of serum renalase can be
lost during the onset of acute pancreatitis and ii) renalase is
useful as a disease biomarker.
[0026] FIG. 8, comprising FIGS. 8A-8C, depicts the results of
experiments showing renalase deficiency worsens cerulean
pancreatitis in vivo. Wild-type (WT) mice or renalase deficient
knock-out (KO) mice were treated with 6 hourly IP injections of
cerulein (CER, 40 mcg/kg). (FIG. 8A) Pancreatic trypsin activation
measured with fluorogenic substrate, normalized to total protein.
(FIG. 8B) Edema measured as % wet weight. Pancreas H&E sections
(FIG. 8C) showed more edema and vacuolization compared with WT.
*p<0.05 vs. WT-CER, +/-SEM, n=5.
[0027] FIG. 9, comprising FIGS. 9A-9E, depicts the results of
experiments that show recombinant exogenous renalase reduce zymogen
activation and injury in vitro in mice. (FIG. 9A) Isolated WT mouse
acini were pretreated for 30 minutes with recombinant renalase (25
.mu.g/mL; 0.1 .mu.M) and then treated with either (FIG. 9A, 9D, 9E)
physiologic (0.1 nM) and supraphysiologic (100 nM) cerulein or
(FIG. 9B) physiologic (1 .mu.M) or supraphysiologic (1000 1 .mu.M)
carbachol for 30 minutes. Trypsin activity (FIGS. 9A-9C), MTT
accumulation (FIG. 9D), and LDH release (FIG. 9E) were assayed
using a fluorogenic substrate. For FIG. 9C, isolated acini from
renalase .sup.-/.sup.- mice were treated pretreated with
recombinant renalase (25 .mu.g/mL) and then treated with cerulein
(0.1 nM or 100 nM) for 30 minutes. *p<0.05 vs. supraphysiologic
cerulein or carbachol, #p<0.05 vs. WT, n=3, +/-SEM.
[0028] FIG. 10, comprising FIGS. 10A-10G, depicts the results of
experiments showing that pretreatment with renalase peptide
(RP-220) ameliorates cerulein-induced pancreatitis in vivo. WT mice
were treated with 6 hourly IP injections of cerulein (CER, 40
mcg/kg). IP renalase peptide, RP-220 (renalase, 1.3 mg/kg) was
given 1 hour before first cerulein injection. (FIG. 10A) Pancreatic
trypsin activation measured with fluorogenic substrate, normalized
to total protein. (FIG. 10B) Edema measured as % wet weight. (FIG.
10C) Apoptosis evaluated by TUNEL positive cells. (FIG. 10D)
Neutrophils (Ly-6B) and (FIG. 10E) macrophages (F4/80) were
quantified. Pancreas H&E sections (FIG. 10G) were evaluated for
edema, pyknotic nucei, and vacuoles to derive morphological score
(FIG. 10F). *p<0.05 vs. WT-CER, +/-SEM, n=5.
[0029] FIG. 11 depicts the results of experiments showing that
treatment with renalase 2 hours after the onset of cerulein
pancreatitis significantly reduces injury. A single dose of
renalase was given intraperitoneally (100 .mu.g peptide/25 g body
weight) 2 hours after inducing cerulein-pancreatitis. Six hourly
injections IP of 50 .mu.g/kg and animals were sacrificed at hour 7.
Histologic pancreatitis severity was evaluated by two blinded
reviewers. This therapeutic treatment decreased pancreatitis in
each category and the total histologic scores (bottom graph) was
significantly less in the renalase treated cerulein (CER) vs. CER
alone. *p<0.05 Wilcoxon rank sum. N=4 mice in each group.
[0030] FIG. 12, comprising FIGS. 12A-12G, depicts the results of
experiments showing renalase increases Ca.sup.2+ efflux through
PCMA. Changes in whole cell cytosolic Ca.sup.2+ were recorded with
the Ca.sup.2+ dye Fluo-4 (HK2 cells) or Fluo-5 (acinar cell) by
time lapse confocal microscopy. FIGS. 12A, 12B, 12D, and 12E are
representative plots of fluorescence over time in HK2 cells (FIGS.
12A and 12B) or mouse acinar cells (FIGS. 12D and 12E). Cells were
pretreated with 30 .mu.M cyclopiazonic acid and perfused with
Ca.sup.2+-free medium. Cells were briefly stimulated with 10 nM
angiotensin IV (Ang in HK2 cells) or 100 nM cerulein (Cer in acinar
cells) in the presence of 1.3 mM Ca.sup.2+. Next, cells were again
perfused with Ca.sup.2+-free media in the absence or presence of
recombinant renalase, and Ca.sup.2+ efflux was observed (box). All
buffers in HK2 cells were also Na.sup.+-free. For FIGS. 12C and
12F, the rate of Ca.sup.2+ efflux was quantified from 30
cells/treatment group. *p<0.05 vs. no renalase. Mean+/-SEM.
[0031] FIG. 13 comprising FIGS. 13A-13C, depicts the results of
experiments showing caloxin blocks the protective effects of
renalase. Isolated WT mouse acini were pretreated for 30 minutes
with caloxin 1b (100 .mu.M) and/or recombinant renalase (25
.mu.g/mL) and then treated with supraphysiologic (100 nM) cerulein
(CER) and assayed for trypsin activity (FIG. 13A) or MTT conversion
(FIG. 13B). *p<0.05 vs. CER, #p<0.05 vs. CER+renalase. WT,
n=3, +/-SEM. H&E sections (FIG. 13C) show that caloxin reverses
the protective effects of renalase on edema and vacuole formation
(arrows).
DETAILED DESCRIPTION
[0032] The present invention relates to the discovery that
modulators of renalase receptor PMCA4b are useful the treatment or
prevention of various diseases or disorders. In one embodiment, the
PMCA4b is a PMCA4b activator. Thus, the present invention relates
to compositions comprising a PMCA4b activator and methods for
treating and preventing renal and pancreatic diseases or disorders.
In some embodiments, the renal disease or disorder treated or
prevented using the compositions and methods of the invention is
acute kidney injury (AKI) or chronic kidney disease (CKD). In some
embodiments, the pancreatic disease or disorder treated or
prevented using the compositions and methods of the invention is
acute pancreatitis or chronic pancreatitis. In another embodiment,
the PMCA4b is a PMCA4b inhibitor. Therefore, the present invention
also relates to compositions comprising a PMCA4b inhibitor and
methods for treating and preventing cancer.
[0033] The present invention also relates to the discovery that
renalase, and fragments thereof, are useful for the treatment or
prevention of diseases or disorders, such as pancreatitis. Thus,
the invention relates to compositions comprising renalase, or
fragments thereof, and methods for treating and preventing diseases
and disorders, including pancreatic disease or disorders.
[0034] The compositions and methods of the invention comprise
recombinant renalase, or fragments thereof. In one embodiment, the
renalase of the invention is a polypeptide comprising the amino
acid sequence of SEQ ID NO: 8. In another embodiment, the renalase
of the invention is a polypeptide comprising the amino acid
sequence of SEQ ID NO: 9. In some embodiments, the renalase of the
invention is a renalase fragment comprising at least a portion of
the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some
embodiments, the renalase fragment is a peptide that retains its
AKI protective activity, but does not exhibit detectable NADH
oxidase activity. In some embodiments, the renalase fragment is a
peptide that retains its protective activity, but does not exhibit
detectable amine oxidase activity. In a particular embodiment, the
renalase fragment is a peptide comprising the amino acid sequence
of SEQ ID NO: 3. In another particular embodiment, the renalase
fragment is a peptide comprising the amino acid sequence of SEQ ID
NO: 4. In another particular embodiment, the renalase fragment is a
peptide comprising the amino acid sequence of SEQ ID NO: 5.
[0035] In one embodiment, the invention is a method of diagnosing a
pancreatic disease or disorder, such as pancreatitis, of a subject
by assessing the level of renalase in a biological sample of the
subject. In one embodiment, a change (i.e., increase or decrease)
in the level of renalase compared with a comparator is a marker for
the diagnosis of a pancreatic disease or disorder, such as
pancreatitis, as well as for monitoring the effectiveness of a
treatment of a pancreatic disease or disorder.
Definitions
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein. In describing and claiming the present invention, the
following terminology will be used.
[0037] It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
[0038] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0039] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass non-limiting variations of .+-.40% or .+-.20% or .+-.10%,
.+-.5%, .+-.1%, or .+-.0.1% from the specified value, as such
variations are appropriate.
[0040] The term "abnormal" when used in the context of organisms,
tissues, cells or components thereof, refers to those organisms,
tissues, cells or components thereof that differ in at least one
observable or detectable characteristic (e.g., age, treatment, time
of day, etc.) from those organisms, tissues, cells or components
thereof that display the "normal" (expected) respective
characteristic. Characteristics which are normal or expected for
one cell or tissue type, might be abnormal for a different cell or
tissue type.
[0041] As used herein, to "alleviate" or "treat" a disease means
reducing the frequency or severity of at least one sign or symptom
of a disease or disorder.
[0042] As used herein the terms "alteration," "defect,"
"variation," or "mutation," refers to a mutation in a gene in a
cell that affects the function, activity, expression (transcription
or translation) or conformation of the polypeptide that it
encodes.
[0043] Mutations encompassed by the present invention can be any
mutation of a gene in a cell that results in the enhancement or
disruption of the function, activity, expression or conformation of
the encoded polypeptide, including the complete absence of
expression of the encoded protein and can include, for example,
missense and nonsense mutations, insertions, deletions, frameshifts
and premature terminations. Without being so limited, mutations
encompassed by the present invention may alter splicing the mRNA
(splice site mutation) or cause a shift in the reading frame
(frameshift).
[0044] By the term "applicator," as the term is used herein, is
meant any device including, but not limited to, a hypodermic
syringe, a pipette, an iontophoresis device, a patch, and the like,
for administering the compositions of the invention to a
subject.
[0045] As used herein, the term "marker" or "biomarker" is meant to
include a parameter which is useful according to this invention for
determining the presence and/or severity of a disease or disorder,
such as AKI or pancreatitis.
[0046] The level of a marker or biomarker "significantly" differs
from the level of the marker or biomarker in a reference sample if
the level of the marker in a sample from the patient differs from
the level in a sample from the reference subject by an amount
greater than the standard error of the assay employed to assess the
marker, and preferably at least 10%, and more preferably 25%, 50%,
75%, or 100%.
[0047] "Cancer," as used herein, refers to the abnormal growth or
division of cells. Generally, the growth and/or life span of a
cancer cell exceeds, and is not coordinated with, that of the
normal cells and tissues around it. Cancers may be benign,
pre-malignant or malignant. Cancer occurs in a variety of cells and
tissues, including the oral cavity (e.g., mouth, tongue, pharynx,
etc.), digestive system (e.g., esophagus, stomach, small intestine,
colon, rectum, liver, bile duct, gall bladder, pancreas, etc.),
respiratory system (e.g., larynx, lung, bronchus, etc.), bones,
joints, skin (e.g., basal cell, squamous cell, meningioma, etc.),
breast, genital system, (e.g., uterus, ovary, prostate, testis,
etc.), urinary system (e.g., bladder, kidney, ureter, etc.), eye,
nervous system (e.g., brain, etc.), endocrine system (e.g.,
thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma,
leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia,
acute myeloid leukemia, chronic myeloid leukemia, etc.).
[0048] The term "coding sequence," as used herein, means a sequence
of a nucleic acid or its complement, or a part thereof, that can be
transcribed and/or translated to produce the mRNA and/or the
polypeptide or a fragment thereof. Coding sequences include exons
in a genomic DNA or immature primary RNA transcripts, which are
joined together by the cell's biochemical machinery to provide a
mature mRNA. The anti-sense strand is the complement of such a
nucleic acid, and the coding sequence can be deduced therefrom. In
contrast, the term "non-coding sequence," as used herein, means a
sequence of a nucleic acid or its complement, or a part thereof,
that is not translated into amino acid in vivo, or where tRNA does
not interact to place or attempt to place an amino acid. Non-coding
sequences include both intron sequences in genomic DNA or immature
primary RNA transcripts, and gene-associated sequences such as
promoters, enhancers, silencers, and the like.
[0049] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For
example, the sequence "A-G-T," is complementary to the sequence
"T-C-A." Complementarity may be "partial," in which only some of
the nucleic acids' bases are matched according to the base pairing
rules. Or, there may be "complete" or "total" complementarity
between the nucleic acids. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, as well as
detection methods that depend upon binding between nucleic
acids.
[0050] A "disease" is a state of health of an animal wherein the
animal cannot maintain homeostasis, and wherein if the disease is
not ameliorated then the animal's health continues to deteriorate.
In contrast, a "disorder" in an animal is a state of health in
which the animal is able to maintain homeostasis, but in which the
animal's state of health is less favorable than it would be in the
absence of the disorder. Left untreated, a disorder does not
necessarily cause a further decrease in the animal's state of
health.
[0051] An "effective amount" as used herein, means an amount which
provides a therapeutic or prophylactic benefit.
[0052] "Encoding" refers to the inherent property of specific
sequences of nucleotides in a polynucleotide, such as a gene, a
cDNA, or an mRNA, to serve as templates for synthesis of other
polymers and macromolecules in biological processes having either a
defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a
defined sequence of amino acids and the biological properties
resulting therefrom. Thus, a gene encodes a protein if
transcription and translation of mRNA corresponding to that gene
produces the protein in a cell or other biological system. Both the
coding strand, the nucleotide sequence of which is identical to the
mRNA sequence and is usually provided in sequence listings, and the
non-coding strand, used as the template for transcription of a gene
or cDNA, can be referred to as encoding the protein or other
product of that gene or cDNA.
[0053] As used herein, the term "fragment," as applied to a nucleic
acid, refers to a subsequence of a larger nucleic acid. A
"fragment" of a nucleic acid can be at least about 15 nucleotides
in length; for example, at least about 50 nucleotides to about 100
nucleotides; at least about 100 to about 500 nucleotides, at least
about 500 to about 1000 nucleotides; at least about 1000
nucleotides to about 1500 nucleotides; about 1500 nucleotides to
about 2500 nucleotides; or about 2500 nucleotides (and any integer
value in between). As used herein, the term "fragment," as applied
to a protein, polypeptide or peptide, refers to a subsequence of a
larger protein or peptide. A "fragment" of a protein, polypeptide,
or peptide can be at least about 5 amino acids in length; for
example, at least about 10 amino acids in length; at least about 20
amino acids in length; at least about 50 amino acids in length; at
least about 100 amino acids in length; at least about 200 amino
acids in length; or at least about 300 amino acids in length (and
any integer value in between).
[0054] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that includes coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., mRNA). The
polypeptide may be encoded by a full length coding sequence or by
any portion of the coding sequence so long as the desired activity
or functional property (e.g., enzymatic activity, ligand binding,
signal transduction, immunogenicity, etc.) of the full-length or
fragment is retained. The term also encompasses the coding region
of a structural gene and the sequences located adjacent to the
coding region on both the 5' and 3' ends for a distance of about 2
kb or more on either end such that the gene corresponds to the
length of the full-length mRNA and 5' regulatory sequences which
influence the transcriptional properties of the gene. Sequences
located 5' of the coding region and present on the mRNA are
referred to as 5'-untranslated sequences. The 5'-untranslated
sequences usually contain the regulatory sequences. Sequences
located 3' or downstream of the coding region and present on the
mRNA are referred to as 3'-untranslated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or clone of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene that are
transcribed into nuclear RNA (hnRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0055] "Homologous" refers to the sequence similarity or sequence
identity between two polypeptides or between two nucleic acid
molecules. When a position in both of the two compared sequences is
occupied by the same base or amino acid monomer subunit, e.g., if a
position in each of two DNA molecules is occupied by adenine, then
the molecules are homologous at that position. The percent of
homology between two sequences is a function of the number of
matching or homologous positions shared by the two sequences
divided by the number of positions compared X 100. For example, if
6 of 10 of the positions in two sequences are matched or homologous
then the two sequences are 60% homologous. By way of example, the
DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a
comparison is made when two sequences are aligned to give maximum
homology.
[0056] "Instructional material," as that term is used herein,
includes a publication, a recording, a diagram, or any other medium
of expression which can be used to communicate the usefulness of
the nucleic acid, peptide, polypeptide, and/or compound of the
invention in the kit for identifying or alleviating or treating the
various diseases or disorders recited herein. Optionally, or
alternately, the instructional material may describe one or more
methods of identifying or alleviating the diseases or disorders in
a cell or a tissue of a subject. The instructional material of the
kit may, for example, be affixed to a container that contains the
nucleic acid, polypeptide, and/or compound of the invention or be
shipped together with a container that contains the nucleic acid,
polypeptide, and/or compound. Alternatively, the instructional
material may be shipped separately from the container with the
intention that the recipient uses the instructional material and
the compound cooperatively.
[0057] "Isolated" means altered or removed from the natural state.
For example, a nucleic acid or a polypeptide naturally present in a
living animal is not "isolated," but the same nucleic acid or
polypeptide partially or completely separated from the coexisting
materials of its natural state is "isolated." An isolated nucleic
acid or protein can exist in substantially purified form, or can
exist in a non-native environment such as, for example, a host
cell.
[0058] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment which has been separated from sequences which flank it
in a naturally occurring state, e.g., a DNA fragment which has been
removed from the sequences which are normally adjacent to the
fragment, e.g., the sequences adjacent to the fragment in a genome
in which it naturally occurs. The term also applies to nucleic
acids which have been substantially purified from other components
which naturally accompany the nucleic acid, e.g., RNA or DNA or
proteins, which naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence.
[0059] The term "label" when used herein refers to a detectable
compound or composition that is conjugated directly or indirectly
to a probe to generate a "labeled" probe. The label may be
detectable by itself (e.g., radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a substrate compound or composition that is
detectable (e.g., avidin-biotin). In some instances, primers can be
labeled to detect a PCR product.
[0060] By the term "modulating," as used herein, is meant mediating
a detectable increase or decrease in the activity and/or level of a
mRNA, polypeptide, or a response in a subject compared with the
activity and/or level of a mRNA, polypeptide or a response in the
subject in the absence of a treatment or compound, and/or compared
with the activity and/or level of a mRNA, polypeptide, or a
response in an otherwise identical but untreated subject. The term
encompasses activating, inhibiting and/or otherwise affecting a
native signal or response thereby mediating a beneficial
therapeutic response in a subject, preferably, a human.
[0061] A "mutation," as used herein, refers to a change in nucleic
acid or polypeptide sequence relative to a reference sequence
(which is preferably a naturally-occurring normal or "wild-type"
sequence), and includes translocations, deletions, insertions, and
substitutions/point mutations. A "mutant" as used herein, refers to
either a nucleic acid or protein comprising a mutation.
[0062] A "nucleic acid" refers to a polynucleotide and includes
poly-ribonucleotides and poly-deoxyribonucleotides. Nucleic acids
according to the present invention may include any polymer or
oligomer of pyrimidine and purine bases, preferably cytosine,
thymine, and uracil, and adenine and guanine, respectively. (See
Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth
Pub. 1982) which is herein incorporated in its entirety for all
purposes). Indeed, the present invention contemplates any
deoxyribonucleotide, ribonucleotide or peptide nucleic acid
component, and any chemical variants thereof, such as methylated,
hydroxymethylated or glucosylated forms of these bases, and the
like. The polymers or oligomers may be heterogeneous or homogeneous
in composition, and may be isolated from naturally occurring
sources or may be artificially or synthetically produced. In
addition, the nucleic acids may be DNA or RNA, or a mixture
thereof, and may exist permanently or transitionally in
single-stranded or double-stranded form, including homoduplex,
heteroduplex, and hybrid states.
[0063] An "oligonucleotide" or "polynucleotide" is a nucleic acid
ranging from at least 2, preferably at least 8, 15 or 25
nucleotides in length, but may be up to 50, 100, 1000, or 5000
nucleotides long or a compound that specifically hybridizes to a
polynucleotide. Polynucleotides include sequences of
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or mimetics
thereof which may be isolated from natural sources, recombinantly
produced or artificially synthesized. A further example of a
polynucleotide of the present invention may be a peptide nucleic
acid (PNA). (See U.S. Pat. No. 6,156,501 which is hereby
incorporated by reference in its entirety.) The invention also
encompasses situations in which there is a nontraditional base
pairing such as Hoogsteen base pairing which has been identified in
certain tRNA molecules and postulated to exist in a triple helix.
"Polynucleotide" and "oligonucleotide" are used interchangeably in
this disclosure. It will be understood that when a nucleotide
sequence is represented herein by a DNA sequence (e.g., A, T, G,
and C), this also includes the corresponding RNA sequence (e.g., A,
U, G, C) in which "U" replaces "T".
[0064] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal, or cells
thereof whether in vitro or in situ, amenable to the methods
described herein. In certain non-limiting embodiments, the patient,
subject or individual is a human.
[0065] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method of K. B. Mullis (U.S. Pat. Nos. 4,683,195
4,683,202, and 4,965,188, hereby incorporated by reference) for
increasing the concentration of a segment of a target sequence in a
mixture of genomic DNA without cloning or purification. This
process for amplifying the target sequence consists of introducing
a large excess of two oligonucleotide primers to the DNA mixture
containing the desired target sequence, followed by a precise
sequence of thermal cycling in the presence of a DNA polymerase.
The two primers are complementary to their respective strands of
the double stranded target sequence. To effect amplification, the
mixture is denatured and the primers then annealed to their
complementary sequences within the target molecule. Following
annealing, the primers are extended with a polymerase so as to form
a new pair of complementary strands. The steps of denaturation,
primer annealing and polymerase extension can be repeated many
times (i.e., denaturation, annealing and extension constitute one
"cycle"; there can be numerous "cycles") to obtain a high
concentration of an amplified segment of the desired target
sequence. The length of the amplified segment of the desired target
sequence is determined by the relative positions of the primers
with respect to each other, and therefore, this length is a
controllable parameter. By virtue of the repeating aspect of the
process, the method is referred to as the "polymerase chain
reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified". As used herein, the terms "PCR product," "PCR
fragment," "amplification product" or "amplicon" refer to the
resultant mixture of compounds after two or more cycles of the PCR
steps of denaturation, annealing and extension are complete. These
terms encompass the case where there has been amplification of one
or more segments of one or more target sequences.
[0066] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, that is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences.
[0067] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
that can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, of which there are many types. "Polypeptides" include,
for example, biologically active fragments, substantially
homologous polypeptides, oligopeptides, homodimers, heterodimers,
variants of polypeptides, modified polypeptides, derivatives,
analogs, fusion proteins, among others. The polypeptides include
natural peptides, recombinant peptides, synthetic peptides, or a
combination thereof.
[0068] As used herein, "polynucleotide" includes cDNA, RNA, DNA/RNA
hybrid, antisense RNA, ribozyme, genomic DNA, synthetic forms, and
mixed polymers, both sense and antisense strands, and may be
chemically or biochemically modified to contain non-natural or
derivatized, synthetic, or semi-synthetic nucleotide bases. Also,
contemplated are alterations of a wild type or synthetic gene,
including but not limited to deletion, insertion, substitution of
one or more nucleotides, or fusion to other polynucleotide
sequences.
[0069] To "prevent" a disease or disorder as the term is used
herein, means to reduce the severity or frequency of at least one
sign or symptom of a disease or disorder being experienced by a
subject.
[0070] "Sample" or "biological sample" as used herein means a
biological material isolated from a subject. The biological sample
may contain any biological material suitable for detecting a mRNA,
polypeptide or other marker of a physiologic or pathologic process
in a subject, and may comprise fluid, tissue, cellular and/or
non-cellular material obtained from the individual.
[0071] As used herein, "substantially purified" refers to being
essentially free of other components. For example, a substantially
purified polypeptide is a polypeptide which has been separated from
other components with which it is normally associated in its
naturally occurring state.
[0072] As used herein, the terms "therapy" or "therapeutic regimen"
refer to those activities taken to prevent, alleviate or alter a
disorder or disease state, e.g., a course of treatment intended to
reduce or eliminate at least one sign or symptom of a disease or
disorder using pharmacological, surgical, dietary and/or other
techniques. A therapeutic regimen may include a prescribed dosage
of one or more compounds or surgery. Therapies will most often be
beneficial and reduce or eliminate at least one sign or symptom of
the disorder or disease state, but in some instances the effect of
a therapy will have non-desirable or side-effects. The effect of
therapy will also be impacted by the physiological state of the
subject, e.g., age, gender, genetics, weight, other disease
conditions, etc.
[0073] The term "therapeutically effective amount" refers to the
amount of the subject compound that will elicit the biological or
medical response of a tissue, system, or subject that is being
sought by the researcher, veterinarian, medical doctor or other
clinician. The term "therapeutically effective amount" includes
that amount of a compound that, when administered, is sufficient to
prevent development of, or alleviate to some extent, one or more of
the signs or symptoms of the disorder or disease being treated. The
therapeutically effective amount will vary depending on the
compound, the disease and its severity and the age, weight, etc.,
of the subject to be treated.
[0074] To "treat" a disease or disorder as the term is used herein,
means to reduce the frequency or severity of at least one sign or
symptom of a disease or disorder experienced by a subject.
[0075] As used herein, the term "wild-type" refers to a gene or
gene product isolated from a naturally occurring source. A
wild-type gene is that which is most frequently observed in a
population and is thus arbitrarily designed the "normal" or
"wild-type" form of the gene. In contrast, the term "modified" or
"mutant" refers to a gene or gene product that displays
modifications in sequence and/or functional properties (i.e.,
altered characteristics) when compared to the wild-type gene or
gene product. It is noted that naturally occurring mutants can be
isolated; these are identified by the fact that they have altered
characteristics (including altered nucleic acid sequences) when
compared to the wild-type gene or gene product.
[0076] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
Description
[0077] The present invention includes the unexpected identification
of the renalase receptor PMCA4b, a plasma membrane calcium ATPase.
As demonstrated herein, PMCA4b co-localizes with renalase in HK-2
cells, while inhibition of PMCA4b enzymatic activity was found to
prevent renalase-mediated mitogen-activated protein kinase (MAPK)
signaling in these cells. Therefore the interaction between PMCA4b
and recombinant renalase or renalase peptide fragments appears to
be critical for initiating a pattern of MAPK signaling, which
results in the observed cytoprotective effect of renalase and
renalase peptides. Thus, the present invention relates to the
discovery that the protective effect of renalase against a disease
or disorder is mediated by the interaction of renalase or a short
renalase peptide with a renalase receptor. While the preferred
renalase receptor is PMCA4b, the invention should be construed to
encompass any other receptor that binds to renalase, a renalase
fragment, or combinations thereof.
[0078] In one aspect of the invention, the compositions and methods
of the invention comprise a PMCA4b activator. The compositions and
methods of the invention include compositions and methods for
treating or preventing disorders and diseases where an increased
activity or level of PMCA4b is desirable. In various embodiments,
the diseases and disorders where an increased activity or level of
PMCA4b is desirable which can be treated or prevented with the
compositions and methods of the invention include acute kidney
injury (AKI), chronic kidney disease (CKD), renal ischemic injury,
renal reperfusion injury, renal ischemic-reperfusion injury, toxic
renal injury, renal tubular necrosis, renal tubular inflammation,
renal tubular apoptosis, hypertension, pancreatitis, and any
combination thereof.
[0079] In another aspect of the invention, the compositions and
methods of the invention comprise a PMCA4b inhibitor. The
compositions and methods of the invention include compositions and
methods for treating or preventing disorders and diseases where a
decreased activity or level of PMCA4b is desirable. In various
embodiments, a disease or disorder where a decreased activity or
level of PMCA4b is desirable which can be treated or prevented with
the compositions and methods of the invention includes cancer. In
one embodiment, the cancer is selected from the group consisting of
brain cancer, bladder cancer, breast cancer, cervical cancer,
colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia,
lung cancer, melanoma, metastatic melanoma, mesothelioma,
neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer,
renal cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's
lymphoma, Hodgkin's lymphoma, uterine cancer, and any combination
thereof.
[0080] In another embodiment, the invention is a method of
identifying a modulator of a renalase receptor, such as PMCA4b.
[0081] In some embodiments, the compositions and methods of the
invention comprise renalase, or a fragment thereof, for use in the
treatment or prevention of a pancreatic disease or disorder, such
as pancreatitis. In some embodiments, the renalase of the invention
is a polypeptide comprising the amino acid sequence of SEQ ID NO: 8
or SEQ ID NO: 9. In some embodiments, the renalase of the invention
is a renalase fragment comprising at least a portion of the amino
acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments,
the renalase fragment is a peptide that retains its protective
activity, but does not exhibit detectable NADH oxidase activity. In
some embodiments, the renalase fragment is a peptide that retains
its protective activity, but does not exhibit detectable amine
oxidase activity. In a particular embodiment, the renalase fragment
is a peptide comprising the amino acid sequence of SEQ ID NO: 3. In
another particular embodiment, the renalase fragment is a peptide
comprising the amino acid sequence of SEQ ID NO: 4. In another
particular embodiment, the renalase fragment is a peptide
comprising the amino acid sequence of SEQ ID NO: 5. In another
particular embodiment, the renalase fragment is a peptide
consisting of the amino acid sequence of SEQ ID NO: 3. In another
particular embodiment, the renalase fragment is a peptide
consisting of the amino acid sequence of SEQ ID NO: 4. In another
particular embodiment, the renalase fragment is a peptide
consisting of the amino acid sequence of SEQ ID NO: 5.
[0082] The compositions and methods of the invention comprise
recombinant renalase, or fragments thereof. The compositions and
methods of the invention include compositions and methods for
treating or preventing disorders and diseases where an increased
activity or level of renalase is desirable. In various embodiments,
the disorders and diseases where an increased activity or level of
renalase is desirable which can be treated or prevented with the
compositions and methods of the invention include acute
pancreatitis or chronic pancreatitis.
[0083] In another embodiment, the invention is a method of
diagnosing a pancreatic disease or disorder of a subject by
assessing the level of renalase in a biological sample of the
subject. In one embodiment, a change (i.e., increase or decrease)
in the level of renalase compared with a comparator is a marker for
the diagnosis of a pancreatic disease or disorder, such as
pancreatitis, as well as for monitoring the effectiveness of a
treatment of a pancreatic disease or disorder, such as
pancreatitis.
PMCA4b Activator Compositions and Methods of Treatment and
Prevention
[0084] In various embodiments, the present invention includes
PMCA4b activator compositions and methods of increasing the level
or activity of PMCA4b in a subject, a tissue, or an organ in need
thereof. In various embodiments, the PMCA4b activator compositions
and methods of treatment of the invention increase the amount of
PMCA4b polypeptide, the amount of PMCA4b mRNA, the amount of PMCA4b
enzymatic activity, the amount of PMCA4b substrate binding
activity, or a combination thereof. In one embodiment, the PMCA4b
activator composition comprises a PMCA4b activator. In various
embodiments, the diseases and disorders wherein activation of
PMCA4b may improve therapeutic outcome include, but are not limited
to, AKI, chronic kidney disease (CKD), myocardial necrosis, heart
failure, congestive heart failure, cardiac ischemic injury, cardiac
reperfusion injury, cardiac ischemic-reperfusion injury, toxic
cardiac injury, renal ischemic injury, renal reperfusion injury,
renal ischemic-reperfusion injury, toxic renal injury, renal
tubular necrosis, renal tubular inflammation, renal tubular
apoptosis, ischemic brain injury, reperfusion brain injury,
ischemic-reperfusion brain injury, toxic brain injury, ischemic
liver injury, reperfusion liver injury, ischemic-reperfusion liver
injury, toxic liver injury, pancreatitis, acute pancreatitis,
chronic pancreatitis, preservation of organs harvested for
transplantation, pheochromocytoma, and hypertension. In some
embodiments, the compositions and methods of the invention are
useful for controlling or maintaining blood pressure. In some
embodiments, the compositions and methods of the invention are
useful for treating or preventing sympathetic nervous system
diseases and disorders, such as, by way of a non-limiting examples,
anxiety, post-traumatic stress disorder (PTSD) and attention
deficit hyperactivity disorder (ADHD).
[0085] In one aspect, the methods of the present invention include
a method of treating or preventing a disease or disorder in a
subject in need thereof. In one embodiment, the method comprises
administering to the subject a therapeutically effective amount of
a composition comprising at least one PMCA4b activator. In one
embodiment, the disease or disorder is selected from the group
consisting of acute kidney injury (AKI), chronic kidney disease
(CKD), renal ischemic injury, renal reperfusion injury, renal
ischemic-reperfusion injury, toxic renal injury, renal tubular
necrosis, renal tubular inflammation, renal tubular apoptosis,
hypertension, pancreatitis, and any combination thereof.
[0086] It will be understood by one skilled in the art, based upon
the disclosure provided herein, that an increase in the level of
PMCA4b encompasses the increase in PMCA4b expression, including
transcription, translation, or both. The skilled artisan will also
appreciate, once armed with the teachings of the present invention,
that an increase in the level of PMCA4b includes an increase in
PMCA4b activity (e.g., enzymatic activity, substrate binding
activity, etc.). Thus, increasing the level or activity of PMCA4b
includes, but is not limited to, increasing the amount of PMCA4b
polypeptide, and increasing transcription, translation, or both, of
a nucleic acid encoding PMCA4b; and it also includes increasing any
activity of a PMCA4b polypeptide as well. The PMCA4b activator
compositions and methods of the invention can selectively activate
PMCA4b, or can activate both PMCA4b and another molecule.
[0087] Thus, the present invention relates to the prevention and
treatment of a disease or disorder by administration of a
therapeutically effective amount of a PMCA4b activator composition
to a subject in need thereof, for the treatment or prevention of a
disease or disorder, or its associated signs, symptoms or
pathologies.
[0088] It is understood by one skilled in the art, that an increase
in the level of PMCA4b encompasses an increase in the amount of
PMCA4b. Additionally, the skilled artisan would appreciate, that an
increase in the level of PMCA4b includes an increase in PMCA4b
activity. Thus, increasing the level or activity of PMCA4b
includes, but is not limited to, the administration of a renalase
activator, as well as increasing transcription, translation, or
both, of a nucleic acid encoding PMCA4b; and it also includes
increasing any activity of PMCA4b as well.
[0089] The increased level or activity of PMCA4b can be assessed
using a wide variety of methods, including those disclosed herein,
as well as methods well-known in the art or to be developed in the
future. That is, the routineer would appreciate, based upon the
disclosure provided herein, that increasing the level or activity
of PMCA4b can be readily assessed using methods that assess the
level of a nucleic acid encoding PMCA4b (e.g., mRNA), the level of
PMCA4b polypeptide, and/or the level of PMCA4b activity in a
biological sample obtained from a subject.
[0090] One skilled in the art, based upon the disclosure provided
herein, would understand that the invention is useful in subjects
who, in whole (e.g., systemically) or in part (e.g., locally,
tissue, organ), are being or will be, treated for a disease or
disorder associated with a diminished level or activity of PMCA4b.
The skilled artisan will appreciate, based upon the teachings
provided herein, that the diseases and disorders treatable by the
compositions and methods described herein encompass any disease or
disorder where in an increase in PMCA4b will promote a positive
therapeutic outcome.
[0091] One of skill in the art will realize that in addition to
activating PMCA4b directly, diminishing the amount or activity of a
molecule that itself diminishes the amount or activity of PMCA4b
can serve to increase the amount or activity of PMCA4b. Thus, a
PMCA4b activator can include, but should not be construed as being
limited to, a chemical compound, a protein, a peptidomemetic, an
antibody, a ribozyme, an allosteric modulator, and an antisense
nucleic acid molecule. One of skill in the art would readily
appreciate, based on the disclosure provided herein, that a PMCA4b
activator encompasses a chemical compound that increases the level,
enzymatic activity, or substrate binding activity of PMCA4b.
Additionally, a PMCA4b activator encompasses a chemically modified
compound, fusion, conjugate, and/or derivatives, as is well known
to one of skill in the chemical arts.
[0092] It will be understood by one skilled in the art, based upon
the disclosure provided herein, that an increase in the level of
PMCA4b encompasses the increase in PMCA4b expression, including
transcription, translation, or both. The skilled artisan will also
appreciate, once armed with the teachings of the present invention,
that an increase in the level of PMCA4b includes an increase in
PMCA4b activity (e.g., enzymatic activity, substrate binding
activity, etc.). Thus, increasing the level or activity of PMCA4b
includes, but is not limited to, increasing the amount of PMCA4b
polypeptide, increasing transcription, translation, or both, of a
nucleic acid encoding PMCA4b; and it also includes increasing any
activity of a PMCA4b polypeptide as well. The PMCA4b activator
compositions and methods of the invention can selectively activate
PMCA4b, or can activate both PMCA4b and another molecule. Thus, the
present invention relates to administration of a composition
comprising a PMCA4b activator.
[0093] The PMCA4b activator compositions and methods of the
invention that increase the level or activity (e.g., enzymatic
activity, substrate binding activity, etc.) of PMCA4b include, but
should not be construed as being limited to, a chemical compound, a
protein, a peptide, a peptidomemetic, an antibody, a ribozyme, a
small molecule chemical compound, an allosteric modulator, an
antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.), or
combinations thereof. One of skill in the art would readily
appreciate, based on the disclosure provided herein, that a PMCA4b
activator composition encompasses a chemical compound that
increases the level or activity of PMCA4b. Additionally, a PMCA4b
activator composition encompasses a chemically modified compound,
fusion, conjugate, and/or derivatives, as is well known to one of
skill in the chemical arts.
[0094] The PMCA4b activator compositions and methods of the
invention that increase the level or activity (e.g., enzymatic
activity, substrate binding activity, etc.) of PMCA4b include
antibodies. The antibodies of the invention include a variety of
forms of antibodies including, for example, polyclonal antibodies,
monoclonal antibodies, intracellular antibodies ("intrabodies"),
Fv, Fab and F(ab)2, single chain antibodies (scFv), heavy chain
antibodies (such as camelid antibodies), synthetic antibodies,
chimeric antibodies, and a humanized antibodies. In one embodiment,
the antibody of the invention is an antibody that specifically
binds to PMCA4b. In some embodiments, the activator is an
activating renalase antibody.
[0095] In some embodiments, the activator is a renalase
polypeptide, or an analogue or homolog thereof. In one embodiment,
the activator is a renalase polypeptide comprising the amino acid
sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In another embodiment,
the activator is a renalase polypeptide comprising at least a
portion of the amino acid sequence of renalase. In some
embodiments, the activator is a renalase polypeptide comprising the
amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
In some embodiments, the activator is a renalase polypeptide
comprising at least a portion of the amino acid sequence of SEQ ID
NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In a particular embodiment,
the activator is a renalase polypeptide comprising the amino acid
of SEQ ID NO: 4. In some embodiments, the renalase polypeptide, or
fragment thereof, is conjugated to another molecule.
[0096] Further, one of skill in the art would, when equipped with
this disclosure and the methods exemplified herein, appreciate that
a PMCA4b activator includes such activators as discovered in the
future, as can be identified by well-known criteria in the art of
pharmacology, such as the physiological results of activation of
PMCA4b as described in detail herein and/or as known in the art.
Therefore, the present invention is not limited in any way to any
particular PMCA4b activator as exemplified or disclosed herein;
rather, the invention encompasses those activators that would be
understood by the routineer to be useful as are known in the art
and as are discovered in the future.
[0097] Further methods of identifying and producing a PMCA4b
activator are well known to those of ordinary skill in the art,
including, but not limited, obtaining an activator from a naturally
occurring source (e.g., Streptomyces sp., Pseudomonas sp.,
Stylotella aurantium, etc.). Alternatively, a PMCA4b activator can
be synthesized chemically. Further, the routineer would appreciate,
based upon the teachings provided herein, that a PMCA4b activator
can be obtained from a recombinant organism. Compositions and
methods for chemically synthesizing PMCA4b activators and for
obtaining them from natural sources are well known in the art and
are described in the art.
[0098] One of skill in the art will appreciate that an activator
can be administered as a small molecule chemical, a protein, an
antibody, a nucleic acid construct encoding a protein, or
combinations thereof. In some embodiments, the activator is an
allosteric activator. Numerous vectors and other compositions and
methods are well known for administering a protein or a nucleic
acid construct encoding a protein to cells or tissues. Therefore,
the invention includes a method of administering a protein or a
nucleic acid encoding an protein that is an activator of PMCA4b.
(Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York).
[0099] One of skill in the art will realize that diminishing the
amount or activity of a molecule that itself diminishes the amount
or activity of PMCA4b can serve to increase the amount or activity
of PMCA4b. Antisense oligonucleotides are DNA or RNA molecules that
are complementary to some portion of a mRNA molecule. When present
in a cell, antisense oligonucleotides hybridize to an existing mRNA
molecule and inhibit translation into a gene product. Inhibiting
the expression of a gene using an antisense oligonucleotide is well
known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as
are methods of expressing an antisense oligonucleotide in a cell
(Inoue, U.S. Pat. No. 5,190,931). The methods of the invention
include the use of antisense oligonucleotide to diminish the amount
of a molecule that causes a decrease in the amount or activity of
PMCA4b, thereby increasing the amount or activity of PMCA4b.
Contemplated in the present invention are antisense
oligonucleotides that are synthesized and provided to the cell by
way of methods well known to those of ordinary skill in the art. As
an example, an antisense oligonucleotide can be synthesized to be
between about 10 and about 100, more preferably between about 15
and about 50 nucleotides long. The synthesis of nucleic acid
molecules is well known in the art, as is the synthesis of modified
antisense oligonucleotides to improve biological activity in
comparison to unmodified antisense oligonucleotides (Tullis, 1991,
U.S. Pat. No. 5,023,243).
[0100] Similarly, the expression of a gene may be inhibited by the
hybridization of an antisense molecule to a promoter or other
regulatory element of a gene, thereby affecting the transcription
of the gene. Methods for the identification of a promoter or other
regulatory element that interacts with a gene of interest are well
known in the art, and include such methods as the yeast two hybrid
system (Bartel and Fields, eds., In: The Yeast Two Hybrid System,
Oxford University Press, Cary, N.C.).
[0101] Alternatively, inhibition of a gene expressing a protein
that diminishes the level or activity of PMCA4b can be accomplished
through the use of a ribozyme. Using ribozymes for inhibiting gene
expression is well known to those of skill in the art (see, e.g.,
Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989,
Biochemistry 28: 4929; Altman et al., U.S. Pat. No. 5,168,053).
Ribozymes are catalytic RNA molecules with the ability to cleave
other single-stranded RNA molecules. Ribozymes are known to be
sequence specific, and can therefore be modified to recognize a
specific nucleotide sequence (Cech, 1988, J. Amer. Med. Assn.
260:3030), allowing the selective cleavage of specific mRNA
molecules. Given the nucleotide sequence of the molecule, one of
ordinary skill in the art could synthesize an antisense
oligonucleotide or ribozyme without undue experimentation, provided
with the disclosure and references incorporated herein.
[0102] One of skill in the art will appreciate that a PMCA4b
activator can be administered singly or in any combination thereof.
One of skill in the art will also appreciate administration can be
acute (e.g., over a short period of time, such as a day, a week or
a month) or chronic (e.g., over a long period of time, such as
several months or a year or more). Further, a PMCA4b activator can
be administered singly or in any combination thereof in a temporal
sense, in that they may be administered simultaneously, before,
and/or after each other. One of ordinary skill in the art will
appreciate, based on the disclosure provided herein, that a PMCA4b
activator can be used, and that an activator can be used alone or
in any combination with another PMCA4b activator to effect a
therapeutic result.
[0103] It will be appreciated by one of skill in the art, when
armed with the present disclosure including the methods detailed
herein, that the invention is not limited to treatment of a disease
or disorder once is established. Particularly, the symptoms of the
disease or disorder need not have manifested to the point of
detriment to the subject; indeed, the disease or disorder need not
be detected in a subject before treatment is administered. That is,
significant pathology from disease or disorder does not have to
occur before the present invention may provide benefit. Therefore,
the present invention, as described more fully herein, includes a
method for preventing diseases and disorders in a subject, in that
a PMCA4b activator, as discussed elsewhere herein, can be
administered to a subject prior to the onset of the disease or
disorder, thereby preventing the disease or disorder from
developing.
[0104] One of skill in the art, when armed with the disclosure
herein, would appreciate that the prevention of a disease or
disorder in a subject encompasses administering to a subject a
PMCA4b activator as a preventative measure against a disease or
disorder.
[0105] As more fully discussed elsewhere herein, methods of
increasing the level or activity of PMCA4b encompass a wide
plethora of techniques for increasing not only PMCA4b activity, but
also for increasing expression of a nucleic acid encoding PMCA4b.
Additionally, as disclosed elsewhere herein, one skilled in the art
would understand, once armed with the teaching provided herein,
that the present invention encompasses a method of preventing a
wide variety of diseases or disorders where increased expression
and/or activity of PMCA4b mediates, treats or prevents a disease or
disorder. Further, the invention encompasses treatment or
prevention of such diseases or disorders discovered in the
future.
[0106] The invention encompasses administration of a PMCA4b
activator to practice the methods of the invention; the skilled
artisan would understand, based on the disclosure provided herein,
how to formulate and administer the appropriate PMCA4b activator to
a subject. However, the present invention is not limited to any
particular method of administration or treatment regimen. This is
especially true where it would be appreciated by one skilled in the
art, equipped with the disclosure provided herein, including the
reduction to practice using an art-recognized model of
ischemia-reperfusion injury, that methods of administering a PMCA4b
activator can be determined by one of skill in the pharmacological
arts.
[0107] As used herein, the term "pharmaceutically-acceptable
carrier" means a chemical composition with which an appropriate
PMCA4b modulator may be combined and which, following the
combination, can be used to administer the appropriate PMCA4b
modulator thereof, to a subject.
PMCA4b Inhibitor Compositions and Methods of Treatment and
Prevention
[0108] In various embodiments, the present invention includes
PMCA4b inhibitor compositions and methods of treating or preventing
a disease or disorder where a diminished activity or level of
PMCA4b is desired. One non-limiting example of a disease or
disorder where a diminished activity or level of PMCA4b is desired
which can be treated or prevented with the compositions and methods
of the invention includes cancer. In various embodiments, the
PMCA4b inhibitor compositions and methods of treatment or
prevention of the invention diminish the amount of PMCA4b
polypeptide, the amount of PMCA4b mRNA, the amount of PMCA4b
enzymatic activity, the amount of PMCA4b substrate binding
activity, or a combination thereof. In one embodiment, the PMCA4b
inhibitor is caloxin 1b, or an analogue or derivative thereof. In
another embodiment, the PMCA4b inhibitor is cisplatin, or an
analogue or derivative thereof.
[0109] In one aspect, the methods of the present invention include
a method of treating cancer in a subject in need thereof. In one
embodiment, the method comprises administering to the subject a
therapeutically effective amount of a composition comprising at
least one PMCA4b inhibitor. In one embodiment, the cancer is
selected from the group consisting of brain cancer, bladder cancer,
breast cancer, cervical cancer, colorectal cancer, liver cancer,
kidney cancer, lymphoma, leukemia, lung cancer, melanoma,
metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer,
prostate cancer, pancreatic cancer, renal cancer, skin cancer,
thymoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma,
uterine cancer, and any combination thereof
[0110] It will be understood by one skilled in the art, based upon
the disclosure provided herein, that a decrease in the level of
PMCA4b encompasses the decrease in PMCA4b expression, including
transcription, translation, or both. The skilled artisan will also
appreciate, once armed with the teachings of the present invention,
that a decrease in the level of PMCA4b includes a decrease in
PMCA4b activity (e.g., enzymatic activity, substrate binding
activity, etc.). Thus, decreasing the level or activity of PMCA4b
includes, but is not limited to, decreasing transcription,
translation, or both, of a nucleic acid encoding PMCA4b; and it
also includes decreasing any activity of a PMCA4b polypeptide as
well. The PMCA4b inhibitor compositions and methods of the
invention can selectively inhibit PMCA4b, or can inhibit both
PMCA4b and another molecule.
[0111] Inhibition of PMCA4b can be assessed using a wide variety of
methods, including those disclosed herein, as well as methods known
in the art or to be developed in the future. That is, the routineer
would appreciate, based upon the disclosure provided herein, that
decreasing the level or activity of PMCA4b can be readily assessed
using methods that assess the level of a nucleic acid encoding
PMCA4b (e.g., mRNA), the level of a PMCA4b polypeptide present in a
biological sample, the level of PMCA4b activity (e.g., enzymatic
activity, substrate binding activity, etc.), or combinations
thereof.
[0112] One skilled in the art, based upon the disclosure provided
herein, would understand that the invention is useful in treating
or preventing in a subject in need thereof, whether or not the
subject is also being treated with other medication or therapy.
Further, the skilled artisan would further appreciate, based upon
the teachings provided herein, that the disease or disorders
treatable by the compositions and methods described herein
encompass any disease or disorder where PMCA4b plays a role and
where diminished PMCA4b level or activity will promote a positive
therapeutic outcome.
[0113] The PMCA4b inhibitor compositions and methods of the
invention that decrease the level or activity (e.g., enzymatic
activity, substrate binding activity, etc.) of PMCA4b include, but
should not be construed as being limited to, a chemical compound, a
protein, a peptide, a peptidomemetic, an antibody, a ribozyme, a
small molecule chemical compound, an antisense nucleic acid
molecule (e.g., siRNA, miRNA, etc.), or combinations thereof. In
some embodiments, the inhibitor is an allosteric inhibitor. One of
skill in the art would readily appreciate, based on the disclosure
provided herein, that a PMCA4b inhibitor composition encompasses a
chemical compound that decreases the level or activity of PMCA4b.
Additionally, a PMCA4b inhibitor composition encompasses a
chemically modified compound, and derivatives, as is well known to
one of skill in the chemical arts.
[0114] The PMCA4b inhibitor compositions and methods of the
invention that decrease the level or activity (e.g., enzymatic
activity, substrate binding activity, etc.) of PMCA4b include
antibodies. The antibodies of the invention include a variety of
forms of antibodies including, for example, polyclonal antibodies,
monoclonal antibodies, intracellular antibodies ("intrabodies"),
Fv, Fab and F(ab)2, single chain antibodies (scFv), heavy chain
antibodies (such as camelid antibodies), synthetic antibodies,
chimeric antibodies, and a humanized antibodies. In one embodiment,
the antibody of the invention is an antibody that specifically
binds to PMCA4b.
[0115] Further, one of skill in the art, when equipped with this
disclosure and the methods exemplified herein, would appreciate
that a PMCA4b inhibitor composition includes such inhibitors as
discovered in the future, as can be identified by well-known
criteria in the art of pharmacology, such as the physiological
results of inhibition of PMCA4b as described in detail herein
and/or as known in the art. Therefore, the present invention is not
limited in any way to any particular PMCA4b inhibitor composition
as exemplified or disclosed herein; rather, the invention
encompasses those inhibitor compositions that would be understood
by the routineer to be useful as are known in the art and as are
discovered in the future.
[0116] Further methods of identifying and producing PMCA4b
inhibitor compositions are well known to those of ordinary skill in
the art, including, but not limited, obtaining an inhibitor from a
naturally occurring source (e.g., Streptomyces sp., Pseudomonas
sp., Stylotella aurantium, etc.). Alternatively, a PMCA4b inhibitor
can be synthesized chemically. Further, the routineer would
appreciate, based upon the teachings provided herein, that a PMCA4b
inhibitor composition can be obtained from a recombinant organism.
Compositions and methods for chemically synthesizing PMCA4b
inhibitors and for obtaining them from natural sources are well
known in the art and are described in the art.
[0117] One of skill in the art will appreciate that an inhibitor
can be administered as a small molecule chemical, a protein, an
antibody, a nucleic acid construct encoding a protein, an antisense
nucleic acid, a nucleic acid construct encoding an antisense
nucleic acid, or combinations thereof. Numerous vectors and other
compositions and methods are well known for administering a protein
or a nucleic acid construct encoding a protein to cells or tissues.
Therefore, the invention includes a method of administering a
protein or a nucleic acid encoding a protein that is an inhibitor
of PMCA4b. (Sambrook et al., 2012, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al.,
1997, Current Protocols in Molecular Biology, John Wiley &
Sons, New York).
[0118] One of skill in the art will realize that diminishing the
amount or activity of a molecule that itself increases the amount
or activity of PMCA4b can serve in the compositions and methods of
the present invention to decrease the amount or activity of
PMCA4b.
[0119] Antisense oligonucleotides are DNA or RNA molecules that are
complementary to some portion of an RNA molecule. When present in a
cell, antisense oligonucleotides hybridize to an existing RNA
molecule and inhibit translation into a gene product. Inhibiting
the expression of a gene using an antisense oligonucleotide is well
known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as
are methods of expressing an antisense oligonucleotide in a cell
(Inoue, U.S. Pat. No. 5,190,931). The methods of the invention
include the use of an antisense oligonucleotide to diminish the
amount of PMCA4b, or to diminish the amount of a molecule that
causes an increase in the amount or activity of PMCA4b, thereby
decreasing the amount or activity of PMCA4b.
[0120] Contemplated in the present invention are antisense
oligonucleotides that are synthesized and provided to the cell by
way of methods well known to those of ordinary skill in the art. As
an example, an antisense oligonucleotide can be synthesized to be
between about 10 and about 100, more preferably between about 15
and about 50 nucleotides long. The synthesis of nucleic acid
molecules is well known in the art, as is the synthesis of modified
antisense oligonucleotides to improve biological activity in
comparison to unmodified antisense oligonucleotides (Tullis, 1991,
U.S. Pat. No. 5,023,243).
[0121] Similarly, the expression of a gene may be inhibited by the
hybridization of an antisense molecule to a promoter or other
regulatory element of a gene, thereby affecting the transcription
of the gene. Methods for the identification of a promoter or other
regulatory element that interacts with a gene of interest are well
known in the art, and include such methods as the yeast two hybrid
system (Bartel and Fields, eds., In: The Yeast Two Hybrid System,
Oxford University Press, Cary, N.C.).
[0122] Alternatively, inhibition of a gene expressing PMCA4b, or of
a gene expressing a protein that increases the level or activity of
PMCA4b, can be accomplished through the use of a ribozyme. Using
ribozymes for inhibiting gene expression is well known to those of
skill in the art (see, e.g., Cech et al., 1992, J. Biol. Chem.
267:17479; Hampel et al., 1989, Biochemistry 28: 4929; Altman et
al., U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA
molecules with the ability to cleave other single-stranded RNA
molecules. Ribozymes are known to be sequence specific, and can
therefore be modified to recognize a specific nucleotide sequence
(Cech, 1988, J. Amer. Med. Assn. 260:3030), allowing the selective
cleavage of specific mRNA molecules. Given the nucleotide sequence
of the molecule, one of ordinary skill in the art could synthesize
an antisense oligonucleotide or ribozyme without undue
experimentation, provided with the disclosure and references
incorporated herein.
[0123] One of skill in the art will appreciate that inhibitors of
PMCA4b can be administered acutely (e.g., over a short period of
time, such as a day, a week or a month) or chronically (e.g., over
a long period of time, such as several months or a year or more).
One of skill in the art will appreciate that inhibitors of PMCA4b
can be administered singly or in any combination with other agents.
Further, PMCA4b inhibitors can be administered singly or in any
combination in a temporal sense, in that they may be administered
concurrently, or before, and/or after each other. One of ordinary
skill in the art will appreciate, based on the disclosure provided
herein, that PMCA4b inhibitor compositions can be used to treat or
prevent a disease or disorder in a subject in need thereof, and
that an inhibitor composition can be used alone or in any
combination with another inhibitor to effect a therapeutic
result.
[0124] In various embodiments, any of the inhibitors of PMCA4b of
the invention described herein can be administered alone or in
combination with other inhibitors of other molecules associated
with cancer.
[0125] It will be appreciated by one of skill in the art, when
armed with the present disclosure including the methods detailed
herein, that the invention is not limited to treatment of a disease
or disorder that is already established. Particularly, the disease
or disorder need not have manifested to the point of detriment to
the subject; indeed, the disease or disorder need not be detected
in a subject before treatment is administered. That is, significant
disease or disorder does not have to occur before the present
invention may provide benefit. Therefore, the present invention
includes a method for preventing a disease or disorder in a
subject, in that a PMCA4b inhibitor composition, as discussed
previously elsewhere herein, can be administered to a subject prior
to the onset of the disease or disorder, thereby preventing the
disease or disorder from developing. The preventive methods
described herein also include the treatment of a subject that is in
remission for the prevention of a recurrence of a disease or
disorder.
[0126] One of skill in the art, when armed with the disclosure
herein, would appreciate that the prevention of a disease or
disorder encompasses administering to a subject a PMCA4b inhibitor
composition as a preventative measure against the disease or
disorder. As more fully discussed elsewhere herein, methods of
decreasing the level or activity of PMCA4b encompass a wide
plethora of techniques for decreasing not only PMCA4b activity, but
also for decreasing expression of a nucleic acid encoding PMCA4b,
including either a decrease in transcription, a decrease in
translation, or both.
[0127] Additionally, as disclosed elsewhere herein, one skilled in
the art would understand, once armed with the teaching provided
herein, that the present invention encompasses a method of
preventing a wide variety of diseases, disorders and pathologies
where a decrease in expression and/or activity of PMCA4b mediates,
treats or prevents the disease, disorder or pathology. Methods for
assessing whether a disease relates to the levels or activity of
PMCA4b are known in the art. Further, the invention encompasses
treatment or prevention of such diseases discovered in the
future.
[0128] The invention encompasses administration of an inhibitor of
PMCA4b to practice the methods of the invention; the skilled
artisan would understand, based on the disclosure provided herein,
how to formulate and administer the appropriate PMCA4b inhibitor to
a subject. However, the present invention is not limited to any
particular method of administration or treatment regimen.
Methods of Identifying a Modulator of a Renalase Receptor
[0129] The current invention relates to a methods of identifying a
compound that modulates the activity of a renalase receptor, such
as, for example, PMCA4b. In some embodiments, the method of
identifying of the invention identifies an inhibitor compound that
decreases the activity of a renalase receptor, such as, for
example, PMCA4b. In other embodiments, the method of identifying of
the invention identifies an activator compound that increases the
activity of a renalase receptor, such as, for example, PMCA4b.
[0130] Other methods, as well as variation of the methods disclosed
herein will be apparent from the description of this invention. In
various embodiments, the test compound concentration in the
screening assay can be fixed or varied. A single test compound, or
a plurality of test compounds, can be tested at one time. In some
embodiments, the method of identifying is a high-throughput screen.
Suitable test compounds that may be used include, but are not
limited to, proteins, nucleic acids, antisense nucleic acids,
shRNA, small molecules, antibodies and peptides.
[0131] The invention relates to a method for screening test
compounds to identify a modulator compound by its ability to
modulate (i.e., increase or decrease) the level of activity of a
renalase receptor, such as, for example, PMCA4b, in the presence
and absence of the test compound. The activity of the renalase
receptor that is assessed can be any measurable activity of the
renalase receptor. In one embodiment, the activity of the renalase
receptor that is assessed is the level of mitogen-activated protein
kinase (MAPK) signaling. In another embodiment, the activity of the
renalase receptor that is assessed is the level of ATPase
activity.
[0132] Test compounds that can be assessed in the methods of the
invention include a chemical compound, a protein, a peptide, a
peptidomemetic, an antibody, a nucleic acid, an antisense nucleic
acid, an siRNA, a miRNA, a shRNA, a ribozyme, an allosteric
modulator, and a small molecule chemical compound.
[0133] The test compounds can be obtained using any of the numerous
approaches in combinatorial library methods known in the art,
including: biological libraries; spatially addressable parallel
solid phase or solution phase libraries; synthetic library methods
requiring deconvolution; the "one-bead one-compound" library
method; and synthetic library methods using affinity chromatography
selection. The biological library approach is limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam et al., 1997, Anticancer Drug Des. 12:45).
[0134] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example, in: DeWitt et al., 1993,
Proc. Natl. Acad. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678;
Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem.
37:1233.
[0135] Libraries of compounds may be presented in solution (e.g.,
Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991,
Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA
89:1865-1869) or on phage (Scott and Smith, 1990, Science
249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al.,
1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J.
Mol. Biol. 222:301-310; and Ladner supra).
[0136] In situations where "high-throughput" modalities are
preferred, it is typical to that new chemical entities with useful
properties are generated by identifying a chemical compound (called
a "lead compound") with some desirable property or activity,
creating variants of the lead compound, and evaluating the property
and activity of those variant compounds.
[0137] In one embodiment, high throughput screening methods involve
providing a library containing a large number of test compounds
potentially having the desired activity. Such "combinatorial
chemical libraries" are then screened in one or more assays, as
described herein, to identify those library members (particular
chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
Compositions and Methods of Treatment and Prevention
[0138] In various embodiments, the present invention includes
renalase activator compositions and methods of increasing the level
or activity of renalase, or a fragment thereof, in a subject, a
tissue, or an organ in need thereof. In various embodiments, the
renalase activator compositions and methods of treatment of the
invention increase the amount of renalase polypeptide, the amount
of renalase mRNA, the amount of renalase enzymatic activity, the
amount of renalase substrate binding activity, or a combination
thereof. In various embodiments, the diseases and disorders where
in increase in renalase may improve therapeutic outcome include,
but are not limited to, pancreatic diseases or disorders such as
pancreatitis.
[0139] It will be understood by one skilled in the art, based upon
the disclosure provided herein, that an increase in the level of
renalase encompasses the increase in renalase expression, including
transcription, translation, or both. The skilled artisan will also
appreciate, once armed with the teachings of the present invention,
that an increase in the level of renalase includes an increase in
renalase activity (e.g., enzymatic activity, substrate binding
activity, etc.). Thus, increasing the level or activity of renalase
includes, but is not limited to, increasing the amount of renalase
polypeptide, and increasing transcription, translation, or both, of
a nucleic acid encoding renalase; and it also includes increasing
any activity of a renalase polypeptide as well. The renalase
activator compositions and methods of the invention can selectively
activate renalase, or can activate both renalase and another
molecule.
[0140] Thus, the present invention relates to the prevention and
treatment of a disease or disorder by administration of a
therapeutically effective amount of a renalase polypeptide, a
recombinant renalase polypeptide, an active renalase polypeptide
fragment (i.e., renalase peptide), or an activator of renalase
expression or activity, to a subject in need thereof, for the
treatment or prevention of a disease or disorder, or its associated
signs, symptoms or pathologies. In some embodiments, the renalase
polypeptide comprises the amino acid of SEQ ID NO: 8 or SEQ ID NO:
9, or an analogue or homolog thereof. In some embodiments, the
renalase of the invention is a renalase polypeptide fragment
comprising at least a portion of the amino acid sequence of SEQ ID
NO: 8 or SEQ ID NO: 9, or an analogue or homolog thereof. In some
embodiments, the renalase polypeptide fragment is a peptide that
retains its protective activity, but does not exhibit detectable
NADH oxidase activity. In some embodiments, the renalase
polypeptide fragment is a peptide that retains its protective
activity, but does not exhibit detectable amine oxidase activity.
In a particular embodiment, the renalase polypeptide fragment is a
peptide comprising the amino acid sequence of SEQ ID NO: 3, or an
analogue or homolog thereof. In another particular embodiment, the
renalase polypeptide fragment is a peptide comprising the amino
acid sequence of SEQ ID NO: 4, or an analogue or homolog thereof.
In another particular embodiment, the renalase polypeptide fragment
is a peptide comprising the amino acid sequence of SEQ ID NO: 5, or
an analogue or homolog thereof. In another particular embodiment,
the renalase polypeptide fragment is a peptide consisting of the
amino acid sequence of SEQ ID NO: 3, or an analogue or homolog
thereof. In another particular embodiment, the renalase polypeptide
fragment is a peptide consisting of the amino acid sequence of SEQ
ID NO: 4, or an analogue or homolog thereof. In another particular
embodiment, the renalase polypeptide fragment is a peptide
consisting of the amino acid sequence of SEQ ID NO: 5, or an
analogue or homolog thereof. In some embodiments, the renalase
polypeptide, or fragment thereof, is conjugated to another
molecule.
[0141] It is understood by one skilled in the art, that an increase
in the level of renalase encompasses an increase in the amount of
renalase, or fragment thereof (e.g., by administration of renalase
or a fragment thereof, by increasing renalase protein expression,
etc.). Additionally, the skilled artisan would appreciate, that an
increase in the level of renalase includes an increase in renalase
activity. Thus, increasing the level or activity of renalase
includes, but is not limited to, the administration of renalase or
a fragment thereof, as well as increasing transcription,
translation, or both, of a nucleic acid encoding renalase; and it
also includes increasing any activity of renalase as well.
[0142] The increased level or activity of renalase can be assessed
using a wide variety of methods, including those disclosed herein,
as well as methods well-known in the art or to be developed in the
future. That is, the routineer would appreciate, based upon the
disclosure provided herein, that increasing the level or activity
of renalase can be readily assessed using methods that assess the
level of a nucleic acid encoding renalase (e.g., mRNA), the level
of renalase polypeptide, and/or the level of renalase activity in a
biological sample obtained from a subject.
[0143] One skilled in the art, based upon the disclosure provided
herein, would understand that the invention is useful in subjects
who, in whole (e.g., systemically) or in part (e.g., locally,
tissue, organ), are being or will be, treated for a disease or
disorder associated with a diminished level or activity of
renalase. The skilled artisan will appreciate, based upon the
teachings provided herein, that the diseases and disorders
treatable by the compositions and methods described herein
encompass any disease or disorder where in an increase in renalase
will promote a positive therapeutic outcome.
[0144] One of skill in the art will realize that in addition to
activating renalase directly, diminishing the amount or activity of
a molecule that itself diminishes the amount or activity of
renalase can serve to increase the amount or activity of renalase.
Thus, a renalase activator can include, but should not be construed
as being limited to, a chemical compound, a protein, a
peptidomemetic, an antibody, a ribozyme, and an antisense nucleic
acid molecule. In some embodiments the renalase activator is an
allosteric activator. One of skill in the art would readily
appreciate, based on the disclosure provided herein, that a
renalase activator encompasses a chemical compound that increases
the level, enzymatic activity, or substrate binding activity of
renalase. Additionally, a renalase activator encompasses a
chemically modified compound, and derivatives, as is well known to
one of skill in the chemical arts.
[0145] It will be understood by one skilled in the art, based upon
the disclosure provided herein, that an increase in the level of
renalase encompasses the increase in renalase expression, including
transcription, translation, or both. The skilled artisan will also
appreciate, once armed with the teachings of the present invention,
that an increase in the level of renalase includes an increase in
renalase activity (e.g., enzymatic activity, substrate binding
activity, etc.). Thus, increasing the level or activity of renalase
includes, but is not limited to, increasing the amount of renalase
polypeptide, increasing transcription, translation, or both, of a
nucleic acid encoding renalase; and it also includes increasing any
activity of a renalase polypeptide as well. The renalase activator
compositions and methods of the invention can selectively activate
renalase, or can activate both renalase and another molecule. Thus,
the present invention relates to administration of a renalase
polypeptide, a recombinant renalase polypeptide, an active renalase
polypeptide fragment, or an activator of renalase expression or
activity.
[0146] Further, one of skill in the art would, when equipped with
this disclosure and the methods exemplified herein, appreciate that
a renalase activator includes such activators as discovered in the
future, as can be identified by well-known criteria in the art of
pharmacology, such as the physiological results of activation of
renalase as described in detail herein and/or as known in the art.
Therefore, the present invention is not limited in any way to any
particular renalase activator as exemplified or disclosed herein;
rather, the invention encompasses those activators that would be
understood by the routineer to be useful as are known in the art
and as are discovered in the future.
[0147] Further methods of identifying and producing a renalase
activator are well known to those of ordinary skill in the art,
including, but not limited, obtaining an activator from a naturally
occurring source (e.g., Streptomyces sp., Pseudomonas sp.,
Stylotella aurantium, etc.). Alternatively, a renalase activator
can be synthesized chemically. Further, the routineer would
appreciate, based upon the teachings provided herein, that a
renalase activator can be obtained from a recombinant organism.
Compositions and methods for chemically synthesizing renalase
activators and for obtaining them from natural sources are well
known in the art and are described in the art.
[0148] One of skill in the art will appreciate that an activator
can be administered as a small molecule chemical, a protein, a
nucleic acid construct encoding a protein, or combinations thereof.
Numerous vectors and other compositions and methods are well known
for administering a protein or a nucleic acid construct encoding a
protein to cells or tissues. Therefore, the invention includes a
method of administering a protein or a nucleic acid encoding a
protein that is an activator of renalase. (Sambrook et al., 2012,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York; Ausubel et al., 1997, Current Protocols in
Molecular Biology, John Wiley & Sons, New York).
[0149] One of skill in the art will realize that diminishing the
amount or activity of a molecule that itself diminishes the amount
or activity of renalase can serve to increase the amount or
activity of renalase. Antisense oligonucleotides are DNA or RNA
molecules that are complementary to some portion of a mRNA
molecule. When present in a cell, antisense oligonucleotides
hybridize to an existing mRNA molecule and inhibit translation into
a gene product. Inhibiting the expression of a gene using an
antisense oligonucleotide is well known in the art (Marcus-Sekura,
1988, Anal. Biochem. 172:289), as are methods of expressing an
antisense oligonucleotide in a cell (Inoue, U.S. Pat. No.
5,190,931). The methods of the invention include the use of
antisense oligonucleotide to diminish the amount of a molecule that
causes a decrease in the amount or activity renalase, thereby
increasing the amount or activity of renalase. Contemplated in the
present invention are antisense oligonucleotides that are
synthesized and provided to the cell by way of methods well known
to those of ordinary skill in the art. As an example, an antisense
oligonucleotide can be synthesized to be between about 10 and about
100, more preferably between about 15 and about 50 nucleotides
long. The synthesis of nucleic acid molecules is well known in the
art, as is the synthesis of modified antisense oligonucleotides to
improve biological activity in comparison to unmodified antisense
oligonucleotides (Tullis, 1991, U.S. Pat. No. 5,023,243).
[0150] Similarly, the expression of a gene may be inhibited by the
hybridization of an antisense molecule to a promoter or other
regulatory element of a gene, thereby affecting the transcription
of the gene. Methods for the identification of a promoter or other
regulatory element that interacts with a gene of interest are well
known in the art, and include such methods as the yeast two hybrid
system (Bartel and Fields, eds., In: The Yeast Two Hybrid System,
Oxford University Press, Cary, N.C.).
[0151] Alternatively, inhibition of a gene expressing a protein
that diminishes the level or activity of renalase can be
accomplished through the use of a ribozyme. Using ribozymes for
inhibiting gene expression is well known to those of skill in the
art (see, e.g., Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel
et al., 1989, Biochemistry 28: 4929; Altman et al., U.S. Pat. No.
5,168,053). Ribozymes are catalytic RNA molecules with the ability
to cleave other single-stranded RNA molecules. Ribozymes are known
to be sequence specific, and can therefore be modified to recognize
a specific nucleotide sequence (Cech, 1988, J. Amer. Med. Assn.
260:3030), allowing the selective cleavage of specific mRNA
molecules. Given the nucleotide sequence of the molecule, one of
ordinary skill in the art could synthesize an antisense
oligonucleotide or ribozyme without undue experimentation, provided
with the disclosure and references incorporated herein.
[0152] One of skill in the art will appreciate that a renalase
activator, renalase polypeptide, a recombinant renalase
polypeptide, or an active renalase polypeptide fragment can be
administered singly or in any combination thereof. One of skill in
the art will also appreciate administration can be acute (e.g.,
over a short period of time, such as a day, a week or a month) or
chronic (e.g., over a long period of time, such as several months
or a year or more). Further, a renalase polypeptide, a recombinant
renalase polypeptide, or an active renalase polypeptide fragment
can be administered singly or in any combination thereof in a
temporal sense, in that they may be administered simultaneously,
before, and/or after each other. One of ordinary skill in the art
will appreciate, based on the disclosure provided herein, that a
renalase polypeptide, a recombinant renalase polypeptide, or an
active renalase polypeptide fragment can be used, and that an
activator can be used alone or in any combination with another
renalase polypeptide, recombinant renalase polypeptide, active
renalase polypeptide fragment, or renalase activator to effect a
therapeutic result. One of ordinary skill in the art will also
appreciate, based on the disclosure provided herein, that a
renalase polypeptide, a recombinant renalase polypeptide, or an
active renalase polypeptide fragment can be can be used alone or in
any combination with any other composition, drug, or treatment
useful in treating a pancreatic disease or disorder, such as
pancreatitis. In some embodiments, the administered renalase
polypeptide, recombinant renalase polypeptide, active renalase
polypeptide fragment, or combinations thereof, are administered in
combination with another composition, drug, or treatment that
reduces, inhibits, or blocks the metabolism or degradation of the
administered renalase polypeptide, recombinant renalase
polypeptide, active renalase polypeptide fragment, or combination
thereof.
[0153] It will be appreciated by one of skill in the art, when
armed with the present disclosure including the methods detailed
herein, that the invention is not limited to treatment of a disease
or disorder once is established. Particularly, the symptoms of the
disease or disorder need not have manifested to the point of
detriment to the subject; indeed, the disease or disorder need not
be detected in a subject before treatment is administered. That is,
significant pathology from disease or disorder does not have to
occur before the present invention may provide benefit. Therefore,
the present invention, as described more fully herein, includes a
method for preventing diseases and disorders in a subject, in that
a renalase molecule (e.g., polypeptide, peptide, etc.), or a
renalase activator, as discussed elsewhere herein, can be
administered to a subject prior to the onset of the disease or
disorder, thereby preventing the disease or disorder from
developing.
[0154] One of skill in the art, when armed with the disclosure
herein, would appreciate that the prevention of a disease or
disorder in a subject encompasses administering to a subject a
renalase polypeptide, a recombinant renalase polypeptide, an active
renalase polypeptide fragment, or renalase activator as a
preventative measure against a disease or disorder.
[0155] As more fully discussed elsewhere herein, methods of
increasing the level or activity of a renalase encompass a wide
plethora of techniques for increasing not only renalase activity,
but also for increasing expression of a nucleic acid encoding
renalase. Additionally, as disclosed elsewhere herein, one skilled
in the art would understand, once armed with the teaching provided
herein, that the present invention encompasses a method of
preventing a wide variety of diseases or disorders where increased
expression and/or activity of renalase mediates, treats or prevents
a disease or disorder. Further, the invention encompasses treatment
or prevention of such diseases or disorders discovered in the
future.
[0156] The invention encompasses administration of a renalase
polypeptide, a recombinant renalase polypeptide, an active renalase
polypeptide fragment, or a renalase activator to practice the
methods of the invention; the skilled artisan would understand,
based on the disclosure provided herein, how to formulate and
administer the appropriate renalase polypeptide, recombinant
renalase polypeptide, active renalase polypeptide fragment, or
renalase activator to a subject. However, the present invention is
not limited to any particular method of administration or treatment
regimen. This is especially true where it would be appreciated by
one skilled in the art, equipped with the disclosure provided
herein, including the reduction to practice using an art-recognized
model of ischemia-reperfusion injury, that methods of administering
a renalase polypeptide, a recombinant renalase polypeptide, an
active renalase polypeptide fragment, or renalase activator can be
determined by one of skill in the pharmacological arts.
[0157] As used herein, the term "pharmaceutically-acceptable
carrier" means a chemical composition with which an appropriate
renalase modulator may be combined and which, following the
combination, can be used to administer the appropriate renalase
modulator thereof, to a subject.
Methods of Diagnosis
[0158] In some embodiments, a change (i.e., increase or decrease)
in the level of renalase compared with a comparator is used in the
methods of the invention as marker for the diagnosis of a
pancreatic disease or disorder, as well as for monitoring the
treatment the effectiveness of a pancreatic disease or
disorder.
[0159] In one embodiment, the invention is a method of diagnosing a
pancreatic disease or disorder of a subject by assessing the level
of renalase in a biological sample of the subject. In one
embodiment, the biological sample of the subject is a bodily fluid.
Non-limiting examples of bodily fluids in which the level of
renalase can be assessed include, but are not limited to, blood,
serum, plasma and urine. In various embodiments, the level of
renalase in the biological sample of the subject is compared with
the renalase level in a comparator. Non-limiting examples of
comparators include, but are not limited to, a negative control, a
positive control, an expected normal background value of the
subject, a historical normal background value of the subject, an
expected normal background value of a population that the subject
is a member of, or a historical normal background value of a
population that the subject is a member of.
[0160] In another embodiment, the invention is a method of
monitoring the progression of a pancreatic disease or disorder of a
subject by assessing the level of renalase in a biological sample
of the subject. In one embodiment, the biological sample of the
subject is a bodily fluid. Non-limiting examples of bodily fluids
in which the level of renalase can be assessed include, but are not
limited to, blood, serum, plasma and urine. In various embodiments,
the level of renalase in the biological sample of the subject is
compared with the renalase level in a comparator. Non-limiting
examples of comparators include, but are not limited to, a negative
control, a positive control, an expected normal background value of
the subject, a historical normal background value of the subject,
an expected normal background value of a population that the
subject is a member of, or a historical normal background value of
a population that the subject is a member of.
[0161] In a further embodiment, the invention is a method of
assessing the severity of a pancreatic disease or disorder of a
subject by assessing the level of renalase in a biological sample
of the subject. In one embodiment, the biological sample of the
subject is a bodily fluid. Non-limiting examples of bodily fluids
in which the level of renalase can be assessed include, but are not
limited to, blood, serum, plasma and urine. In various embodiments,
the level of renalase in the biological sample of the subject is
compared with the renalase level in a comparator. Non-limiting
examples of comparators include, but are not limited to, a negative
control, a positive control, an expected normal background value of
the subject, a historical normal background value of the subject,
an expected normal background value of a population that the
subject is a member of, or a historical normal background value of
a population that the subject is a member of.
[0162] In another embodiment, the invention is a method of
selecting a treatment regimen to treat a pancreatic disease or
disorder of a subject by assessing the level of renalase in a
biological sample of the subject. In one embodiment biological
sample of the subject is a bodily fluid. Non-limiting examples of
bodily fluids in which the level of renalase can be assessed
include, but are not limited to, blood, serum, plasma and urine. In
various embodiments, the level of renalase in the biological sample
of the subject is compared with the renalase level in a comparator.
Non-limiting examples of comparators include, but are not limited
to, a negative control, a positive control, an expected normal
background value of the subject, a historical normal background
value of the subject, an expected normal background value of a
population that the subject is a member of, or a historical normal
background value of a population that the subject is a member
of.
[0163] In another embodiment, the invention is a method of
monitoring the effect of a treatment of a pancreatic disease or
disorder of a subject by assessing the level of renalase in a
biological sample of the subject. In one embodiment, the biological
sample of the subject is a bodily fluid. Non-limiting examples of
bodily fluids in which the level of renalase can be assessed
include, but are not limited to, blood, serum, plasma and urine. In
various embodiments, the level of renalase in the biological sample
of the subject is compared with the renalase level in a comparator.
Non-limiting examples of comparators include, but are not limited
to, a negative control, a positive control, an expected normal
background value of the subject, a historical normal background
value of the subject, an expected normal background value of a
population that the subject is a member of, or a historical normal
background value of a population that the subject is a member
of.
[0164] In various embodiments, the subject is a human subject, and
may be of any race, sex and age. Representative subjects include
those who are suspected of having experienced a pancreatic disease
or disorder, such as pancreatitis, those who have been diagnosed as
having experienced a pancreatic disease or disorder, such as
pancreatitis, those who have been diagnosed as having a disease or
disorder associated with a pancreatic disease or disorder, and
those who are at risk of developing a pancreatic disease or
disorder, such as pancreatitis.
[0165] Information obtained from the methods of the invention
described herein can be used alone, or in combination with other
information (e.g., disease status, disease history, vital signs,
blood chemistry, etc.) from the subject or from the biological
sample obtained from the subject.
[0166] In the diagnostic methods of the invention, a biological
sample obtained from a subject is assessed for the level of
renalase contained therein. In one embodiment, the biological
sample is a sample containing at least a fragment of a renalase
polypeptide useful in the methods described herein.
[0167] In other various embodiments of the methods of the
invention, the level of renalase is determined to be reduced when
the level of renalase is reduced by at least 10%, by at least 20%,
by at least 30%, by at least 40%, by at least 50%, by at least 60%,
by at least 70%, by at least 80%, by at least 90%, or by at least
100%, when compared to with a comparator control. In various
embodiments, a reduced level of renalase is indicative of a disease
or disorder.
[0168] In other various embodiments of the methods of the
invention, the level of renalase is determined to be increased when
the level of renalase is increased by at least 10%, by at least
20%, by at least 30%, by at least 40%, by at least 50%, by at least
60%, by at least 70%, by at least 80%, by at least 90%, or by at
least 100%, when compared to with a comparator control. In various
embodiments, an increased level of renalase is indicative of a
disease or disorder.
[0169] In the methods of the invention, a biological sample from a
subject is assessed for the level of renalase in the biological
sample obtained from the patient. The level of renalase in the
biological sample can be determined by assessing the amount of
renalase polypeptide in the biological sample, the amount of
renalase mRNA in the biological sample, the amount of renalase
enzymatic activity in the biological sample, or a combination
thereof.
[0170] In various embodiments of the methods of the invention,
methods of measuring renalase levels in a biological sample
obtained from a patient include, but are not limited to, an
immunochromatography assay, an immunodot assay, a Luminex assay, an
ELISA assay, an ELISPOT assay, a protein microarray assay, a
Western blot assay, a mass spectrophotometry assay, a
radioimmunoassay (RIA), a radioimmunodiffusion assay, a liquid
chromatography-tandem mass spectrometry assay, an ouchterlony
immunodiffusion assay, reverse phase protein microarray, a rocket
immunoelectrophoresis assay, an immunohistostaining assay, an
immunoprecipitation assay, a complement fixation assay, FACS, an
enzyme-substrate binding assay, an enzymatic assay, an enzymatic
assay employing a detectable molecule, such as a chromophore,
fluorophore, or radioactive substrate, a substrate binding assay
employing such a substrate, a substrate displacement assay
employing such a substrate, and a protein chip assay (see also,
2007, Van Emon, Immunoassay and Other Bioanalytical Techniques, CRC
Press; 2005, Wild, Immunoassay Handbook, Gulf Professional
Publishing; 1996, Diamandis and Christopoulos, Immunoassay,
Academic Press; 2005, Joos, Microarrays in Clinical Diagnosis,
Humana Press; 2005, Hamdan and Righetti, Proteomics Today, John
Wiley and Sons; 2007).
Therapeutic Inhibitor Compositions and Methods
[0171] In various embodiments, the present invention includes
renalase inhibitor compositions and methods of treating or
preventing a disease or disorder where a diminished activity or
level of renalase is desired. One non-limiting example of a disease
or disorder where a diminished activity or level of renalase is
desired which can be treated or prevented with the compositions and
methods of the invention includes cancer. In various embodiments,
the renalase inhibitor compositions and methods of treatment or
prevention of the invention diminish the amount of renalase
polypeptide, the amount of renalase mRNA, the amount of renalase
enzymatic activity, the amount of renalase substrate binding
activity, or a combination thereof.
[0172] It will be understood by one skilled in the art, based upon
the disclosure provided herein, that a decrease in the level of
renalase encompasses the decrease in renalase expression, including
transcription, translation, or both. The skilled artisan will also
appreciate, once armed with the teachings of the present invention,
that a decrease in the level of renalase includes a decrease in
renalase activity (e.g., enzymatic activity, substrate binding
activity, etc.). Thus, decreasing the level or activity of renalase
includes, but is not limited to, decreasing transcription,
translation, or both, of a nucleic acid encoding renalase; and it
also includes decreasing any activity of a renalase polypeptide as
well. The renalase inhibitor compositions and methods of the
invention can selectively inhibit renalase, or can inhibit both
renalase and another molecule.
[0173] Inhibition of renalase can be assessed using a wide variety
of methods, including those disclosed herein, as well as methods
known in the art or to be developed in the future. That is, the
routineer would appreciate, based upon the disclosure provided
herein, that decreasing the level or activity of renalase can be
readily assessed using methods that assess the level of a nucleic
acid encoding renalase (e.g., mRNA), the level of a renalase
polypeptide present in a biological sample, the level of renalase
activity (e.g., enzymatic activity, substrate binding activity,
etc.), or combinations thereof.
[0174] One skilled in the art, based upon the disclosure provided
herein, would understand that the invention is useful in treating
or preventing in a subject in need thereof, whether or not the
subject is also being treated with other medication or therapy.
Further, the skilled artisan would further appreciate, based upon
the teachings provided herein, that the disease or disorders
treatable by the compositions and methods described herein
encompass any disease or disorder where renalase plays a role and
where diminished renalase level or activity will promote a positive
therapeutic outcome.
[0175] The renalase inhibitor compositions and methods of the
invention that decrease the level or activity (e.g., enzymatic
activity, substrate binding activity, etc.) of renalase include,
but should not be construed as being limited to, a chemical
compound, a protein, a peptide, a peptidomemetic, an antibody, a
ribozyme, a small molecule chemical compound, an antisense nucleic
acid molecule (e.g., siRNA, miRNA, etc.), or combinations thereof.
In some embodiments, the inhibitor is an allosteric inhibitor. One
of skill in the art would readily appreciate, based on the
disclosure provided herein, that a renalase inhibitor composition
encompasses a chemical compound that decreases the level or
activity of renalase. Additionally, a renalase inhibitor
composition encompasses a chemically modified compound, and
derivatives, as is well known to one of skill in the chemical
arts.
[0176] The renalase inhibitor compositions and methods of the
invention that decrease the level or activity (e.g., enzymatic
activity, substrate binding activity, etc.) of renalase include
antibodies. The antibodies of the invention include a variety of
forms of antibodies including, for example, polyclonal antibodies,
monoclonal antibodies, intracellular antibodies ("intrabodies"),
Fv, Fab and F(ab)2, single chain antibodies (scFv), heavy chain
antibodies (such as camelid antibodies), synthetic antibodies,
chimeric antibodies, and a humanized antibodies. In one embodiment,
the antibody of the invention is an antibody that specifically
binds to renalase.
[0177] Further, one of skill in the art, when equipped with this
disclosure and the methods exemplified herein, would appreciate
that a renalase inhibitor composition includes such inhibitors as
discovered in the future, as can be identified by well-known
criteria in the art of pharmacology, such as the physiological
results of inhibition of renalase as described in detail herein
and/or as known in the art. Therefore, the present invention is not
limited in any way to any particular renalase inhibitor composition
as exemplified or disclosed herein; rather, the invention
encompasses those inhibitor compositions that would be understood
by the routineer to be useful as are known in the art and as are
discovered in the future.
[0178] Further methods of identifying and producing renalase
inhibitor compositions are well known to those of ordinary skill in
the art, including, but not limited, obtaining an inhibitor from a
naturally occurring source (e.g., Streptomyces sp., Pseudomonas
sp., Stylotella aurantium, etc.). Alternatively, a renalase
inhibitor can be synthesized chemically. Further, the routineer
would appreciate, based upon the teachings provided herein, that a
renalase inhibitor composition can be obtained from a recombinant
organism. Compositions and methods for chemically synthesizing
renalase inhibitors and for obtaining them from natural sources are
well known in the art and are described in the art.
[0179] One of skill in the art will appreciate that an inhibitor
can be administered as a small molecule chemical, a protein, an
antibody, a nucleic acid construct encoding a protein, an antisense
nucleic acid, a nucleic acid construct encoding an antisense
nucleic acid, or combinations thereof. Numerous vectors and other
compositions and methods are well known for administering a protein
or a nucleic acid construct encoding a protein to cells or tissues.
Therefore, the invention includes a method of administering a
protein or a nucleic acid encoding a protein that is an inhibitor
of renalase. (Sambrook et al., 2012, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel
et al., 1997, Current Protocols in Molecular Biology, John Wiley
& Sons, New York).
[0180] One of skill in the art will realize that diminishing the
amount or activity of a molecule that itself increases the amount
or activity of renalase can serve in the compositions and methods
of the present invention to decrease the amount or activity of
renalase.
[0181] Antisense oligonucleotides are DNA or RNA molecules that are
complementary to some portion of an RNA molecule. When present in a
cell, antisense oligonucleotides hybridize to an existing RNA
molecule and inhibit translation into a gene product. Inhibiting
the expression of a gene using an antisense oligonucleotide is well
known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as
are methods of expressing an antisense oligonucleotide in a cell
(Inoue, U.S. Pat. No. 5,190,931). The methods of the invention
include the use of an antisense oligonucleotide to diminish the
amount of renalase, or to diminish the amount of a molecule that
causes an increase in the amount or activity of renalase, thereby
decreasing the amount or activity of renalase.
[0182] Contemplated in the present invention are antisense
oligonucleotides that are synthesized and provided to the cell by
way of methods well known to those of ordinary skill in the art. As
an example, an antisense oligonucleotide can be synthesized to be
between about 10 and about 100, more preferably between about 15
and about 50 nucleotides long. The synthesis of nucleic acid
molecules is well known in the art, as is the synthesis of modified
antisense oligonucleotides to improve biological activity in
comparison to unmodified antisense oligonucleotides (Tullis, 1991,
U.S. Pat. No. 5,023,243).
[0183] Similarly, the expression of a gene may be inhibited by the
hybridization of an antisense molecule to a promoter or other
regulatory element of a gene, thereby affecting the transcription
of the gene. Methods for the identification of a promoter or other
regulatory element that interacts with a gene of interest are well
known in the art, and include such methods as the yeast two hybrid
system (Bartel and Fields, eds., In: The Yeast Two Hybrid System,
Oxford University Press, Cary, N.C.).
[0184] Alternatively, inhibition of a gene expressing renalase, or
of a gene expressing a protein that increases the level or activity
of renalase, can be accomplished through the use of a ribozyme.
Using ribozymes for inhibiting gene expression is well known to
those of skill in the art (see, e.g., Cech et al., 1992, J. Biol.
Chem. 267:17479; Hampel et al., 1989, Biochemistry 28: 4929; Altman
et al., U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA
molecules with the ability to cleave other single-stranded RNA
molecules. Ribozymes are known to be sequence specific, and can
therefore be modified to recognize a specific nucleotide sequence
(Cech, 1988, J. Amer. Med. Assn. 260:3030), allowing the selective
cleavage of specific mRNA molecules. Given the nucleotide sequence
of the molecule, one of ordinary skill in the art could synthesize
an antisense oligonucleotide or ribozyme without undue
experimentation, provided with the disclosure and references
incorporated herein.
[0185] One of skill in the art will appreciate that inhibitors of
renalase can be administered acutely (e.g., over a short period of
time, such as a day, a week or a month) or chronically (e.g., over
a long period of time, such as several months or a year or more).
One of skill in the art will appreciate that inhibitors of renalase
can be administered singly or in any combination with other agents.
Further, renalase inhibitors can be administered singly or in any
combination in a temporal sense, in that they may be administered
concurrently, or before, and/or after each other. One of ordinary
skill in the art will appreciate, based on the disclosure provided
herein, that renalase inhibitor compositions can be used to treat
or prevent a disease or disorder in a subject in need thereof, and
that an inhibitor composition can be used alone or in any
combination with another inhibitor to effect a therapeutic
result.
[0186] In various embodiments, any of the inhibitors of renalase of
the invention described herein can be administered alone or in
combination with other inhibitors of other molecules associated
with cancer.
[0187] It will be appreciated by one of skill in the art, when
armed with the present disclosure including the methods detailed
herein, that the invention is not limited to treatment of a disease
or disorder that is already established. Particularly, the disease
or disorder need not have manifested to the point of detriment to
the subject; indeed, the disease or disorder need not be detected
in a subject before treatment is administered. That is, significant
disease or disorder does not have to occur before the present
invention may provide benefit. Therefore, the present invention
includes a method for preventing a disease or disorder in a
subject, in that a renalase inhibitor composition, as discussed
previously elsewhere herein, can be administered to a subject prior
to the onset of the disease or disorder, thereby preventing the
disease or disorder from developing. The preventive methods
described herein also include the treatment of a subject that is in
remission for the prevention of a recurrence of a disease or
disorder.
[0188] One of skill in the art, when armed with the disclosure
herein, would appreciate that the prevention of a disease or
disorder encompasses administering to a subject a renalase
inhibitor composition as a preventative measure against the disease
or disorder. As more fully discussed elsewhere herein, methods of
decreasing the level or activity of renalase encompass a wide
plethora of techniques for decreasing not only renalase activity,
but also for decreasing expression of a nucleic acid encoding
renalase, including either a decrease in transcription, a decrease
in translation, or both.
[0189] Additionally, as disclosed elsewhere herein, one skilled in
the art would understand, once armed with the teaching provided
herein, that the present invention encompasses a method of
preventing a wide variety of diseases, disorders and pathologies
where a decrease in expression and/or activity of renalase
mediates, treats or prevents the disease, disorder or pathology.
Methods for assessing whether a disease relates to the levels or
activity of renalase are known in the art. Further, the invention
encompasses treatment or prevention of such diseases discovered in
the future.
[0190] The invention encompasses administration of an inhibitor of
renalase to practice the methods of the invention; the skilled
artisan would understand, based on the disclosure provided herein,
how to formulate and administer the appropriate renalase inhibitor
to a subject. However, the present invention is not limited to any
particular method of administration or treatment regimen.
Kits
[0191] The present invention also pertains to kits useful in the
methods of the invention. Such kits comprise various combinations
of components useful in any of the methods described elsewhere
herein, including for example, a PMCA4b modulator, a renalase
activator, a renalase inhibitor, materials for quantitatively
analyzing renalase polypeptide or renalase nucleic acid, materials
for assessing the activity of a renalase polypeptide or a renalase
nucleic acid, and instructional material. For example, in one
embodiment, the kit comprises components useful for the
quantification of renalase nucleic acid in a biological sample. In
another embodiment, the kit comprises components useful for the
quantification of renalase polypeptide in a biological sample. In a
further embodiment, the kit comprises components useful for the
assessment of the activity (e.g., enzymatic activity, substrate
binding activity, etc.) of a renalase polypeptide in a biological
sample.
[0192] In a further embodiment, the kit comprises the components of
method for diagnosing a disease or disorder, or for monitoring the
effectiveness of a treatment administered to a subject in need
thereof, containing instructional material and the components for
determining whether the level of renalase in a biological sample
obtained from the subject is modulated during or after
administration of the treatment. In various embodiments, to
determine whether the level of renalase is modulated in a
biological sample obtained from the subject, the level of renalase
is compared with the level of at least one comparator control
contained in the kit, such as a positive control, a negative
control, a historical control, a historical norm, or the level of
another reference molecule in the biological sample. In certain
embodiments, the ratio of renalase and a reference molecule is
determined to aid in the monitoring of the treatment.
Pharmaceutical Compositions and Administration
[0193] Compositions comprising a PMCA4b modulator can be formulated
and administered to a subject, as now described. In one embodiment,
the PMCA4b modulator is a PMCA4b activator. In another embodiment,
the PMCA4b modulator is a PMCA4b inhibitor. For example,
compositions identified as useful PMCA4b modulators for the
treatment and/or prevention of a disease or disorder can be
formulated and administered to a subject, as now described.
[0194] Compositions comprising a renalase polypeptide, a renalase
polypeptide fragment, an activator of renalase level or activity,
or an inhibitor of renalase level or activity can be formulated and
administered to a subject, as now described. By way of non-limiting
examples, a composition identified as a useful renalase active or
activator, including renalase polypeptides, recombinant renalase
polypeptides, and active renalase polypeptide fragments, for the
treatment and/or prevention of a disease or disorder can be
formulated and administered to a subject, as now described. By way
of more non-limiting examples, a composition identified as a useful
renalase inhibitor, including a chemical compound, a protein, a
peptide, a peptidomemetic, an antibody, a ribozyme, a small
molecule chemical compound, an antisense nucleic acid molecule
(e.g., siRNA, miRNA, etc.), for the treatment and/or prevention of
a disease or disorder can be formulated and administered to a
subject, as now described.
[0195] The invention encompasses the preparation and use of
pharmaceutical compositions comprising a composition useful for the
treatment or prevention of a disease or disorder, disclosed herein
as an active ingredient. Such a pharmaceutical composition may
consist of the active ingredient alone, in a form suitable for
administration to a subject, or the pharmaceutical composition may
comprise the active ingredient and one or more pharmaceutically
acceptable carriers, one or more additional ingredients, or some
combination of these. The active ingredient may be present in the
pharmaceutical composition in the form of a physiologically
acceptable ester or salt, such as in combination with a
physiologically acceptable cation or anion, as is well known in the
art. In various embodiments, the active ingredient is a PMCA4b
modulator, as elsewhere described herein.
[0196] As used herein, the term "pharmaceutically-acceptable
carrier" means a chemical composition with which an appropriate
PMCA4b modulator thereof, may be combined and which, following the
combination, can be used to administer the appropriate PMCA4b
modulator thereof, to a subject.
[0197] The pharmaceutical compositions useful for practicing the
invention may be administered to deliver a dose of between about
0.1 ng/kg/day and 100 mg/kg/day, or more.
[0198] In various embodiments, the pharmaceutical compositions
useful in the methods of the invention may be administered, by way
of example, systemically, parenterally, or topically, such as, in
oral formulations, inhaled formulations, including solid or
aerosol, and by topical or other similar formulations. In addition
to the appropriate therapeutic composition, such pharmaceutical
compositions may contain pharmaceutically acceptable carriers and
other ingredients known to enhance and facilitate drug
administration. Other possible formulations, such as nanoparticles,
liposomes, resealed erythrocytes, and immunologically based systems
may also be used to administer an appropriate modulator thereof,
according to the methods of the invention.
[0199] As used herein, the term "physiologically acceptable" ester
or salt means an ester or salt form of the active ingredient which
is compatible with any other ingredients of the pharmaceutical
composition, which is not deleterious to the subject to which the
composition is to be administered.
[0200] The formulations of the pharmaceutical compositions
described herein may be prepared by any method known or hereafter
developed in the art of pharmacology. In general, such preparatory
methods include the step of bringing the active ingredient into
association with a carrier or one or more other accessory
ingredients, and then, if necessary or desirable, shaping or
packaging the product into a desired single- or multi-dose
unit.
[0201] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for ethical administration to
humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to animals
of all sorts. Modification of pharmaceutical compositions suitable
for administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design and
perform such modification with merely ordinary, if any,
experimentation.
[0202] Pharmaceutical compositions that are useful in the methods
of the invention may be prepared, packaged, or sold in formulations
suitable for oral, rectal, vaginal, parenteral, topical, pulmonary,
intranasal, buccal, intravenous, transdermal, subcutaneous,
intramuscular, ophthalmic, intrathecal and other known routes of
administration. Other contemplated formulations include projected
nanoparticles, liposomal preparations, resealed erythrocytes
containing the active ingredient, and immunologically-based
formulations.
[0203] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in bulk, as a single unit dose, or as a
plurality of single unit doses. As used herein, a "unit dose" is
discrete amount of the pharmaceutical composition comprising a
predetermined amount of the active ingredient. The amount of the
active ingredient is generally equal to the dosage of the active
ingredient which would be administered to a subject or a convenient
fraction of such a dosage such as, for example, one-half or
one-third of such a dosage.
[0204] The relative amounts of the active ingredient, the
pharmaceutically acceptable carrier, and any additional ingredients
in a pharmaceutical composition of the invention will vary,
depending upon the identity, size, and condition of the subject
treated and further depending upon the route by which the
composition is to be administered. By way of example, the
composition may comprise between 0.1% and 100% (w/w) active
ingredient.
[0205] In addition to the active ingredient, a pharmaceutical
composition of the invention may further comprise one or more
additional pharmaceutically active agents.
[0206] Controlled- or sustained-release formulations of a
pharmaceutical composition of the invention may be made using
conventional technology.
[0207] A formulation of a pharmaceutical composition of the
invention suitable for oral administration may be prepared,
packaged, or sold in the form of a discrete solid dose unit
including, but not limited to, a tablet, a hard or soft capsule, a
cachet, a troche, or a lozenge, each containing a predetermined
amount of the active ingredient. Other formulations suitable for
oral administration include, but are not limited to, a powdered or
granular formulation, an aqueous or oily suspension, an aqueous or
oily solution, or an emulsion.
[0208] Pharmaceutically acceptable excipients used in the
manufacture of pharmaceutical compositions include, but are not
limited to, inert diluents, granulating and disintegrating agents,
binding agents, and lubricating agents. Known dispersing agents
include, but are not limited to, potato starch and sodium starch
glycollate. Known surface active agents include, but are not
limited to, sodium lauryl sulphate. Known diluents include, but are
not limited to, calcium carbonate, sodium carbonate, lactose,
microcrystalline cellulose, calcium phosphate, calcium hydrogen
phosphate, and sodium phosphate. Known granulating and
disintegrating agents include, but are not limited to, corn starch
and alginic acid. Known binding agents include, but are not limited
to, gelatin, acacia, pre-gelatinized maize starch,
polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known
lubricating agents include, but are not limited to, magnesium
stearate, stearic acid, silica, and talc.
[0209] Liquid formulations of a pharmaceutical composition of the
invention may be prepared, packaged, and sold either in liquid form
or in the form of a dry product intended for reconstitution with
water or another suitable vehicle prior to use.
[0210] Liquid suspensions may be prepared using conventional
methods to achieve suspension of the active ingredient in an
aqueous or oily vehicle. Aqueous vehicles include, for example,
water and isotonic saline. Oily vehicles include, for example,
almond oil, oily esters, ethyl alcohol, vegetable oils such as
arachis, olive, sesame, or coconut oil, fractionated vegetable
oils, and mineral oils such as liquid paraffin. Liquid suspensions
may further comprise one or more additional ingredients including,
but not limited to, suspending agents, dispersing or wetting
agents, emulsifying agents, demulcents, preservatives, buffers,
salts, flavorings, coloring agents, and sweetening agents. Oily
suspensions may further comprise a thickening agent.
[0211] Known suspending agents include, but are not limited to,
sorbitol syrup, hydrogenated edible fats, sodium alginate,
polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose
derivatives such as sodium carboxymethylcellulose, methylcellulose,
and hydroxypropylmethylcellulose. Known dispersing or wetting
agents include, but are not limited to, naturally-occurring
phosphatides such as lecithin, condensation products of an alkylene
oxide with a fatty acid, with a long chain aliphatic alcohol, with
a partial ester derived from a fatty acid and a hexitol, or with a
partial ester derived from a fatty acid and a hexitol anhydride
(e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol,
polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan
monooleate, respectively). Known emulsifying agents include, but
are not limited to, lecithin and acacia. Known preservatives
include, but are not limited to, methyl, ethyl, or
n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid.
Known sweetening agents include, for example, glycerol, propylene
glycol, sorbitol, sucrose, and saccharin. Known thickening agents
for oily suspensions include, for example, beeswax, hard paraffin,
and cetyl alcohol.
[0212] Liquid solutions of the active ingredient in aqueous or oily
solvents may be prepared in substantially the same manner as liquid
suspensions, the primary difference being that the active
ingredient is dissolved, rather than suspended in the solvent.
Liquid solutions of the pharmaceutical composition of the invention
may comprise each of the components described with regard to liquid
suspensions, it being understood that suspending agents will not
necessarily aid dissolution of the active ingredient in the
solvent. Aqueous solvents include, for example, water and isotonic
saline. Oily solvents include, for example, almond oil, oily
esters, ethyl alcohol, vegetable oils such as arachis, olive,
sesame, or coconut oil, fractionated vegetable oils, and mineral
oils such as liquid paraffin.
[0213] Powdered and granular formulations of a pharmaceutical
preparation of the invention may be prepared using known methods.
Such formulations may be administered directly to a subject, used,
for example, to form tablets, to fill capsules, or to prepare an
aqueous or oily suspension or solution by addition of an aqueous or
oily vehicle thereto. Each of these formulations may further
comprise one or more of dispersing or wetting agent, a suspending
agent, and a preservative. Additional excipients, such as fillers
and sweetening, flavoring, or coloring agents, may also be included
in these formulations.
[0214] A pharmaceutical composition of the invention may also be
prepared, packaged, or sold in the form of oil-in-water emulsion or
a water-in-oil emulsion. The oily phase may be a vegetable oil such
as olive or arachis oil, a mineral oil such as liquid paraffin, or
a combination of these. Such compositions may further comprise one
or more emulsifying agents such as naturally occurring gums such as
gum acacia or gum tragacanth, naturally-occurring phosphatides such
as soybean or lecithin phosphatide, esters or partial esters
derived from combinations of fatty acids and hexitol anhydrides
such as sorbitan monooleate, and condensation products of such
partial esters with ethylene oxide such as polyoxyethylene sorbitan
monooleate. These emulsions may also contain additional ingredients
including, for example, sweetening or flavoring agents.
[0215] Methods for impregnating or coating a material with a
chemical composition are known in the art, and include, but are not
limited to methods of depositing or binding a chemical composition
onto a surface, methods of incorporating a chemical composition
into the structure of a material during the synthesis of the
material (i.e., such as with a physiologically degradable
material), and methods of absorbing an aqueous or oily solution or
suspension into an absorbent material, with or without subsequent
drying.
[0216] As used herein, "parenteral administration" of a
pharmaceutical composition includes any route of administration
characterized by physical breaching of a tissue of a subject and
administration of the pharmaceutical composition through the breach
in the tissue. Parenteral administration thus includes, but is not
limited to, administration of a pharmaceutical composition by
injection of the composition, by application of the composition
through a surgical incision, by application of the composition
through a tissue-penetrating non-surgical wound, and the like. In
particular, parenteral administration is contemplated to include,
but is not limited to, cutaneous, subcutaneous, intraperitoneal,
intravenous, intramuscular, intracisternal injection, and kidney
dialytic infusion techniques.
[0217] Formulations of a pharmaceutical composition suitable for
parenteral administration comprise the active ingredient combined
with a pharmaceutically acceptable carrier, such as sterile water
or sterile isotonic saline. Such formulations may be prepared,
packaged, or sold in a form suitable for bolus administration or
for continuous administration. Injectable formulations may be
prepared, packaged, or sold in unit dosage form, such as in ampules
or in multi-dose containers containing a preservative. Formulations
for parenteral administration include, but are not limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles,
pastes, and implantable sustained-release or biodegradable
formulations. Such formulations may further comprise one or more
additional ingredients including, but not limited to, suspending,
stabilizing, or dispersing agents. In one embodiment of a
formulation for parenteral administration, the active ingredient is
provided in dry (i.e., powder or granular) form for reconstitution
with a suitable vehicle (e.g., sterile pyrogen-free water) prior to
parenteral administration of the reconstituted composition.
[0218] The pharmaceutical compositions may be prepared, packaged,
or sold in the form of a sterile injectable aqueous or oily
suspension or solution. This suspension or solution may be
formulated according to the known art, and may comprise, in
addition to the active ingredient, additional ingredients such as
the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations may be
prepared using a non-toxic parenterally-acceptable diluent or
solvent, such as water or 1,3-butane diol, for example. Other
acceptable diluents and solvents include, but are not limited to,
Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as synthetic mono- or di-glycerides. Other
parentally-administrable formulations which are useful include
those which comprise the active ingredient in microcrystalline
form, in a liposomal preparation, or as a component of a
biodegradable polymer systems. Compositions for sustained release
or implantation may comprise pharmaceutically acceptable polymeric
or hydrophobic materials such as an emulsion, an ion exchange
resin, a sparingly soluble polymer, or a sparingly soluble
salt.
[0219] Formulations suitable for topical administration include,
but are not limited to, liquid or semi-liquid preparations such as
liniments, lotions, oil-in-water or water-in-oil emulsions such as
creams, ointments or pastes, and solutions or suspensions.
Topically-administrable formulations may, for example, comprise
from about 1% to about 10% (w/w) active ingredient, although the
concentration of the active ingredient may be as high as the
solubility limit of the active ingredient in the solvent
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0220] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for pulmonary
administration via the buccal cavity. Such a formulation may
comprise dry particles which comprise the active ingredient and
which have a diameter in the range from about 0.5 to about 7
nanometers, and preferably from about 1 to about 6 nanometers. Such
compositions are conveniently in the form of dry powders for
administration using a device comprising a dry powder reservoir to
which a stream of propellant may be directed to disperse the powder
or using a self-propelling solvent/powder-dispensing container such
as a device comprising the active ingredient dissolved or suspended
in a low-boiling propellant in a sealed container. Preferably, such
powders comprise particles wherein at least 98% of the particles by
weight have a diameter greater than 0.5 nanometers and at least 95%
of the particles by number have a diameter less than 7 nanometers.
More preferably, at least 95% of the particles by weight have a
diameter greater than 1 nanometer and at least 90% of the particles
by number have a diameter less than 6 nanometers. Dry powder
compositions preferably include a solid fine powder diluent such as
sugar and are conveniently provided in a unit dose form.
[0221] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50 to 99.9% (w/w)
of the composition, and the active ingredient may constitute 0.1 to
20% (w/w) of the composition. The propellant may further comprise
additional ingredients such as a liquid non-ionic or solid anionic
surfactant or a solid diluent (preferably having a particle size of
the same order as particles comprising the active ingredient).
[0222] Pharmaceutical compositions of the invention formulated for
pulmonary delivery may also provide the active ingredient in the
form of droplets of a solution or suspension. Such formulations may
be prepared, packaged, or sold as aqueous or dilute alcoholic
solutions or suspensions, optionally sterile, comprising the active
ingredient, and may conveniently be administered using any
nebulization or atomization device. Such formulations may further
comprise one or more additional ingredients including, but not
limited to, a flavoring agent such as saccharin sodium, a volatile
oil, a buffering agent, a surface active agent, or a preservative
such as methylhydroxybenzoate. The droplets provided by this route
of administration preferably have an average diameter in the range
from about 0.1 to about 200 nanometers. The formulations described
herein as being useful for pulmonary delivery are also useful for
intranasal delivery of a pharmaceutical composition of the
invention. Another formulation suitable for intranasal
administration is a coarse powder comprising the active ingredient
and having an average particle from about 0.2 to 500
micrometers.
[0223] Such a formulation is administered in the manner in which
snuff is taken i.e. by rapid inhalation through the nasal passage
from a container of the powder held close to the nares.
Formulations suitable for nasal administration may, for example,
comprise from about as little as 0.1% (w/w) and as much as 100%
(w/w) of the active ingredient, and may further comprise one or
more of the additional ingredients described herein.
[0224] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for buccal
administration. Such formulations may, for example, be in the form
of tablets or lozenges made using conventional methods, and may,
for example, contain 0.1 to 20% (w/w) active ingredient, the
balance comprising an orally dissolvable or degradable composition
and, optionally, one or more of the additional ingredients
described herein. Alternately, formulations suitable for buccal
administration may comprise a powder or an aerosolized or atomized
solution or suspension comprising the active ingredient. Such
powdered, aerosolized, or aerosolized formulations, when dispersed,
preferably have an average particle or droplet size in the range
from about 0.1 to about 200 nanometers, and may further comprise
one or more of the additional ingredients described herein.
[0225] A pharmaceutical composition of the invention may be
prepared, packaged, or sold in a formulation suitable for
ophthalmic administration. Such formulations may, for example, be
in the form of eye drops including, for example, a 0.1-1.0% (w/w)
solution or suspension of the active ingredient in an aqueous or
oily liquid carrier. Such drops may further comprise buffering
agents, salts, or one or more other of the additional ingredients
described herein. Other opthalmically-administrable formulations
which are useful include those which comprise the active ingredient
in microcrystalline form or in a liposomal preparation.
[0226] As used herein, "additional ingredients" include, but are
not limited to, one or more of the following: excipients; surface
active agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the invention are
known in the art and described, for example in Genaro, ed., 1985,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., which is incorporated herein by reference. Typically dosages
of the compound of the invention which may be administered to an
animal, preferably a human, range in amount from about 0.01 mg to
about 1000 mg per kilogram of body weight of the animal. The
precise dosage administered will vary depending upon any number of
factors, including, but not limited to, the type of animal and type
of disease or disorder being treated, the age of the animal and the
route of administration. Preferably, the dosage of the compound
will vary from about 1 mg to about 100 mg per kilogram of body
weight of the animal. The compound can be administered to an animal
as frequently as several times daily, or it can be administered
less frequently, such as once a day, once a week, once every two
weeks, once a month, or even less frequently, such as once every
several months or even once a year or less. The frequency of the
dose will be readily apparent to the skilled artisan and will
depend upon any number of factors, such as, but not limited to, the
type and severity of the disease or disorder being treated, the
type and age of the animal, etc.
EXPERIMENTAL EXAMPLES
[0227] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0228] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the present
invention and practice the claimed methods. The following working
examples therefore, specifically point out the preferred
embodiments of the present invention, and are not to be construed
as limiting in any way the remainder of the disclosure.
Example 1
Identification of a Receptor for Extracellular Renalase
[0229] The results described herein identify PMCA4b as a renalase
receptor, and a key mediator of renalase dependent MAPK signaling.
Using biotin transfer studies with RP-220 in the human proximal
tubular cell line HK-2 and protein identification by mass
spectrometry, PMCA4b was identified as a renalase binding protein.
This previously characterized plasma membrane ATPase is involved in
cell signaling and cardiac hypertrophy. Co-immunoprecipitation and
co-immunolocalization confirmed protein-protein interaction between
endogenous renalase and PMCA4b. Down-regulation of endogenous
PMCA4b expression by siRNA transfection, or inhibition of its
enzymatic activity by the specific peptide inhibitor caloxin1b each
abrogated RP-220 dependent MAPK signaling and cytoprotection. In
control studies, these maneuvers had no effect on epidermal growth
factor mediated signaling, confirming specificity of the
interaction between PMCA4b and renalase.
[0230] The materials and methods employed in these experiments are
now described.
Materials and Methods
Synthesis and Analysis of Renalase and Renalase Peptides
[0231] Renalase peptides (SEQ ID NOs 1-7; FIG. 1A) were acetylated
at the amino terminus and purified to 98% homogeneity (United
Peptides, Herndon, Va.). Recombinant renalase was synthesized as
previously described (Desir et al., 2012, J. Am. Heart Assoc.
1:e002634). Renalase expression was detected using an anti-renalase
monoclonal antibody generated against the renalase peptide RP-220
(amino acid 220-239 of hRenalase1; SEQ ID NO: 4)
Renalase Receptor Cross Linking and Identification
[0232] Potential disulfide bonds formed by the single, amino
terminal cysteine present in renalase peptide 220 (RP-220; SEQ ID
NO: 4), and in the scrambled renalase peptide (RP-Scr220, control
peptide; SEQ ID NO: 7) (FIG. 1A) were disrupted using an
immobilized reducing column (#77701, Pierce Biotechnology,
Rockford, Ill.). The reduced peptides were recovered by elution,
and the concentration of free sulfhydryl groups was estimated using
the Ellman's Reagent (#22582, Pierce Biotechnology, Rockford,
Ill.).
[0233] Reduced RP-220 (SEQ ID NO: 4) and RP-Scr220 (SEQ ID NO: 7)
were conjugated to a tri-functional cross-linker, Mts-Atf-Biotin
(#33093, Pierce Biotechnology, Rockford, Ill.) according to the
manufacturer's instructions. Conjugation was achieved through the
sulfhydryl-reactive methanethiosulfonate (Mts) moiety, and the
spacer arm between Mts, and the photoactivatable tetrafluorophenyl
azide (Atf) moiety was 11 .ANG.. Coupling efficiency was estimated
by dot blot using streptavidin-HRP to measure biotin incorporation
(#21130, Pierce Biotechnology, Rockford, Ill.). The labeled probes
were protected from light and stored in 50 .mu.l aliquots, at
-80.degree. C. until use.
[0234] HK-2 cells (human proximal tubular cell line), obtained from
American Type Culture Collection (ATCC, Manassas, Va.) were grown
at 37.degree. C. to 80% confluence in 150 mm dishes in DMEM/F12
media supplemented with glutamine, 10% FBS, antibiotics, and 5%
CO.sub.2. Cells were cooled to 4.degree. C. to prevent probe
internalization, and then incubated for 16 hrs with 50 .mu.g of
either Mts-Atf-Biotin labeled RP220 or RP-Scr220. Cross-linking of
the probes was initiated by exposing the cells to ultraviolet light
for five minutes using a Stratalinker 2500 (Stratagene, Agilent
Technologies, Santa Clara, Calif.).
[0235] The cells were suspended in phosphate buffered saline (PBS)
with protease inhibitors (Complete Ultra #05892953001, Roche
Diagnostics, Indianapolis, Ind.) and subjected to 3 cycles of
freeze-thawing. The membrane fraction was collected by
centrifugation (180,000 g for 1 hour) and solubilized in RIPA
buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na.sub.2EDTA, 1 mM
EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium
pyrophosphate, 1 mM glycerophosphate, 1 mM Na.sub.3VO.sub.4, 1
.mu.g/ml leupeptin) (Cell Signaling Technologies, Danvers, Mass.)
for 4 hours at 4.degree. C.
[0236] The biotinylated proteins were purified using streptavidin
agarose resin (Pierce Biotechnology, Rockford, Ill.) according to
the manufacturer's instructions. The proteins were separated by SDS
PAGE using a 4-20% gradient gel (Bio-Rad, Hercules, Calif.) and
stained by Western-blotting using streptavidin-HRP to identify
proteins bands found predominantly cross-linked to the renalase
bait RP-220. These bands and the corresponding bands from the
samples cross-linked to the control bait RP-Scr220 were cut out of
coomassie blue stained gels, and individual proteins were
identified by mass spectrometry (Yale Keck Biotechnology Resource
Laboratory). Plasma membrane protein(s) consistently present in the
samples cross-linked to RP-220, and absent from those cross-linked
to the control bait RP-Scr220 were evaluated further.
Downregulation of PMCA4b Expression using siRNA
[0237] ATP2B4 (PMCA4b) specific siRNAs and non-targeting controls
(SMARTpool ON-TARGETplus ATP2B4 siRNA, L-006118-00-0005) were
purchased from Thermo Fischer Scientific (Waltham, Mass.), and
transfected into HK-2 cells using Lipofectamine 2000 (Invitrogen,
Life Technologies Grand Island N.Y.). HK-2 cells were grown to 70%
confluence in DMEM/F12 medium supplemented with 10% FBS. PMCA4b
protein expression was assessed by western immunoblot using an
anti-PMCA4 mouse monoclonal antibody (#H00000493-M07, Novus
Biologicals, Littleton, Colo.).
Detection of Endogenous Co-Expression of PMCA4b and Renalase
[0238] HK-2 cells, which highly express renalase and PMCA4b
endogenously, were fixed, permeabilized, and incubated with a goat
polyclonal anti-renalase antibody (#AF5350, R&D Systems,
Minneapolis, Minn.), and an anti-PMCA4 mouse monoclonal antibody
(#H00000493-M07, Novus Biologicals, Littleton, Colo.) for 2 hours
at room temperature. Following the application of two labeled
secondary antibodies (Alexa488-rabbit anti-goat to detect renalase
and Alexa555-goat anti-mouse to detect PMCA4, Molecular Probes,
Life Technologies Grand Island N.Y.), cells were then examined
using a Zeiss LSM 510 confocal imaging system.
Co-Immunoprecipitation of Endogenous PMCA4b and Renalase
[0239] HK-2 cells were lysed using ice-cold RIPA buffer (Cell
Signaling Technologies, Danvers, Mass.), and the lysate was
centrifuged 14,000 g for 15 min. The supernatant was collected and
incubated with protein A/G agarose beads (Santa Cruz Biotechnology,
Santa Cruz, Calif.) to reduce non-specific binding.
Immunoprecipitation was carried out using A/G agarose beads and
either goat polyclonal anti-renalase antibody or anti-PMCA4 mouse
monoclonal antibody, and proteins were visualized by western
blotting.
Gene Expression Analysis
[0240] Total RNA from tissue samples was extracted using RNeasy
Plus kit (Qiagen, Valencia, Calif.) and converted into cDNA using
an Omniscript RT kit (Qiagen, Valencia, Calif.). PMCA4b mRNA in
kidney was assessed in renalase KO mice by real-time PCR and
normalized to the corresponding levels in WT samples (defined as
1.0). Target cDNA was amplified using Qiagen Dr_atp2b4_1_SG
QuantiTect primer assay and Platinum SYBR Green qPCR superMix-UDG.
Standard cycling conditions were run with a Step-One-Plus real time
PCR system (Applied Biosystems), and the resulting Ct values
analyzed using the 2-.DELTA..DELTA.CT method.
In Vitro Model of Cisplatin Toxicity
[0241] HK-2 cells (human proximal tubular line) obtained from ATTC
(Manassas, Va., USA) were cultured in DMEM/F12 supplemented with
glutamine, 10% FBS and antibiotics, and were maintained at
37.degree. C. in 5% CO.sub.2. Cells were exposed to cisplatin (20
.mu.M) in the presence or absence of recombinant renalase or
renalase peptides for 24 hrs, and cell viability was assessed by
the WST1 method (Roche Applied Science, Germany).
[0242] To examine renalase dependent MAPKs signaling, cells treated
with RP-220 (15 ug/ml) or EGF (100 ng/ml) as a positive control
were harvested in RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1 mM Na.sub.2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5
mM sodium pyrophosphate, 1 mM .alpha.-glycerophosphate, 1 mM
Na.sub.3VO.sub.4, 1 .mu.g/ml leupeptin) supplemented with a
protease and phosphatase inhibitor cocktail (Roche Applied Science,
Germany). Proteins were separated by SDS-PAGE and immunoblotting
was carried out using the following antibodies: anti-renalase
monoclonal antibodies (Desir et al., 2012, J. Am. Heart Assoc.
1:e002634; Lee et al., 2013, J. Am. Soc. Nephrol. 24:445-455), and
antibodies specific for total and phosphorylated ERK, p38, and JNK
(Cell Signaling Technology, MA, USA). The following inhibitors were
obtained from Sigma-Aldrich Corp (St. Louis, Mo.): U0126
(1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]) for ERK1/2,
and SB203580
(4-[4'-Fluorophenyl]-2-[4'-methylsulfinylphenyl]-5-[4'-pyridyl]--
imidazole) for p38 .alpha.-.beta..
Statistical Analysis
[0243] When appropriate, the Kruskal-Wallis one-way analysis of
variance by ranks was used to evaluate statistical significance.
When the Kruskal-Wallis test revealed statistical significance, the
Mann-Whitney test was used for pairwise comparisons. All data are
mean.+-.SEM, and values of P<0.05 were accepted as a
statistically significant difference. Statistical analysis was
carried out using GraphPad Prism (GraphPad Software, Inc.).
[0244] The results of the experiments are now described.
Protection by RP-220 Against Cisplatin Toxicity Linked to p38 MAPK
Activation
[0245] It has previously been shown that in renalase KO mice,
renalase deficiency worsens ischemic AKI, while the administration
of recombinant renalase significantly attenuates AKI (Lee et al.,
2013, J. Am. Soc. Nephrol. 24:445-455). Additional studies indicate
that the protective effect of renalase against ischemic and
cisplatin AKI does not depend on the enzymatic activity of
renalase, but rather is mediated by the interaction of renalase or
short renalase peptides (RP-224, RP-220 and RP-H220, FIG. 1a) with
a receptor(s). A scrambled renalase peptide, RP-Scr220, did not
activate MAPK signaling, and failed to protect cells (Wang et al.,
2014, J. Am. Soc. Nephrol. DOI:10.1681/asn.2013060665). The
protective effects of renalase peptides corresponded to the
activation of intracellular signaling that promotes cell survival
(Wang et al., 2014, J. Am. Soc. Nephrol.
DOI:10.1681/asn.2013060665).
[0246] In order to determine if RP-220 could be used as probe to
identify the receptor(s) for extracellular renalase, its
specificity was examined by comparing its protective effect against
cisplatin toxicity to that of mutated RP-220 (RP-A220; SEQ ID NO:
6), and of renalase peptides RP-19 (SEQ ID NO: 1) and RP-128 (SEQ
ID NO: 2; FIG. 1A). Renalase peptides RP-19 and RP-128 were chosen
because they contain putative ERK docking (D) domains (Motif Scan)
(Roux and Blenis, 2004, Microbiol. Mol. Biol. Rev. 68:320-344).
RP-220 significantly protected HK-2 cells exposed to cisplatin for
24 hrs in a dose dependent manner (FIG. 1B). Control peptides
RP-19, RP-128, and RP-A220 did not improve cell survival, and at
the highest concentration (80 .mu.g/ml), RP-A220 actually
exacerbated cisplatin toxicity. Recombinant renalase (80 .mu.g/ml),
RP-224 (100 .mu.g/ml), RP-220 (15 .mu.g/ml), and RP-H220 (15
.mu.g/ml) protected HK-2 cells to a similar degree (FIG. 1C). The
concentration dependent effects for RP-220 and RP-H220 indicated
that both peptides are equipotent at protecting HK-2 cells against
cisplatin cytotoxicity, and RP-220 (15 .mu.g/ml) was used for all
subsequent studies (FIG. 1D).
[0247] It has previously been demonstrated that both Renalase1 and
RP-220 treatments rapidly increased ERK and p38 phosphorylation in
HK-2 cell (Wang et al., 2014, J. Am. Soc. Nephrol. DOI:
10.1681/asn.2013060665). To test if protection of HK-2 cells
against cisplatin toxicity by RP-220 was linked to MAPK activation,
the patterns of MAPK signaling were examined at early time points
(1-60 min) in HK-2 cells exposed to cisplatin with and without
RP-220. Cisplatin alone modestly increased ERK and p38
phosphorylation (FIGS. 2A-2C). This effect on p38 phosphorylation
was markedly enhanced (5 fold) by the addition of RP-220 (FIG. 2C).
However, only a slight increase in ERK activation (0.5 fold) was
noted with RP-220 over that elicited by cisplatin alone (FIG. 2B).
Although not wishing to be bound to any particular theory, these
results suggest that RP-220's cytoprotective action may be due to
its activation of p38 and not ERK.
[0248] To determine the relative importance of ERK and p38 as
mediators of RP-220's protective effect, their activities were
chemically inhibited. Reducing the activity of ERK1/2 (U0126), or
p38.alpha.-.beta. (SB203580) was examined in HK-2 cells under
control conditions. In control studies, ERK and p38 inhibition had
no deleterious effect and were not found to reduce HK-2 cell
survival (FIG. 3A). In HK-2 cells treated with both cisplatin and
RP-220, ERK inhibition did not diminish the protection conferred by
RP-H220 (FIG. 3B). In marked contrast, p38 inhibition completely
abrogated the RP-220's protection (FIG. 3B). This result provides
strong support for the hypothesis that p38 is a key mediator of
RP-220's defense against cisplatin toxicity.
Extracellular Renalase Binds to the Plasma Membrane
Ca.sup.++-ATPase Isoform PMCA4b
[0249] RP-220 was used as a probe to identify a plasma membrane
protein(s) that interact with extracellular renalase. The biotin
label transfer method was used with the label transfer reagent
Mts-Atf-Biotin, which was linked to the single cysteine located at
the N terminus of RP-220. Labeled RP-220 was incubated with HK-2
cells for 24 hrs at 4.degree. C. to minimize internalization, and
was cross-linked to the interacting protein(s) by exposure to UV
light. The biotin-labeled proteins were purified using a
streptavidin column and identified by mass spectrometry. The plasma
membrane calcium-ATPase isoform, PMCA4b, was reproducibly
cross-linked to RP-220 (FIG. 4A). PMCA4b was abundantly expressed
in HK-2 cells (FIG. 4B) and co-localized with renalase at the
plasma membrane (arrows), and within the cytoplasm of HK-2 cells
(FIG. 4C).
[0250] In vitro interaction between endogenously expressed PMCA4b
and RP-220 was evaluated by co-immunoprecipitation. Agarose beads
coated with either PMCA4b or renalase antibody depleted whole cell
lysates of renalase (FIG. 4D, upper blot, lanes 1 vs 2 and 3), and
renalase could be eluted from both sets of beads (FIG. 4D, upper
blot, lanes 4-5). The blot was re-probed with an anti-PMCA4b
antibody, and likewise, PMCA4b could be eluted from both sets of
beads (FIG. 4D, bottom blot, lanes 4-5). Of note, endogenous
expression of PMCA4b is significantly higher than that of renalase
(FIG. 4D, upper and lower blots, lanes 1), and as a consequence
less PMCA4b protein is eluted from beads coated with the renalase
antibody than from those coated with the PMCA4b antibody. (FIG. 4D,
upper and lower blots, lanes 4 vs 5). In addition, PMCA4b coated
beads completely depleted renalase from the supernatant. Although
not wishing to be bound to any particular theory, this result
suggests that extracellular renalase is largely bound to
PMCA4b.
PMCA4b Mediates Renalase's Action on Cell Signaling and
Cytoprotection
[0251] It was next determined whether inhibition of PMCA4b activity
modulated renalase mediated MAPK signaling. Caloxin1b, a peptide
inhibitor of PMCA4b (Pande et al., 2008, J. Cell. Mol. Med.
12:1049-1060, abrogated renalase-mediated ERK and p38 MAPKs
phosphorylation in HK-2 cells (FIG. 5A). Since Caloxin1b is
reported to also inhibit PMCA1, additional evidence regarding
PMCA4b's role in renalase mediated signaling was obtained by
specifically down-regulating PMCA4b expression using siRNA. In
control studies, non-targeting siRNAs affected neither PMCA4b
expression nor RP-220 mediated p38 (FIG. 5B, left panel). In
contrast, PMCA4b-targeting siRNAs decreased protein expression by
more than 90% and prevented RP-220 mediated p38 phosphorylation
(FIG. 5B, middle and right panels). The specificity of the
interaction between RP-220 and PMCA4b was examined by testing if
PMCA4b downregulation also affected epidermal growth factor (EGF)
mediated MAPK activation. As shown in FIG. 5C, inhibition of PMCA4b
expression had no effect on EGF dependent ERK, p38 and JNK
phosphorylation.
[0252] Down-regulation of PMCA4b expression abrogated the
protective effect of RP-220 and RP-H220 against cisplatin
cytotoxicity (FIG. 5d). It has previously been shown that in marked
contrast to what is observed in WT mice, neither renalase nor
RP-220 is effective at activating MAPK signaling and at protecting
renalase KO mice against mild or severe ischemic AKI (Wang et al.,
2014, J. Am. Soc. Nephrol. DOI:10.1681/asn.2013060665). Based on
these results, it was hypothesized that the interaction of RP-220
with its cognate receptor was disrupted in the renalase KO, perhaps
due to a decrease in receptor gene or protein expression brought
about by deletion of the renalase gene. PMCA4b gene expression in
the renalase KO mouse was measured by quantitative PCR, and found
to be 11.4 fold lower than in WT control (n=6, p<0.03).
Expression of PMCA4b protein in WT and renalase KO kidneys was
evaluated by western immunoblotting. As shown in FIG. 5E
(representative blot), PMCA4b expression in the renalase KO was
63.5.+-.7.5% lower than in WT (n=6, p<0.03). These data provide
strong support for the hypothesis that PMCA4b functions as a
renalase receptor, and is critical in mediating the signaling and
cytoprotective effects of renalase.
TABLE-US-00001 Renalase polypeptide and peptide sequences RP 19
(SEQ ID NO: 1) LLRRQTSGPLYLAVWDKAED RP 128 (SEQ ID NO: 2)
FRHRVTQINLRDDKWEVSKQ RP 224 (SEQ ID NO: 3) CVSIDNKKRNI RP 220 (SEQ
ID NO: 4) CIRFVSIDNKKRNIESSEIG RP H220 (SEQ ID NO: 5)
HHHHHHCIRFVSIDNKKRNIESSEIG RP A220 (SEQ ID NO: 6)
IRFVSIDNAAANIESSEIG RP 220 SCRAMBLED (SEQ ID NO: 7)
CSKRIFKVISSIEDNNERG Renalase (NP_001026879.2)- (SEQ ID NO: 8)
MAQVLIVGAGMTGSLCAALLRRQTSGPLYLAVWDKAEDSGGRMTTACSPH
NPQCTADLGAQYITCTPHYAKKHQRFYDELLAYGVLRPLSSPIEGMVMKE
GDCNFVAPQGISSIIKHYLKESGAEVYFRHRVTQINLRDDKWEVSKQTGS
PEQFDLIVLTMPVPEILQLQGDITTLISECQRQQLEAVSYSSRYALGLFY
EAGTKIDVPWAGQYITSNPCIRFVSIDNKKRNIESSEIGPSLVIHTTVPF
GVTYLEHSIEDVQELVFQQLENILPGLPQPIATKCQKWRHSQVTNAAANC
PGQMTLHHKPFLACGGDGFTQSNFDGCITSALCVLEALKNYI Renalase (NP_060833.1)-
(SEQ ID NO: 9) MAQVLIVGAGMTGSLCAALLRRQTSGPLYLAVWDKAEDSGGRMTTACSPH
NPQCTADLGAQYITCTPHYAKKHQRFYDELLAYGVLRPLSSPIEGMVMKE
GDCNFVAPQGISSIIKHYLKESGAEVYFRHRVTQINLRDDKWEVSKQTGS
PEQFDLIVLTMPVPEILQLQGDITTLISECQRQQLEAVSYSSRYALGLFY
EAGTKIDVPWAGQYITSNPCIRFVSIDNKKRNIESSEIGPSLVIHTTVPF
GVTYLEHSIEDVQELVFQQLENILPGLPQPIATKCQKWRHSQVPSAGVIL
GCAKSPWMMAIGFPI
Example 2
Modulation of the Activity of PMCA4b Mediates Renalase's
Cytoprotective Action
[0253] The effect of renalase on the ATPase activity of PMCA4b is
examined, including its effect on Vmax, Km, and constitutive
activation. The local and/or global effect of renalase's
interaction with PMCA4b on calcium dynamics is also examined.
Whether the disruption of the PMCA4b macromolecular complex with
RAS SF-1 modulates the action of renalase (MAPK signaling and
cytoprotection) is further examined. Whether PMCA4b knockout mice
respond differently than wild type mice to renal ischemia or
exposure to cisplatin is examined, as is whether renalase modifies
the extent of renal injury.
Example 3
Assessment of Renalase in Pancreatitis
[0254] As described elsewhere herein, the region of renalase that
affects pancreatitis and cell signaling responses has been isolated
in a region of the molecule that lacks enzyme activity. The peptide
containing this activity, RP-220, is conserved in all renalase
isoforms, but lacks the amine oxidase activity of recombinant
renalase and instead acts only as a signaling molecule. In the
studies described herein, renalase is administered as both a
recombinant full-length human renalase or as the 20 amino acid
peptide (RP-220). RP-220 dramatically decreases inflammatory,
ischemic acute kidney injury in WT mice independent of its
enzymatic activity.
Cytosolic Ca.sup.2+ Signaling Mediates Pancreatitis
[0255] The acinar events that lead to pancreatitis are
Ca.sup.2+-dependent, associated with pathologic cytoplasmic calcium
signaling, and can be modified by the plasma membrane Ca.sup.2+
ATPase (PMCA). Under physiologic conditions, coordinated,
oscillatory Ca.sup.2+ signals that originate in the apical region
of the acinar cell are linked to the secretion of inactive zymogens
from acinar cell into the pancreatic duct (Williams, 2001, Annu Rev
Physiol 63, 77-97). In pancreatitis, a different acinar cell
Ca.sup.2+ signaling pattern is observed. Instead of the physiologic
increase in oscillations and several-fold increases in cytosolic
Ca.sup.2+, pancreatitis is associated with a global, peak-plateau
pattern, with much larger rise in Ca.sup.2+ followed by a sustained
elevation at a lower level (Matozaki et al., 1990, JMV-180. J Biol
Chem 265, 6247-6254). Pathologic Ca.sup.2+ signaling and Ca.sup.2+
overload have been linked to most of the early events in the
pathogenesis of acute pancreatitis, including zymogen activation,
inhibition of secretion, inflammation, and necrosis (Raraty et al.,
2000, Proc Natl Acad Sci USA 97, 13126-13131; Muallem et al., 1995,
J Cell Biol 128, 589-598; Criddle et al., 2006, Gastroenterology
130, 781-793; Gerasimenko et al., 2002, J Cell Sci 115, 485-497;
Huang et al., 2013, Gut; Awla et al., 2012, Gastroenterology 143,
1352-1360 e1357).
[0256] Physiologic levels of cytosolic acinar cell Ca.sup.2+ are
maintained through the actions of several Ca.sup.2+ pumps and
channels, including the plasma membrane Ca.sup.2+ ATPase (PMCA).
PMCA, which actively pumps Ca.sup.2+ from the cell, is the main
Ca.sup.2+ efflux pathway in the acinar cell, which lacks functional
Na.sup.+/Ca.sup.2+ exchanger (Tepikin et al., 1992, J Biol Chem
267, 3569-3572; Petersen, 2003, Cell Calcium 33, 337-344). PMCA is
a key regulator of baseline Ca.sup.2+ levels and defender against
pathological increases in intracellular Ca.sup.2+. PMCA exists in
four isoforms. PMCA1 and PMCA4 are ubiquitously expressed, while
PMCA2 and PMCA3 are tissue specific and expressed mainly in neurons
(Lopreiato et al., 2014, J Biol Chem 289, 10261-10268). PMCA is
activated by calmodulin (Falchetto et al., 1992, Protein Sci 1,
1613-1621) and regulated through phosphorylation by several protein
kinases (Wang et al., 1991, J Biol Chem 266, 9078-9085; Smallwood
et al., 1988, J Biol Chem 263, 2195-2202; James et al., 1989,
Biochemistry 28, 4253-4258). Inhibition of PMCA in acinar cells
inhibits Ca.sup.2+ efflux induced by supramaximal acetylcholine and
worsens antioxidant-induced necrosis in acinar cells in vitro
(Ferdek et al., 2012, Curr Biol 22, 1241-1246). Additionally, the
protective effects of insulin on pancreatitis responses are
mediated by its ability to preserve PMCA activity and prevent
pathological increases in Ca.sup.2+ (Samad et al., 2014, J Biol
Chem 289, 23582-23595; Mankad et al., 2012, J Biol Chem 287,
1823-1836).
[0257] These observations are consistent with the explanation that
PMCA is a key mediator of Ca.sup.2+ extrusion from the acinar cell
and reduces cell injury by lowering cytoplasmic Ca.sup.2+
levels.
[0258] The plasma membrane Ca.sup.2+ ATPase (PMCA) is the receptor
for renalase (Wang et al., 2015, Identification of a Receptor for
Extracellular Renalase. PLoS ONE 10, e0122932). As described
elsewhere herein, the protective effects of renalase exhibit
saturable kinetics, consistent with the explanation that it acts
through a plasma membrane receptor. Affinity crosslinking of a
biotinylated renalase peptide (RP-220) to membrane proteins in
human proximal tubular cell line (HK-2) and protein identification
by mass spectrometry identified the PMCA4b as the major renalase
binding protein (FIG. 6A). Co-localization (FIG. 6B) and
co-immunoprecipitation (FIG. 6C) confirmed protein-protein
interaction between endogenous renalase and PMCA4b. PMCA4b is a
widely expressed plasma-membrane Ca.sup.2+-extruder that is
localized to the basolateral membrane of polarized cells (Antalffy
et al., 2012, Cell Calcium 51, 171-178; Lopreiato et al., 2014, J
Biol Chem 289, 10261-10268). The data described herein are
consistent with the explanation that renalase activates PMCA to
decrease cytoplasmic Ca.sup.2+ levels (FIGS. 12 and 13).
Additionally, FIG. 6D shows that down-regulation of endogenous
PMCA4b expression by siRNA transfection blocks the protective
effects of RP-220 in HK-2 cells.
Renalase is Present in Mouse Acinar Cells and its Levels are
Reduced in the Serum After Inducing Acute Experimental
Pancreatitis
[0259] FIGS. 7A and 7B show that renalase is expressed in the
pancreatic acinar cell, but at lower levels than the kidney. Since
renalase is present in the serum (FIG. 6C), renalase could affect
the acinar cells through autocrine/paracrine pathways and also as a
hormone. Serum renalase levels decrease in a time-dependent manner
in mice after initiating cerulein-induced pancreatitis, falling by
about 70% after 7 hrs of cerulein pancreatitis (FIG. 7C). These
observations have important implications: i) the protective effects
of serum renalase are lost during the onset of acute pancreatitis
and ii) renalase is useful as a disease biomarker.
Genetic Deletion of Renalase Leads to Worsening of Acute
Pancreatitis
[0260] Since renalase has a protective effect, the genetic deletion
of endogenous renalase is associated with greater injury. This
issue was examined in mice with genetic deletion of renalase. FIG.
7B shows the loss of renalase protein in the knockout mouse. Though
the knockouts (renalase -/-) breed slowly and have slight
elevations in their resting blood pressure, they have no other
overt phenotype. FIG. 8 shows that renalase knockout (-/-) mice
exhibit more severe cerulein-induced pancreatitis, as measured by
zymogen activation, edema, and histologic severity than wild-type
(WT). This finding is consistent with the explanation that
endogenous renalase has a protective effect in acute pancreatitis.
Thus, a reduction in renalase levels at the onset of acute
pancreatitis may increase disease severity.
Renalase Reduces Injury in Pancreatic Acini
[0261] Next, it was assessed whether pretreatment with recombinant
human renalase reduces injury in pancreatitis. This was first
assessed in isolated groups of murine acinar cells (acini) that
were exposed to supraphysiologic concentrations of cerulein and
carbachol (which induces early acinar cell pancreatitis responses)
in isolated WT mouse acini and acini isolated from renalase
deficient mice in vitro (FIG. 9). The recombinant renalase reduced
trypsinogen activation induced by either cerulein (FIG. 9A) or
carbachol (FIG. 9B). Acini from renalase knockout (KO) mice also
showed reduced trypsinogen activation after renalase addition (FIG.
9C). Renalase had no effect on untreated acini or acini treated
with physiologic stimulation. Recombinant full-length renalase and
the truncated renalase peptide (RP-220) showed similar potency in
protecting acini from injury.
Pre-Treatment with Exogenous Recombinant Renalase Decreased
Symptoms of Pancreatitis Injury in Vitro (Acini) and in Vivo
[0262] To determine whether the protective of renalase observed in
isolated acini was relevant in vivo, WT mice were pretreated (1
hour) with a single dose of the renalase peptide (RP-220)
intraperitoneally (100 .mu.g peptide/25 g body weight) prior to
inducing cerulein-pancreatitis (6 hourly injections IP of 50
.mu.g/kg). This prophylactic pretreatment reduced all measures of
pancreatitis severity including, zymogen activation, edema,
apoptosis, inflammatory cell infiltrate, and histologic severity 7
hours after inducing disease (FIG. 10). Next, we examined whether
giving renalase after disease onset could reduce disease
severity.
Post-Treatment with Exogenous Recombinant Renalase After the Onset
of in Vivo Pancreatitis Reduced Symptoms of Injury
[0263] The same protocol was used as in the experiments in FIG. 10
to induce cerulein pancreatitis in WT mice, but renalase was not
given until 2 hours after initiating disease (FIG. 11). Individual
and aggregate histologic scores demonstrated that renalase
significantly decreased injury. The data shown in FIG. 10 and FIG.
11 indicate that exogenous renalase can reduce the severity of
pancreatitis in both a prophylactic and therapeutic context.
Exogenous Renalase Affects Cytosolic Ca.sup.2+-Signaling and
Appears to Activate the Plasma Membrane Ca.sup.2+ ATPase (PMCA)
[0264] PMCA selectively binds renalase (FIG. 6). To determine if
renalase affects the activity of PMCA4b (FIG. 6), an important
mediator of Ca.sup.2+ extrusion in many cell types, renalase
effects on Ca.sup.2+ extrusion were examined. Since the protective
effects of renalase were first characterized in HK-2 cells (human
kidney cell line) (Lee et al., 2013, J Am Soc Nephrol 24, 445-455;
Wang et al., 2014, Journal of the American Society of Nephrology:
JASN), its effects on Ca.sup.2+ signals were evaluated in these
cells and subsequently in mouse acinar cells (FIG. 12). Renalase
did not change baseline Ca.sup.2+ levels in either cell type. To
isolate effects on Ca.sup.2+ efflux through PMCA, other Ca.sup.2+
channels/pumps were blocked so that changes in cytosolic Ca.sup.2+
were solely due to PMCA. This included inhibition of re-uptake into
the endoplasmic reticulum and, in the case of HK2 cells, inhibition
of the plasma-membrane Na--Ca.sup.2+ exchanger that, similar to
PMCA, extrudes Ca.sup.2+ but is not present in acinar cells. To
prevent Ca.sup.2+ influx after stimulation, cells were perfused
with Ca.sup.2+ free medium with 1 mM EDTA. Cells were also
pretreated with cyclopiazonic acid to deplete ER stores. To inhibit
the Na.sup.+/Ca.sup.2+ exchangers present in in HK2 cells, but not
acinar cells (Muallem et al., 1988, J Membr Biol 102, 153-162),
Na.sup.+-free medium was used in HK2 cells (but not in acinar
cells). HK2 cells and isolated mouse acini were perfused with
Ca.sup.2+-free medium and then briefly stimulated with 10 nM
angiotensin IV (HK2 cells) or 100 nM cerulein (acinar cells) in the
presence of 1.3 mM Ca.sup.2+ to increase intracellular Ca.sup.2+.
Perfusion buffer was then switched back to Ca.sup.2+-free medium in
the presence or absence of renalase, and cytosolic Ca.sup.2+ levels
were monitored over time and the rates of Ca.sup.2+ efflux were
calculated (FIG. 12). In both HK2 cells and acinar cells, renalase
increased the rate of Ca.sup.2+ efflux.
[0265] Since PMCA has been identified as a renalase receptor and
renalase enhances Ca.sup.2+ efflux, it was assessed whether PMCA
inhibition would block the protective effects of renalase. FIG. 13
shows that pretreatment of acini with caloxin 1b1, a selective
peptide inhibitor of PMCA 4b (inhibition of
PMCA4b>PMCA1>PMCA2>PMCA3) that binds to its extracellular
loops (Strehler et al., 2013, Journal of pharmacy &
pharmaceutical sciences : a publication of the Canadian Society for
Pharmaceutical Sciences, Societe canadienne des sciences
pharmaceutiques 16, 190-206; Pande et al., 2008, Journal of
cellular and molecular medicine 12, 1049-1060), eliminated the
protective effect of renalase on zymogen activation, cellular
injury by MTT and histology. Together (FIG. 6, FIG. 12, FIG. 13),
the results described herein are consistent with the explanation
that renalase stimulates PMCA and increases the rates of calcium
efflux and that this effect is related to the protective effects of
renalase on acinar cell injury.
[0266] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
Sequence CWU 1
1
9120PRTHomo sapiens 1Leu Leu Arg Arg Gln Thr Ser Gly Pro Leu Tyr
Leu Ala Val Trp Asp 1 5 10 15 Lys Ala Glu Asp 20 220PRTHomo sapiens
2Phe Arg His Arg Val Thr Gln Ile Asn Leu Arg Asp Asp Lys Trp Glu 1
5 10 15 Val Ser Lys Gln 20 311PRTHomo sapiens 3Cys Val Ser Ile Asp
Asn Lys Lys Arg Asn Ile 1 5 10 420PRTHomo sapiens 4Cys Ile Arg Phe
Val Ser Ile Asp Asn Lys Lys Arg Asn Ile Glu Ser 1 5 10 15 Ser Glu
Ile Gly 20 526PRTHomo sapiens 5His His His His His His Cys Ile Arg
Phe Val Ser Ile Asp Asn Lys 1 5 10 15 Lys Arg Asn Ile Glu Ser Ser
Glu Ile Gly 20 25 619PRTHomo sapiens 6Ile Arg Phe Val Ser Ile Asp
Asn Ala Ala Ala Asn Ile Glu Ser Ser 1 5 10 15 Glu Ile Gly
719PRTHomo sapiens 7Cys Ser Lys Arg Ile Phe Lys Val Ile Ser Ser Ile
Glu Asp Asn Asn 1 5 10 15 Glu Arg Gly 8342PRTHomo sapiens 8Met Ala
Gln Val Leu Ile Val Gly Ala Gly Met Thr Gly Ser Leu Cys 1 5 10 15
Ala Ala Leu Leu Arg Arg Gln Thr Ser Gly Pro Leu Tyr Leu Ala Val 20
25 30 Trp Asp Lys Ala Glu Asp Ser Gly Gly Arg Met Thr Thr Ala Cys
Ser 35 40 45 Pro His Asn Pro Gln Cys Thr Ala Asp Leu Gly Ala Gln
Tyr Ile Thr 50 55 60 Cys Thr Pro His Tyr Ala Lys Lys His Gln Arg
Phe Tyr Asp Glu Leu 65 70 75 80 Leu Ala Tyr Gly Val Leu Arg Pro Leu
Ser Ser Pro Ile Glu Gly Met 85 90 95 Val Met Lys Glu Gly Asp Cys
Asn Phe Val Ala Pro Gln Gly Ile Ser 100 105 110 Ser Ile Ile Lys His
Tyr Leu Lys Glu Ser Gly Ala Glu Val Tyr Phe 115 120 125 Arg His Arg
Val Thr Gln Ile Asn Leu Arg Asp Asp Lys Trp Glu Val 130 135 140 Ser
Lys Gln Thr Gly Ser Pro Glu Gln Phe Asp Leu Ile Val Leu Thr 145 150
155 160 Met Pro Val Pro Glu Ile Leu Gln Leu Gln Gly Asp Ile Thr Thr
Leu 165 170 175 Ile Ser Glu Cys Gln Arg Gln Gln Leu Glu Ala Val Ser
Tyr Ser Ser 180 185 190 Arg Tyr Ala Leu Gly Leu Phe Tyr Glu Ala Gly
Thr Lys Ile Asp Val 195 200 205 Pro Trp Ala Gly Gln Tyr Ile Thr Ser
Asn Pro Cys Ile Arg Phe Val 210 215 220 Ser Ile Asp Asn Lys Lys Arg
Asn Ile Glu Ser Ser Glu Ile Gly Pro 225 230 235 240 Ser Leu Val Ile
His Thr Thr Val Pro Phe Gly Val Thr Tyr Leu Glu 245 250 255 His Ser
Ile Glu Asp Val Gln Glu Leu Val Phe Gln Gln Leu Glu Asn 260 265 270
Ile Leu Pro Gly Leu Pro Gln Pro Ile Ala Thr Lys Cys Gln Lys Trp 275
280 285 Arg His Ser Gln Val Thr Asn Ala Ala Ala Asn Cys Pro Gly Gln
Met 290 295 300 Thr Leu His His Lys Pro Phe Leu Ala Cys Gly Gly Asp
Gly Phe Thr 305 310 315 320 Gln Ser Asn Phe Asp Gly Cys Ile Thr Ser
Ala Leu Cys Val Leu Glu 325 330 335 Ala Leu Lys Asn Tyr Ile 340
9315PRTHomo sapiens 9Met Ala Gln Val Leu Ile Val Gly Ala Gly Met
Thr Gly Ser Leu Cys 1 5 10 15 Ala Ala Leu Leu Arg Arg Gln Thr Ser
Gly Pro Leu Tyr Leu Ala Val 20 25 30 Trp Asp Lys Ala Glu Asp Ser
Gly Gly Arg Met Thr Thr Ala Cys Ser 35 40 45 Pro His Asn Pro Gln
Cys Thr Ala Asp Leu Gly Ala Gln Tyr Ile Thr 50 55 60 Cys Thr Pro
His Tyr Ala Lys Lys His Gln Arg Phe Tyr Asp Glu Leu 65 70 75 80 Leu
Ala Tyr Gly Val Leu Arg Pro Leu Ser Ser Pro Ile Glu Gly Met 85 90
95 Val Met Lys Glu Gly Asp Cys Asn Phe Val Ala Pro Gln Gly Ile Ser
100 105 110 Ser Ile Ile Lys His Tyr Leu Lys Glu Ser Gly Ala Glu Val
Tyr Phe 115 120 125 Arg His Arg Val Thr Gln Ile Asn Leu Arg Asp Asp
Lys Trp Glu Val 130 135 140 Ser Lys Gln Thr Gly Ser Pro Glu Gln Phe
Asp Leu Ile Val Leu Thr 145 150 155 160 Met Pro Val Pro Glu Ile Leu
Gln Leu Gln Gly Asp Ile Thr Thr Leu 165 170 175 Ile Ser Glu Cys Gln
Arg Gln Gln Leu Glu Ala Val Ser Tyr Ser Ser 180 185 190 Arg Tyr Ala
Leu Gly Leu Phe Tyr Glu Ala Gly Thr Lys Ile Asp Val 195 200 205 Pro
Trp Ala Gly Gln Tyr Ile Thr Ser Asn Pro Cys Ile Arg Phe Val 210 215
220 Ser Ile Asp Asn Lys Lys Arg Asn Ile Glu Ser Ser Glu Ile Gly Pro
225 230 235 240 Ser Leu Val Ile His Thr Thr Val Pro Phe Gly Val Thr
Tyr Leu Glu 245 250 255 His Ser Ile Glu Asp Val Gln Glu Leu Val Phe
Gln Gln Leu Glu Asn 260 265 270 Ile Leu Pro Gly Leu Pro Gln Pro Ile
Ala Thr Lys Cys Gln Lys Trp 275 280 285 Arg His Ser Gln Val Pro Ser
Ala Gly Val Ile Leu Gly Cys Ala Lys 290 295 300 Ser Pro Trp Met Met
Ala Ile Gly Phe Pro Ile 305 310 315
* * * * *