U.S. patent application number 15/197228 was filed with the patent office on 2016-10-20 for e-prostanoid receptor, ptger3, as a novel anti-diabetic therapeutic target.
The applicant listed for this patent is Wisconsin Alumni Research Foundation. Invention is credited to Alan Attie, Mark Keller, Michelle Kimple.
Application Number | 20160303059 15/197228 |
Document ID | / |
Family ID | 49158183 |
Filed Date | 2016-10-20 |
United States Patent
Application |
20160303059 |
Kind Code |
A1 |
Keller; Mark ; et
al. |
October 20, 2016 |
E-PROSTANOID RECEPTOR, PTGER3, AS A NOVEL ANTI-DIABETIC THERAPEUTIC
TARGET
Abstract
Provided herein are methods for increasing insulin secretion
from beta cells. Also provide herein are methods comprising
administering to a subject in need of increased insulin secretion a
composition comprising a compound that directly or indirectly
activates adenylate cyclase and an E prostanoid 3 (EP3) receptor
antagonist that attenuates G alpha-i-subfamily (GSIS)-mediated
adenylate cyclase inhibition.
Inventors: |
Keller; Mark; (McFarland,
WI) ; Attie; Alan; (Madison, WI) ; Kimple;
Michelle; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wisconsin Alumni Research Foundation |
Madison |
WI |
US |
|
|
Family ID: |
49158183 |
Appl. No.: |
15/197228 |
Filed: |
June 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13774516 |
Feb 22, 2013 |
9381176 |
|
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15197228 |
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61602837 |
Feb 24, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4985 20130101;
A61K 31/404 20130101; A61P 3/10 20180101; A61P 5/50 20180101; A61P
7/12 20180101; A61K 38/26 20130101; A61P 3/08 20180101; A61K 31/18
20130101; A61K 38/26 20130101; A61K 2300/00 20130101; A61K 31/18
20130101; A61K 2300/00 20130101; A61K 31/404 20130101; A61K 2300/00
20130101; A61K 31/4985 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/18 20060101
A61K031/18; A61K 31/404 20060101 A61K031/404; A61K 31/4985 20060101
A61K031/4985; A61K 38/26 20060101 A61K038/26 |
Goverment Interests
STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
DK066369, DK058037, and DK080845 awarded by the National Institutes
of Health. The government has certain rights in the invention.
Claims
1-6. (canceled)
7. A method for increasing insulin secretion from beta cells of a
diabetic individual, the method comprising administering to the
individual a therapeutic combination comprising therapeutically
effective amounts of (1) a compound that directly or indirectly
activates adenylate cyclase and (2) an E prostanoid 3 receptor
antagonist that attenuates G alpha-i-subfamily-mediated adenylate
cyclase inhibition, whereby the beta cells secrete more insulin
after the therapeutic combination is administered than before, and
wherein the amount of insulin secreted by the beta cells is
increased relative to that secreted by beta cells of a diabetic
individual not receiving the combination or receiving either the
adenylate cyclase-activating compound or the E prostanoid 3
receptor antagonist alone.
8. The method of claim 7, wherein the compound that directly or
indirectly activates adenylate cyclase is selected from the group
consisting of a compound that activates a glucagon-like peptide-1
(GLP-1) receptor, a compound that activates a gastric inhibitory
peptide (GIP) receptor, and a compound that activates a pituitary
adenylate cyclase-activating peptide (PACAP) receptor.
9. The method of claim 8, wherein the compound that activates the
GLP-1 receptor is selected from the group consisting of a DPP-4
inhibitor and an incretin mimetic.
10. The method of claim 7, wherein the E prostanoid 3 receptor
antagonist is selected from the group consisting of L-798,106 and
DG-041.
11. The method of claim 7, wherein the compound that directly or
indirectly activates adenylate cyclase is sitagliptin and the E
prostanoid 3 receptor antagonist is L-798,106.
12. The method of claim 7, wherein the compound that directly or
indirectly activates adenylate cyclase is sitagliptin and the E
prostanoid 3 receptor antagonist is DG-041.
13. A method of treating diabetes in an individual, the method
comprising administering to a diabetic individual in need of
increased insulin secretion a therapeutic combination comprising
therapeutically effective amounts of (1) a compound that directly
or indirectly activates adenylate cyclase and (2) an E prostanoid 3
receptor antagonist that attenuates G alpha-i-subfamily-mediated
adenylate cyclase inhibition, whereby beta cells of the individual
secrete more insulin after the therapeutic combination is
administered than before, wherein insulin secretion is increased
relative to that of beta cells of a diabetic individual not
receiving the combination or receiving either the adenylate
cyclase-activating compound or the E prostanoid 3 receptor
antagonist alone, and whereby the increased insulin secretion
treats diabetes in the individual.
14. The method of claim 13, wherein the compound that directly or
indirectly activates adenylate cyclase is selected from the group
consisting of a compound that activates a GLP-1 receptor, a
compound that activates a GIP receptor, and a compound that
activates a PACAP receptor.
15. The method of claim 14, wherein the compound that activates the
GLP-1 receptor is selected from the group consisting of a DPP-4
inhibitor and an incretin mimetic.
16. The method of claim 13, wherein the E prostanoid 3 receptor
antagonist is selected from the group consisting of L-798,106 and
DG-041.
17. The method of claim 13, wherein the compound that directly or
indirectly activates adenylate cyclase is sitagliptin and the E
prostanoid 3 receptor antagonist is L-798,106.
18. The method of claim 13, wherein the compound that directly or
indirectly activates adenylate cyclase is sitagliptin and the E
prostanoid 3 receptor antagonist is DG-041.
19. The method of claim 13, wherein the diabetes is Type II
diabetes.
20. The method of claim 13, wherein the therapeutic combination
comprises more than one compound that directly or indirectly
activates adenylate cyclase.
21. The method of claim 13, wherein the therapeutic combination
comprises more than one E prostanoid 3 receptor antagonist that
attenuates G alpha-i-subfamily-mediated adenylate cyclase
inhibition.
22. The method of claim 7, wherein the compound that directly or
indirectly activates adenylate cyclase is a GLP-1 mimetic or a
DPP-4 inhibitor.
23. The method of claim 7, wherein the therapeutically effective
amounts are lower than effective amounts of the adenylate
cyclase-activating compound or the E prostanoid 3 receptor
antagonist when either is administered alone.
24. A composition comprising therapeutically effective amounts of
(1) a compound that directly or indirectly activates adenylate
cyclase and (2) an E prostanoid 3 receptor antagonist that
attenuates G alpha-i-subfamily-mediated adenylate cyclase
inhibition, and a pharmaceutically acceptable carrier.
25. The composition of claim 24, wherein the compound that directly
or indirectly activates adenylate cyclase is selected from the
group consisting of a compound that activates a GLP-1 receptor, a
compound that activates a GIP receptor, and a compound that
activates a PACAP receptor.
26. The composition of claim 25, wherein the compound that
activates the GLP-1 receptor is selected from the group consisting
of a DPP-4 inhibitor and an incretin mimetic.
27. The composition of claim 24, wherein the E prostanoid 3
receptor antagonist is selected from the group consisting of
L-798,106 and DG-041.
28. The composition of claim 24, wherein the compound that directly
or indirectly activates adenylate cyclase is sitagliptin and the E
prostanoid 3 receptor antagonist is L-798,106.
29. The composition of claim 24, wherein the compound that directly
or indirectly activates adenylate cyclase is sitagliptin and the E
prostanoid 3 receptor antagonist is DG-041.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 13/774,516, filed Feb. 22, 2013, which claims
the benefit of U.S. Provisional Application No. 61/602,837, filed
on Feb. 24, 2012, each of which is incorporated by reference herein
in its entirety.
BACKGROUND OF THE INVENTION
[0003] Type 2 diabetes mellitus (T2D) is a disease in which a
person has high blood sugar as a result of resistance of the body's
tissues to the glucose-lowering effects of insulin and failure of
the beta cells of the islets of Langerhans to produce enough
insulin. The American Diabetes Association reports that there are
18.8 million Americans with diagnosed T2D, 7.0 million individuals
with undiagnosed T2D, and another 79 million potential candidates
with pre-diabetes. An annual expenditure of $174 billion is
attributed to the disease; this figure from 2007 is expected to
rise. Complications of T2D are the third leading cause of death in
the United States; in 2007 T2D was listed as a contributing factor
to over 200,000 deaths. Prolonged untreated diabetes leads to heart
diseases, stroke, kidney disease, blindness, and loss of limbs from
advanced peripheral vascular disease. Combined, these facts
underscore the critical need for increased understanding of and
treatments for T2D.
[0004] The standard of care for T2D management in children and
adults is healthy eating, portion control, increased physical
activity, and glucose-lowering medications. However, few of the
available medications have been approved for use in children or
adolescents; thus, physicians are hesitant to prescribe these
medications, and in doing so, fail to prevent further beta cell
destruction if hyperglycemia persists.
[0005] Obesity is a risk factor for T2D because it is usually
associated with insulin resistance. However, although most people
with T2D are obese, most obese people do not develop T2D because
they compensate for insulin resistance by secreting more insulin.
When obese people progress to develop T2D, it is because their beta
cells are unable to satisfy the increased demand for insulin. Thus,
most of the newer T2D treatments in the clinic or under development
target beta cell dysfunction and not insulin sensitivity.
[0006] Drugs that target G protein complexes are used as T2D
therapeutics. GTP-binding proteins (G proteins) are
membrane-associated signaling molecules whose activity is regulated
by the cycle of GTP binding (active state) and GTP hydrolysis to
GDP (inactive state), followed by GDP dissociation and re-binding
of GTP. Heterotrimeric G proteins are composed of a
beta-gamma-dimer and a catalytically-active alpha-subunit that are
tightly associated with a transmembrane G protein-coupled receptor
(GPCR) in their inactive state. Upon activation by receptor-ligand
interaction these G protein-GPCR complexes dissociate in order to
transmit signals to downstream effectors (i.e., signal
transduction).
[0007] Of the four subfamilies of heterotrimeric G protein
alpha-subunits (G alpha-s, G alpha-i, G alpha-q, and G alpha-12),
only those in the G alpha-s subfamily can positively regulate the
catalytic activity of adenylate cyclase, increasing the conversion
of ATP to cAMP. cAMP is a known potentiator of beta cell function,
having been shown to augment glucose-stimulated insulin secretion
(GSIS) by numerous mechanisms (Lang, Eur. J. Biochem.
259:3-17(1999); Furman and Pyne, Curr. Opin. Investig. Drugs
7:898-905 (2006); Shibasaki et al., Proc. Natl. Acad. Sci. USA
104:19333-19338 (2007)). Furthermore, cAMP has also been shown to
have proliferative and anti-apoptotic effects on beta cells (Li et
al., J. Biol. Chem. 278:471-478 (2003)).
[0008] Drugs that target a specific GPCR are widely used as T2D
therapeutics. The hormone glucagon-like peptide 1 (GLP-1) is
secreted by specialized cells in the intestine in response to the
presence of nutrients from food. Sugars, proteins and fats can all
cause GLP-1 release from the gut cells. GLP-1 activates a G
alpha-s-coupled receptor on beta cells to stimulate cAMP production
and potentiate GSIS from beta cells of the islets of Langerhans.
Although GLP-1 is rapidly degraded by the enzyme dipeptidyl
peptidase (DPP)-4, stable GLP-1 analogs exenatide (Byetta.RTM.,
Lilly) and liraglutide (Victoza.RTM., Novo-Nordisk) have been shown
to be clinically effective for the treatment of T2D (Furman and
Pyne (2006); Triplitt, Am. J. Manag. Care 13:S47-54 (2007)). In
addition, inhibitors of DPP-4, including sitagliptin (Januvia.RTM.,
Merck), saxagliptin (Onglyza.RTM., Bristol-Myers Squibb),
vildagliptin (Galvus.RTM., Novartis), and linagliptin
(Tradjenta.RTM., Eli Lilly) also lead to improved beta cell
function and glucose clearance in T2D patients (Furman and Pyne,
supra; Triplitt, supra).
[0009] Of all the current diabetes therapeutics, agents that
stimulate beta cell cAMP production, including DPP-4 inhibitors and
GLP-1 analogs, are the only ones that positively impact beta cell
replication, neogenesis, and/or survival in rodent models (Xu et
al., Diabetes 48:2260-2276 (1999); Wang and Brubaker Diabetologia
45:1263-1273 (2002); Sturis et al., Br. J. Pharmacol. 140-123-132
(2003); Perfetti et al., Endocrinology 141:4600-4605 (2000);
Gedulin et al., Endocrinology 146:2069-2076 (2005); Farilla et al.,
Endocrinology 143:2069-2076 (2002)) or human islets (Farilla et
al., Endocrinology 144:5149-5158 (2003)). Interestingly, GLP-1
treatment can protect both rodent and human beta cells from
immune-mediated apoptosis (Sano et al., Biochem. Biophys. Res.
Commun. 404:756-761 (2011); Pugazhenthi et al., Diabetologia
53:2357-2368 (2010)).
[0010] Unfortunately, not all diabetic patients respond to
GLP-1-based treatments. Approximately 35-60% of diabetic patients
treated with sitagliptin fail to achieve a glycosylated hemoglobin
(i.e., HbAlc) target of <7% (Raz et al., Diabetologia
49:2564-2571 (2006); Aschner et al., Diabetes Care 29:2632-2637
(2006); Nonaka et al., Diabetes Res. Clin. Prac. 79:291-298
(2008)). HbAlc.fwdarw.6.5% is a criterion for the diagnosis of
diabetes, according to the American Diabetes Association. Further,
there exists a controversy in the literature about whether agents
that target GLP-1 action or breakdown also stimulate pancreatitis,
a risk factor for the later development of pancreatic cancer
(Anderson and Trujillo, Ann. Pharmacother. 44:904-909 (2010);
Elashoff et al., Gastroenterology 141:150-156 (2011)).
[0011] There remains a need in the art for additional treatments
for increasing insulin secretion from beta cells in individuals
with T2D and in individuals at risk for developing T2D.
BRIEF SUMMARY
[0012] The invention relates generally to methods for increasing
insulin secretion from beta cells and, more particularly, to
methods comprising administering to a subject in need of increased
insulin secretion a composition comprising a compound that
activates adenylate cyclase and an E prostanoid 3 receptor
antagonist that attenuates G alpha-i-subfamily (GSIS)-mediated
adenylate cyclase inhibition. EP3 (gene symbol: Ptger3) is a
cellular receptor for E-series prostanoids such as PGE1 and
PGE2.
[0013] In one aspect, the present invention is summarized as a
method for increasing insulin secretion from beta cells. The method
includes the steps of administering to a subject in need of
increased insulin secretion a composition comprising a compound
that directly or indirectly activates adenylate cyclase and an E
prostanoid 3 receptor antagonist that attenuates G
alpha-i-subfamily (GSIS)-mediated adenylate cyclase inhibition. In
some embodiments, the compound that leads to activation of
adenylate cyclase is selected from the group consisting of a
compound that activates a glucagon-like peptide-1 (GLP-1) receptor,
a gastric inhibitory peptide (GIP) receptor, or a pituitary
adenylate cyclase-activating peptide (PACAP) receptor. In some of
these embodiments, the compound that activates the GLP-1 receptor
is selected from the group consisting of DPP-4 inhibitors and
incretin mimetics. In some embodiments, a compound that modulates
EP3 is L-798,106. In other embodiments, an E prostanoid 3 receptor
antagonist is DG-041 or another EP3 antagonist known in the art. In
a preferred embodiment of the present invention, a compound that
directly or indirectly activates adenylate cyclase is sitagliptin
and an E prostanoid 3 receptor antagonist is L-798,106.
[0014] In a further aspect, the invention is summarized as a method
of treating or preventing type II diabetes in an individual. The
method includes the steps of administering to a subject in need of
increased insulin secretion a comprising a compound that directly
or indirectly activates adenylate cyclase and an E prostanoid 3
receptor antagonist that attenuates G alpha-i-subfamily
(GSIS)-mediated adenylate cyclase inhibition. In some embodiments,
the compound that directly or indirectly activates adenylate
cyclase is selected from the group consisting of a compound that
activates a glucagon-like peptide-1 (GLP-1) receptor, a gastric
inhibitory peptide (GIP) receptor, or a pituitary adenylate
cyclase-activating peptide (PACAP) receptor. In some of these
embodiments the compound that activates the GLP-1 receptor is
selected from the group consisting of DPP-4 inhibitors and incretin
mimetics. In some embodiments, a compound that attenuates
GSIS-mediated adenylate cyclase inhibition is L-798,106. In a
preferred embodiment of the present invention, a compound that
directly or indirectly activates adenylate cyclase is sitagliptin
and the E prostanoid 3 receptor antagonist that attenuates
GSIS-mediated adenylate cyclase inhibition is L-798,106.
[0015] In another aspect, the invention is summarized as a method
of treating or preventing type I diabetes in an individual. The
method includes the steps of administering to a subject in need of
increased insulin secretion a compound that directly or indirectly
activates adenylate cyclase and an E prostanoid 3 receptor
antagonist that attenuates G alpha-i-subfamily (GSIS)-mediated
adenylate cyclase inhibition by modulating EP3. In some cases, the
method comprises administering to a subject in need of increased
insulin secretion any pairwise combination of a compound that
directly or indirectly activates adenylate cyclase and a compound
that attenuates GSIS-mediated adenylate cyclase inhibition
described herein. In some cases, the method comprises administering
more than one compound that directly or indirectly activates
adenylate cyclase or more than one E prostanoid 3 receptor
antagonist that attenuates G alpha-i-subfamily (GSIS)-mediated
adenylate cyclase inhibition by modulating EP3.
[0016] In a further aspect, the invention is summarized as a
composition comprising a compound that directly or indirectly
activates adenylate cyclase and an E prostanoid 3 receptor
antagonist that attenuates G alpha-i-subfamily (GSIS)-mediated
adenylate cyclase inhibition by modulating EP3. The composition can
comprise any pairwise combination of a compound that directly or
indirectly activates adenylate cyclase and a compound that
attenuates GSIS-mediated adenylate cyclase inhibition described
herein.
[0017] These and other features, objects, and advantages of the
present invention will become better understood from the
description that follows. In the description, reference is made to
the accompanying drawings, which form a part hereof and in which
there is shown by way of illustration, not limitation, embodiments
of the invention. The description of preferred embodiments is not
intended to limit the invention to cover all modifications,
equivalents and alternatives. Reference should therefore be made to
the claims recited herein for interpreting the scope of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The present invention will be better understood and
features, aspects and advantages other than those set forth above
will become apparent when consideration is given to the following
detailed description thereof. Such detailed description makes
reference to the following drawings, wherein:
[0019] FIGS. 1A-1B illustrate using BTBR-Ob mice as a model for
identifying novel therapeutics that might prevent or reverse beta
cell de-compensation in T2D. Depicted are fasting plasma glucose
levels (A) and insulin levels (B) in plasma samples obtained from
genetically obese (Leptin ob/ob) B6 and BTBR mice. Results are
shown as a function of age in weeks.
[0020] FIGS. 2A-2B illustrate that diabetes and Ptger3 gene
expression are highly correlated. FIG. 2A depicts fasting plasma
glucose values in plasma samples obtained from B6 and BTBR mice at
4 and 10 weeks of age, when mice were either lean (L) or obese (0).
Circles show individual mice (n=5 for each condition). Horizontal
bars show mean+/-SEM. The convergence of strain, age and obesity
revealed the diabetes-susceptible nature of BTBR mice. FIG. 2B
depicts islet expression profiling of the same group of mice
illustrated in FIG. 2A. 10-week-old BTBR-Ob mice were diabetic; all
of the other mouse groups were non-diabetic. Diabetic BTBR-Ob mice
were the only group that displayed significantly increased Ptger3
expression on an Agilent mouse gene microarray.
[0021] FIG. 3 illustrates that both the receptor for E prostanoids
(EP3) and the enzymes responsible for their synthesis are
up-regulated in diabetic islets. Quantitative real-time (qRT) PCR
was performed on cDNA samples generated from non-diabetic and
diabetic mouse islets. EP3 total represents a primer set to a
region common in all three splice variants (alpha, beta, and
gamma), all of which were elevated in diabetic islets. Also tested
were primers against the other E prostanoid receptors (EP1, EP2,
and EP4). Primers against the prostaglandin-endoperoxidase
synthases (Ptgs1, Ptgs2, and Ptgs3), as well as the prostaglandin E
synthases (Ptges and Ptges2) were also tested. The expression of
both Ptgs1 and Ptgs2, the rate limiting step in PGE2 synthesis from
arachidonic acid, are both elevated in diabetic islets.
[0022] FIGS. 4A-4B depict analyses of the gene promoters of the
mouse, rat, and human Ptger3 gene. The genomic sequence 10,000 base
pairs upstream of the transcription start site was used as the
promoter region, and was manually analyzed in Matlnspector
(Genomatix) using the consensus sequences for carbohydrate response
element binding protein (ChREBP) (E-boxes). The mouse and rat
promoter sequences are conserved and contain two identical E-boxes.
While the human promoter is not conserved among species, it
contains at least five degenerate E-box sequences.
[0023] FIG. 5 illustrates that a Ptger3 antagonist can normalize
insulin secretion from islets obtained from diabetic mice. Insulin
secretion from islets isolated from either 10 week old lean
(non-diabetic; left side, unshaded bars) or ob/ob (diabetic; right
side, shaded bars) BTBR mice. High glucose (16.7 mM) was used to
stimulate insulin secretion in the absence (-) or presence (+) of
the EP3-selective agonist PGE1 (5 .mu.M), or the EP3-specific
antagonist L-798,106 (20 .mu.M); 1 hour, static incubation. High
glucose was significantly different than low glucose (1.7 mM) for
all conditions (p<0.03). PGE1 and L-798,106 significantly
altered insulin secretion from islets obtained from diabetic mice,
but not islets obtained from non-diabetic mice. The data are
representative of three independent experiments.
[0024] FIG. 6 illustrates that a Ptger3 antagonist can enhance
insulin secretion from diabetic human islets. Insulin secretion
assays were performed with islets obtained from human cadaveric
organ donors that were either non-diabetic or from patients
confirmed to have T2D. High glucose (16.7 mM) was used to stimulate
insulin secretion in the absence or presence of the L-798,106 (20
.mu.M) in a 45 minute static incubation. Data are represented as
the fold potentiation of insulin secretion relative to high-glucose
alone. Means and error bars represent the experimental mean+/-SEM
for each human islet sample. Islets obtained from non-diabetic
donors 5-7, which were non-responsive to L-798-106, were incubated
overnight in low glucose medium (8 mM) containing 0.5 mM xylitol to
mimic high glucose exposure without the confounding effects of
chronic insulin secretion. Under these conditions, L-798,106 was
effective to promote glucose dependent insulin secretion.
[0025] FIG. 7 illustrates that PGE2 production is elevated in
islets from diabetic mice or diabetic humans. Islets were cultured
for 24 hours in medium containing 8 mM glucose. The amount of PGE2
secreted into the medium was determined by a specific assay and was
normalized to the total number of islets used for each
measurement.
[0026] FIG. 8 illustrates that both the natural ligand of EP3
(PGE2) and a specific agonist (sulprostone) dose-dependently
suppresses insulin secretion from beta cells. The EP3 antagonist
L-798,106 suppresses the effects of PGE2, restoring insulin
secretion to levels observed in the absence of the EP3 agonists.
Insulin secretion was monitored from the rat insulinoma beta cell
line Ins-1 832/3 in response to high glucose (16.7 mM). Increasing
concentrations of PGE2 resulted in .about.80% maximal reduction in
insulin secretion, indicating functional Ptger3 receptor
expression. Parallel studies show a high level of Ptger3 expression
in Ins-1 cells. The approximate IC50 for PGE2 was .about.10 nM. In
the presence of 50 nM PGE2, increasing concentrations of L-798,106
restored maximal insulin secretion, demonstrating effective
inhibition of Ptger3. The approximate EC50 for L-798,106 in the
presence of 50 nM (maximal inhibitory concentration) was 100 nM.
Similar results were observed for suprostone-mediated suppression
of insulin secretion and the reversal of this suppression by
L-798,106. All studies were 2 hour static incubations. Data are the
mean of at least three independent studies.
[0027] FIG. 9 illustrates that a Ptger3 antagonist can augment the
effect of GLP-1 on insulin secretion from islets obtained from
diabetic mice. Insulin secretion was measured from islets obtained
from non-diabetic BTBR mice (left panel) and from diabetic
BTBR-ob/ob mice (right panel) in response to intermediate glucose
(11.1 mM) alone, or in the presence of GLP-1 (50 nM) (+,-),
L-798,106 (10 .mu.M) (-,+), or the combination of GLP-1 and
L-798,106 (+,+). The Ptger3 antagonist augments GLP-1 dependent
insulin secretion from diabetic, but not non-islets, demonstrating
a synergism between EP3 antagonism and GLP-1 mediated insulin
secretion exclusively from diabetic islets.
[0028] FIG. 10 illustrates the model of inverse regulation of the
cAMP signaling pathway in beta cells by EP3 (Ptger3 gene product)
and GLP-1. GLP-1 analogs and inhibitors of the DPP-4 enzyme that
degrades endogenous GLP-1 all function in the beta cell by
increasing cAMP production mediated by AC. cAMP can positively
impact both insulin secretion and, potentially, beta cell mass, by
activating beta cell proliferation, growth, and survival pathways.
The EP3 isoform of the E prostanoid receptor (Ptger3) blocks cAMP
production via a Gi-coupled pathway; thus, activated EP3 may oppose
all of the functions of GLP-1-based therapeutics in beta cells.
Antagonizing the Ptger3 pathway may prove to be a superior
therapeutic for T2D, alone or in combination with GLP-1 receptor
agonism.
[0029] FIG. 11 illustrates the molecular structure of L-798,106;
chemical name:
(2E)-N-[(5-bromo-2-methoxyphenyl)sulfonyl]-3-[2-(2-naphthalenylmeth-
yl)phenyl]-2-propenamide.
[0030] FIG. 12 illustrates the molecular structure of DG-041;
chemical name:
(2E)-3-[1-[(2,4-dichlorophenyl)methyl]-5-fluoro-3-methyl-1H-indol-7-
-yl]-N-[(4,5-dichloro-2-thienyl)sulfonyl]-2-propenamide
(DG-041).
[0031] While the present invention is susceptible to various
modifications and alternative forms, exemplary embodiments thereof
are shown by way of example in the drawings and are herein
described in detail. It should be understood, however, that the
description of exemplary embodiments is not intended to limit the
invention to the particular forms disclosed, but on the contrary,
the intention is to cover all modifications, equivalents and
alternatives falling within the spirit and scope of the invention
as defined by the appended claims.
DESCRIPTION OF THE INVENTION
[0032] Broadly, provided herein are methods for increasing insulin
secretion from beta cells. More specifically, this document
provides methods of administering to a subject in need of increased
insulin a composition comprising a compound that leads to
activation of adenylate cyclase and a compound that inhibits G
alpha-i-mediated inhibition of adenylate cyclase by modulating EP3
activity (gene symbol: Ptger3). Dosage of the composition, and the
agents in the composition, can be administered in an amount
sufficient to achieve GSIS from beta cells at a level
characteristic of that secreted by a beta cell obtained from a
non-diabetic subject. One of skill in the art in possession of this
disclosure can optimize the dosages as appropriate. The present
invention is based, at least in part, on the Inventors' discovery
that diabetes is associated with increased expression of Ptger3 in
pancreatic islets. Through experiments described in the Examples
below, the Inventors found that: (i) increased Ptger3 expression in
diabetic mouse islet cells results in E-prostanoid-mediated
negative regulation of insulin secretion from beta cells; and (ii)
when EP3 activity is suppressed in islets from diabetic subjects,
insulin secretion can be elevated to levels comparable to that
observed from non-diabetic subjects. Further, the Inventors found
that treating islets obtained from diabetic mice with both GLP-1
and an EP3 antagonist increased GSIS to a level higher than that
achieved by treatment with either GLP-1 or the EP3 antagonist
alone. This finding is particularly important given that T2D drugs
that target the GLP-1 pathway are inefficacious in a significant
portion of T2D patients (Raz et al., supra; Aschner et al., supra;
Nonaka et al., supra).
[0033] The methods disclosed have valuable applications including
treatment or prevention of type 1 diabetes (T1D) or T2D. In several
aspects, the present invention involves increasing insulin
secretion by increasing cyclic AMP (cAMP) production mediated by
adenylate cyclase (AC). cAMP can positively impact both insulin
secretion and, potentially, beta cell mass by activating beta cell
proliferation, growth, and survival pathways. Some agents currently
used to increase insulin secretion by increasing cAMP production
target the GLP-1 pathway, which is known to activate AC in beta
cells.
[0034] In contrast, the methods of the present invention increase
insulin secretion by increasing cAMP production by targeting two
pathways that modulate adenylate cyclase in beta cells: (i) a
hormonal-mediated positive regulatory pathway, such as GLP-1, and
(ii) the negative regulation mediated by the E prostanoid receptor
EP3. The Inventors found that, when EP3 is activated in beta cells,
insulin secretion from beta cells is inhibited, likely through
cAMP. EP3 expression and/or E prostanoid production are elevated
only in subjects with diabetes. Blocking EP3 activity (for example,
using an EP3-specific antagonistic compound) enhances GSIS from
diabetic pancreatic islets to a level characteristic of a
non-diabetic islet. Advantageously, since EP3 expression and
activity are only elevated in response to diabetes, molecules that
are developed to inhibit Ptger3 should only be effective in
diabetic patients, yielding a disease-specific drug profile.
[0035] The Inventors' observation that EP3 activity modulates GSIS,
and the fact that EP3 can inhibit cAMP production in beta cells,
are relevant to the disclosed method. Knowing that T2D drugs that
target hormonal pathways such as GLP-1 that signal downstream to
adenylate cyclase-mediated cAMP production in beta cells, the
Inventors predicted that treating islets obtained from diabetic
subjects with a composition containing both a compound that
directly or indirectly activates adenylate cyclase and, in
response, stimulates AC-mediated cAMP production in beta cells and
a compound that inhibits EP3 activity would lead to increased GSIS
from the treated islets. Without being bound to any particular
theory, a composition described herein is effective for increasing
GSIS in islets obtained from diabetic mice, at least in part
because the adenylate cyclase-activating and EP3-inhibiting
compounds of each composition simultaneously relieve a tonic
inhibition on adenylate cyclase caused by increased endogenous E
prostanoid signaling through increased EP3 receptor and also
stimulate AC through the stimulatory GLP-1 receptor.
[0036] Adenylate cyclase is an enzyme capable of integrating
positive and negative signals that act directly from G
protein-coupled receptors (GPCRs) through stimulation of the
G-protein alpha and beta/gamma subunits or indirectly via
intracellular signaling by, for example, a
calcium/calmodulin-dependent protein kinase (CaMK) or a Protein
Kinase C (PKC) isoenzyme. As used herein, adenylate cyclase is
"activated" when any process (e.g., a conformational change
stimulated by a G-protein) initiates the activity of an inactive
adenylate cyclase enzyme.
[0037] In some embodiments of the present invention, the methods
can be used to treat diabetic subjects exhibiting sub-optimal GSIS
in response to treatment with GLP-1 mimetics or DPP-4 inhibitors.
As used herein, "sub-optimal" means that GSIS levels in the subject
are lower than those exhibited by a non-diabetic subject. One of
skill in the art can develop appropriate dosing regimens sufficient
to balance adenylate cyclase-mediated cAMP production and
subsequent insulin secretion using the methods of the present
invention. GLP-1 mimetics suitable for use in the present invention
include, but are not limited to exentatide, liraglutide, and
taspoglutide. DPP-4 inhibitors suitable for use in the present
invention include, but are not limited to sitagliptin, saxagliptin,
vildagliptin, and linagliptin. In some embodiments of the present
invention, the methods can be used to treat subjects who react
adversely to GLP-1 mimetics or DPP-4 inhibitors. As used herein,
"react adversely" means that the subject exhibits symptoms or
characteristics, physiological or otherwise, that are unfavorable
in terms of the subject's health. In such cases, the required
effective amount of GLP-1 mimetics or DPP-4 inhibitors might be
lower when administered in combination with a Ptger3 antagonist
relative to administration of the former alone, at least in part
because increasing the ratio of Ptger3 antagonist to GLP-1 agonist
would likely result in increased insulin secretion from beta cells.
One of skill in the art can develop appropriate dosing regimens for
such situations.
[0038] It is contemplated that the present invention can also be
practiced using a combination of an EP3 antagonist and a compound
targeted to a hormonal pathway other than GLP-1 that stimulates
adenylate cyclase-mediated cAMP production in beta cells. Examples
of such hormones are gastric inhibitory polypeptide (GIP),
pituitary adenylate cyclase-activating peptide (PACAP), and
vasoactive intestinal peptide (VIP). Like GLP-1, GIP, PACAP, and
VIP are classified as "incretins." Incretins function in beta cells
to potentiate GSIS. Furthermore, receptors for GLP-1, GIP, PACAP,
and VIP are in the same subfamily as the GLP-1 receptor (Subfamily
B1 of the Secretin family of GPCRs, also referred to as Class B or
Class 2). Thus, it is contemplated that any agent having the
capacity to potentiate GLP-1 function in a cell could also
potentiate other incretin functions in said cell if these incretin
receptors were still expressed on the cell surface in the T2D
state.
[0039] It is contemplated that in some cases the present invention
can be practiced using any pairwise combination of a compound that
directly or indirectly activates adenylate cyclase and a compound
that attenuates GSIS-mediated adenylate cyclase inhibition
described herein. For example, a method described herein can
comprise administering to a subject in need of increased insulin
secretion more than one compound that directly or indirectly
activates adenylate cyclase. In some cases, the method can
alternatively or additionally comprise administering to a subject
in need of increased insulin secretion more than one E prostanoid 3
receptor antagonist that attenuates G alpha-i-subfamily
(GSIS)-mediated adenylate cyclase inhibition by modulating EP3.
[0040] It is contemplated that the compositions of the present
invention can be administered to subjects in need of increased
insulin secretion from beta cells. The composition can be
administered to the subject via an appropriate delivery route and
device. One of skill in the art can develop appropriate dose
delivery methods. The composition can be provided as part of a kit.
Such a kit could include a composition as described and claimed
herein and a delivery device to administer the composition to the
subject. It is contemplated that the present invention can be
practiced using a variety of EP3 antagonists. L-798,106
(5-Bromo-2-methoxy-N-[3-(2-naphthalen-2-ylmethyl-phenyl)-acryloyl]-benzen-
esulfonamide) is one EP3 antagonist that is exemplified herein.
See, for example, Jones et al., Fundam. Clin. Pharmacol. 22 (Suppl.
2):P078, 2008; Clarke et al., Br. J. Pharmacol. 141:600-609, 2004.
In some cases, other EP3 antagonists can be used to practice the
methods provided herein. For example, DG-041 is an EP3 antagonist
that can be used to practice the methods provided herein (Singh et
al., ACS Chem. Biol. 4:115-126, 2009). Additional selective EP3
receptor ligands appropriate for use according to the methods
provided herein are described in Juteau et al., Bioorg. Med. Chem.
9:1977-1984, 2001(a); Juteau et al., Bioorg. Med. Chem. Lett.
11:747-749, 2001(b); Belley et al., Bioorg. Med. Chem. Lett.
15:527-530, 2005; Belley et al., Bioorg. Med. Chem. Lett.
16:5639-5642, 2006; Jin et al., ACS Med. Chem. Lett. 1:316-320,
2010; Gallant et al., 2002 (Bioorg. Med. Chem. Lett. 12:2583-2586);
Zhou et al., Bioorg Med Chem Lett. 19:123-126, 2009(a); Zhou et
al., Bioorg. Med. Chem. Lett. 19:1528-153, 2009(b); Zhou et al.,
Bioorg. Med. Chem. Lett. 20:2658-2664, 2010; O'Connell et al.,
Bioorg. Med. Chem. Lett. 19:778-782, 2009; Mishra and Singh, J.
Chem. Inf. Model. 50:1502-1509, 2010; Hategan et al., Bioorg. Med.
Chem. Lett. 19:6797-6800, 2009; Li et al., Bioorg. Med. Chem. Lett.
20:6744-6747, 2010; Morales-Ramos et al., Bioorg. Med. Chem. Lett.
21:2806-2811, 2011; Hilfiker et al., Bioorg. Med. Chem. Lett.
19:4292-4295, 2009, each of which is incorporated herein by
reference as if set forth in its entirety.
[0041] The Inventors were the first to recognize that increased EP3
activity negatively modulates insulin secretion in pancreatic
islets obtained from diabetic subjects. Previously, the Inventors
profiled gene expression in six tissues of lean and obese B6 and
BTBR mice before and after the onset of diabetes (Keller et al.,
Genome Res. 18:706-716 (2008)). Although it was not mentioned in
Keller et al. (2008), data provided by the aforementioned study
indicates that the expression level of the prostaglandin E receptor
3 (subtype EP3), encoded by Ptger3, is elevated >30-fold in the
islets of diabetic BTBR mice relative to non-diabetic mice.
[0042] Prostaglandin E receptor 3 is a member of the G-protein
coupled receptor family. It is one of four receptors identified for
E-series prostanoids such as PGE1 and PGE2, and the only receptor
that couples primarily to G alpha i-subfamily proteins. In a
rat-derived beta cell line, the endogenous E prostanoid receptor
signals exclusively through G alpha-z, a member of the G alpha-i
subfamily of heterotrimeric G proteins, to block insulin secretion
(Kimple et al., J. Biol. Chem. 280:31708-31713 (2005)). Because of
the observed negative effect of PGE1 on insulin secretion, the
identity of the E prostanoid receptor was presumed to be EP3.
Further, loss of expression of G alpha-z caused constitutive
increases in pancreatic islet cAMP production and GSIS (Kimple et
al., J. Biol. Chem. 283:4560-4567 (2008)), suggesting a role for G
alpha-i-subfamily signaling in regulating pancreatic beta cell
function and biology. In view of this information and the finding
that Ptger3 expression is upregulated in the islets of diabetic
mice relative to non-diabetic mice, the Inventors hypothesized that
a PGE2-EP3-G alpha-i pathway is upregulated in animals with T2D.
Thus, dysfunctional signaling by the PGE2-EP3-G alpha-i subfamily
pathway caused by increased expression of the receptor could
negatively affect cAMP production, leading to diminished beta cell
function and mass. The Inventors hypothesized that antagonism of
the EP3 receptor might prevent or reverse the beta cell
pathobiology characteristic of T2D.
[0043] 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 to or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and materials are
described herein.
[0044] In describing the embodiments and claiming the invention,
the following terminology will be used in accordance with the
definitions set out below.
[0045] As used herein, "about" means within 5% of a stated
concentration range or within 5% of a stated time frame.
[0046] As used herein, "administering" or "administration" includes
any means for introducing a compound of the present invention into
the body, preferably into the systemic circulation. Examples
include but are not limited to oral, nasal, otic, ophthalmic,
buccal, sublingual, pulmonary, transdermal, transmucosal, as well
as subcutaneous, intraperitoneal, intravenous, epidural and
intramuscular injection.
[0047] As used herein, "agonist" means a chemical, compound, or
molecule that binds to a cellular receptor and activates it to
cause a response. Agonists can be naturally occurring or chemical
mimics.
[0048] As used herein, "analog" means a chemical mimic of a
naturally-occurring agonist. As drugs, analogs might be used for
many reasons, including, but not limited to, increased stability,
longevity, activation of a cellular receptor, or access
to/restriction from a particular body tissue.
[0049] As used herein, "antagonist" means a chemical, compound or
molecule that binds to a cellular receptor but does not provoke the
biological response seen when an agonist binds to the receptor.
Antagonists can compete with agonists for binding to the same site
on the receptor, or might bind to a separate site, causing the
receptor to undergo changes that preclude agonist binding.
Antagonists can be used therapeutically to block an overactive
agonist/receptor interaction that is causing an undesired
biological consequence.
[0050] As used herein, "effective amount" means an amount of an
agent sufficient to evoke a specified cellular effect according to
the present invention.
[0051] As used herein, "obesity" means a medical condition in which
excess body fat has accumulated to the extent that it may have a
negative effect on health. One measurement of body fat is the body
mass index (BMI), a measurement that compares weight and height,
defines obesity as greater than 30 kg (weight) per m.sup.2
(height.sup.2). Obesity increases the likelihood of various
diseases, particularly heart disease and T2D.
[0052] A "therapeutically effective amount" means an amount of a
compound that, when administered to a subject for treating a
disorder, condition, or disease, is sufficient to effect such
treatment for the disorder, or condition, or disease. The
"therapeutically effective amount" will vary depending on the
compound, the disorder, or condition, or disease state being
treated, the severity or the disorder, or condition, or disease
treated, the age and relative health of the subject, the route and
form of administration, the judgment of the attending medical or
veterinary practitioner, and other factors.
[0053] For purposes of the present invention, "treating" or
"treatment" describes the management and care of a patient for the
purpose of combating the disease, condition, or disorder. The terms
embrace both preventative, i.e., prophylactic, and palliative
treatments. Treating includes the administration of a compound of
present invention to prevent the onset of the symptoms or
complications, alleviating the symptoms or complications, or
eliminating the disease, condition, or disorder.
[0054] The invention will be more fully understood upon
consideration of the following non-limiting Examples. It is
specifically contemplated that the methods disclosed are suited for
treatment of Types I and II Diabetes generally. All papers and
patents disclosed herein are hereby incorporated by reference as if
set forth in their entirety.
EXAMPLES
Example 1
Materials and Methods
[0055] Animals.
[0056] C57B1/6 and BTBR mice, both lean and harboring the
Leptin.sup.Ob mutation, were derived from in-house breeding
colonies in the University of Wisconsin Biochemistry Department, as
described in Keller et al. (2008).
[0057] Plasma Measurements.
[0058] Blood samples were collected by retroorbital puncture from
mice fasted for 4 hours (8 am-noon) in EDTA-coated tubes to
generate plasma samples. Plasma glucose was measured by the glucose
oxidase method using a commercially available kit (Sigma-Aldrich).
For lean mice, insulin was measured by radioimmunoassay (RIA;
RI-13K, Linco Research). For ob mice, insulin was measured by an
in-house developed ELISA using a pair of anti-insulin/proinsulin
antibodies (clones D6C4 and D3E7-BT) purchased from Research
Diagnostics. Briefly, half-area 96-well high-binding plates
(Corning) were coated overnight with 3 .mu.g/mL D6C4/PBS to a total
volume of 50 .mu.L/well. After aspiration of D6C4, plates were
blocked with PBS containing 4% RIA-grade BSA (Sigma) for 1 hour
(100 .mu.L/well) and then incubated for 1 hour with 25 .mu.L
insulin standards (Linco Research, 0.1-10 ng/mL) or plasma samples.
25 .mu.l D3E7-BT, at 1 .mu.g/ml in PBS/1% BSA was added, gently
mixed, and incubated for an additional hour. After washing each
well three times with wash buffer (50 mM Tris, 0.2% Tween-20, pH
8.0), 1 .mu.g/mL of streptavidin-HRP (Pierce) in PBS/0.1% BSA was
added (50 .mu.L/well) and incubated for 30 minutes. Following an
additional three washes, 16 .mu.mol/mL of o-phenylenediamine
(Sigma), dissolved in citrate buffer (0.1 M citrate-phosphate,
0.03% H.sub.2O.sub.2 at pH 5.0), was added (50 .mu.L/well) and
incubated for 30 minutes, followed by an equal volume of 0.18 M
sulfuric acid to quench the reaction. Absorbance at 492 nm was
determined by a plate reader (Ultra 384 TECAN).
[0059] Mouse Islet Isolation & Culture.
[0060] Intact pancreatic islets were isolated from 10-12-week-old
BTBR lean and ob/ob mice using a known collagenase digestion
protocol (Rabaglia et al., Am. J. Physiology Endo. Metab.
289:E218-224 (2005)). Instead of CO.sub.2 asphyxiation and
decapitation, mice were Avertin-anesthetized and exsanguinated
immediately prior to cannulation of the bile duct and inflation of
the pancreas with a collagenase solution. Islets were cultured
overnight in 5 ml RPMI 1640 containing 11.1 mM glucose and 10%
heat-inactivated FBS with 1.times. penicillin/streptomycin prior to
in vitro assay for insulin secretion and PGE2 production.
[0061] Mouse Islet Insulin Secretion and PGE2 Production
Assays.
[0062] Insulin secretion assays were performed in mesh-bottomed
glass tubes essentially as in Rabaglia et al. (2005). Modifications
include: 4 islets/replicate were picked into Krebs Ringer
Bicarbonate Buffer (KRBB) containing 1.7 mM glucose and 0.25% BSA.
Replicates were pre-incubated for 1 hour in fresh 1.7 mM KRBB
before being transferred to stimulation medium containing the
desired concentrations of glucose and drug treatment (e.g., PGE1,
PGE2, and sulprostone [Sigma Aldrich] or L-798,106 [Tocris]).
Insulin secretion as a percentage of total insulin content was
determined using an in-house insulin ELISA, as described above.
[0063] The Prostaglandin E.sub.2 EIA Kit (Monoclonal) was obtained
from Cayman Chemical Company. Overnight culture medium was
subjected to PGE2 concentration analysis as recommended in the
manufacturer's protocol. Media samples were diluted 1:2 in assay
buffer before analysis. PGE2 concentration was normalized to the
total number of cultured islets and the incubation time to obtain
PGE2 production/islet/h.
[0064] Human Islet Culture and Insulin Secretion Assays.
[0065] Human islets were obtained through the Integrated Islet
Distribution Program. On the day of arrival, islets were cultured
overnight in RPMI containing 8 mM glucose, 10% heat-inactivated
FBS, and 1.times. penicillin/streptomycin to confirm viability and
sterility. Islets were cultured in 6 cm Petri dishes with
approximately 1000 islet equivalents plated per dish. Islets were
hand-picked and incubated an additional day in medium containing
the desired concentration of glucose and/or xylitol before assaying
for insulin secretion. Insulin secretion assays on human islets
were performed identically to those described for mouse islets,
except that 10 islets were used per replicate instead of 4.
[0066] INS1 Cell Culture and Insulin Secretion Assays.
[0067] The glucose responsive rat beta cell line, INS1 (832/3) was
cultured in RPMI 1640 (11.1 mM glucose) supplemented with
NaHCO.sub.3, HEPES, heat-inactivated FBS, L-glutamine, sodium
pyruvate and .beta.-mercaptoethanol. 3 days prior to assay,
1.times.10.sup.5 cells were plated per well in 96-well cell culture
plates. Twenty-four hours prior to the assay, the medium was
aspirated and fresh growth medium added. On the day of the assay,
confluent Ins-1 832/3 cells were washed once with sterile PBS and
pre-incubated for 2 hours in KRBB supplemented with 25 mM HEPES and
containing 1.7 mM glucose and 0.2% BSA. Cells were then stimulated
for 2 hours with KRBB containing 16.7 mM glucose and the desired
concentrations of agonists/antagonists. Secretion buffer was
collected and the secreted and cellular insulin content determined
by acid/ethanol extraction, as described above.
[0068] Quantitative Real-Time PCR.
[0069] cDNA was generated from islet RNA samples obtained from lean
and obese 10-week old C57B1/6 as described in as described in
Keller et al., (2008). Four nanograms (ng) of template cDNA was
subjected to a PCR cycling protocol (95 degrees C., 10 min; 40
cycles of 95 degrees, 30 sec.; 55 degrees, 30 sec.; and 68 degrees,
30 sec.) using Sybr Green as a read-out for double stranded DNA
production. Fluorescence was measured during the 68 degree
extension step. The threshold for fluorescence detection was set
automatically using the instrument control software (Applied
Biosystems). Gene-specific primers were designed and ordered from
IDT. Primers against mouse .beta.-actin were used in separate
reactions as the housekeeping gene control. Ptger3 mRNA expression
was represented as the difference in cycle times between the
.beta.-actin and Ptger3 primer sets. Melting curves and 1.5%
agarose gels of PCR products were performed to ensure primer
efficiency and specificity.
[0070] Statistical Analysis.
[0071] Data are expressed as means+/-standard error of the means
when applicable. Statistical significance was judged by computing a
Student's t-test, with a threshold for significance of
p<0.05.
Example 2
Genetics Determine Diabetes Susceptibility
[0072] A murine model of T2D was generated by utilizing two mouse
strains that differ in their susceptibility to obesity-induced
diabetes, caused by beta cell decompensation. The C57B1/6 (B6)
mouse strain is diabetes-resistant when challenged with morbid
obesity imposed by the Leptie mutation. In contrast, BTBR mice
develop severe diabetes in response to the same obesity challenge,
resulting in blood glucose levels that can exceed 600 mg/dL.
[0073] Strain-dependent differences in diabetes susceptibility in
B6 and BTBR mice were exploited to identify key regulatory genes
expressed in pancreatic islets that might contribute to the
pathogenesis of T2D. Under fasting conditions, genetically obese B6
mice maintain euglycemia due to a large compensatory increase in
plasma insulin (FIGS. 1A-B). In contrast, BTBR mice develop severe
diabetes, beginning as early as 6 weeks of age, eventually leading
to beta cell decompensation (FIGS. 1A-B).
[0074] Previously, the Inventors have shown that mechanistically,
islets from BTBR-Ob mice are unable to mount the increase in islet
function and mass necessary to compensate for the insulin
resistance resulting from morbid obesity, eventually leading to
beta cell decompensation (Clee et al., Am. J. Ther. 12:491-498
(2005)). Further, the Inventors showed that whole pancreas insulin
content, a measure of beta cell mass, is reduced by .about.80% in
diabetic BTBR-Ob mice compared with non-diabetic B6-Ob mice. This
loss in beta cell mass underlies an inability to secrete sufficient
insulin to properly regulate blood glucose. Uncontrolled blood
glucose levels ultimately leads to beta cell death.
[0075] Collectively, the results shown in FIGS. 1A-1B, FIG. 2A, and
in Clee et al. (2005) suggest that BTBR-Ob mice are an ideal model
for the identification of novel therapeutics that might prevent or
reverse beta cell decompensation in T2D.
Example 3
Diabetes and Ptger3 Expression are Highly Correlated
[0076] Previously, the Inventors profiled gene expression in six
tissues of surveyed gene expression of lean and obese B6 and BTBR
mice aged 4 weeks and 10 weeks, before and after the onset of
diabetes (Keller et al., 2008) (FIG. 2A). The focus of the 2008
publication was a group of genes related to progression through the
cell cycle, thus explaining possible differences in diabetes
susceptibility between the B6-Ob and BTBR-Ob mouse strains. Recent
analysis of the islet gene expression profiling provided by Keller
et al. (2008) indicated that the gene expression level of an
isoform of a G protein coupled receptor, Ptger3, is greatly
elevated in the islets of diabetic BTBR mice relative to
non-diabetic mice (FIG. 2B). Quantitative real-time (qRT) PCR
confirmed that Ptger3 mRNA expression is increased significantly in
10 week-old diabetic BTBR mice relative to lean BTBR mice. Further,
all three mouse Ptger3 mRNA splice variants (.alpha., .beta., and
.gamma.) were elevated >30-fold (p<10-8) in response to
diabetes (FIG. 3). These mRNA splice variants encode proteins
EP3.alpha., EP3.beta., and EP3.gamma. that are 90% identical,
varying only in the C-terminal tail. Each of the mouse EP3 variants
can couple to inhibitory G proteins of the G alpha-i subfamily,
which, when stimulated in the .beta.-cell, would result in a net
inhibition of GSIS.
[0077] Extreme hyperglycemia is a pathological phenotype of T2D in
BTBR mice. Accordingly, the Inventors analyzed the promoter of the
mouse Ptger3 gene for carbohydrate response elements (i.e.,
E-boxes) that are activated by carbohydrate response element
binding protein (ChREBP, also referred to as MLXIPL, a basic
helix-loop-helix transcription factor of the Myc/Max/Mad
superfamily (Minn et al., Endocrinology. 146:2397-2405 (2005)). The
murine Ptger3 promoter contains four consensus ChREBP binding
sites, which might explain the increased Ptger3 mRNA expression
exhibited by diabetic mice (FIG. 4). Similar analyses of rat and
human Ptger3 gene promoters reveal three consensus E-boxes in the
rat promoter and five degenerate E-boxes in the human promoter.
Example 4
Ptger3 Antagonist Normalizes Insulin Secretion from Islets Obtained
from Diabetic Mice
[0078] As a first step in testing their hypothesis that the EP3
signaling pathway contributes to the pathophysiology of T2D, the
Inventors subjected islets isolated from 10-week-old BTBR lean and
Ob mice to treatment with a selective agonist (PGE1) or specific
antagonist (L-798,106) of the EP3 receptor to monitor the impact on
GSIS. Briefly, mouse islets were incubated in medium containing 1.7
mM glucose (non-stimulatory towards insulin secretion) or 16.7 mM
glucose (stimulatory towards insulin secretion), with and without
the addition of 5 .mu.M PGE1 or 20 .mu.M L-798,106. PGE1 had
already been shown to effectively blunt GSIS through G-alpha-i
proteins in a beta cell line (Kimple et al. (2005)), and to be
relatively selective for EP3 (Kiriyama et al., Br. J. of
Pharmacology 122: 217-224 (1997)); thus, the Inventors chose PGE1
as the EP3 agonist in this initial experiment. The results show
that PGE1 reduced GSIS from islets obtained from diabetic BTBR
mice, but had no effect on islets obtained from non-diabetic mice
(FIG. 5). Similarly, the EP3-specific small molecule antagonist
L-798,106 (Tocris) enhanced GSIS from islets obtained from diabetic
mice, restoring GSIS to that observed in islets obtained from
non-diabetic mice, yet had no effect on GSIS from islets obtained
from non-diabetic mice. These results indicate: (1) the EP3
signaling pathway results in a blockade of GSIS and is functionally
upregulated in diabetic BTBR mice, and (2) that an endogenous
agonist for EP3 is being synthesized by the diabetic islets
themselves, as the antagonist would have no effect if there were no
agonist with which to compete for receptor binding.
[0079] qRT-PCR analysis of PGE2 synthetic pathway components
revealed that two prostaglandin-endoperoxidase synthases (Ptgs1 and
Ptgs2, also referred to as COX-1 and COX-2) are up-regulated in
islets from diabetic BTBR mice relative to islets from lean BTBR
mice (FIG. 3). This finding is consistent with the increased
expression of PGE2 synthetic enzymes observed in mouse and human
subjects with T2D.
[0080] Taken together, the Inventors' functional and expression
analyses indicate that both the endogenous ligand for the EP3
receptor (PGE2) and expression of the receptor itself are
up-regulated specifically in diabetic mice relative to lean mice,
suggesting a possible mechanism for islet dysfunction in T2D.
Example 5
EP3 Antagonist Enhances Insulin Secretion from Human Islets
Obtained from Diabetic Subjects
[0081] To determine if the EP3 signaling pathway regulates insulin
secretion in diabetic humans, islets were obtained from cadaveric
donors that were either non-diabetic (n=7) or confirmed T2D
patients (n=4). The average age of human donors was .about.52 years
and was not significantly different between non-diabetics and
diabetics. The average BMI was significantly different (p<0.04)
between diabetics (BMI=41) and non-diabetics (BMI=30), although
both groups would be classified as obese.
[0082] Non-diabetic and T2D human islets were cultured for 48 hours
in 8 mM glucose. A subset of the non-diabetic donor islets were
also incubated with 0.5 mM xylitol, a glucose analog that is
non-metabolizable beyond glucose-6 phosphate, during the final 24
hour culture period. This concentration of xylitol in combination
with sub-stimulatory glucose has been shown to increase expression
of genes that are regulated by ChREBP. Xylitol activates the FoxO1
transcription factor via glucose-6 phosphate formation, providing
an alternative mechanism for up-regulating gene expression.
Following 48 hours in culture, cellular insulin secretion was
stimulated for 45 minutes with 16.7 mM glucose in the absence and
presence of 10 .mu.M L-798,106. Secreted insulin was normalized to
total insulin content in order to determine the fractional release
of insulin. Data from each human islet set was normalized to its
own response in 16.7 mM glucose alone to isolate the effect of
L-798,106 on GSIS (FIG. 6). Responses of T2D donor islets and
xylitol-incubated non-diabetic donor islets to L-798,106 both
differed significantly from responses of non-diabetic islets
incubated in 8 mM glucose alone. Measurement of PGE2 production by
non-diabetic and confirmed T2D human islets revealed significant
increase in PGE2 production by T2D islets relative to non-diabetic
islets (FIG. 7). These data suggest that (1) hyperglycemia
up-regulates one or more of the components of the EP3 signaling
pathway in human islets, similar to diabetic mouse islets, and (2)
that this up-regulation can be mimicked by stimulation of
glucose-responsive transcriptional activators.
[0083] Taken together, the Inventors' results from non-diabetic and
diabetic mouse and human islets suggest that: (1) EP3 expression
and activity are induced in parallel with diabetes in both mouse
and human islets, and (2) as a cell-surface receptor, EP3 is a
potentially druggable target for anti-diabetic therapeutics.
Example 6
EP3 Antagonist Enhances Insulin Secretion from a Rat-Derived
Insulinoma Cell Line
[0084] Rat Ins-1 beta cell lines stably expressing the human
proinsulin gene are used as a models of physiological insulin
secretion in response to glucose. The rat Ins-1 beta cell line
Ins-1 832/3 responds strongly to both glucose and cyclic AMP (cAMP)
potentiation of GSIS and demonstrates high EP3 expression relative
to the other E prostanoid receptor isoforms (e.g., EP1, EP2, and
EP4) (data not shown). Thus, Ins-1 832/3 is a suitable model for
elucidating EP3 agonist/antagonist binding kinetics on the GSIS
response.
[0085] Insulin secretion assays in Ins-1 832/3 cells were performed
wherein the dose of PGE2 (a physiological agonist of EP3) or
sulprostone (an EP3-selective agonist) were varied over a wide
concentration range. Both PGE2 and sulprostone dose-dependently
inhibited GSIS. The minimum dose of sulprostone yielding maximum
effect on GSIS inhibition was approximately 10 nM and the minimum
dose of PGE2 was approximately 50 nM. Dose response analysis of
L-798,106 in medium containing stimulatory glucose and 10 nM
sulprostone or medium containing 50 nM PGE2 revealed that both
PGE2- and sulprostone-dependent suppression of GSIS in Ins-1 cells
was reversed in a dose-dependent manner (FIG. 8). These results
suggest that the combination of elevated EP3 expression and
endogenous E-prostanoid production are required to yield maximal
responsiveness to the antagonist, L-798,106.
Example 7
Synergy of GLP-1 and L-798,106 in Improving Diabetic Islet
Function
[0086] GLP-1-based therapies are used in T2D therapy. To test the
possibility that EP3 antagonism can augment the effect of GLP-1
agonism, islets from diabetic BTBR mice were treated with
stimulatory glucose, with and without the addition of 50 nM GLP-1
and/or 10 L-198,106, and the level of insulin secretion monitored
(FIG. 9). GLP-1 treatment alone had a significant impact on insulin
secretion. However, the combination of GLP-1 and L-798,106 had a
stronger positive effect on insulin secretion than GLP-1 alone.
Example 8
Predicted Role of Ptger3 in Insulin Secretion from Islets Obtained
from Diabetic Subjects
[0087] GLP-1 analogs and inhibitors of the enzyme that degrades
endogenous GLP-1 and DPP-4 all function in the beta cell by
increasing cyclic AMP (cAMP) production mediated by adenylate
cyclase (AC). cAMP can positively impact both insulin secretion
and, potentially, beta cell mass, by activating beta cell
proliferation, growth, and survival pathways. E prostanoid
signaling in the Ins-1 832/13 beta cell line is known to proceed
through a member of the G alpha-i subfamily to block GSIS.
Activation of EP3 through G alpha-i subfamily members would
predictably lead to decreased cAMP production. Thus, the Inventors
hypothesized that activated EP3 might oppose all of the functions
of GLP-1-based therapeutics in beta cells (FIG. 10). The Inventors
predicted that antagonizing the EP3 pathway, alone or in
combination with GLP-1 receptor agonism, might prove to be a
superior therapeutic for T2D relative to known therapeutics,
especially those that target GLP-1.
[0088] Sitagliptin, a T2D therapeutic, is a DPP-4 inhibitor that
stimulates beta cell cAMP production. However, sitagliptin is
ineffective for preventing or treating diabetes in a subset of
treated individuals (Raz et al., supra; Aschner et al., supra;
Nonaka et al., supra).
[0089] The Inventors predict that sitagliptin cannot treat or
prevent T2D in individuals in whom Ptger3 expression is at such a
level that Ptger3-mediated suppression of adenylate cyclase
inhibits cAMP production, thereby preventing insulin secretion from
proceeding at physiologically-appropriate levels (FIG. 10).
[0090] The Inventors predict that other peptide hormones and small
molecules that bind to and activate receptors in the same family as
GLP-1 receptor, Subfamily B1, of the secretin receptor family,
would also synergize with EP3 antagonists to promote beta cell
function if those receptors were expressed on the beta cell. These
hormones include other known potentiators of glucose-stimulated
insulin secretion (i.e., incretins), including glucose-dependent
insulinotropic peptide (or gastric inhibitory peptide (GIP)),
Pituitary adenylate cyclase-activating polypeptide (PACAP), and
vasoactive intestinal peptide (VIP). Furthermore, agents that act
through an alternative stimulatory pathway, such as cholecystokinin
(CCK), which on the beta cell binds to a G alpha-q-coupled
receptor, might also synergize with Ptger3 antagonists and
incretins.
Example 9
Treating Type I Diabetes with a Composition Comprising a Compound
that Leads to Activation of Adenylate Cyclase and a Compound that
Modulates EP3
[0091] It is contemplated that a composition comprising a compound
that directly or indirectly activates adenylate cyclase (e.g., a
compound that activates a GLP-1 receptor, GIP receptor, or PACAP
receptor) and a compound that modulates EP3 (e.g., L-798,106;
DG-041, or another EP3 antagonist) could be useful for preventing
or treating Type I Diabetes (T1D). The molecular structures of
L-798,06 and DG-041 are presented in FIG. 11 and FIG. 12,
respectively. The composition can comprise any pairwise combination
of a compound that directly or indirectly activates adenylate
cyclase and a compound that attenuates GSIS-mediated adenylate
cyclase inhibition described herein.
[0092] Type I Diabetes occurs when immune-mediated pancreatic beta
cell destruction leads to near-absolute endogenous insulin
deficiency (Mathis et al., Nature 414:792-798 (2001); Devendra et
al., BMJ 328:750-754 (2004)). The residual beta cell function
observed in early T1D indicates the presence of a pool of
potentially expandable beta cells. A recent strategy for T1D
therapy comes from the obesity- and insulin-resistance-linked T2D
field. In both T1DM and late-stage T2DM, beta cells fail to
maintain sufficient mass and function to properly regulate blood
glucose levels (Cnop et al., Diabetes 54 Suppl. 2:S97-107 (2005)).
Beta cell dysfunction is at least as important as insulin
resistance in the pathogenesis of T2DM, if not more so. Thus,
nearly all of the newer T2DM treatments in the clinic or under
development target beta cell dysfunction and not insulin
sensitivity.
[0093] As discussed above, agents that stimulate beta cell cAMP
production, including DPP-4 inhibitors and GLP-1 analogs, can
positively impact beta cell replication, neogenesis and/or survival
in rodent models. GLP-1 receptor agonism positively impacts
replication and neogenesis in human islets, and GLP-1 treatment can
protect both rodent and human beta cells from immune-mediated
destruction (Sano et al., supra; Pugazhenthi et al., supra).
Testing of GLP-1 agonists and DPP-4 inhibitors in rodent models of
T1D and in human T1D patients and pancreatic islet transplant
recipients show prolonged survival, improved glycemia, and
maintenance of graft function for a longer duration ((Yanay et al.,
J. Gene Med. 12:538-544 (2010)); Kielgast et al., Curr. Diabetes
Rev. 5:266-275 (2009); Faradji et al., Cell Transplant 18:1247-1259
(2009)).
[0094] These studies suggest that T1D could be prevented or treated
with therapeutics that act through cAMP production, including the
compositions and methods of the present invention.
[0095] The invention has been described in connection with what are
presently considered to be the most practical and preferred
embodiments. However, the present invention has been presented by
way of illustration and is not intended to be limited to the
disclosed embodiments. Accordingly, those skilled in the art will
realize that the invention is intended to encompass all
modifications and alternative arrangements within the spirit and
scope of the invention as set forth in the appended claims.
Sequence CWU 1
1
13117DNAMus musculus 1cacttgccta acatgtg 17217DNAMus musculus
2cactaggaaa gcagaag 17317DNAMus musculus 3caaaagcaat tcaagtg
17417DNAMus musculus 4caagatcttg ccaggtg 17517DNAMus musculus
5caggtggcct tcaccag 17617DNARattus norvegicus 6cagatgccct tcaacag
17717DNARattus norvegicus 7caaaagcaat tcaagtg 17817DNARattus
norvegicus 8cacgtgtcct tcaccag 17917DNAHomo sapiens 9caggtgcgcc
tcggcag 171017DNAHomo sapiens 10ctaaaggact tcaggag 171117DNAHomo
sapiens 11ctgttgatac tcaagag 171217DNAHomo sapiens 12ctgctgagcc
acaggag 171317DNAHomo sapiens 13cacgtcggct ccacctg 17
* * * * *