U.S. patent application number 11/123895 was filed with the patent office on 2005-12-29 for methods for identifying compounds for treating diabetes mellitus.
Invention is credited to Efendic, Suad.
Application Number | 20050287557 11/123895 |
Document ID | / |
Family ID | 34969127 |
Filed Date | 2005-12-29 |
United States Patent
Application |
20050287557 |
Kind Code |
A1 |
Efendic, Suad |
December 29, 2005 |
Methods for identifying compounds for treating diabetes
mellitus
Abstract
The invention is directed to methods for identifying candidate
beta cell-sensitizing compounds comprising providing a pancreatic
beta islet cell population derived from a diabetic subject,
contacting the beta islet cell population with one or more
somatostatin receptor 5-binding compounds, and identifying those
somatostatin receptor 5-binding compounds that promote insulin
secretion from the pancreatic beta islet cell population at a
higher rate than from a control cell population. The invention is
further directed to methods for treating type II diabetes
comprising administering a beta cell-sensitizing compound
identified by the first method of the invention to a diabetic
patient in need of treatment.
Inventors: |
Efendic, Suad; (Lidingo,
SE) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE
32ND FLOOR
CHICAGO
IL
60606
US
|
Family ID: |
34969127 |
Appl. No.: |
11/123895 |
Filed: |
May 6, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60569834 |
May 10, 2004 |
|
|
|
Current U.S.
Class: |
435/6.13 ;
435/366 |
Current CPC
Class: |
G01N 2800/042 20130101;
G01N 33/507 20130101; A61P 5/48 20180101; G01N 2333/655 20130101;
G01N 2333/726 20130101 |
Class at
Publication: |
435/006 ;
435/366 |
International
Class: |
C12Q 001/68; C12N
005/08 |
Claims
I claim:
1. A method for identifying candidate pancreatic beta
cell-sensitizing compounds comprising: (a) providing a pancreatic
beta-islet cell population derived from a diabetic subject, wherein
the pancreatic beta islet cell population comprises pancreatic beta
islet cells expressing somatostatin receptor 5; (b) contacting the
pancreatic beta-islet cell population with one or more somatostatin
receptor 5-binding test compounds; and (c) identifying those
somatostatin receptor 5 binding test compounds that promote insulin
secretion from the pancreatic beta-islet cell population at a
higher rate than from a control cell population, wherein such
compounds are candidate pancreatic beta cell-sensitizing
compounds.
2. The method of claim 1, wherein the somatostatin receptor
5-binding test compounds are somatostatin receptor 5
antagonists.
3. The method of claim 1, wherein the contacting of the pancreatic
beta-islet cell population is performed at an elevated glucose
concentration.
4. The method of claim 3, wherein the control cell population
comprises pancreatic beta cells from a normoglycemic subject.
5. The method of claim 1, further comprising: (d) testing the
pancreatic beta cell-sensitizing compounds for somatostatin
receptor 2 agonist activity.
6. The method of claim 1 further comprising synthesizing the
candidate pancreatic beta cell-sensitizing compounds.
7. A method for treating type II diabetes, comprising administering
to a diabetic patient in need of treatment a beta cell-sensitizing
compound identified by the method of claim 1 in an amount
sufficient to improve glycemic control in the patient.
8. The method of claim 7, further comprising administering a
somatostatin receptor 2 agonist to the diabetic patient.
9. The method of claim 7, wherein the beta cell-sensitizing
compound comprises both somatostatin receptor 5 antagonist activity
and somatostatin receptor 2 agonist activity.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority from the co-pending
provisional application Ser. No. 60/569,834 filed May 10, 2004,
incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] Diabetes mellitus is a complex chronic disorder that results
primarily either from partial or complete lack of insulin secretion
by the pancreas. The endocrine pancreas consists of the islets of
Langerhans, which include several different cell types, including
beta cells that are responsible for insulin secretion, alpha cells
that produce glucagon, and delta cells that produce somatostatin
(Luft, Efendic et al. Med Biol 52:428-430, 1974). Insulin and
glucagon secreted from the beta and alpha cells serve to regulate
blood glucose levels.
[0003] In diabetic patients, the pancreas produces insufficient or
no insulin, the hormone which is responsible for the uptake of
glucose into cells. As a result, the level of glucose in the blood
becomes abnormally high. There are two main types of diabetes
mellitus. In Type I diabetes mellitus, pancreatic beta cells are
destroyed and insulin production ceases almost completely. Without
regular injections of insulin the sufferer lapses into a coma and
dies. Individuals suffering from Type I diabetes are totally
insulin-dependent.
[0004] In patients with Type II diabetes, insufficient insulin is
produced to maintain normal blood glucose levels. Often the body is
resistant to the effects of insulin as the transduction of insulin
signal is impaired. In most cases, insulin-replacement injections
are not initially required. The combination of dietary measures,
weight reduction and oral medication can keep the condition under
control for a period of time, but most people with Type II diabetes
ultimately require insulin injections.
[0005] While diabetes may be controlled with insulin and in some
cases through careful diet or combinations of diet and oral
medication, blood sugar levels fluctuate, sometimes dramatically,
in diabetic patients irrespective of therapy. The pathological
complications of diabetes are fundamentally related to
hyperglycemia. If the diabetes is poorly controlled it can lead to
diabetic complications. Such diabetic complications include
nephropathy, neuropathy, retinopathy, heart disease,
atherosclerosis, high blood pressure, stroke, and neurodegenerative
conditions.
[0006] Intensive insulin treatment or treatments that produce
insulin, such as sulfonylureas and glinides, increase the risk of
hypoglycemia (or insulin shock), which occurs if blood glucose
levels fall below normal. To date, FDA approved drugs that promote
insulin secretion do so indiscriminately, at both high and low
blood glucose levels, which can lead to hypoglycemia, discouraging
the use of such drugs. Ongoing efforts in the pharmaceutical
industry have identified genes and target molecules that
participate in insulin expression and secretion. However, the
identification of key molecular processes that regulate
glucose-induced insulin expression and regulation requires
knowledge of the complex inter-relationships of the metabolic
pathways that govern this process.
[0007] Somatostatins are ubiquitous polypeptides known to affect
basic biological processes such as growth, development, metabolism,
and cell differentiation in vertebrates (US 20010025097). There are
two major forms of somatostatin in mammals, SS-14, a 14 amino acid
polypeptide
(Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys) (SEQ ID
NO:1), and SS-28
(Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gl-
y-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys) (SEQ ID NO:2), a
28 amino acid polypeptide. Both SS-14 and SS-28 are produced by
proteolytic cleavage from larger precursors, and SS-14 is conserved
among such mammals as monkeys, rats, cows, sheep, chickens and
humans (US 20010025097).
[0008] The biological effects of somatostatin are apparently all
inhibitory in nature, and are elicited upon receptor binding on a
target cell. For example, somatostatin inhibits the pancreatic
secretion of glucagon and insulin.
[0009] The effects of somatostatin on target cells are mediated by
at least 5 classes of somatostatin receptors, SSTR1-SSTR5. SSTR2
and SSTR5 are the predominant subtypes in peripheral tissues. In
rodents, somatostatin inhibits glucagon and insulin release via
SSTR2 and SSTR5, respectively. Previous work has demonstrated that
SS-14 interacts with SSTR1-5 with similar affinity, while SSTR5
displays a 10-fold higher affinity for SS-28 than SSTR1-4. SS-28
has been shown to preferentially inhibit insulin secretion from
beta cells, and SS-14 inhibits the release of glucagon more
potently than that of insulin.
[0010] Previous work has shown that an SSTR5 selective agonist
inhibits glucose-stimulated insulin secretion from pancreatic
islets isolated from normal rats and from normal mice (see, for
example, (Rossowski et al., Biochem Biophys Res Commun 205:341-346,
1994). SSTR5 knockout mice indicate a role for SSTR5 in mediating
pancreatic insulin secretion (Strowski et al., Mol Endocrinol
17:93-106, 2003). Antagonists for the somatostatin receptors, such
as PRL-3195 (an SSTR5 antagonist) are known in the art. (Rajeswaran
et al., J Med Chem 44:1305-1311, 2001).
[0011] Compounds that preferentially promote insulin release in
response to hyperglycemia in Type II diabetes would represent a new
class of drugs that minimize hypoglycemic episodes, and provide a
pharmacological strategy with greater application and acceptance to
improve glycemic control in Type II diabetic patients, and possibly
decrease risk of developing diabetic complications, including but
not limited to nephropathy, neuropathy, retinopathy, heart disease,
atherosclerosis, high blood pressure, stroke, and neurodegenerative
conditions, caused or exacerbated by poor glycemic control in
diabetic patients.
SUMMARY OF THE INVENTION
[0012] The inventors of the present invention have unexpectedly
found that SSTR5-selective antagonists selectively promote insulin
secretion at elevated glucose levels in pancreatic islets isolated
from Type II diabetic rats to a greater degree than they promote
insulin release from these pancreatic islets isolated from
normoglycemic animals. Thus, SSTR5-selective antagonists can act as
"beta-cell sensitizers" and be used, for example, to promote
glucose-induced insulin release in diabetic subjects while
minimizing hypoglycemic episodes caused by indiscriminate promotion
of insulin secretion regardless of glucose concentration.
[0013] In a first aspect, the present invention provides methods
for identifying candidate pancreatic beta cell-sensitizing
compounds comprising:
[0014] (a) providing a pancreatic beta-islet cell population
derived from a diabetic subject, wherein the pancreatic beta islet
cell population comprises pancreatic beta islet cells expressing
somatostatin receptor 5;
[0015] (b) contacting the pancreatic beta-islet cell population
with one or more somatostatin receptor 5-binding test compounds;
and
[0016] (c) identifying those somatostatin receptor 5 binding test
compounds that promote insulin secretion from the pancreatic
beta-islet cell population at a higher rate than from a control
cell population, wherein such compounds are candidate pancreatic
beta cell-sensitizing compounds.
[0017] In a preferred embodiment of this first aspect of the
invention, the somatostatin receptor 5-binding test compounds are
somatostatin receptor 5 antagonists.
[0018] In a further preferred embodiment of this first aspect of
the invention, the contacting is carried out at an elevated glucose
concentration.
[0019] In another preferred embodiment of this first aspect of the
invention, the control cell population comprises pancreatic beta
cells from a normoglycemic subject.
[0020] In another preferred embodiment, the method comprises
identifying those candidate beta cell-sensitizing compounds that
are also agonists of the somatostatin 2 receptor.
[0021] In a second aspect, the invention provides methods for
treating type II diabetes comprising administering to a diabetic
patient in need of treatment a beta cell-sensitizing compound
identified by the methods of the first aspect of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 presents the effect of PRL-3195 on glucose induced
insulin release in batch incubated islets of Wistar and
GK-rats.
[0023] FIG. 2 provides an example of PRL-3195 stimulation of
glucose-induced insulin release in perifused islets from diabetic
(GK) and normoglycemic (Wistar) rats.
DETAILED DESCRIPTION OF THE INVENTION
[0024] All references cited are herein incorporated by reference in
their entirety.
[0025] Within this application, unless otherwise stated, the
techniques utilized may be found in any of several well-known
references such as: Molecular Cloning: A Laboratory Manual
(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene
Expression Technology (Methods in Enzymology, Vol. 185, edited by
D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to
Protein Purification" in Methods in Enzymology (M. P. Deutshcer,
ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to
Methods and Applications (Innis, et al. 1990. Academic Press, San
Diego, Calif.), Culture of Animal Cells: A Manual of Basic
Technique, 2.sup.nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York,
N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.
J. Murray, The Humana Press Inc., Clifton, N. J.), and the Ambion
1998 Catalog (Ambion, Austin, Tex.).
[0026] As used herein, the singular forms "a", "an" and "the"
include plural referents unless the context clearly dictates
otherwise.
[0027] In one aspect, the present invention provides methods for
identifying candidate pancreatic beta cell-sensitizing compounds
comprising:
[0028] (a) providing a pancreatic beta-islet cell population
derived from a diabetic subject, wherein the pancreatic beta islet
cell population comprises pancreatic beta islet cells expressing
somatostatin receptor 5;
[0029] (b) contacting the pancreatic beta-islet cell population
with one or more somatostatin receptor 5-binding test compounds;
and
[0030] (c) identifying those somatostatin receptor 5 binding test
compounds that promote insulin secretion from the pancreatic
beta-islet cell population at a higher rate in Type 2 diabetes than
from a control cell population, wherein such compounds are
candidate pancreatic beta cell-sensitizing compounds.
[0031] As used herein, the term "pancreatic beta cell sensitizing
compounds" means compounds that preferentially promote insulin
release from pancreatic beta cells from diabetic subjects as
compared to pancreatic beta cells from normoglycemic subjects. Any
selective amount of increased insulin release in pancreatic beta
cells from diabetic subjects, as opposed to indiscriminate insulin
release, would be of great benefit in treating diabetic patients,
while reducing the risk of hypoglycemia. In a preferred embodiment,
insulin release is increased at least 5%, and preferably at least
10%, 15%, 20%, 25%, 30%, 40%, 45%, or 50% greater, or more,
relative to control.
[0032] As used herein, the diabetic subject can be any mammal,
preferably human.
[0033] As used herein, the diabetic subject suffers from type II
diabetes, wherein the pancreatic beta cells still can produce
insulin.
[0034] As used herein, the control cell population can be
pancreatic beta cells from a non-diabetic subject contacted with
test compounds, pancreatic beta cells from a diabetic subject
contacted with control compounds, historical standards, or any
other relevant control.
[0035] In a preferred embodiment, the contacting is performed at
elevated glucose concentration, and the control comprises
pancreatic beta cells from a normoglycemic subject.
[0036] As used herein the term "elevated glucose levels" means
glucose concentrations of at least 6 mM, preferably between 6 mM
and 50 mM, more preferably between 6 mM and 40 mM, more preferably
between 7 mM and 30 mM, more preferably between 8 mM and 30 mM, and
even more preferably between 8 mM and 25 mM. As used herein "normal
glucose levels" means glucose concentrations of less than 6 mM,
preferably between 3-5 mM.
[0037] As used herein the term "pancreatic beta islet cell
population" means any population of cells (i.e.: two or more cells)
that contains pancreatic beta islet cells. Such pancreatic beta
islet cell populations include the pancreas, isolated pancreatic
islets of Langerhans ("pancreatic islets"), and isolated pancreatic
beta islet cells. Methods for the isolation of pancreas are well
known in the art, and methods for isolating pancreatic islets, can
be found, for example, in Cejvan et al., Diabetes 52:1176-1181,
2003; Zambre et al., Biochem. Pharmacol. 57:1159-1164, 1999; and
Fagan et al., Surgery 124:254-259, 1998, and references cited
therein. As known by those of skill in the art, pancreatic beta
cells express SSTR5 (See, for example, Fagan et al., 1998). In a
preferred embodiment, isolated pancreatic islets are used.
[0038] As used herein the term "contacting" means in vivo or in
vitro, and (when in vitro) under suitable conditions for
maintaining the pancreatic beta cell populations in organ cultures
or tissue cultures, using appropriate media and/or perfusate
conditions to promote binding of the SSTR5 antagonist to SSTR5.
Such techniques are known to those of skill in the art. As used
herein the "contacting" can occur at the time of initiating the
culturing, or any time subsequent to initiating the culturing of
the pancreatic beta cells. Such contacting can comprise adding the
test compounds to a perfusate, adding the test compounds to
cell/organ culture medium, or any other technique known in the art
for contacting cells/organ cultures.
[0039] As used herein, compounds that "promote insulin secretion"
are those that induce an increase in insulin secretion from the
pancreatic beta cells compared to insulin secretion from the
pancreatic beta cells seen in the absence of the test compounds.
Any increase in insulin secretion over baseline levels is
beneficial in diabetic patients in a hyperglycemic state, and thus
the method does not require a specific amount of increase in
insulin secretion from the pancreatic beta cells, so long as the
compound(s) promotes insulin secretion from the pancreatic beta
cells at a higher rate than control. As used herein "at a higher
rate" means an increased amount of insulin secreted during the
course of the experiment, preferably a statistically significant
increase as indicated by standard statistical calculations.
[0040] Methods for conducting perifusion studies with pancreatic
islets and perfusion studies with isolated pancreas, can be found,
for example, in Cejvan et al., Diabetes 52:1176-1181, 2003, and
references cited therein. For example, the pancreas from a diabetic
rat may be isolated by standard methods and connected to an open
non-recycling perfusion system to administer perfusion medium to
the pancreas via a cannula inserted into the abdominal aorta, as
described in Cejvan et al., 2003. Alternatively, pancreatic beta
islets can be isolated from a pancreas from a diabetic rat by
collagenase digestion, as described in Cejvan et al., 2003 and
subject to perifusion as described in Cejvan et al., 2003. The
resulting population of cells comprises pancreatic beta islet cells
expressing somatostatin receptor 5. In either case, at least a
first population of such cells and a second population of such
cells is provided, one cultured at elevated glucose levels, and the
other cultured at normal glucose levels, as defined above. Test
compounds can be added to the cultures at the time of culturing, or
subsequent to establishment of the cultures, as discussed above.
The compounds can be added at various concentrations or for various
time periods in order to assess different aspects of test compound
activity, as is known by those of skill in the art. At desired time
points, the insulin secretion from the first and second populations
of cells is determined. Such methods for measuring insulin
secretion are known in the art. See, for example, Fagan et al.,
1998, and references cited therein.
[0041] The methods of the invention are used to identify SSTR5
antagonists that can act to selectively promote insulin secretion
from pancreatic beta cells from diabetic subjects. However, the
method of the invention does not require that only SSTR5
antagonists be used in the assay. For example, test compounds shown
to bind to SSTR5 can be used in the assay, and those that are
agonists are expected to inhibit insulin secretion, rather than
promote insulin secretion, and thus will be selected against by the
assay. Any method known in the art for identifying compounds that
bind SSTR5 can be used, including but not limited to the use of
membrane preparations from cells transfected to express SSTR5 at
the cell surface. (See, for example, Baumbach et al., Mol Pharmacol
54:864-873, 1998; Rohrer et al, Science 282:737-740, 1998; U.S.
20020128206; U.S. Pat. No. 5,846,934). In a preferred embodiment,
the binding of the test compounds against membrane preparations
from cells expressing SSTR1-4 is also assessed, and those that show
significantly higher binding affinities to SSTR5 than to any of
SSTR1-4 are selected for. In a further preferred embodiment, the
cells used are those that express little/no detectable SSTR5 prior
to transfection, such as Chinese hamster ovary cells
[0042] Alternatively, the test compounds used in the assay can
include only those previously identified as antagonists of SSTR5.
Any method known in the art for identifying a compound as an
antagonist of SSTR5 can be used, including competitive binding
assays in which various concentrations of test compounds are used
to compete with SS-14 or SS-28 for binding to membrane preparations
from SSTR5 expressing cells, such as recombinant cells expressing
SSTR5. (See, for example, U.S. 20020128206; U.S. 20010025097; U.S.
Pat. No. 5,846,934). In a preferred embodiment, the ability of test
compounds to compete with SS-14 or SS-28 for binding to membrane
preparations from cells expressing one of SSTR1-4 would also be
assessed, and those that exhibit increased ability to compete SS-14
and/or SS-28 binding to SSTR5 than to any of SSTR1-4 would be
selected for. In a further preferred embodiment, the cells used are
those that express little/no detectable SSTR5 prior to
transfection, such as Chinese hamster ovary cells. In a further
preferred embodiment, SS-28 is used for competition
experiments.
[0043] In further embodiments, SSTR5 antagonists are identified
using methods including but not limited to the following (which can
be used alone, or together with the methods disclosed above):
[0044] Identification of test compounds that bind to SSTR5 and that
promote cAMP accumulation in test cells expressing SSTR5 compared
to cAMP accumulation in the same cell types in the absence of the
test compound(s). See, for example, Strowski et al., Mol Endocrinol
17:93-106, 2003; U.S. Pat. No. 5,846,934; Baumbach et al.,
1998.
[0045] Identification of test compounds that bind to SSTR5 and that
inhibit phospholipase C activity (Reubi et al., Proc Natl Acad Sci
USA 97:13973-978, 2000) and/or inositol phosphate formation
(Wilkinson et al., Br J Pharmacol 118:445-447, 1996) compared to
phospholipase C activity and/or inositol phosphate formation in the
same cell types in the absence of the test compound(s).
[0046] In a further embodiment, the SSTR5 antagonist compounds
identified as beta cell sensitizers are also tested for SSTR2
agonist activity, and those with both types of activity are
identified as a preferred class of beta cell sensitizers. Any
method known in the art for identifying a compound as an agonist of
SSTR2 can be used, including but not limited to the use of membrane
preparations from cells transfected to express SSTR2 at the cell
surface. (See, for example, Rohrer et al, Science 282:737-740,
1998; U.S. 20020128206; U.S. 20010025097; U.S. Pat. No.
5,846,934).
[0047] In a further embodiment, the method comprises synthesizing
the compounds identified in the assay, using standard methods in
the art.
[0048] In a further aspect, the present invention provides methods
for treating type II diabetes, comprising administering to a
diabetic patient a beta cell sensitizer identified by the assay
disclosed above, in any embodiment thereof, to promote improved
glycemic control in the diabetic patient. In one embodiment, the
method comprising administering an SSTR5 antagonist to the patient.
In a further embodiment, the method comprises administering an
SSTR5 antagonist and an SSTR2 agonist to the patient. In this
embodiment, the SSTR5 antagonist and the SSTR2 agonist can be
separate compounds, or they can be the same compound, identified as
disclosed above.
EXAMPLES
[0049] Animals
[0050] Male Wistar rats, aged 2-3 months, were obtained from
B&K Universal (Sollentuna, Sweden). The animals were fed ad
libitum with free access to water and placed in rooms with
alternate 12 hour periods of light and darkness. GK rats came from
Stockholm's colony which was established 1992
[0051] Isolation of Pancreatic Islets, Batch Incubation, and
Perifusion Studies.
[0052] Islets were isolated by digestion with collagenase
(Hoffmann-La Roche, Basel, Switzerland) and cultured for 20-22 h in
RPMI-1640 medium supplemented with 11 mmol/l glucose and 10%
(vol/vol) FCS. For both batch and perifusion studies, the islets
were first preincubated for 35 min in Krebs-Ringer bicarbonate
buffer (KRBB) containing 3.3 mmol/l glucose and 2 g/l bovine plasma
albumin (Sigma, St. Louis, Mo.).
[0053] For batch incubations, batches of ten islets were then
incubated for 1 h in 350 .mu.l KRBB-albumin-glucose. As a stimulus
for hormone release, 3.3 to 25 mM glucose or 20 mM arginine were
used. PRL-3195 (Rajeswaran et al., J. Med. Chem. 44:1305-1311,
2001) was added to incubations with glucose and arginine at a
concentration of 10 .mu.M. After each incubation, 100 .mu.l
aliquots of incubation medium were stored at -20.degree. C. for
subsequent radioimmunoassay of insulin (Herbert V et al. J Clin
Endocrinol Metab 25:1375-1384, 1965).
[0054] For perifusion, following culture and preincubation, 100
islets were added to each of two perifusion chambers by layering
between inert polystyrene beads (Bio-Gel 200-400 mesh; Bio-Rad
Laboratories, Richmond, Calif.). This perifusion system has been
previously described (Kanatsuna T et al. Diabetes 30:231-234,
1981)., The KRBB-albumin-glucose with the flow rate of 0.4 ml/2 min
was used as a basal perifusion medium. The perifusion protocol was
started by a 30-min equilibration period with basal medium,
followed by a 20-min stimulation period, and finally by a 10-min
reperifusion with the basal medium. Glucose at a concentration 16.7
mM was used as the stimulus for insulin release. Simultaneously
with 16.7 mM glucose was administered somatostatin receptor 5
antagonist (PRL-3195) at 10 or 50 .mu.M. Samples were collected at
2-min intervals, ice-chilled immediately, frozen, and kept at
-20.degree. C. for subsequent radioimmunoassay of insulin (Herbert
V et al. J Clin Endocrinol Metab 25:1375-1384, 1965).
[0055] Insulin Release from Isolated Islets in Batch
Incubations.
[0056] When batch-incubated islets from normoglycemic (Wistar) rats
were incubated at increasing glucose concentrations, there was a
clear stimulation of insulin release (Table 1). In rats with Type
II diabetes (GK), insulin release was not significantly different
from the normoglycemic (Wistar) rats at 3.3 mM glucose. With
increasing glucose concentrations, however, insulin release from GK
rat islets increased, but to an extent that was markedly less than
that of Wistar rats. The addition of the SSTR5 antagonist PRL-3195
at a concentration of 10 .mu.M to islets from Wistar and GK rats
increased insulin release at all glucose concentrations. This
effect of PRL-3195 was, however, more pronounced in GK rats. (See
FIG. 1)
[0057] At 3.3 mM glucose, 20 mM arginine significantly stimulated
insulin release from Wistar and GK rat islets. This response was,
however, significantly less for GK rat islets (P<0.001). The
addition of PRL-3195 significantly stimulated insulin release in
both cases.
[0058] Insulin Release from Isolated Perifused Islets.
[0059] To examine insulin release under more physiological
conditions, the effect of the SSTR5 antagonist PRL-3195 was tested
in perifused islets. In perifused islets from normoglycemic rats
(Wistar), there was a marked and biphasic insulin response when the
glucose concentration was increased from 3.3 to 16.7 mM. This
response was, however, at 9.14 pmol/100 islets, significantly less
(p=0.02) in GK rat islets than in Wistar islets which released 19.8
pmol/100 islets during the stimulation period (FIG. 2). When 10
.mu.M PRL-3195 was added to the perifusion medium, the insulin
response to glucose was not significantly increased in islets from
either Wistar or GK rats. However, 50 .mu.M PRL-3195 significantly
enhanced the insulin release evoked by 16.7 mM glucose in perifused
GK islets to 15.3 pmol/100 islets (p=0.049), whereas in islets from
Wistar rats there was no significant effect.
[0060] The results from the batch incubation and perifusion studies
indicate that an SSTR5 antagonist is more effective at promoting
glucose-induced insulin secretion from islet cells derived from
Type II diabetic rats as compared to islets from Wistar rats.
Importantly the antagonist is less effective at promoting insulin
secretion from islet cells derived from both normoglycemic rats and
Type II diabetic rats when the islet cells are exposed to normal
glucose concentrations. Thus, SSTR5-selective antagonists are
promising compounds for the treatment of diabetes. These
SSTR5-selective antagonists are able to promote glucose-induced
insulin release in diabetic subjects while minimizing hypoglycemic
episodes caused by indiscriminate promotion of insulin secretion
regardless of glucose concentration.
[0061] The complete disclosures of all patents, patent applications
including provisional patent applications, and publications cited
herein are incorporated by reference. The foregoing detailed
description and examples have been provided for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown
and described; many variations will be apparent to one skilled in
the art and are intended to be included within the invention
defined by the claims.
Sequence CWU 1
1
2 1 14 PRT artificial sequence synthetic peptide 1 Ala Gly Cys Lys
Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 1 5 10 2 28 PRT artificial
sequence synthetic peptide 2 Ser Ala Asn Ser Asn Pro Ala Met Ala
Pro Arg Glu Arg Lys Ala Gly 1 5 10 15 Cys Lys Asn Phe Phe Trp Lys
Thr Phe Thr Ser Cys 20 25
* * * * *