U.S. patent application number 15/749351 was filed with the patent office on 2018-08-16 for compositions for use in the treatment of diabetes.
The applicant listed for this patent is University of Ulster. Invention is credited to Peter Raymond FLATT, Aine Maire McKillop.
Application Number | 20180228742 15/749351 |
Document ID | / |
Family ID | 54062975 |
Filed Date | 2018-08-16 |
United States Patent
Application |
20180228742 |
Kind Code |
A1 |
McKillop; Aine Maire ; et
al. |
August 16, 2018 |
Compositions for use in the treatment of diabetes
Abstract
This invention relates to compositions for use in the treatment
of diabetes, for example type-2 diabetes; obesity; and/or metabolic
syndrome. Specifically, the invention relates to a composition for
use in the treatment of diabetes, the composition comprising at
least one of diindolylmethane; indole-3-carbinol; embelin;
[6]-gingerol; and [6]-shogaol, or combinations each thereof. Also
disclosed is use in the treatment of obesity and use in the
treatment of metabolic syndrome.
Inventors: |
McKillop; Aine Maire;
(Coleraine, GB) ; FLATT; Peter Raymond;
(Coleraine, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Ulster |
Coleraine, County Londonderry |
|
GB |
|
|
Family ID: |
54062975 |
Appl. No.: |
15/749351 |
Filed: |
July 29, 2016 |
PCT Filed: |
July 29, 2016 |
PCT NO: |
PCT/EP2016/068183 |
371 Date: |
January 31, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/405 20130101;
A61P 3/04 20180101; A61P 3/10 20180101; A61K 2300/00 20130101; A61K
31/122 20130101; A61K 31/7004 20130101; A61K 31/405 20130101; A61K
2300/00 20130101; A61K 31/122 20130101; A61K 2300/00 20130101; A61K
31/7004 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/122 20060101
A61K031/122; A61P 3/04 20060101 A61P003/04; A61P 3/10 20060101
A61P003/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 2015 |
GB |
1513543.7 |
Claims
1. A method of treating diabetes, the method comprising
administering a pharmaceutical composition comprising at least one
of diindolylmethane; indole-3-carbinol; embelin; [6]-gingerol; and
[6]-shogaol, or combinations of each thereof to a subject in need
thereof.
2. The method of claim 1, wherein the composition comprises
embelin.
3. The method of claim 1, wherein the composition comprises a
pharmaceutically acceptable amount of embelin.
4. The method of claim 1, wherein the composition comprises from
10.sup.-12 to 10.sup.-4 mol/L of embelin.
5. The method of claim 1, wherein the composition comprises
10.sup.-4 mol/L of embelin.
6. The method of claim 1, wherein the composition further comprises
glucose.
7. The method of claim 6, wherein the composition comprises 5.6 mM
glucose.
8. The method of claim 6, wherein the composition comprises 16.7 mM
glucose.
9. The method of claim 2, further comprising administering glucose
before the composition comprising embelin.
10. The method of claim 2, further comprising co-administering
glucose with the composition comprising embelin.
11. The method of claim 2, further comprising administering glucose
after the composition comprising embelin.
12. The method of claim 2, wherein the composition is administered
in an amount such that the embelin is administered in an amount of
0.1 .mu.mol/kg to 50 .mu.mol/kg body weight of said subject.
13. The method of claim 2, wherein the composition is administered
in combination with glucose in an amount such that the glucose is
administered in an amount of 18 mmol/kg body weight of said
subject.
14. A method of treating obesity, the method comprising
administering a pharmaceutical composition comprising embelin to a
subject in need thereof.
15. A method of treating metabolic syndrome, the method comprising
administering a pharmaceutical composition comprising embelin to a
subject in need thereof.
16-18. (canceled)
Description
FIELD OF THE INVENTION
[0001] This invention relates to compositions for use in the
treatment of diabetes, for example type-2 diabetes; obesity; and/or
metabolic syndrome.
BACKGROUND TO THE INVENTION
[0002] Diabetes is a major public health challenge with: at least
180 million reported cases of diabetes worldwide--a figure set to
more than double by 2030 according to the World Health Organisation
(WHO), consumption of 10% of Western healthcare budgets, and around
3.2 million deaths per year resulting from related complications.
This alarming increase in incidence, coupled with the failure of
established anti-diabetic drugs to tightly manage or control
diabetes, demonstrates the market need for new innovation.
[0003] The worldwide increase in the incidence of obesity,
metabolic syndrome, and type-2 diabetes demands the development of
new drugs for safe and effective treatment, limiting the
progression to long-term diabetic complications.
[0004] G-protein coupled receptor 84, also known as GPR84, (herein,
GPR-a1), or inflammation-related G-protein coupled receptor EX33;
is a receptor that has been identified on a number of tissues and
is activated by medium chain fatty acids. GPR84 is a chemokine
receptor that has been identified on peripheral blood leucocytes
(neutrophils, T-lymphocytes, B-lymphocytes), spleen, adipocytes,
bone marrow and lungs. GPR84 gene knockout in mice has found that
the receptor has a role in interleukin-4 (IL-4) gene expression,
highlighting the potential of GPR84 as a new therapeutic target,
and opening new avenues, such as identification of new specific
agonists for GPR84 as new effective treatments for diabetes.
SUMMARY OF THE INVENTION
[0005] According to a first aspect of the present invention, there
is provided a composition for use in the treatment of diabetes, the
composition comprising at least one of diindolylmethane;
indole-3-carbinol; embelin; [6]-gingerol; and [6]-shogaol, or
combinations each thereof.
[0006] According to a second aspect of the present invention, there
is provided use of at least one of diindolylmethane;
indole-3-carbinol; embelin; [6]-gingerol; and [6]-shogaol, or
combinations each thereof in the manufacture of a medicament
composition for the treatment of diabetes.
[0007] According to a third aspect of the present invention, there
is provided a method for the treatment of diabetes, the method
comprising the steps of administering a composition comprising at
least one of diindolylmethane; indole-3-carbinol; embelin;
[6]-gingerol; and [6]-shogaol, or combinations each thereof.
[0008] Also disclosed is a composition for use in altering,
optionally increasing, insulin release, the composition comprising
at least one of diindolylmethane; indole-3-carbinol; embelin;
[6]-gingerol; and [6]-shogaol, or combinations each thereof.
[0009] Also disclosed is use of at least one of diindolylmethane;
indole-3-carbinol; embelin; [6]-gingerol; and [6]-shogaol, or
combinations each thereof in the manufacture of a medicament
composition for altering, optionally increasing, insulin
release.
[0010] Also disclosed is a method for altering, optionally
increasing, insulin release, the method comprising the steps of
administering a composition comprising at least one of
diindolylmethane; indole-3-carbinol; embelin; [6]-gingerol; and
[6]-shogaol, or combinations each thereof.
[0011] Optionally, the method comprises the step of administering a
pharmaceutically acceptable amount of the composition comprising at
least one of diindolylmethane; indole-3-carbinol; embelin;
[6]-gingerol; and [6]-shogaol, or combinations each thereof.
[0012] Further optionally, the method comprises the step of
administering a pharmaceutically acceptable amount of the
composition comprising at least one of diindolylmethane;
indole-3-carbinol; embelin; [6]-gingerol; and [6]-shogaol, or
combinations each thereof to a subject in need thereof.
[0013] Further optionally, the method comprises the step of
administering a pharmaceutically acceptable amount of the
composition comprising at least one of diindolylmethane;
indole-3-carbinol; embelin; [6]-gingerol; and [6]-shogaol, or
combinations each thereof to a subject suffering from diabetes.
[0014] Optionally, the composition comprises a pharmaceutically
acceptable amount at least one of diindolylmethane;
indole-3-carbinol; embelin; [6]-gingerol; and [6]-shogaol, or
combinations each thereof.
[0015] Further optionally, the composition comprises from
10.sup.-12 to 10.sup.-4 mol/L of at least one of diindolylmethane;
indole-3-carbinol; embelin; [6]-gingerol; and [6]-shogaol, or of a
combination each thereof.
[0016] Still further optionally, the composition comprises
10.sup.-4 mol/L of at least one of diindolylmethane;
indole-3-carbinol; embelin; [6]-gingerol; and [6]-shogaol, or of a
combination each thereof.
[0017] Optionally, the composition further comprises glucose.
[0018] Further optionally, the composition further comprises 5.6 mM
glucose.
[0019] Alternatively, the composition further comprises 16.7 mM
glucose.
[0020] Optionally, the glucose is administered before the at least
one of diindolylmethane; indole-3-carbinol; embelin; [6]-gingerol;
and [6]-shogaol, or the combination each thereof.
[0021] Optionally or additionally, the glucose is co-administered
with the at least one of diindolylmethane; indole-3-carbinol;
embelin; [6]-gingerol; and [6]-shogaol, or the combination each
thereof.
[0022] Optionally or additionally, the glucose is administered
after the at least one of diindolylmethane; indole-3-carbinol;
embelin; [6]-gingerol; and [6]-shogaol, or the combination each
thereof.
[0023] Optionally, the composition comprises diindolylmethane
(3,3'-methanediylbis(1H-indole)).
[0024] Further optionally, the composition comprises a
pharmaceutically acceptable amount of diindolylmethane
(3,3'-methanediylbis(1H-indole)).
[0025] Still further optionally, the composition comprises from
10.sup.-9 to 10.sup.-4 mol/L of diindolylmethane
(3,3'-methanediylbis(1H-indole)).
[0026] Still further optionally, the composition comprises from
10.sup.-8 to 10.sup.-4 mol/L of diindolylmethane
(3,3'-methanediylbis(1H-indole)) and 5.6 mM glucose.
[0027] Further alternatively, the composition comprises from
10.sup.-9 to 10.sup.-4 mol/L of diindolylmethane
(3,3'-methanediylbis(1H-indole)) and 16.7 mM glucose.
[0028] Optionally or additionally, the composition comprises
indole-3-carbinol (1H-Indol)-3-ylmethanol).
[0029] Further optionally, the composition comprises a
pharmaceutically acceptable amount of indole-3-carbinol
(1H-Indol-3-ylmethanol).
[0030] Still further optionally, the composition comprises
10.sup.-7-10.sup.-4 mol/L of indole-3-carbinol
(1H-Indol)-3-ylmethanol) and 5.6 mM glucose.
[0031] Still further optionally, the composition comprises
10.sup.-6-10.sup.-4 mol/L of indole-3-carbinol
(1H-Indol)-3-ylmethanol) and 16.7 mM glucose.
[0032] Optionally or additionally, the composition comprises
embelin (2,5-dihydroxy-3-undecylcyclohexa-2,5-diene-1,4-dione).
[0033] Further optionally, the composition comprises a
pharmaceutically acceptable amount of embelin
(2,5-dihydroxy-3-undecylcyclohexa-2,5-diene-1,4-dione).
[0034] Still further optionally, the composition comprises from
10.sup.-10 to 10.sup.-4 mol/L of embelin
(2,5-dihydroxy-3-undecylcyclohexa-2,5-diene-1,4-dione).
[0035] Still further optionally, the composition comprises from
10.sup.-9 to 10.sup.-4 mol/L of embelin
(2,5-dihydroxy-3-undecylcyclohexa-2,5-diene-1,4-dione) and 5.6 mM
glucose.
[0036] Alternatively, the composition comprises from 10.sup.-10 to
10.sup.-4 mol/L of embelin
(2,5-dihydroxy-3-undecylcyclohexa-2,5-diene-1,4-dione) and 16.7 mM
glucose.
[0037] Optionally or additionally, the composition comprises
[6]-gingerol
((5S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one).
[0038] Further optionally, the composition comprises a
pharmaceutically acceptable amount of [6]-gingerol
((5S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one).
[0039] Still further optionally, the composition comprises from
10.sup.-9 to 10.sup.-4 mol/L of [6]-gingerol
((5S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one).
[0040] Still further optionally, the composition comprises from
10.sup.-9 to 10.sup.-4 mol/L of [6]-gingerol
((5S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one) and 5.6
mM glucose.
[0041] Alternatively, the composition comprises from 10.sup.-9 to
10.sup.-4 mol/L of [6]-gingerol
((5S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one) and 16.7
mM glucose.
[0042] Optionally or additionally, the composition comprises
[6]-shogaol ((E)-1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one).
[0043] Further optionally, the composition comprises a
pharmaceutically acceptable amount of [6]-shogaol
((E)-1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one).
[0044] Still further optionally, the composition comprises a
pharmaceutically acceptable amount of [6]-shogaol
((E)-1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one) and 5.6 mM
glucose.
[0045] Still further optionally, the composition comprises a
pharmaceutically acceptable amount of [6]-shogaol
((E)-1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one) and 16.7 mM
glucose.
[0046] Optionally, the composition is administered in an amount
such that the at least one of diindolylmethane; indole-3-carbinol;
embelin; [6]-gingerol; and [6]-shogaol, or combinations each
thereof is administered in an amount of 0.1 .mu.mol/kg to 50
.mu.mol/kg body weight.
[0047] Further optionally, the composition is administered in
combination with glucose in an amount such that the glucose is
administered in an amount of 18 mmol/kg body weight.
[0048] According to a further aspect of the present invention,
there is provided a composition for use in the treatment of
obesity, the composition comprising at least one of
diindolylmethane; indole-3-carbinol; embelin; [6]-gingerol; and
[6]-shogaol, or combinations each thereof.
[0049] According to a still further aspect of the present
invention, there is provided use of at least one of
diindolylmethane; indole-3-carbinol; embelin; [6]-gingerol; and
[6]-shogaol, or combinations each thereof in the manufacture of a
medicament composition for the treatment of obesity.
[0050] According to a still further aspect of the present
invention, there is provided a method for the treatment of obesity,
the method comprising the steps of administering a composition
comprising at least one of diindolylmethane; indole-3-carbinol;
embelin; [6]-gingerol; and [6]-shogaol, or combinations each
thereof.
[0051] According to a further aspect of the present invention,
there is provided a composition for use in the treatment of
metabolic syndrome, the composition comprising at least one of
diindolylmethane; indole-3-carbinol; embelin; [6]-gingerol; and
[6]-shogaol, or combinations each thereof.
[0052] According to a still further aspect of the present
invention, there is provided use of at least one of
diindolylmethane; indole-3-carbinol; embelin; [6]-gingerol; and
[6]-shogaol, or combinations each thereof in the manufacture of a
medicament composition for the treatment of metabolic syndrome.
[0053] According to a still further aspect of the present
invention, there is provided a method for the treatment of
metabolic syndrome, the method comprising the steps of
administering a composition comprising at least one of
diindolylmethane; indole-3-carbinol; embelin; [6]-gingerol; and
[6]-shogaol, or combinations each thereof.
[0054] According to a still further aspect of the present
invention, there is provided a composition for use in the treatment
of any one of diabetes, obesity, or metabolic syndrome; the
composition comprising at least one of capric acid (decanoic acid),
undecanoic acid, lauric acid (dodecanoic acid), and tridecanoic
acid; or combinations each thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] Embodiments of the present invention will now be
demonstrated by way of non-limiting examples and in reference to
the accompanying drawings in which:
[0056] FIG. 1 illustrates double immunofluorescence of GPR84
co-localised with insulin in clonal pancreatic BRIN-BD11 cells;
[0057] FIG. 2 illustrates cellular localisation of GPR84 with
insulin and glucagon in mouse pancreatic tissue;
[0058] FIG. 3 illustrates the effect of diindolylmethane on insulin
secretion and LDH release from clonal BRIN-BD11 cells at 5.6 mM
glucose;
[0059] FIG. 4 illustrates the effect of diindolylmethane on insulin
secretion and LDH release from clonal BRIN-BD11 cells at 16.7 mM
glucose;
[0060] FIG. 5 illustrates the effect of embelin on insulin
secretion and LDH release from clonal BRIN-BD11 cells at 5.6 mM
glucose;
[0061] FIG. 6 illustrates the effect of embelin on insulin
secretion and LDH release from clonal BRIN-BD11 cells at 16.7 mM
glucose;
[0062] FIG. 7 illustrates the effect of compositions of the present
invention and alanine on intracellular Ca2+ from clonal BRIN-BD11
cells at 5.6 mM glucose;
[0063] FIG. 8 illustrates the effect of medium chain fatty acids
and alanine on intracellular Ca2+ from clonal BRIN-BD11 cells at
5.6 mM glucose;
[0064] FIG. 9 illustrates the effect of medium chain fatty acids
and alanine on intracellular Ca2+ from clonal BRIN-BD11 cells at
16.7 mM glucose;
[0065] FIG. 10 illustrates the effect of compositions of the
present invention and alanine on intracellular Ca2+ from clonal
BRIN-BD11 cells at 16.7 mM glucose;
[0066] FIG. 11 illustrates the effect of compositions of the
present invention and glucagon-like peptide-1 (GLP-1) on cAMP
production from clonal BRIN-BD11 cells at 11.1 mM glucose;
[0067] FIG. 12 illustrates acute effects of compositions of the
present invention on plasma glucose in NIH Swiss mice on normal
chow and high fat diet following glucose load;
[0068] FIG. 13 illustrates acute effects of medium chain fatty
acids on plasma glucose in NIH Swiss mice on normal chow and high
fat diet following glucose load;
[0069] FIG. 14 illustrates acute effects of compositions of the
present invention on plasma glucose and plasma insulin in NIH Swiss
mice on normal chow and high fat diet following glucose load;
[0070] FIG. 15 illustrates the effects of daily administration of
(A) Embelin and (B) Diindolylmethane on non-fasting blood glucose
in mice fed on a high-fat diet;
[0071] FIG. 16 illustrates the effects of daily administration of
(A) Embelin and (B) Diindolylmethane on non-fasting plasma insulin
in mice fed on a high-fat diet;
[0072] FIG. 17 illustrates the effects of daily administration of
(A) Embelin and (B) Diindolylmethane on body weight in mice fed on
a high-fat diet;
[0073] FIG. 18 illustrates the effects of daily administration of
(A) Embelin and (B) Diindolylmethane on energy intake in mice fed
on a high-fat diet;
[0074] FIG. 19 illustrates the effects of daily administration of
(A) Embelin and (B) Diindolylmethane on fluid intake in mice fed on
a high-fat diet;
[0075] FIG. 20 illustrates the effects of daily administration of
(A) Embelin and (B) Diindolylmethane on blood glucose following
oral administration of glucose to mice fed on a high-fat diet;
[0076] FIG. 21 illustrates the effects of daily administration of
(A) Embelin and (B) Diindolylmethane on plasma insulin following
oral administration of glucose to mice fed on a high-fat diet;
[0077] FIG. 22 illustrates the effects of daily administration of
Diindolylmethane and Indole-3-carbinol on pancreatic weight (A) and
pancreatic insulin content (B) in mice fed on a high-fat diet;
[0078] FIG. 23 illustrates the effects of daily administration of
(A) Embelin and (B) Diindolylmethane on insulin sensitivity in mice
fed on a high-fat diet;
[0079] FIG. 24 illustrates the effects of daily administration of
(A) Embelin and (B) Diindolylmethane on plasma GLP-1 concentration
following oral administration of glucose to mice fed on a high-fat
diet;
[0080] FIG. 25 illustrates plasma GLP-1 concentrations in lean mice
and high-fat fed mice administered orally with saline (controls) or
compositions of the present invention for 21 days;
[0081] FIG. 26 illustrates the effects of daily administration of
Diindolylmethane and Embelin on triglycerides total cholesterol
(B), HDL cholesterol (C) and LDL cholesterol (D) in mice fed on a
high-fat diet;
[0082] FIG. 27 illustrates the effects of daily administration of
Embelin on lean body mass (A), body fat content (B and C) in mice
fed on a high-fat diet;
[0083] FIG. 28 illustrates the effects of daily administration of
Diindolylmethane on lean body mass (A), body fat content (B and C)
in mice fed on a high-fat diet;
[0084] FIG. 29 illustrates the effects of daily administration of
Embelin on bone mineral density (A), bone mineral content (B) and
bone area (C) in mice fed on a high-fat diet; and
[0085] FIG. 30 illustrates the effects of daily administration of
Diindolylmethane on bone mineral density (A), bone mineral content
(B) and bone area (C) in mice fed on a high-fat diet.
[0086] For the avoidance of doubt, the following terms are used
synonymously herein: [0087] GPR-a1 GPR84, inflammation-related
G-protein coupled receptor EX33 [0088] MCFA1 Alanine [0089] MCFA2
Tridecanoic acid [0090] MCFA3 Lauric acid (dodecanoic acid) [0091]
MCFA4 Undecanoic acid [0092] MCFA5 Capric acid (decanoic acid)
[0093] Agonist-1 DIM, Diindolylmethane [0094] Agonist-2
Indole-3-carbinol [0095] Agonist-3 Embelin [0096] Agonist-4
[6]-Gingerol [0097] Agonist-5 [6]-Shogaol
Materials and Methods
Insulin Secretion
[0098] Insulin-secreting BRIN-BD11 cells were cultured with
RPMI-1640 media (11.1 mM glucose) containing antibiotics (100 U/ml
penicillin and 0.1 mg/ml streptomycin) and 10% foetal calf serum at
37.degree. C. in an atmosphere of 95% air and 5% carbon dioxide.
For acute insulin secretion studies, cells were detached using
trypsin/EDTA and incubated overnight in 24 well plates with 150,000
cells per well. Cells were then pre-incubated for 40 minutes at 1.1
mmol/l glucose in Krebs buffer (comprising 4.7 mmol/l KCL, 115
mmol/l NaCl, 1.28 mmol/CaCl2, 10 mmol/l NaHCO3, 5 g/l BSA, 1.2
mmol/l KH2PO4, 1.2 mmol/l MgSO47H2O pH 7.4). Test incubations were
then performed at 37.degree. C. for 20 minutes. DIM,
indole-3-carbinol, embelin, [6]-gingerol, and [6]-shogaol at
10.sup.-12-10.sup.-4 mol/L were tested at both 5.6 mmol/L and 16.7
mmol/L glucose, as indicated in the accompanying drawings.
Supernatants were removed, evaluated for lactate dehydrogenase
(LDH) release as an indicator of cytotoxicity (as per
manufacturer's protocol) or frozen at -20.degree. C. until
determination of insulin by radioimmunoassay.
[0099] Pancreatic islets were isolated from normal mice derived
from the colony maintained at the University of Ulster, UK by
collagenase digestion. After overnight culture as above, groups of
10 islets were incubated for 1 hour at 37.degree. C. in 1 ml of 1.1
mmol/l glucose Krebs. Test incubations were then carried out for 1
hour at 11.1 mmol/l glucose with various GPR120 agonists
(10.sup.-10-10.sup.-4 mol/L). Insulin release and insulin content
of islets, treated overnight with 1 ml acid ethanol, were
determined by radioimmunoassay.
Intracellular Ca2+ and cAMP
[0100] For intracellular Ca2+ measurement, monolayers of BRIN-BD11
cells were seeded overnight at a density of 80,000 cells per well
in a 96-well black walled clear bottom plate. Cells were washed
with 100 .mu.l of Krebs buffer and incubated for 1 hour with Flex
calcium assay kit reagent at 37.degree. C. GPR84 at 10.sup.-4 mol/L
were added at 5.6 mmol/L and 16.7 mmol/L glucose. Fluorometric data
were obtained using the FLEX Station scanner and test solutions
were transferred using fluid transfer workstation at a wavelength
of 525 nm (Molecular Devices). For cAMP determination, BRIN-BD11
cells were seeded in a 96-well plate at a density of 30,000 cells
per well. Cells were washed with 300 .mu.l Krebs buffer for 40 min
and 150 .mu.l of compositions of the present invention at
10.sup.-10-10.sup.-4 mol/L were tested at 11.1 mol/L glucose. After
20 minutes, test solutions were removed and 0.1M HCL (150 .mu.L)
was added to lyse the cells. Total cAMP production in the cell
supernatants were measured using cAMP enzyme immunoassay kit
according to the manufacturer's protocol (Sigma, Poole, UK).
Histology
[0101] BRIN-BD11 cells were allowed to attach overnight to
polylysine-coated slides and fixed using 4% paraformaldehyde/PBS.
Antigen retrieval was achieved by incubation in sodium citrate (50
mmol/l) at 90.degree. C. for 20 minutes. Pancreatic tissues from
normal mice were fixed in 4% PFA/PBS, embedded in paraffin wax and
sections cut at 8 .mu.m. Sections were mounted onto
polylysine-coated slides and dried on a hot plate. Pancreatic
sections were de-waxed and antigen retrieval performed as described
above. Slides were incubated overnight at 4.degree. C. with guinea
pig anti-insulin (1:500), guinea pig anti-glucagon (1:500) and
rabbit anti-GPR84 antibodies (1:100). After washing in PBS,
sections were incubated with Alexa Fluor 488 fluorescein goat
anti-rabbit or anti-guinea pig IgG and anti-guinea pig or
anti-rabbit Alexa 594 nm IgG (1:400; Molecular Probes (Life
Technologies Ltd, Paisley, UK)) for 45 minutes at 37.degree. C. and
DAPI nuclear stain for 15 minutes at 37.degree. C. Finally slides
were washed in PBS, mounted and analysed using a BX51 Olympus
microscope equipped with an Olympus XM10 digital camera. Relative
GPR84 quantification analysis was performed on BRIN-BD11 cells
after exposure to compositions of the present invention at
10.sup.-4 mol/L at 11.1 mmol/L glucose for 20 minutes. GPR84 and
insulin immunofluorescence staining was performed as described
above. Analysis was performed by Cell-F software (closed polygon
icon), with >200 cells per treatment group. All slides were
blinded and a negative control slide was performed to ensure
antibody specificity with omission of the primary antibody.
Statistics
[0102] Data are expressed as the mean .+-.the standard error of the
mean (SEM). Results were compared using the Student's t-test or
one-way ANOVA on Prism graph pad version 5.0. Differences in data
were considered to be statistically significant for p<0.05.
Animals
[0103] Adult male (20-22 week) NIH Swiss mice (Harlan UK Ltd) were
individually housed in an air-conditioned room at 22.+-.2.degree.
C. with 12 hour light: 12 hour darkness cycles. Drinking water and
standard rodent maintenance diet (Trouw Nutrition, Cheshire, UK)
were supplied ad libitum. Non-fasted NIH Swiss mice (n=6) received
an oral injection of glucose alone (18 mmol/kg body weight) or in
combination with compositions of the present invention (50
.mu.mol/kg body weight). Blood samples were obtained from the cut
tip from tail vein of conscious mice and centrifuged at 13,000 rpm
for 3 minutes at 4.degree. C. Plasma glucose was measured by an
automated glucose oxidase procedure using a Beckman glucose
analyser and insulin determined by radioimmunoassay. All animal
experiments were carried out in accordance with the UK Animal
(Scientific Procedures) Act 1986.
Administration in High Fat Fed Diabetic Mice
[0104] Daily oral administration of compositions of the present
invention (0.1 .mu.mol/kg body weight) or saline vehicle (0.9% w/v
NaCl) were utilised in a long term study (28 days) examining their
effects on high fat fed diabetic NIH Swiss mice. In order to
confirm diabetes, an oral glucose tolerance test (OGTT) was
performed. Food intake, fluid intake, body weight, non-fasted
plasma glucose and insulin concentrations were monitored every 2-
to 4-days as indicated in the accompanying drawings. At the end of
the study, glucose tolerance (18 mmol/kg body weight) and insulin
sensitivity (25 U/kg body weight) were assessed. Mice were
anesthetised by isoflurane and killed by cervical dislocation. Dual
energy X-ray absorption (DEXA) scanning was performed after prior
calibration and quality control with the aluminium/lucite phantom
(0.069 g/cm2, 12.0% fat) using a PIXImus system (software version
1.4x).
EXAMPLES
Example 1
Expression of GPR84 in BRIN-BD11 Cells and Mouse Islets
[0105] Distribution of insulin and GPR84 were investigated in
BRIN-BD11 cells. DAPI (blue) stained nuclei (FIG. 1A), and insulin
(green) were distributed across the BRIN-BD11 cells (FIG. 1B) with
a similar staining pattern to GPR84 (red) (FIG. 1C). Double
immunofluorescence combination of insulin with GPR84 indicated
areas of co-localisation (yellow) (FIG. 1D), demonstrating the
presence of GPR-a1 in pancreatic beta cells. The distribution of
DAPI, insulin, glucagon and GPR84 in mouse pancreatic islets are
shown in FIG. 2. DAPI (blue) displayed the nuclei in pancreatic
islets (FIGS. 2A, B), GPR84 (green) was expressed throughout the
islet with a similar staining pattern to insulin (red) (FIGS. 2C,
D). Merge of insulin and GPR84 indicated that insulin secreting
beta cells express GPR84 (FIG. 2G) while there was no evidence of
the GPR84 receptor in glucagon secreting alpha cells (FIG. 2H).
While no co-localisation was displayed on glucagon secreting alpha
cells, the effect of GPR84 on beta cells may result in paracrine
effects on other pancreatic islet cells.
Example 2
Effects of Compositions of the Present Invention on Insulin
Secretion from BRIN-BD11 Cells
[0106] Insulin releasing properties of compositions of the present
invention at 10.sup.-12-10.sup.-4 mol/L were assessed in clonal
BRIN-BD11 cells at 5.6 mM and 16.7 mM glucose. Diindolylmethane at
10.sup.-8-10.sup.-4 mol/L enhanced insulin release (EC50
1.3.times.10.sup.-7 mol/L) (p<0.05-p<0.001) (FIG. 3A) at 5.6
mM basal glucose concentrations. At stimulatory glucose
concentrations (16.7 mM glucose), Diindolylmethane significantly
enhanced insulin release at 10.sup.-9-10.sup.-4 mol/L (EC50
1.0.times.10.sup.-6 mol/L) (p<0.05-p<0.001) (FIG. 4A). No
cytotoxicity was found with Diindolylmethane at 5.6 mM glucose
(FIG. 3B) and 16.7 mM glucose (FIG. 4B).
[0107] Embelin at 10.sup.-9-10.sup.-4 mol/L enhanced insulin
release (p<0.05-p<0.001) (EC50 of 7.3.times.10.sup.-7 mol/L)
(FIG. 5A) at 5.6 mM basal glucose concentrations. At stimulatory
glucose concentrations (16.7 mM glucose), Embelin enhanced insulin
release at 10.sup.-10-10.sup.-4 mol/L (p<0.01-p<0.001) (EC50
of 2.1.times.10.sup.-7 mol/L) (FIG. 6A). No cytotoxicity was found
with Embelin at 5.6 mM glucose (FIG. 5B) and 16.7 mM glucose (FIG.
6B).
[0108] Indole-3-carbinol at 10.sup.-7-10.sup.-4 mol/L enhanced
insulin release (p<0.05-p<0.01) (EC50 of 1.5.times.10.sup.-6
mol/L) at 5.6 mM basal glucose concentrations. At stimulatory
glucose concentrations (16.7 mM glucose), Indole-3-carbinol
enhanced insulin release at 10.sup.-6-10.sup.-4 mol/L
(p<0.05-p<0.01) (EC50 of 4.0.times.10.sup.-7 mol/L). No
cytotoxicity was observed.
[0109] [6]-gingerol at 10.sup.-8-10.sup.-4 mol/L enhanced insulin
release (p<0.05-p<0.001) (EC50 of 1.9.times.10.sup.-6 mol/L)
at 5.6 mM basal glucose concentrations. At stimulatory glucose
concentrations (16.7 mM glucose), [6]-gingerol enhanced insulin
release at 10.sup.-8-10.sup.-4 mol/L (p<0.05-p<0.001) (EC50
of 2.8.times.10.sup.-6 mol/L). No cytotoxicity was observed.
[0110] Medium chain fatty acids (10.sup.-7-10.sup.-4M) resulted in
increased insulin secretion (p<0.05-p<0.001) with EC50
ranging from 4.5.times.10.sup.-8 mol/L-2.0.times.10.sup.-5 mol/l at
5.6 mM glucose; and EC50 of 6.4.times.10.sup.-8
mol/L-1.3.times.10.sup.-7 mol/L at 16.7 mM glucose.
[0111] All compositions of the present invention tested at 5.6 mM
or 16.7 mM glucose resulted in no LDH release indicating no adverse
effects on clonal BRIN-BD11 cells.
Example 3
Effect of Compositions of the Present Invention on Intracellular
Ca2+ and cAMP in BRIN-BD11 Cells
[0112] For confirmation of the stimulatory ability of compositions
of the present invention on insulin secretion in pancreatic islets
and to examine the mechanism of action, beta stimulus coupling
pathways and changes in intracellular calcium concentrations and
cAMP production in pancreatic BRIN-BD11 cells were examined.
[0113] At both basal and stimulatory glucose concentrations,
compositions of the present invention (10.sup.-4 mol/L) augmented
intracellular Ca2+ concentrations at 5.6 mM glucose
(p<0.05-p<0.001) (FIGS. 7-10) with the exception of
Indole-3-carbinol and [6]-gingerol. At 16.7 mM glucose,
Diindolylmethane, Embelin and [6]-shogaol increased intracellular
Ca2+ concentrations (p<0.001) (FIG. 10).
[0114] As shown in FIG. 11, the stimulatory action of
Diindolylmethane and Embelin on the insulin secretory pathway
involves the cAMP-dependent pathway in pancreatic islets.
[6]-shogaol and medium chain fatty acids act through the cAMP
pathway.
Example 4
In Vivo Acute Effects of Compositions of the Present Invention on
Plasma Glucose and Insulin in NIH Swiss Mice on Lean and High Fat
Diet
[0115] Administration of compositions of the present invention
resulted in a decrease in circulating glucose in vivo (FIG. 12).
Diindolylmethane and Embelin decreased circulating glucose
(p<0.05) in high fat fed mice. Plasma glucose was also
significantly decreased (p<0.001) following administration of
Indole-3-carbinol in high fat fed mice. [6]-gingerol also resulted
in a significant decrease (p<0.001) in glucose in acute studies
carried out in NIH Swiss mice.
[0116] The acute effects of medium chain fatty acids on plasma
glucose were studied in NIH Swiss mice on normal chow and high fat
diet following a glucose load (FIG. 13). The medium chain fatty
acid ligands of GPR84 decreased plasma glucose after a glycaemic
excursion (p<0.001) (FIG. 13).
[0117] Diindolylmethane (p<0.01) and Embelin (p<0.01)
administration resulted in an increase in insulin release in high
fat fed mice (FIG. 14).
Example 5
Long Term In Vivo Effects of Compositions of the Present Invention
in NIH Swiss Mice on Lean and High Fat Diet
[0118] Effects of Diindolylmethane and Embelin on food intake,
fluid intake, body weight, non-fasting plasma glucose, insulin,
glucagon and pancreatic insulin content were measured.
[0119] Embelin administration resulted in a significant decrease in
plasma glucose in high fat fed mice after 9 days of treatment
(p<0.05-p<0.01) (FIG. 15). Area under the curve results
demonstrated a significant decrease (p<0.05, 17%) in plasma
glucose over the 21 days.
[0120] Diindolylmethane administration resulted in a decrease in
plasma glucose (p<0.01) at 21 days and area under the curve
results demonstrated a significant decrease (p<0.01, 18%
decrease) over the 21 day period.
[0121] Plasma insulin was augmented after 21 days by Embelin
(p<0.05) and Diindolylmethane (p<0.05) (FIG. 16).
[0122] Daily oral administration of Embelin for 21 days had no
effect on body weight while Diindolylmethane decreased body weight
after 15 days (FIG. 17). Diindolylmethane decreased energy intake
(p<0.05-p<0.001) (FIG. 18) and had no effect on fluid intake
(FIG. 19).
[0123] Following long-term administration of Embelin, plasma
glucose was significantly decreased (p<0.001), as demonstrated
following a glucose load (FIG. 20). Plasma glucose was also
attenuated by Diindolylmethane (p<0.001) (FIG. 20). Daily
administration of Embelin (p<0.001) and Diindolylmethane
(p<0.001) resulted in decreased plasma insulin (FIG. 21).
Pancreatic insulin content was increased in the HFF mouse model
(FIG. 22) due to islet size and insulin resistance. Embelin
(p<0.01) and Diindolylmethane (p<0.05) reduced pancreatic
insulin content in the HFF model (FIG. 22) which may have resulted
in the decrease in circulating insulin. Insulin sensitivity was
decreased with Embelin (p<0.05) and Diindolylmethane
(p<0.001) (FIG. 23).
[0124] Interestingly, Diindolylmethane (p<0.01) and Embelin
(p<0.05) increased GLP-1 secretion over the long term study,
with a 61% and 45% increase in circulating GLP-1 at 30 min (FIG.
24). Plasma GLP-1 was raised in vivo by Diindolylmethane
(p<0.01) and Embelin (p<0.05) at the end of the study (FIG.
25).
[0125] Embelin reduced total cholesterol (p<0.05) and
low-density lipoprotein (LDL) cholesterol (p<0.05) in high fat
fed mice (FIG. 26). Daily administration of Embelin resulted in no
change in total body fat (FIG. 27) whilst Diindolylmethane
decreased body fat (p<0.01) in high fat fed mice (FIG. 28).
[0126] Embelin and Diindolylmethane resulted in no change in body
mineral density, bone mineral content and bone area in HFF mice
(FIG. 29-30).
[0127] The present inventors have, for the first time, identified
expression of GPR84 on pancreatic islets. This work has shown GPR84
distribution in pancreatic BRIN-BD11 cells and in mouse pancreatic
tissue with GPR84 predominately co-expressed with insulin. This
research has clearly demonstrated the expression of GPR84 in
pancreatic islets and also, for the first time demonstrated the
importance of GPR84 in islet cell function. In this study, the
immunocytochemical cell work was complimented by studies
demonstrating the effect of compositions of the present invention
on insulin secretion in pancreatic islets. All compositions of the
present invention exhibited enhanced potency in the clonal
BRIN-BD11 cells and isolated mouse islets, and demonstrated that
glucose sensitises insulin-secreting cells to the pharmacological
actions of compositions of the present invention.
[0128] GPR84 has been shown in the use of the present invention to
have an effect on the beta cell stimulus-secretion coupling pathway
in pancreatic islets. The mechanism of action of GPR84
agonist-induced insulin release, examined intracellular Ca2+ and
cAMP production in BRIN-BD11 cells. The compositions of the present
invention caused a prompt augmentation in intracellular Ca2+,
indicating modulation of insulin secretion is attributed partly
through Ca2+ dependent pathways. In the use of the present
invention, compositions of the present invention caused a moderate
increase in total cAMP production, indicating that the compositions
of the present invention predominately work through the Ca2+
activated pathway and to a lesser extent the cAMP dependent
pathway.
[0129] The invention is not limited to the embodiment(s) described
herein but can be amended or modified without departing from the
scope of the present invention.
* * * * *