Method for screening for diabetes mellitus therapeutic agents

Abstract

Provided are a pharmaceutical composition including geraniol or a pharmaceutically acceptable salt thereof as an active ingredient for use in preventing or treating diabetes mellitus, and a method of preventing or treating diabetes mellitus or a complication due to diabetes mellitus of an individual, in which the method includes administering to the individual a pharmaceutical composition comprising geraniol or a pharmaceutically acceptable salt thereof as an active ingredient and inducing an olfactory stimulation to the individual using the pharmaceutical composition. Also provided is a screening method for an antidiabetic agent that may include contacting a cell expressing an olfactory receptor with a test material; measuring a level of expression of glucagon-like peptide-1 (GLP-1) secreted from the cell; and determining that the test material, when the test material promotes expression of GLP-1, as a candidate material for an antidiabetic agent. In addition, a quasi-drug composition and a cosmetic composition including geraniol for uses are provided.

Claims

1. A method of screening a test material to determine if the test material is a candidate for an antidiabetic agent for administration by inhalation, the method comprising: (a) contacting (i) an NCI-H716 cell, or a culture thereof, expressing human olfactory receptors OR1A1 and OR with the test material; and (ii) an NCI-H716 cell, or a culture thereof, for which the OR1A1 and/or OR1G1 expression has been knocked down with the test material; (b) measuring an expression level of glucagon-like peptide-1 (GLP-1) secreted from the NCI-H716 cell, or the culture thereof, of step (a)(i) expressing human olfactory receptors OR1A1 and OR1G1; (c) measuring an expression level of glucagon-like peptide-1 (GLP-1) secreted from the NCI-H716 cell, or the culture thereof, of step (a)(ii) for which the OR1A1 and/or OR1G1 expression has been knocked down; and (d) if the expression level of GLP-1 measured in the step (b) increases compared with before contacting with the test material, and the expression level of GLP-1 measured in the step (c) does not increase compared with before contacting with the test material, determining that the test material is a candidate material for an antidiabetic agent for administration by inhalation.

2. The method of claim 1, wherein the test material is an odorant.

3. The method of claim 2, wherein the odorant comprises a monoterpenoid.

4. The method of claim 2, wherein the odorant comprises geraniol.

5. The method of claim 1, wherein the contacting comprises incubating the NCI-H716 cell or the culture thereof, of step (a)(i) expressing human olfactory receptors OR1A1 and OR1G1, and the NCI-H716 cell, or the culture thereof, of step (a)(ii) for which the OR1A1 and/or OR1G1 expression has been knocked down with the test material at a concentration of 10 μM to 2000 μM.

6. The method of claim 1, wherein the measuring is identifying an increase of an amount of GLP-1 by an enzyme-linked immunosorbent assay (ELISA), a multiplex assay (GLP-1 multiplex assay), a radioimmunoassay, a quantitative immunofluorescence assay (fluorescent antibody method), or a latex agglutination assay.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A is a result of verification of GLP-1 secretion promoting effect by geraniol in a NCI-H716 cell, i.e., a human enteroendocrine cell. It was found that geraniol dose-dependently promotes GLP-1 secretion in a human enteroendocrine cell, as compared with the isomer thereof, nerol.

(2) FIG. 1B is a graph showing that geraniol stimulates an olfactory receptor, OR1A1, promoting GLP-1 secretion.

(3) FIG. 1C is a graph showing that geraniol stimulates an olfactory receptor, OR1G1, promoting GLP-1 secretion.

(4) FIG. 2 is a graph showing change of calcium concentration in a human enteroendocrine cell after geraniol treatment. 100 μM of geraniol was treated after 1 minute elapsed from the start of the experiment, and the calcium concentration in the cell was found to be the highest after 4 minutes elapsed.

(5) FIG. 3 is a graph of measurement of change amount of cAMP in a cell versus time after geraniol treatment. After 30 minutes elapsed from 100 μM geraniol treatment, the concentration of cAMP in the cell was the highest, and then decreased thereafter.

(6) FIG. 4 is a graph of measurement of change amount of cAMP in a cell versus time after treatment of forskolin, which is a cAMP activator. FIG. 4 is a graph of measurement of change amount of cAMP after 10 minutes elapsed from 10 μM forskolin treatment. Forskolin treatment was found to show a similar pattern as the geraniol treatment.

(7) FIG. 5A is a graph of hypoglycemic effect in a type II diabetes mellitus mouse model at an oral glucose tolerance test, after gavage of geraniol of 150 mg/weight Kg.

(8) FIG. 5B is a graph of hypoglycemic effect in a type II diabetes mellitus mouse model at an oral glucose tolerance test, after gavage of geraniol of 500 mg/weight Kg.

(9) FIG. 5C is a graph of hypoglycemic effect in a type II diabetes mellitus mouse model at an oral glucose tolerance test, after gavage of metformin, which is used as an antidiabetic agent, of 300 mg/weight Kg. The effect thereof may be compared with that of geraniol.

(10) FIG. 6A is a graph of plasma GLP-1 increase effect in a type II diabetes mellitus mouse model at an oral glucose tolerance test, after gavage of geraniol of 150 mg/weight Kg.

(11) FIG. 6B is a graph of plasma insulin increase effect in a type II diabetes mellitus mouse model at an oral glucose tolerance test, after gavage of geraniol of 150 mg/weight Kg.

(12) FIG. 6C is a graph of plasma GLP-1 increase effect in a type II diabetes mellitus mouse model at an oral glucose tolerance test, after gavage of geraniol of 300 mg/weight Kg.

(13) FIG. 6D is a graph of plasma insulin increase effect in a type II diabetes mellitus mouse model at an oral glucose tolerance test, after gavage of geraniol of 300 mg/weight Kg.

(14) FIGS. 7A and 7B are images that verify the presence of olfactory receptors in the small intestine of human and mouse through immunofluorescent staining.

(15) FIG. 7A A to C are results that verify GLP-1 co-expression with OR1A1 (olfactory receptor family 1 subfamily A member 1) or OR1G1 in an ileum tissue of a human through immunofluorescent staining. FIG. 7A D to F are results that verify expression of GLP-1 and OR1A1, OR1G1, or Golf (G protein, olfactory type) in an NCI-H716 cell, i.e., a human enteroendocrine cell.

(16) FIG. 7B G to I are results that verify GLP-1 co-expression with olfactory marker protein (OMP), OR1G1, or OR1A1 in duodenum and ileum tissues of a mouse. FIG. 7B G is for illustrating expressions of GLP-1, OR1A1, and OR1G1 through immunofluorescent staining, in an order from the left to the right.

(17) FIG. 8 is a graph for verifying cytotoxicity of geraniol in a human enteroendocrine cell through MTT assay. The results thereof are shown in cell viability (%) compared with a comparison group treated with PBS, and hydro peroxided (H.sub.2O.sub.2) was used as a positive control group. As a result, it was found that 10 to 1000 μM of geraniol had no cytotoxicity in an NCI-H716 cell, i.e., a human enteroendocrine cell.

(18) FIG. 9 is a graph of hypoglycemic effect in a type II diabetes mellitus mouse model at an oral glucose tolerance test, at an occurrence of smelling of geraniol. The graph shows a blood sugar level reducing due to geraniol olfactory stimulation, as compared with an oral glucose tolerance test of oral administration of metformin, i.e., an antidiabetic agent, at 300 mg/weight Kg, over time.

(19) FIG. 10 is a graph of an increase of amount of GLP-1 in plasma of a type II diabetes mellitus mouse model at an oral glucose tolerance test, at an occurrence of smelling of geraniol. The graph shows a GLP-1 level increasing due to geraniol olfactory stimulation, as compared with an oral glucose tolerance test of oral administration of metformin, i.e., an antidiabetic agent, at 300 mg/weight Kg, over time.

DETAILED DESCRIPTION OF THE DISCLOSURE

(20) The present invention will be described in further detail with reference to the following examples. However, the description is for understanding the present invention only and is not intended to limit the scope of the present invention.

EXAMPLE 1

GLP-1 Secretion Promotion in Enteroendocrine Cell by Geraniol, and Changes of cAMP

(21) (1) Verification of GLP-1 Secretion Promotion in Enteroendocrine Cell by Geraniol

(22) 1-1. Cell Culture

(23) A NCI-H716 cell, i.e., a human enteroendocrine cell, was obtained from Korean Cell Line Bank. The obtained cell was culture in RPMI 1640 medium until the number of obtained cells increased in a proper amount. Then, the cells were moved to a matrigel-coated plate, and then cultured in DMEM medium for 48 hours, for endocrine differentiation.

(24) 1-2. Preparation of Test Material

(25) Geraniol and nerol were purchased from Signa-Aldrich, USA.

(26) 1-3. Treatment of Cell with Test Material

(27) Human enteroendocrine cells were each treated with geraniol at a concentration of 10, 20, 50, 100, 200, 500, 1000, and 2000 μM, respectively, and then cells were cultured for 1 hour. A human enteroendocrine cell was treated with nerol, which is an isomer of geraniol, as a comparison group, i.e., a negative control group, under the same condition as in the treating with geraniol.

(28) 1-4. Analysis of GLP-1 secretion amount

(29) GLP-1 secretion from the cell treated with geraniol and the cell treated with nerol were identified using enzyme-linked immunosorbent assay (ELISA). ELISA was carried out using a GLP-1 ELISA kit, available from Millipore, based on the user manual. The amount of GLP-1 secretion was measured using a fluoroskan ascent microplate reader (Thermo Electron Corp., Finland).

(30) FIG. 1A is a result of verification of GLP-1 secretion promoting effect by geraniol in a NCI-H716 cell, i.e., a human enteroendocrine cell. It was found that geraniol dose-dependently promotes GLP-1 secretion in a human enteroendocrine cell, as compared with nerol, i.e., the isomer thereof. As shown in FIG. 1A, it was found that geraniol promoted GLP-1 secretion in proportion to the treatment amount of geraniol in a human enteroendocrine cell. However, the treatment of nerol, i.e., an isomer thereof, had no GLP-1 secretion promotion effect.

(31) (2) Analysis of GLP-1 Secretion by Geraniol in Enteroendocrine Cell in which Olfactory Receptor is Knocked-Down

(32) In a human enteroendocrine cell, in which an olfactory receptor is knocked-down by small-interference RNA (siRNA), an olfactory receptor signal transduction mechanism was identified.

(33) Human enteroendocrine cells in which olfactory receptors OR1A1 and OR1G1 were knocked-down by siRNAs, respectively, were cultured. SiRNAs for knocking-down OR1A1 (SEQ ID NO. 1 and 2, siRNA No. 1108058) and OR1G1 (SEQ ID NO. 3 and 4, siRNA No. 1108121) are available from Bioneer Co. (South Korea). Lipofectamine 2000 (available from Life technology, USA) was used to carry out intracellular transfection, based on the user manual.

(34) The human enteroendocrine cells in which olfactory receptors were knocked-down and a normal human enteroendocrine cell were each treated with geraniol at a concentration of 100 μM. Then, the cells were cultured for 1 hour at 37° C. at a concentration of 5% CO.sub.2. Thereafter, GLP-1 secretion was identified using ELISA. FIG. 1B is a graph showing that geraniol stimulates OR1A1, an olfactory receptor, promoting GLP-1 secretion. FIG. 1C is a graph showing that geraniol stimulates OR1G1, an olfactory receptor, promoting GLP-1 secretion. As shown in FIGS. 1B and 1C, it was found that geraniol stimulates olfactory receptors, such as OR1A1 and/or OR1G1, promoting GLP-1 secretion. Accordingly, it was found that the incretin hormone secretion mechanism of geraniol in a human enteroendocrine cell was through stimulation of olfactory receptors.

(35) (3) Verification of Change Amount of Calcium Concentration in Human Enteroendocrine Cell after Geraniol Treatment

(36) FIG. 2 is a graph showing the change of calcium concentration in a human enteroendocrine cell after geraniol treatment. 100 μM of geraniol was treated after 1 minute elapsed from the start of the experiment, and the calcium concentration in the cell was found to be the highest after 4 minutes elapsed.

(37) (4) Verification of Change Amount of cAMP after Geraniol Treatment

(38) In a human enteroendocrine cell, the result of geraniol treatment (100 μM) and forskolin treatment (10 μM), which is a cAMP activator, were compared with each other. It was found that stimulation to olfactory receptors by geraniol was via cAMP activation in the GLP-1 secretion mechanism, and geraniol treatment had similar tendency with that of forskolin, which is a cAMP activator, by using cAMP ELISA, based on the user manual (Enzo Life Science, USA).

(39) FIG. 3 is a graph of measurement of the change amount of cAMP in a cell versus time after geraniol treatment. As shown in FIG. 3, after 30 minutes elapsed from 100 μM geraniol treatment, the concentration of cAMP in the cell was the highest, and then decreased thereafter.

(40) Furthermore, FIG. 4 is a graph of measurement of the change amount of cAMP in a cell versus time after treatment of forskolin, which is a cAPM activator. FIG. 4 is a graph of measurement of the change amount of cAMP after 10 minutes elapsed from 10 μM forskolin treatment. Forskolin treatment was found to show a similar pattern as the geraniol treatment.

(41) (5) Verification of Presence of Olfactory Receptor in Small Intestine of Human and Mouse

(42) In order to identify the presence of an olfactory receptor in small intestine of human and mouse, immunofluorescent staining was carried out. The results thereof are shown in FIGS. 7A and 7B.

(43) FIGS. 7A and 7B are images that verify the presence of olfactory receptors in the small intestine of human and mouse through immunofluorescent staining FIG. 7A A to C are results that verify GLP-1 co-expression with OR1A1 (olfactory receptor family 1 subfamily A member 1) or OR1G1 in an ileum tissue of a human through immunofluorescent staining. FIG. 7A D to F are results that verify expression of GLP-1 and OR1A1, OR1G1, or Golf (G protein, olfactory type) in an NCI-H716 cell, i.e., a human enteroendocrine cell.

(44) FIG. 7B G to I are results that verify GLP-1 co-expression with olfactory marker protein (OMP), OR1G1, or OR1A1 in duodenum and small intestine tissues of a mouse. FIG. 7B H illustrates % of GLP-1 positive cells co-expressed with OR1A1. In FIG. 7B H, in an order from the left to the right, the graphs each indicate duodenum and ileum. It was found that the GLP-1 positive cells co-expressed with OR1A1 were found in a higher ratio in ileum than in duodenum. In addition, FIG. 7B I illustrates % of GLP-1 positive cells co-expressed with OR1G1. In FIG. 7B I, in an order from the left to the right, the graphs each indicate duodenum and ileum. It was found that the GLP-1 positive cells co-expressed with OR1A1 were found in a higher ratio in ileum than in duodenum.

EXAMPLE 2

Verification of Hypoglycemic Effect and GLP-1 and Insulin Secretion Effect in Mouse Model in the Case of Oral Administration of Geraniol

(45) (1) Hypoglycemic Effect in Mouse Model

(46) Oral glucose tolerance test was performed on a db/db mouse (leptin receptor knocked-out mice) (35 g to 39 g), which is a type II diabetes mellitus model. A male 6 week-old db/db mouse was purchased from Daehan Biolink Co., Ltd (DBL, South Korea).

(47) A fasting blood sugar level of a db/db mouse of 18 hour-gastric emptying fasted state was measured. Then, the experimental group and the control group were each oral administered with geraniol and metformin, and saline via gavage. Geraniol was administered at 150 or 500 mg/weight Kg, and metformin was administered at 300 mg/weight Kg. Then, each group was administered with glucose of 5 g/weight Kg. Oral glucose tolerance test (OGTT) was performed after 10 minutes, 20 minutes, 40 minutes, 90 minutes, and 120 minutes elapsed. The results thereof are shown in FIG. 5A to 5C.

(48) FIG. 5 are graphs of hypoglycemic effect in a type II diabetes mellitus mouse model in the case of oral administration of geraniol. As shown in FIGS. 5A to 5C, oral administration of geraniol exhibited hypoglycemic effect in a type II diabetes mellitus mouse model, as compared with the group administered with saline. Such hypoglycemic effect were found to be comparable to metformin, i.e., an antidiabetic agent.

(49) (2) GLP-1 and Insulin Secretion Effect in Mouse Model

(50) By using the same method in Example 2 (1), db/db mice of fasted state were each gavaged with geraniol, metformin, or saline. Then, each group were administered with glucose of 2 g/weight Kg. after 10 minutes, 20 minutes, 30 minutes, and 40 minutes elapsed, the amount of GLP-1 and insulin in plasma were measured by using multiplex assay (available from Bio-Rad, USA), based on the user manual, and Bio-Plex MAGPIX multiplex reader (available from Bio-Rad, USA). The results thereof are shown in FIGS. 6A to 6D.

(51) FIGS. 6A to 6D are graphs of increase effect of GLP-1 and insulin in the plasma in a type II diabetes mellitus mouse model at an oral administration of geraniol. As shown in FIGS. 6A to 6D, in the case of oral administration of geraniol, hypoglycemic effect was found due to secretion inducing of GLP-1 and additional insulin, compared with a PBS administered group, in a type II diabetes mellitus mouse model. The effect was verified by comparing with metformin, i.e., an antidiabetic agent.

EXAMPLE 3

Verification of GLP-1 Secretion Promotion Effect and Hypoglycemic Effect in Diabetes Mellitus Mouse Model by Geraniol Smelling

(52) A male 6 week-old db/db mouse was purchased from Daehan Biolink Co., Ltd (DBL, South Korea). A fasting blood sugar level of a db/db mouse of 18 hour-gastric emptying fasted state was measured. Then, the experimental group was stimulated (smelling) with geraniol through olfaction. The control group was oral administered with saline and metformin of 300 mg/weight Kg via gavage. Then, each of the experimental group and the control group were administered with glucose of 2 g/weight Kg. After 10 minutes, 20 minutes, 40 minutes, 90 minutes, and 120 minutes elapsed, the amount of GLP-1 in plasma was verified by using multiplex assay (available from Bio-Rad, USA).

(53) FIG. 9 is a graph of hypoglycemic effect over time for comparison between a group treated with geraniol and a group treated with PBS or metformin.

(54) FIG. 10 is a graph of the amount of GLP-1 over time for comparison between a group treated with geraniol and a group treated with PBS or metformin. As shown in FIG. 8, in the case of the experimental group of geraniol smelling, after 10 minutes elapsed from glucose administration, the amount of GLP-1 increased more than the case of metformin administration, increased to the maximum, and then decreased. From this fact, it was found that olfactory stimulation by geraniol lowered the blood sugar level by increasing the level of GLP-1 in the blood of a type II diabetes mellitus mouse. In addition, it was found that olfactory stimulation by geraniol has similar hypoglycemic effect as metformin, which is currently used as an antidiabetic agent by oral administration.

(55) Conventional antidiabetic agents in the development field of a new treatment method with regard to diabetes mellitus have limitations in that the conventional antidiabetic agents cause side effects, such as liver dysfunction, hypoglycemia, or lacticacidemia. The present disclosure provides a novel method of screening a diabetes mellitus treat candidate material, based on the discovery of a mechanism, which is with regard to promotion of GLP-1 secretion through stimulating an olfactory receptor expressed in a human enteroendocrine cell or direct olfactory stimulation direc. It was found that geraniol screened by the screening method according to the present disclosure may stimulate an olfactory receptor in a human enteroendocrine cell, promoting GLP-1 secretion. In addition, hypoglycemic effect was found, which may be due to GLP-1 and insulin secretion through gavage and direct olfactory stimulation in a type II diabetes mellitus mouse model.

EXAMPLE 4

Cell Viability Assay of Geraniol

(56) In order to verify cytotoxicity of geraniol on a human enteroendocrine cell, MTT assay was carried out as follows. In detail, cell viability assay was carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide (MTT) (available from Invitrogen, Carlsbad, Calif., USA). An endocrine cell, a NCI-H716 cell, was treated with geraniol of different levels of concentration and 6 mM MTT for 1 hour. Dimethyl sulfoxide (DMSO) was added to the cells, and absorbance thereof was measured using Bio-Rad model 680 microplated reader (Bio-Rad, Hercules, Calif., USA) at 540 nm.

(57) FIG. 8 is a graph for verifying cytotoxicity of geraniol in a human enteroendocrine cell through MTT assay. The result thereof are shown in cell viability (%) compared with a comparison group treated with PBS, and hydro peroxided (H.sub.2O.sub.2) was used as a positive control group. As shown in FIG. 8, geraniol of 10 μM to 1000 μM, i.e., geraniol of 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 500 μM, and 1000 μM were found not to have cytotoxicity in a human enteroendocrine cell, i.e., a NCI-H716 cell.

Sequence Listing

Incorporation-By-Reference of Material Submitted Electronically

(58) This application contains a sequence listing. It has been submitted electronically via EFS-Web as an ASCII text file entitled “PX047509_ST25.txt”. The sequence listing is 1,268 bytes in size and was created on Jan. 25, 2016. Applicants state that (1) the computer readable form of the sequence listing submitted herewith is identical to the attached PDF copy; and (2) contains no new matter. It is hereby incorporated by reference in its entirety.