JP2010189298A - Gpr119 agonist - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new antihyperglycemic agent or a new food for improving-preventing hyperglycemia by using a new GPR119 agonist. <P>SOLUTION: The GPR119 agonist contains a culture product of Penicillium sp. NBRC 105016 strain or an extract of the culture product. The GPR119 agonist is used as an ingredient for medicines or foods. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a GPR119 agonist and pharmaceuticals and foods containing the same.

GPR119 (G protein-coupled receptor 119) is a new nuclear receptor discovered in recent years. GPR119 agonists are known to promote cAMP production and promote insulin secretion. Examples of GPR119 agonists include [6- (4-benzenesulfonyl-piperidin-1-yl) -5-nitro-pyrimidin-4-yl]-(4-methanesulfonyl-phenyl) -amine (Patent Document 1), 6 ′. -[4- (2-Methoxycarbonyl-acetyl) -phenoxy] -3'-nitro-3,4,5,6-tetrahydro-2H- [1,2 '] bipyridinyl-4-carboxylic acid ethyl ester (patent document) 2), 3- [6- (4-Methanesulfonyl-phenylamino) -5-nitro-pyrimidin-4-yloxymethyl] -pyrrolidine-1-carboxylic acid tert-butyl ester (Patent Document 3), 4- [ 1- (4-Methanesulfonyl-phenyl) -1H-pyrazolo [3,4-d] pyrimidin-4-yloxy] -piperidine-1-carboxylic acid tert Heteroaryl derivatives are known to substitution, such as butyl ester (patent document 4) (Patent Document 5).
In addition, inhibition of DPP-IV (dipeptidyl peptidase-IV) that degrades incretin such as GLP-1 (glucagon-like peptide-1) is known as a mechanism of insulin secretion promotion not involving a GPR119 agonist. (Patent Document 6). However, inhibition of DPP-IV may excessively lower blood glucose levels and may cause hypoglycemia.

JP-T-2006-516572 JP-T-2006-518863 Special table 2007-528856 gazette Special table 2007-531698 gazette JP 2008-195733 A Special table 2008-540651 gazette

  An object of the present invention is to provide a novel GPR119 agonist. Another object of the present invention is to provide a pharmaceutical for the treatment / prevention of hyperglycemia and a food for the improvement / prevention of hyperglycemia by using the novel GPR119 agonist as a component of the medicine / food. .

The present inventors have found that an extract obtained from a culture of a specific strain classified as Penicillium sp. Acts as a GPR119 agonist, and completed the present invention.
That is, the present invention is as follows.

(1) Penicillium sp. A GPR119 agonist comprising a culture of NBRC 105016 strain or an extract of the culture.
(2) The GPR119 agonist according to (1), wherein the extract is an organic solvent extract. (3) The GPR119 agonist according to (2), wherein the organic solvent is selected from methanol, ethanol and butanol.
(4) The GPR119 agonist according to any one of (1) to (3), wherein the culture is obtained by liquid culture using a malt extract-containing liquid medium.
(5) The extract is a polar organic solvent elution fraction in solid phase extraction using a nonpolar solid phase of a culture solution obtained by removing cells from the culture. GPR119 agonist.
(6) The GPR119 agonist according to (5), wherein the polar organic solvent is selected from methanol and ethanol.
(7) The GPR119 agonist according to (5) or (6), wherein the nonpolar solid phase is an octadecyl group-bonded solid phase.
(8) A medicament comprising the GPR119 agonist according to any one of (1) to (7).
(9) A food comprising the GPR119 agonist according to any one of (1) to (7).
(10) A method for treating / preventing hyperglycemia, comprising administering the GPR119 agonist according to any one of (1) to (7) to a subject in need of treatment / prevention of hyperglycemia.
(11) Use of the GPR119 agonist according to any one of (1) to (7) in the manufacture of a medicament for treatment / prevention of hyperglycemia.

The GPR119 agonist of the present invention has a cAMP production promoting action, an insulin secretion promoting action, and a blood glucose level lowering action. Therefore, the GPR119 agonist of the present invention can be used as a cAMP production promoter, an insulin secretion promoter, and a blood glucose level lowering agent. The GPR119 agonist of the present invention is a drug for the treatment / prevention of hyperglycemia, a drug for the treatment / prevention of complications of diabetes, and the treatment / prevention of cardiovascular diseases (hypertension, hyperlipidemia, etc.) Medicines for improving / preventing hyperglycemia, foods for improving / preventing diabetic complications, and foods for improving / preventing cardiovascular diseases (hypertension, hyperlipidemia, etc.) Suitable as a component.
In particular, the GPR119 agonist of the present invention exerts an insulin secretion promoting action and suppresses an increase in blood glucose level only under hyperglycemic conditions such as during feeding. Therefore, the medicament and food of the present invention have a low risk of causing excessive hypoglycemia under relatively low blood glucose conditions such as when not eating, and are highly safe.

It is a figure which shows the relationship between the dilution rate of the sample containing the extract of Penicillium sp NBRC 105016 obtained in the Example, and GPR119 agonist activity. It is a figure which shows the cAMP production promotion effect of the sample containing the extract of Penicillium sp NBRC 105016 strain | stump | stock obtained in the Example. It is a figure which shows the insulin secretion promotion effect of the sample containing the extract of Penicillium sp NBRC 105016 strain | stump | stock obtained in the Example. It is a figure which shows the blood glucose level raise inhibitory effect of the sample containing the extract of Penicillium sp NBRC 105016 strain | stump | stock obtained in the Example.

The GPR119 agonist of the present invention includes a culture of Penicillium sp. NBRC 105016 strain (hereinafter simply referred to as “NBRC 105016 strain”) or an extract of the culture.
The NBRC 105016 strain is a strain registered in the Biological Genetic Resource Department (NBRC) of the National Institute of Product Evaluation Technology. The NBRC 105016 stock was made publicly available from January 29, 2009, and can be easily obtained using a catalog issued by the institution.

As a medium for obtaining a culture of NBRC 105016 strain, a medium usually used for culturing Penicillium filamentous fungi may be used. For example, potato dextrose agar,
Examples include solid culture using a V8 juice agar medium, malt extract-containing liquid medium, potato dextrose medium, and liquid culture using a V8 juice medium. Liquid culture is preferable, and liquid culture using a malt extract-containing liquid medium is more preferable. The medium preferably contains dextrose, sucrose and the like. The medium preferably contains peptone, yeast extract and the like. As a specific medium, a liquid medium prepared by mixing malt extract agar medium, peptone, dextrose, and water is preferably used.
The culture temperature for obtaining a culture of the NBRC 105016 strain may be within the range of temperatures normally used for culturing Penicillium filamentous fungi, and is, for example, 25 to 37 ° C, preferably 30 to 37 ° C. The culture period is, for example, about 7 to 30 days, preferably about 14 to 21 days.
By extracting a culture of the NBRC 105016 strain thus obtained, an extract of the culture can be obtained. When the culture is solid, solid-liquid extraction can be used, and when the culture is liquid, liquid-liquid extraction and solid-phase extraction can be used. The solvent used for extraction is preferably an organic solvent. The organic solvent is preferably a polar organic solvent. In the case of liquid-liquid extraction, butanol is preferably used, and in the case of solid-liquid extraction and solid-phase extraction, methanol and ethanol are preferably mentioned.

In particular, it is preferable to obtain a liquid culture by culturing using a liquid medium and to obtain an extract of the culture by solid phase extraction. When performing solid-phase extraction, the cells in the culture are removed in advance by centrifugation or the like to obtain a culture solution.
The solid phase extraction is preferably performed using a nonpolar solid phase and a polar organic solvent. As the nonpolar solid phase, an aliphatic hydrocarbon group-bonded solid phase or the like can be used, and it is particularly preferable to use an octadecyl group-bonded solid phase. Examples of the octadecyl group-bonded solid phase include octadecyl silica marketed under a product name such as BOND ELUTE-C18 (Varian).
The method of solid phase extraction is performed as follows, for example. First, the solid phase is conditioned by a conventional method. Then, after the culture medium obtained by removing the cells from the liquid culture is poured into the solid phase, it is thoroughly washed with water, and then the polar organic solvent is flushed and bound to the solid phase with the solvent. The hydrophobic fraction in the culture broth is eluted. In this case, the polar organic solvent is preferably methanol and ethanol.

  The GPR119 agonist of the present invention has a cAMP production promoting action, an insulin secretion promoting action, and a blood glucose level lowering action. Therefore, the GPR119 agonist of the present invention can be used as a cAMP production promoter, an insulin secretion promoter, and a blood glucose level lowering agent.

  The GPR119 agonist of the present invention is a disease caused by inactivation of GPR119, a decrease in cAMP production resulting from this, a decrease in insulin secretion, and an increase in blood glucose level, as it is or when formulated with a carrier commonly used in medicine It can be used as a medicament for the treatment / prevention (hereinafter also referred to as “the medicament of the present invention”). Examples of such diseases include diabetes, diabetic complications, hypertension and circulatory diseases such as hyperlipidemia.

  The content of the culture or extract in the medicament of the present invention is determined by the use of the medicament, the patient's symptoms, body weight, age, sex, and the like. For example, the concentration of the methanol eluate (solvent-removed product) obtained by the method described in the following examples is preferably 5 to 10% by mass, more preferably 5 to 7% by mass. Can do.

  The pharmaceutical dosage form of the present invention is not particularly limited and can be appropriately selected depending on the therapeutic purpose. Specific examples include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, syrups, suppositories, and injections.

The medicament of the present invention is a component having an action as a GPR119 agonist other than the culture or extract of the NBRC 105016 strain, a cAMP production promoter, an insulin secretion promoter, a hypoglycemic agent, etc., and is confirmed to be safe. It may contain what is done.
In addition, excipients, binders, disintegrants, lubricants, stabilizers, preservatives, flavoring agents, diluents and the like may be added during the formulation of the medicament of the present invention.

The pharmaceutical administration method of the present invention may be either oral or parenteral. The dosage of the medicament of the present invention is determined according to the patient's symptoms, body weight, age, sex and the like.
For example, the dose of methanol eluate (solvent-removed product) obtained by the method described in the Examples below is preferably 100 to 1000 mg / kg / day, more preferably 500 to 700 mg / kg / day. Can be administered at a moderate dose.
The pharmaceutical of this invention can be manufactured by obtaining the GPR119 agonist of this invention by the said method, and mix | blending arbitrary components with this as needed.

The GPR119 agonist of the present invention is used for the prevention and improvement of diseases caused by the inactivation of GPR119, the decrease in cAMP production resulting from this, the decrease in insulin secretion, and the increase in blood glucose level by processing together with commonly used food ingredients. It can be used as a food (hereinafter also referred to as “the food of the present invention”). Examples of such diseases include diabetes, diabetic complications, hypertension and circulatory diseases such as hyperlipidemia.
In the present invention, “food” includes foods taken by humans as well as feeds taken by animals other than humans.

  The content of the culture or extract in the food of the present invention is determined according to the use of the food, the form of the food, and the like. For example, the content of the methanol eluate (solvent-removed product) obtained by the method described in the following examples is preferably 5 to 10% by mass, more preferably 5 to 7% by mass. be able to.

  The form of the food of the present invention is not particularly limited. For example, processed foods, such as a drink and confectionery, are mentioned.

  The food of the present invention is a component having an action as a GPR119 agonist other than the culture or extract of the NBRC 105016 strain, a cAMP production promoter, an insulin secretion promoter, a blood glucose lowering agent, etc., and is safe as a food ingredient You may contain what has been confirmed.

The intake of the food of the present invention is determined according to the use of the food, the form of the food, and the like.
For example, the intake of methanol eluate (solvent-removed product) obtained by the method described in the following examples is preferably 100 to 1000 mg / kg / day, more preferably 500 to 700 mg / kg / day. Can be used as a guide.
The food of the present invention can be produced by obtaining the GPR119 agonist of the present invention by the above method and processing it together with the food ingredients.

1. Measurement of GPR119 Agonist Activity (1) Preparation of GPCR Assay Sample Filamentous fungus 466 strain was grown in a dark place at 28 ° C. using a malt extract agar medium (MEA medium, manufactured by Difco). Then, the liquid culture of the filamentous fungi was performed by the following method using the 1/2 malt extract containing medium of the composition shown in the following Table 1.
10 ml of the 1/2 malt extract-containing medium was dispensed into test tubes, and filamentous fungi were inoculated from the MEA medium into the liquid medium in the test tubes with a toothpick. Then, it culture | cultivated for 7 to 10 days at 28 degreeC, the dark place, and 180 rpm (reciprocating shaking).

After the culture, the obtained culture was centrifuged at 8000 rpm for 10 minutes, and divided into a culture solution and cells. The culture solution was collected and extracted by solid phase extraction using a BOND ELUT-C18 (500 mg 6 ml) cartridge (Varian) (octadecyl group-bonded solid phase cartridge).
The specific method is as follows. First, the solid phase of the BOND ELUT-C18 cartridge was conditioned with 6 ml methanol and 6 ml water. Subsequently, the culture solution obtained above was poured into a cartridge, and then washed twice with 3 ml of water. Subsequently, 3 ml of methanol was flowed to collect all the eluted fractions, and the recovered methanol eluate was concentrated to dryness.
This concentrated dried product was used as a sample for the following test.

(2) GPCR assay From the 466 samples obtained above, samples having GPR119 agonist activity were screened in vitro. Screening was performed by [ ³⁵ S] GTP-γ S binding assay. The detailed method is as shown in Biophysics 43 (1) 37-39 (2003).
As a result, strong GPR119 agonist activity was detected in the diluted solution of the sample obtained from the culture solution of Penicillium sp. NBRC 105016 strain. Therefore, a sample obtained from the culture solution of NBRC 105016 strain was further diluted with Hepes buffer to confirm dose response. The results are shown in FIG.
As shown in FIG. 1, a signal of about 18000 cpm was detected in a 10 volume-fold diluted solution of a sample obtained from the culture solution of NBRC 105016 strain. On the other hand, a signal activity of about 19200 cpm was detected in the 1 volume-fold diluted solution of the sample.

2. Measurement of cAMP production promoting action The cAMP production promoting action was measured for the butanol extract of the culture solution of the NBRC 105016 strain.
The specific method is as follows.
120 ml of butanol was added to 90 ml of the culture solution of NBRC 105016 obtained by the same method as described above, and liquid-liquid extraction using a separatory funnel was repeated three times. The resulting butanol extract layer was dried (142 mg) and dissolved in 1.8 ml of DMSO. 2 μl of this DMSO solution was dissolved in 198 μl of KRB (glucose 20 mM) to obtain a sample for activity measurement.
Two days before the activity measurement, MIN6 cells were seeded on a 96-well plate so that the cells became 1.0 × 10 ⁴ [cells / well]. Two days later, the medium was sucked with an aspirator, and the cells were washed with KRB (Krebs-Ringer bicarbonate, see composition below) (glucose 0 mM). Subsequently, 100 μl of KRB (glucose 2.5 mM) was added to the well and incubated for 30 minutes in a CO ₂ incubator. Thereafter, the cells were washed with KRB (glucose 0 mM).
After removing KRB in each well, 100 μl of the prepared sample was added to each well and incubated in a 37 ° C. CO ₂ incubator for 2 hours. As a control, the same volume of KRB (glucose 16 mM) was added.
Thereafter, cAMP activity was measured using a kit (Amersham cAMP Biotrak Enzyme immunoassay System RPN 225) (see the following reference).
<Composition of KRB>
NaCl 119mM
KCl 4.74mM
CaCl ₂ 2.54mM
MgCl ₂ 1.19mM
KH ₂ PO ₄ 1.19mM
NaHCO ₃ 25mM
HEPES (pH 7.4) Adjust pH with 10 mM NaOH BSA 0.05%
<References>
A role for intestinal endocrine cell-expressed g protein-coupled receptor 119 in
glycemic control by enhancing glucagon-like Peptide-1 and glucose-dependent insulinotropic Peptide release.
Chu ZL, Carroll C, Alfonso J, Gutierrez V, He H, Lucman A, Pedraza M, Mondala H,
Gao H, Bagnol D, Chen R, Jones RM, Behan DP, Leonard J.
Endocrinology. 2008 May; 149 (5): 2038-47.

The results are shown in FIG.
As shown in FIG. 2, when a sample dilution obtained from the culture solution of the NBRC 105016 strain was added to the medium, a clearly higher cAMP activity was detected compared to the control. From this, it was found that the culture solution of the NBRC 105016 strain has an effect of promoting cAMP production of cells.

3. Measurement of insulin secretion promoting action The insulin secretion promoting action of the sample obtained by butanol extraction of the culture solution of the NBRC 105016 strain was measured. The specific method is as follows.
Two days before the measurement, MIN6 cells were seeded on a 96-well plate at 1.0 × 10 ⁴ cells / well. The cells were washed with KRB (glucose 0 mM), 100 μl of KRB (glucose 2.5 mM) was added, and incubated in a 37 ° C. CO ₂ incubator for 30 minutes. Subsequently, the cells were washed twice with KRB (glucose 0 mM).
After removing KRB in each well, 200 μl of the sample prepared in the same manner as described above was added to each well and incubated at 37 ° C. in a CO ₂ incubator for 2 hours. As a control, the same volume of KRB (glucose 20 mM) was added and incubated in the same manner.
Subsequently, the amount of secreted insulin was measured using a kit of Shibayagi Levis insulin-mouse (H type). The measurement followed the kit protocol (see below).
<Protocol>
Dilute biotin-conjugated anti-insulin antibody 100 times with attached buffer (1)
Dilute peroxidase-avidin conjugate 100-fold with the supplied buffer (2)
Dilute the supplied cleaning solution 10 times with millQ (3)
Wash the antibody-immobilized plate 4 times with (3) Add 100 μl of (1) 10 μl of sample diluent or control and mix well 2 hours at room temperature Incubate antibody-immobilized plate with (3) 4 Dispense 100 μl of wash (2) and mix well. Incubate for 30 minutes at room temperature. Wash antibody-immobilized plate 4 times with (3). Dispense 100 μl of coloring solution and mix well. Incubate for 30 minutes at room temperature Dispense 100 μl of reaction stop solution and mix well Absorbance measurement with microplate reader (450 nm)

The results are shown in FIG.
As shown in FIG. 3, when a diluted solution of a sample obtained from the culture solution of the NBRC 105016 strain was added to the medium, an insulin secretion amount clearly larger than that of the control was observed. From this, it was found that the culture solution of NBRC 105016 strain has an action of promoting insulin secretion of islet cells.

4). Glucose tolerance test using SDT rats (diabetic model rats) Subsequently, diabetic model rats (strain name: SDT / Jcl 9 weeks old pupae, quantity: 5 animals x 2 groups, 10 animals in total, microbiological grade: SPF) Used to perform a glucose tolerance test (GTT). Rats were raised under the following conditions.
<Conditions>
Temperature: 20-26 ° C
Humidity: 45-70%
Ventilation frequency: 10-15 times / hour Lighting time: Bright 7: 00-19: 00, Dark 19: 00-7: 00
Microbiological grade: SPF
Rearing rack: 1 open rack (MAX30 cage)
Rearing cage: Clean cage (282 x 451 x 157 mm)
Accommodates: 2 x 5 cages Total 10: Drinking water: After filling water bottle (250cc), high pressure steam sterilization (121 ° C, 30 minutes)
Water supply: One bottle of water is laid twice a week: Planar chip (121 ° C, 30 minutes high pressure steam sterilization)
Cage exchange: 1 time / week

The glucose tolerance test was performed by the following method.
First, 1 g of a sample obtained from the culture medium of the NBRC 105016 strain was suspended in 16 ml of Hepes buffer solution, and this was suspended 24 hours before and 2 hours before sugar administration (twice in total) per group (5 animals). Oral administration (pre-administration). The dose was 1.5 ml / dose / animal. As a control, physiological saline was similarly administered to another group (5 animals).
Fasted overnight (17 h) from the day before sugar administration. For sugar administration, “Otsuka sugar solution 50%” (glucose solution) was intraperitoneally administered (IP) at 2.0 g / kg (body weight). Moreover, the body weight was measured twice before fasting and after fasting.
Blood samples were collected from the tail of each group of rats before fasting, after fasting, 15 minutes, 30 minutes, 60 minutes, and 120 minutes after sugar administration, and blood glucose levels were measured. Was calculated. The results are shown in FIG.
As shown in FIG. 4, in the control group, the blood glucose level increased rapidly between 15 and 30 minutes after sugar administration, but in the group pre-administered with a sample dilution obtained from the culture solution of NBRC 105016 strain The increase in blood glucose level after sugar administration was suppressed. On the other hand, before the sugar administration and 120 minutes after the sugar administration, even in the group pre-administered with a sample obtained from the culture solution of the NBRC 105016 strain, the blood glucose level was similar to that in the control group. From the above, it was found that the culture solution of NBRC 105016 strain suppressed the increase in blood glucose level only during hyperglycemia and did not lower the blood glucose level during low blood glucose. There was no change in body weight before and after fasting.

  The present invention is applied to antidiabetic drugs, diabetic foods and the like.

Claims (9)

  Penicillium sp. A GPR119 agonist comprising a culture of NBRC 105016 strain or an extract of the culture.   The GPR119 agonist according to claim 1, wherein the extract is an organic solvent extract.   The GPR119 agonist according to claim 2, wherein the organic solvent is selected from methanol, ethanol and butanol.   The GPR119 agonist according to any one of claims 1 to 3, wherein the culture is obtained by liquid culture using a malt extract-containing liquid medium.   The GPR119 agonist according to claim 4, wherein the extract is a polar organic solvent elution fraction in a solid phase extraction using a nonpolar solid phase of a culture solution obtained by removing cells from the culture.   The GPR119 agonist according to claim 5, wherein the polar organic solvent is selected from methanol and ethanol.   The GPR119 agonist according to claim 5 or 6, wherein the nonpolar solid phase is an octadecyl group-bonded solid phase.   The pharmaceutical containing the GPR119 agonist as described in any one of Claims 1-7.   A food comprising the GPR119 agonist according to any one of claims 1 to 7.

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