JP6990903B2 - Insulin secretagogue containing a compound having a sialidase inhibitory activity, a blood glucose increase inhibitor and a diabetes therapeutic agent, and a screening method for these agents. - Google Patents

Description

The present invention relates to an insulin secretagogue containing a compound having a sialidase inhibitory activity, a blood glucose level increase inhibitor and a diabetes therapeutic agent, and a screening method for these agents.

(Diabetes mellitus)
Diabetes is defined as a condition in which insulin-induced regulation of carbohydrate and lipid metabolism homeostasis is inadequate, leading to an increase in fasting / post-feeding blood glucose levels. If this imbalanced homeostasis does not return to normal and persists for a long period of time, it eventually becomes diabetes.
Diabetes is the most common metabolic abnormality in the world, with a group of syndromes characterized by hyperglycemia, metabolic changes in lipids, carbohydrates and proteins, in addition to an increased risk of complications due to vascular disease. Can be defined. Today, it is a major problem as it is estimated that more than hundreds of millions of people worldwide suffer from diabetes.

The causes of diabetes are broadly divided into type I diabetes, in which insulin secretion is deficient due to the destruction of β cells in the pancreas, and type II diabetes, in which the sensitivity of tissues to insulin is reduced. As a treatment method for diabetes, insulin injection is generally used for type I diabetes, and for type II diabetes, a thiazolidine derivative or a biguanide agent, which is an insulin resistance improving drug, is used.
It is said that the treatment of diabetes does not completely cure.

(Cialidase)
Cialidase, also known as neuraminidase, is an enzyme capable of cleaving N-acetylneuraminic acid from sugar chains. Cialidases are found in various organisms including bacteria, viruses, protozoa, and vertebrates, and are roughly classified into three types: viral sialidases, bacterial sialidases, and mammalian sialidases.
These neuraminidase-producing pathogens are involved in major infectious diseases in humans and other animals, and are particularly problematic with the influenza virus.
Therefore, an inhibitor of sialidase activity (an agent containing a compound having sialidase inhibitory activity as an active ingredient) has been actively studied for the treatment of infection by a virus having sialidase.

(Patent document)
Patent Document 1 discloses "an influenza preventive agent containing a pharmacologically acceptable ester derivative of a compound having a sialidase inhibitory activity as an active ingredient". However, it has not been disclosed or suggested that a compound having a sialidase inhibitory activity has an insulin secretagogue effect, a blood glucose level inhibitory effect, and a diabetes therapeutic effect.
Patent Document 2 discloses "double-stranded RNA having sialidase inhibitory activity". However, in the examples, double-stranded RNA having sialidase inhibitory activity has been disclosed to be effective only for cancer. Furthermore, it has not been disclosed or suggested that a compound having a sialidase inhibitory activity has an insulin secretagogue effect.

JP 2002-12555 International release WO2006-054555

The present inventors provide novel insulin secretagogues, blood glucose elevation inhibitors and diabetes therapeutic agents, and screening methods for these agents.

As a result of research to solve the above problems, the present inventors have newly found that strong sialidase activity is detected in pancreatic islets, and further, a compound having sialidase inhibitory activity has an effect of suppressing an increase in blood glucose level and an increase in insulin secretion. The present invention has been completed by finding that it has an action, particularly an action of increasing insulin secretion in a high-concentration glucose state.
That is, the present invention is the following [1] to [8].

[1] An insulin secretagogue, an inhibitor of blood glucose elevation, or a therapeutic agent for diabetes, which contains a compound having a sialidase inhibitory activity.
[2] The first aspect of the present invention, wherein the compound having a sialidase inhibitory activity is N-acetyl-2,3-didehydro-2-deoxynoylamic acid, a salt thereof, a solvate thereof, or a hydrate thereof. Agent.
[3] The agent according to the above [1] or [2], which is an insulin secretagogue.
[4] The agent according to the above [1] or [2], which is an agent for suppressing an increase in blood glucose level.
[5] The agent according to the above [1] or [2], which is a therapeutic agent for diabetes.
[6] The preceding item is characterized in that it has an effect of suppressing an increase in blood glucose level and an effect of increasing insulin secretion in a high-concentration glucose state, and does not have an effect of suppressing an increase in blood glucose level and an effect of increasing insulin secretion in a low-concentration glucose state. 1] The agent according to any one of [5].
[7] A method for screening an active ingredient of an insulin secretagogue, a blood glucose level increase inhibitor, or a diabetes therapeutic agent, which comprises selecting a test compound that inhibits the glucosidase activity of sialidase.
[8] The screening method according to the above [7], which comprises the following steps.
(1) Step of adding the test compound to the system containing sialidase and the substrate (2) Step of measuring the sialidase activity [9] The substrate is sialic acid to which an insoluble fluorescent substance is added. Screening method.
[10] The agent according to any one of the preceding paragraphs [1] to [5], wherein the sialidase is NEU1, NEU2, NEU3 and / or NEU4.
[11] The screening method according to any one of the preceding paragraphs [6] to [9], wherein the sialidase is NEU1, NEU2, NEU3 and / or NEU4.

The insulin secretagogue, the blood glucose increase inhibitor, and the diabetes therapeutic agent of the present invention have an action of suppressing an increase in blood glucose level and an action of increasing insulin secretion in a high-concentration glucose state.

Left figure: Imaging image of sialidase activity in mouse pancreas, Right figure: H & E stained image. The scale is 0.1 mm. Changes in insulin secretion during the action of sialidase inhibitors in islet β-cells. (A), (B) Changes in insulin secretion in mouse pancreatic islet β cells are shown. The vertical axis shows the insulin concentration. The value indicates the mean value + standard error. *** P <0.001, * P <0.05. Results of changes in blood glucose and insulin secretion during intraperitoneal glucose tolerance test with sialidase inhibitor. (A) Changes in blood glucose level in the intra-abdominal glucose tolerance test are shown. The horizontal axis shows time and the vertical axis shows blood glucose level. The value indicates the mean ± standard error. (B) The blood glucose level at the time of 120 minutes of the intra-abdominal glucose tolerance test is shown. ** P <0.01. (C) The plasma insulin concentration at the time of 120 minutes of the intra-abdominal glucose tolerance test is shown. The value indicates the mean value + standard error. * P <0.05. Changes in blood glucose level and insulin secretion during administration of sialidase inhibitors during hypoglycemia. (A) Shows changes in blood glucose level during administration of a sialidase inhibitor during hypoglycemia. The horizontal axis shows time and the vertical axis shows blood glucose level. (B) The plasma insulin concentration at the time of administration of the sialidase inhibitor during hypoglycemia is shown. The value indicates the mean ± standard error. *** P <0.001. Identification of sialidase isozymes that are highly expressed in islets. The relative expression levels in exocrine tissues, where the expression level of mRNA of various isozymes in pancreatic islets is 1, are shown. The value indicates the mean ± standard error. *** P <0.001.

(Subject of the present invention)
The subject of the present invention relates to an insulin secretagogue containing a compound having a sialidase inhibitory activity, a blood glucose level increase inhibitor and a diabetes therapeutic agent, and a screening method for these agents.

(Compound with sialidase inhibitory activity)
The "compound having sialidase inhibitory activity" of the present invention is not particularly limited as long as it has sialidase inhibitory activity.
The sialidase inhibitory activity may be any of viral sialidase inhibitory activity, bacterial sialidase inhibitory activity, and / or mammalian sialidase inhibitory activity, but mammalian sialidase inhibitory activity is preferable.
The compound having a sialidase inhibitory activity is an active ingredient of the insulin secretagogue, the blood glucose increase inhibitor, and the diabetes therapeutic agent of the present invention.
In the "compound having sialidase inhibitory activity" of the present invention, the preferred compound is N-acetyl-2,3-didehydro-2-deoxynoiramic acid (another Japanese name: 2,3-dehydro-2-deoxy-N-). Acetylneuraminic acid, English notation: 2,3-Dehydro-2-deoxy-N-acetylneuraminic Acid, hereinafter sometimes referred to as "DANA"), its salt (particularly a pharmacologically acceptable salt), its solvent Japanese products, their hydrates, and the like can be exemplified.

(salt)
As the salt in the present invention, a commonly known part such as an amino group is a salt of an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, a salt of an organic carboxylic acid such as trifluoroacetic acid, benzenesulfonic acid and methanesulfonic acid. Examples thereof include those obtained as salts of organic sulfonic acids such as, and compounds in which the carboxylic acid moiety is a metal salt such as sodium, potassium and calcium, and a salt of an organic base such as ammonia and triethylamine, but the present invention is not particularly limited.
In particular, as the salt of the compound of the present invention, a sodium salt is preferable.

(Pharmacologically acceptable salt)
As the pharmacologically acceptable salt in the present invention, when it has an acidic group such as a carboxy group, it is possible to form a generally pharmacologically acceptable base addition salt. Examples of such base addition salts include sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt, ammonium salt, dibenzylamine salt, methylamine, ethylamine, diethylamine, triethylamine, tetraethylammonium, guanidine, ethanolamine, and the like. Phenylglycine alkyl ester salt, ethylenediamine salt, morpholine salt, N-methylglucamine salt, diethylamine salt, lysine, arginine, ornithine, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, diethanolamine Examples thereof include salts, piperazine salts, tetramethylammonium salts, tris (hydroxymethyl) aminomethane salts and the like.
For example, the pharmaceutically acceptable salt of N-acetyl-2,3-didehydro-2-deoxyneuraminic acid is a salt formed by ionization of the carboxylic acid at the 1-position, and is, for example, an inorganic base or an inorganic base. It may be an organic base and a salt.

(Solvate, hydrate)
Examples of the solvate in the present invention include, but are not limited to, organic solvates to which methanol, ethanol, propanol, acetone, N, N-dimethylformamide, benzene and the like are added.
Further, the hydrate of each compound of the present invention includes those produced by absorbing water in the air.

(Pharmacologically acceptable prodrug)
The compounds having sialidase inhibitory activity of the present invention also include pharmacologically acceptable prodrac. For example, it can be converted into a hydroxyl group (prodrug of hydroxyl group), carboxylic acid (prodrug of carboxylic acid), acetamide group (prodrug of acetamide group) or the like by hydrolysis, solvolysis or under physiological conditions.

(Insulin secretagogue)
According to the following examples, the insulin secretagogue of the present invention not only has an effect of increasing insulin secretion, but also has an effect of increasing insulin secretion in a high-concentration glucose state. More specifically, the insulin secretagogue of the present invention does not have an effect of increasing insulin secretion in a low glucose state.
In the present invention, the "high glucose state" is not particularly limited as long as the blood glucose concentration is higher than normal, but in the case of humans, for example, 120 mg / dL or more, preferably 130 mg / dL or more, more preferably. A blood glucose concentration of 140 mg / dL or higher is applicable. The "low glucose state" is not particularly limited as long as the blood glucose concentration is lower than normal, but in the case of humans, for example, 100 mg / dL or less, preferably 90 mg / dL or less, more preferably 80 mg / dL. The following blood glucose levels are applicable.

(Blood sugar level rise inhibitor)
According to the following examples, the blood glucose level increase inhibitor of the present invention not only has a blood glucose level increase inhibitory effect, but also has a blood glucose level increase inhibitory effect particularly in a high-concentration glucose state.

(Diabetes treatment agent)
The therapeutic agent for diabetes of the present invention has an action of increasing insulin secretion in a blood glucose concentration-dependent manner. More specifically, the therapeutic agent for diabetes of the present invention has an action of increasing insulin secretion in a high-concentration glucose state. That is, the therapeutic agent for diabetes of the present invention has a low possibility of developing hypoglycemia and can be used safely.
In addition, the therapeutic agent for diabetes of the present invention has an effect on type I diabetes and / or type II diabetes.

(Screening method for active ingredients of insulin secretagogue, blood glucose increase inhibitor and diabetes treatment agent)
The method for screening the active ingredients of the insulin secretagogue, the blood glucose increase inhibitor and the diabetes therapeutic agent of the present invention is characterized by selecting a test compound that inhibits the glucosidase activity of sialidase.
The process in the screening method of the present invention is not particularly limited as long as the object of the present screening can be achieved.

The screening method of the present invention has, for example, the following steps.
(1) Step of adding the test compound to the system containing the sialidase and the substrate (2) Step of measuring the sialidase activity More specifically, it has the following steps.
(1) A step of adding the test compound to a system containing a substrate having a sialidase and a glycosidic bond (2) A step of measuring the glucosidase activity of the sialidase.

Any substance can be used as the "test compound" used in the present invention. The type of the test compound is not particularly limited, and may be a known therapeutic agent, an individual small molecule synthetic compound (particularly siRNA), a compound present in a natural product extract, or a synthetic peptide.
Alternatively, the test compound may be a compound library, a phage display library or a combinatorial library. The test compound is preferably a small molecule compound, preferably a compound library of small molecule compounds. The construction of the compound library is known to those skilled in the art, and a commercially available compound library can also be used.

The sialidase may be derived from viral sialidases, bacterial sialidases and mammalian sialidases, eg, commercial products from Arthrobacter ureafaciens, commercial products from Vibriocholerae, commercial products from Salmonella Typhimurium, obtained from animal tissues, or molecular biology. Various mammalian sialidase isozymes and the like produced by the above-mentioned method can be used.
The substrate is not particularly limited as long as it is a substance that is decomposed into sialidase, but considering that sialidase is a glycosidase (an enzyme that cleaves the glycosidic bond of neutralic acid), a substrate having a glycosidic bond (eg, N-acetyl). (Including molecular species of various sialic acids such as neuraminic acid, N-glycolylneuraminic acid, and KDN) is preferable.
The "system" may be in vivo or in vitro.
If the sialidase activity is lower than that of the control (a system to which the test compound is not added), the test compound can be determined to be a compound having sialidase inhibitory activity.

For the measurement of the sialidase activity, a commercially available kit or the like known per se can be used, and the following can be exemplified.
1) Colorimetric method 2) Fluorescence method 3) Staining method

As a specific example of the screening method of the present invention, BTP (benzothiazolylphenol) -Neu5Ac (5-bromo-4-chloroindol-3-yl-), which is a sialidase active imaging substrate described in the document PLOS ONE January 2014 Volume 9 Issue 1 e81941. Cialidase activity can be measured by irradiating the system with ultraviolet rays and detecting the fluorescence intensity using aDN-acetylneuraminic acid).

(Agent of the present invention)
The agents of the present invention include suppressing and delaying the onset of the disease, including not only prevention before the disease develops, but also prevention against recurrence of the disease after treatment. The agents of the present invention include curing the disease, ameliorating the symptoms and suppressing the progression of the symptoms.

(Subject to administration of the agent of the present invention)
The administration target of the agent of the present invention is preferably a mammal. As used herein, mammals refer to warm-blooded vertebrates, such as primates such as humans and monkeys, rodents such as mice, rats and rabbits, pet animals such as dogs and cats, and cows, horses and Examples include livestock such as pigs. The agent of the present invention is suitable for administration to primates, especially humans.
The effective amount of the agent of the present invention depends, for example, on the age, weight, severity of the patient, the nature of the pharmaceutical product, and the route of administration. Effective amounts of compounds with sialidase inhibitory activity for the treatment of mammals, especially humans, are typically in the range 0.01-1000 mg / kg / day, eg 0.1-100 mg / kg / day.

(Carrier)
The agent of the present invention comprises a compound having sialidase inhibitory activity and one or more pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier generally refers to an inert, non-toxic, solid or liquid, bulking agent, diluent, encapsulating material, etc. that does not react with the active ingredient of the invention, eg, Examples include water, ethanol, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, solvents such as vegetable oils or dispersion media.

(Form of the agent of the present invention)
The agent of the present invention can be used orally or parenterally, for example, on the skin, subcutaneously, mucous membrane, intravenously, intraarterial, intramuscularly, intraperitoneally, intravaginally, lung, or intracerebral. Administered to the eye, and intranasally. Examples of the orally administered preparation include tablets, granules, fine granules, powders, capsules, chewables, pellets, syrups, liquids, suspensions and inhalants. Parenteral preparations include suppositories, retention enemas, instillations, eye drops, nasal drops, pessaries, injections, oral cleansers and ointments, creams, gels, controlled release patches and patches. Examples include external skin preparations such as. The agents of the invention may be administered parenterally in the form of sustained release subcutaneous implants or in the form of targeted delivery systems (eg, monoclonal antibodies, vector delivery, ion implantation, polymer matrix, liposomes and microspheres). good.

(Additive)
The agent of the present invention may further contain additives commonly used in the pharmaceutical field. Such additives include, for example, excipients, binders, disintegrants, lubricants, antioxidants, colorants, flavoring agents and the like, which can be used as needed.

The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

(Cialidase activity in the pancreas)
In this example, the presence or absence of sialidase activity in the pancreas was confirmed. The details are as follows.

(Method)
Frozen sections of the tail of the pancreas of mice (C57BL / 6J, male, 8 weeks old) were prepared. The frozen sections were stained at room temperature with the sialidase active imaging substrate (1 mM BTP3-Neu5Ac, reference: PLOS ONE January 2014Volume 9 Issue 1 e81941) developed by the present inventors. Furthermore, after washing the stained section, it was observed with a fluorescence microscope. Subsequently, the sections were subjected to H & E staining.

(result)
From the results shown in FIG. 1, it was confirmed that strong sialidase activity was detected in pancreatic islets.

(Changes in insulin secretion in pancreatic islet β cells due to addition of compounds with sialidase inhibitory activity)
In this example, changes in insulin secretion in pancreatic islet β cells were confirmed by adding a compound having sialidase inhibitory activity. The details are as follows.

(Method)
Mouse pancreatic islet β cells cultured in 2.8 mM glucose-containing buffer containing 8.3 mM glucose-containing buffer, 2.8 mM glucose-containing buffer, 2.8 mM glucose-containing buffer containing 10 μM DANA, and 8.3 mM glucose containing 0.1 μM DANA. A buffer, 8.3 mM glucose-containing buffer containing 1.0 μM DANA, or 8.3 mM glucose-containing buffer containing 10 μM DANA was applied.
Then, the supernatant was collected, centrifuged, and the supernatant was stored at −20 ° C. The amount of insulin in the sample was measured by the ELISA method using an ultrasensitive mouse / rat insulin measurement kit (Morinaga, Tokyo, Japan).

(result)
As a result of treating mouse pancreatic islet β cells with a high-concentration glucose solution (8.3 mM), insulin secretion was significantly increased. In addition, the increase in insulin secretion in the high-concentration glucose solution was significantly promoted by the sialidase inhibitor 10 μM DANA (FIGS. 2A and 2B).
In addition, no change was observed in the amount of insulin secreted even when DANA was allowed to act on a low-concentration glucose solution (2.8 mM) (Fig. 2B).
From the above, it was confirmed that the compound having sialidase inhibitory activity has an action of increasing insulin secretion in a high-concentration glucose state.

(Changes in blood glucose level and plasma insulin concentration in intraperitoneal glucose tolerance test due to addition of compound having sialidase inhibitory activity)
In this example, changes in blood glucose level and plasma insulin concentration in the intra-abdominal glucose tolerance test due to the addition of a compound having sialidase inhibitory activity were confirmed. The details are as follows.

(Method)
After the mice were fasted for 24 hours, phosphate buffer (PBS), 7 mM or 70 mM DANA was administered tail vein to 0.1 mL per 10 g body weight. After 30 seconds, a 20% glucose solution was administered intraperitoneally to 0.1 mL per 10 g body weight. Blood glucose levels before (0 minutes) and 30 minutes, 60 minutes, 90 minutes, and 120 minutes after glucose administration were measured with a blood glucose measurement system (Accucheck ST Meter, Roche diagnostics, Tokyo, Japan). In addition, plasma was collected 120 minutes after administration, and the insulin concentration was measured using an ultrasensitive mouse / rat insulin measurement kit (Morinaga, Tokyo, Japan).

(result)
The group to which 70 mM DANA (10 μL / g bw) was administered significantly suppressed the increase in blood glucose level as compared with the control group (FIGS. 3A and 3B). In addition, plasma insulin concentration 120 minutes after glucose loading increased significantly in the group receiving 70 mM DANA (Fig. 3C).
From the above, it was confirmed that the compound having a sialidase inhibitory activity has an effect of suppressing an increase in blood glucose level and an effect of increasing insulin secretion under a high concentration glucose state.

(Effect of sialidase inhibitor administration on blood glucose level and plasma insulin concentration during hypoglycemia)
In this example, the effect of administration of the sialidase inhibitor during hypoglycemia on the blood glucose level and plasma insulin concentration was confirmed. The details are as follows.

(Method)
After the mice were fasted for 24 hours, phosphate buffer (PBS), 7 mM or 70 mM DANA was administered tail vein to 0.1 mL per 10 g of body weight. For a part of the group to which the phosphate buffer was administered (control group), 20% glucose was intraperitoneally administered immediately after the phosphate buffer was administered. Blood glucose or insulin levels were measured before and 30 minutes after tail vein administration.

(result)
As a result of administration of DANA, a sialidase inhibitor, to the tail vein of 24-hour fasted mice, administration was performed in the control group administered with phosphate buffer, or in the group administered with 7 mM or 70 mM DANA (10 μL / g bw) for 30 minutes. No significant change was observed in the subsequent blood glucose level (Fig. 4A). It was also confirmed that the blood glucose level increased when glucose was administered.
Next, as a result of measuring the plasma insulin concentration 30 minutes after the administration, the blood glucose level was significantly changed in the control group to which the phosphate buffer was administered and the group to which 7 mM or 70 mM DANA (10 μL / g bw) was administered. Was not observed (Fig. 4B). It was also confirmed that plasma insulin concentration increased when glucose was administered.
As described above, in cell and animal experiments, it was shown that the promotion of insulin release by DANA has no effect during hypoglycemia. Since hypoglycemia caused by a diabetes drug is one of the most serious side effects of a diabetes drug, DANA is considered to be a highly safe diabetes drug.

(Identification of islets of sialidase highly expressed in pancreatic islets)
In Example 1, since the islets of pancreatic islets were found to have sialidase activity as compared with exocrine tissues, the type of sialidase isozymes highly expressed in the islets was investigated. The details are as follows.

(Method)
Mouse pancreatic islets and exocrine tissues were isolated and the expression levels of various sialidase isozyme mRNAs were quantified by real-time RT-PCR. More specifically, the mRNA expression levels of Neu1 (neuraminidase1), Neu2 (neuraminidase 2), Neu3 (neuraminidase 3) and Neu4 (neuraminidase 4) were quantified.

(result)
Siarydase isozymes Neu1, Neu2, Neu3 and Neu4 were highly expressed in the islets compared to exocrine tissues (Fig. 5). This was consistent with the detection of strong sialidase activity in the islets.

(General)
According to the results of Examples 1 to 5, the compound having a sialidase inhibitory activity has an insulin secretagogue effect, a blood glucose level increase inhibitory effect, and / or a diabetes therapeutic effect. Furthermore, since the compound having a sialidase inhibitory activity has an action of increasing the amount of insulin secretion depending on the glucose concentration in the blood, the onset of hypoglycemia is unlikely to occur and it is safe. In particular, a compound having a sialidase inhibitory activity has an effect of suppressing an increase in blood glucose level and an effect of increasing insulin secretion in a high-concentration glucose state, and does not have an effect of suppressing an increase in blood glucose level and an effect of increasing insulin secretion in a low-concentration glucose state. It is characterized by that.

To provide a novel insulin secretagogue, an inhibitor of blood glucose elevation, a therapeutic agent for diabetes, and a screening method for these agents.

Claims (3)

A preventive agent for recurrence after diabetes treatment, including N-acetyl-2,3-didehydro-2-deoxyneuraminic acid .
The first aspect of claim 1, wherein it has an effect of suppressing an increase in blood glucose level and an effect of increasing insulin secretion in a high-concentration glucose state, and does not have an effect of suppressing an increase in blood glucose level and an effect of increasing insulin secretion in a low-concentration glucose state. Insulin.
A method for screening an active ingredient of a recurrence preventive agent after diabetes treatment , which comprises selecting a test compound that inhibits the glucosidase activity of sialidase.
The method comprises the following steps:
(1) Step of adding a test compound to a system containing a sialidase and a substrate
(2) Steps for measuring sialidase activity, and
The substrate is sialic acid with an insoluble fluorescent substance added, and the sialidase is NEU1, NEU2, NEU3 and / or NEU4.
Method.

Images