JPH0834730A - Amyrin agglutination inhibitor - Google Patents

Abstract

PURPOSE:To obtain a therapeutic or prophylactic agent for diseases caused by cytotoxiclty due to amyrin agglutination, through its action to inhibit the amyrin agglutination. CONSTITUTION:This amyrin agglutination inhibitor contains, as an active ingredient, a rifamycin of formula I [R1 is H, formula II; R2 is OH, OCH2COOH, OCH2CON(C2H5)2; R1 and R2 maybind to form a ring structure of formula III] or formula IV (R3 and R4 may bind to form a carbonyl together with C atoms bonding to these groups, or formula V) or its pharmaceutically permissible salt. The particularly preferred rifamycin is the one of formula I where R1 is a group of formula II and R2 is OH. The diseases caused by cytotoxicity due to amyrin agglutination is type 2 diabetes mellitus, diabetes with lowered glucose resistance, obesity, insulin-resistant diabetes and the rifamycin is particularly useful as a therapeutic or prophylactic agent for type 2 diabetes mellitus.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an amylin aggregation inhibitor. More specifically, it relates to a therapeutic or prophylactic agent for a disease based on cytotoxicity caused by amylin aggregation, which contains rifamycins as active ingredients.

[0002]

Amylin is a 37-amino acid peptide identified as a major constituent of amyloid protein accumulated in pancreatic β cells of diabetic patients (for example, P. Wes.
Termark et al., Proc. Natl. Acad. Sci. USA, Volume 84:
Pp. 3881-3885 (1987), GJS Coope
R et al., Proc. Natl. Acad. Sci. USA, 84: 862.
8-8632 (1987), GJS Cooper et al.
Proc. Natl. Acad. Sci. USA, Volume 85: 7763-7
See page 766 (1988)). The relationship between amylin and the onset of diabetes has not always been clear,
More recently, it has been clarified that amylin aggregates to form fibrous bodies and thereby exert cytotoxicity on pancreatic β cells. (For example, A. Lorenzo et al., Natur
e, Vol. 368, pp. 756-760 (1994), etc.). Therefore, a drug that inhibits amylin aggregation may inhibit the accumulation of amyloid protein in pancreatic β cells caused by amylin aggregation / deposition, and thus may inhibit pancreatic β cell damage caused by amylin aggregates. Expected to be useful as a therapeutic and / or preventive agent for diabetes. However, there are no reports of low molecular weight compounds having such inhibitory activity.

On the other hand, rifamycins are used in Streptomyces
Antibiotics produced by mediterranei, such as rifamycin B and rifamycin S, and their semi-synthetic derivatives, such as rifamycin SV and rifampicin, are known to have strong antibacterial activity against Mycobacterium tuberculosis. Has been. However, it has not been reported that such rifamycins have an activity of inhibiting amylin aggregation.

[0004]

DISCLOSURE OF THE INVENTION The object of the present invention is to provide a drug which inhibits amylin aggregation. Another object of the present invention is to provide a therapeutic or prophylactic agent for a disease based on cytotoxicity due to amylin aggregation, which is mediated by amylin aggregation inhibitory action.

[0005]

Means for Solving the Problems As a result of diligent studies on the possibility of a drug that inhibits amylin aggregation, the present inventors have found that the rifamycins of the present invention have an activity of inhibiting amylin aggregation, and further, amylin. The inventors have found that they have an action of suppressing cytotoxicity due to the above, and thus arrived at the present invention.

[0006]

That is, the present invention has the following formula [I]:

[0007]

[Chemical 7]

(Where R ₁ is a hydrogen atom or the following formula [I
I]

[0009]

Embedded image

Represents a group represented by the following formula, wherein R ₂ is a hydroxyl group and has the following formula [III] or [IV]

[0011]

[Chemical 9]

Or a group represented by the following formula [V] in which R ₁ and R ₂ are taken together.

[0013]

[Chemical 10]

You may represent the cyclic structure represented by. ),
Or the following formula [VI]

[0015]

[Chemical 11]

(Where R ₃ and R ₄ together form a carbonyl group with the carbon atom to which they are attached, or

[0017]

[Chemical 12]

Represents a group represented by ) A rifamycin represented by the formula (1) or a pharmaceutically acceptable salt thereof as an amylin aggregation inhibitor containing as an active ingredient, and rifamycins as an active ingredient, the above formula [I] or the above formula [VI] A therapeutic or prophylactic agent for a disease based on cytotoxicity due to amylin aggregation, which comprises a rifamycin represented by the following formula or a pharmaceutically acceptable salt thereof, via an amylin aggregation inhibitory action.

The rifamycins of the present invention have the above formula [I]
R in the formula [I]₁And R₂Union
For example, R₁Is a hydrogen atom and R₂Is a hydroxyl group, before
A group represented by the above formula [III] or the above formula [IV]
Rifamycins (Rifamycin SV, Rifa
Mycin B, rifamycin B diethylamide), R ₁
Is a group represented by the above formula [II] and R₂Is a hydroxyl group
Amycins (rifampicin), R₁And R₂Together
Rifama representing a cyclic structure represented by the above formula [V]
Isins (rifaximin) and the like can be mentioned.
These R₁And R₂Among the combinations of
Let's go R₁Is a hydrogen atom and R₂Is a hydroxyl group or the above formula
Rifamycins represented by [II], R₁Before
R is a group represented by the formula [II]₂Rifama in which is a hydroxyl group
Isines can be mentioned.

The structural formulas of rifamycins in these preferred embodiments are as follows (1) to (3).

(1) Rifampicin

[0022]

[Chemical 13]

(2) Rifamycin SV

[0024]

Embedded image

(3) Rifamycin B

[0026]

[Chemical 15]

The rifamycins of the present invention have the above formula [VI]
And R ₃ and R ₄ together form a carbonyl group with the carbon atom to which they are bonded, or represent a group represented by the following formula [VII]. Examples of such preferred rifamycins include rifamycin S and rifamycin O, respectively.

Among these, particularly, as the rifamycins represented by the above formula [I] of the present invention, R ₁ in the above formula [I] is a group represented by the above formula [II] and R ₂
Rifampicin in which is a hydroxyl group is preferred.

The rifamycins of the present invention are pharmaceutically capable of forming with known bases or acids (for example, inorganic acids / organic acids) due to the acidic or basic groups when present in the molecule. In the case where a salt acceptable for the above is present, such a salt is also included.

The rifamycins related to the present invention are all known compounds, and, for example, rifampicin, rifamycin SV, rifamycin B and the like are also available as reagents. In addition, rifamycin S is a known method (Mn
It can be easily synthesized by an oxidation reaction using O ₂ or H ₂ O ₂ .

The amylin aggregation inhibitor of the present invention can be used as the amylin aggregation inhibitor of the present invention, preferably by blending a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier in this case include the same as the below-mentioned excipients. Regarding the compounding amount of the compound of the present invention and the carrier in this case,
It depends on the dose of the active ingredient as described below, but is not particularly limited and may be selected from a wide range. Usually, the rifamycin antibiotic of the present invention is 1 to 70% by weight, preferably 5 to 5% by weight in the total composition.
0% by weight. The obtained composition is further prepared by a known method using suitable excipients and the like, oral capsules such as soft capsules, hard capsules, tablets, granules, powders, suspensions, solutions, syrups, and injections. It is provided as an agent, a suppository or an external preparation. Such excipients include vegetable oils (eg corn oil, cottonseed oil, coconut oil, almond oil, peanut oil,
Olive oil), oily esters such as medium-chain fatty acid glyceride oil, mineral oil, glycerin esters such as tricaprylin and triacetin, alcohols such as ethanol, physiological saline, propylene glycol, polyethylene glycol, petrolatum, animal fats and oils, cellulose derivatives (Crystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), polyvinylpyrrolidone, dextrin, lactose, mannitol, sorbitol, starch and the like.

The rifamycins of the present invention or a pharmaceutically acceptable salt thereof has an activity of inhibiting the aggregation of amylin, and therefore suppresses the cytotoxicity due to the aggregation of amylin, and therefore, is based on this cytotoxicity. It is useful as a therapeutic or prophylactic agent for diseases such as type II diabetes, diabetic glucose intolerance, obesity, and insulin resistant diabetes. Among these diseases, the therapeutic or prophylactic agent for diabetes can be mentioned as a preferable therapeutic agent or prophylactic agent for type II diabetes. As these therapeutic or prophylactic agents, rifamycins of the present invention or pharmaceutically acceptable salts thereof are rifamycins represented by the above formula [I] A pharmaceutically acceptable salt thereof, wherein in the formula [I], R ₁ is a hydrogen atom, R ₂ is a hydroxyl group or a group represented by the above formula [III], or R ₁ is the above formula [III]. II] is a group represented by
Those in which ₂ is a hydroxyl group can be mentioned as preferable ones. Among them, the rifamycins as an active ingredient is the rifamycins represented by the above formula [I] or a pharmaceutically acceptable salt thereof, wherein R ₁ is represented by the formula [II] ] Rifamycins in which R ₂ is a hydroxyl group are more preferable.

The dose of the active ingredient depends on the degree of disease and the age of the patient, but is usually about 10 to 1000 mg / day / person, preferably 100 to 600 mg / day / person.
It is preferable to formulate so as to satisfy such conditions.

Various test methods and measuring methods used in the present invention are as follows.

(1) Test Method for Amylin Aggregation Inhibitory Activity It was carried out by a method using thioflavin T (ThT).

Thioflavin T (ThT) binds to the β-sheet structure of aggregated proteins such as amyloid β protein which belongs to amyloid protein like amylin, and emits a new fluorescence (482 nm) which is not shown in the free state. It is reported that the fluorescence intensity is proportional to the degree of aggregation of the bound protein (Harry LeVine III.1).
993, Protein Science 2, 404-410). Therefore, the amylin aggregation inhibitory activity of the drug can be examined by measuring the degree of aggregation of the drug-containing amylin based on the fluorescence intensity of ThT binding thereto.

Specifically, 10 μl was taken from a sample solution of amylin containing a certain drug, and this was added to 1 ml of a ThT solution dissolved in 50 mM sodium phosphate buffer (pH 6.0) at a concentration of 3 μM and stirred. To do. Spectrofluorometer (made by JASCO) immediately after stirring
Is used to measure the fluorescence of the solution at an excitation wavelength of 450 nm and a fluorescence wavelength of 482 nm. If the increase in fluorescence is suppressed for a certain period of time as compared with a solution containing only amylin that does not contain a drug, the drug is determined to have amylin aggregation inhibitory activity.

(2) Amylin fine particles using PC12 cells
Cytotoxicity measurement method (a) Preparation of cells PC12 cells (RCB0009, Lot6) distributed from RIKEN cell bank were used. This cell is a clonal cell derived from rat adrenal medulla, and differentiates into a sympathetic ganglion cell-like by culturing in the presence of NGF (Gree
ne and Tischler, Proc. Natl. Acad. Sci. USA, 73:
2424 to 2428, 1976), it is widely used as a nerve cell model system.

The culture was RPMI containing 10% horse serum (Gibco) and 5% quasi-fetal bovine serum (Mitsubishi Kasei).
Using 1640 (manufactured by Gibco) as a medium,
Performed in the presence of 5% CO ₂ . 5,000 cells / well
96-well culture plate (Sumitomo Bakelite, SUMI
LON CELL LIGHT C-1), the next day, D-glucose-free, 50 ng / ml / NGF
Replace with medium containing (from Sigma, rat submandibular gland)
After 4 days, the medium is exchanged and the culture is continued. After 7 days, the culture is used for the following measurements.

(B) MT of PC12 cells with amylin
Measurement of T-reducing capacity lowering action The PC12 cells on the 7th day of culture in (a) above were replaced with a medium (1
After 80 μl / well, phosphate buffered saline (PB)
20 μl / we of amylin (manufactured by Bachem) dissolved in S)
Add 11. 40 hours later, 5m of MTT (made by Sigma)
25 μl / well of g / ml PBS solution is added, and the culture is continued at 37 ° C. in the presence of 5% CO ₂ . After 4 hours, the medium was sucked up, 0.04N HCl / isopropanol (100 μl / well) was added to dissolve the formazan, and a colorimetric assay (570 to 650) was performed using a microplate reader (Molecular Devices, Vmax).
nm) to measure changes in MTT reducing ability of PC12 cells.

[0041]

The present invention will be described in detail below with reference to examples. In addition, unless otherwise specified,% indications in Examples and the like indicate weight / volume%.

[Example 1] Rifampicin was dissolved in DMSO at 10 mg / ml. Amylin (manufactured by Bachem) was twice dissolved in deionized water to a concentration of 100 μg / ml, and a 0.2 M sodium phosphate buffer solution (pH 7.
The mixture was mixed with 4) at a ratio of 1: 1 to obtain an amylin solution having a final concentration of 50 μg / ml. This solution was dispensed into 6 Eppendorf tubes by 200 μl each. 10mg prepared above
/ Ml of rifampicin solution was added to each of 3 tubes containing amylin solution in an amount of 2 μl (final concentration of rifampicin 100 μg / ml; (B)). This was used as a control without adding anything to the remaining three tubes (A).

The rifapicin-containing amylin solution (B) and the control amylin solution (A) were allowed to stand at room temperature. According to the above-mentioned test method ((1) Test method for amylin aggregation inhibitory activity), a part of each solution was taken over time to measure the degree of amylin aggregation.

As a result, as shown in FIG. 1, in the control amylin solution (A), an increase in fluorescence, that is, amylin aggregation was observed from the second day after the dissolution of amylin.
On the other hand, no increase in fluorescence was observed in the amylin solution (B) containing 100 μg / ml of rifampicin even on the 4th day. The above results indicate that rifampicin suppresses amylin aggregation.

[Example 2] The test method ((2) PC1
Amylin cytotoxicity measurement method using 2 cells),
The rifampicin-containing amylin solution (B) obtained in Example 1 and the control amylin solution (A) were allowed to stand at room temperature for 4 days, and then added to PC12 cells to give amylin PC12 cell MTT reducing ability. The effect on the lowering effect was investigated. That is, the final concentration of amylin was 5 μg / ml, 0.5 μg / ml, 0.0
The cells were added to PC12 cells at 5 μg / ml and cultured for 2 days, and then the MTT reducing ability was examined. As a result, as shown in FIG. 2, amylin showed a concentration-dependent MTT reduction activity-decreasing action on PC12 cells, whereas rifampicin suppressed the MTT reduction ability-decreasing action of amylin.
This result indicates that rifampicin has an effect of suppressing the cytotoxicity of amylin.

[Example 3] Production of capsules 1 capsule is 100 m of rifampicin having the following composition
A capsule containing g was prepared.

Rifampicin 100.00 mg Lactose 50.00 g Ethylcellulose 1.50 g Stearic acid 1.50 g Rifampicin and lactose were mixed and then moistened with a methylene chloride solution of ethylcellulose to finally obtain fine granules. Finely pulverized stearic acid was mixed with this to obtain a powder. The powder thus obtained was filled in a capsule (# 2) to obtain a capsule.

[0048]

The formula [I] or the formula [V of the present invention
The drug containing rifamycins represented by the formula [I] as an active ingredient has an action of suppressing amylin aggregation and further has an action of suppressing cytotoxicity due to amylin. And / or is expected to be useful as a prophylactic agent.

[Brief description of drawings]

1 shows the amylin aggregation-inhibitory effect of rifapicin of the present invention in Example 1. FIG. In the figure, A,
B corresponds to no addition (control) (A) and rifampicin (B), respectively, and each bar graph ae is 0th.
It corresponds to the day, the first day, the second day, the third day, and the fourth day.

FIG. 2 shows the effect of rifampicin of the present invention on the viability of PC12 cells in Example 2. In the figure, among the bar graphs, the dark bar graph shows the viability of PC12 cells when only amylin was added (no rifampicin added), and the light bar graph shows the viability of PC12 cells when a mixed solution of amylin and rifampicin was added (rifampicin added).

Claims (7)

[Claims] 1. The following formula [I]: (Where R ₁ is a hydrogen atom or the following formula [II] R ₂ is a hydroxyl group, and is represented by the following formula [III] or [IV] In represents a group represented by or formula R ₁ and R ₂ together [V] embedded image You may represent the cyclic structure represented by. ) Or the following formula [VI] (Where R ₃ and R ₄ together form a carbonyl group with the carbon atom to which they are attached, or Represents a group represented by. ) An amylin aggregation inhibitor containing a rifamycin represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. 2. In the above formula [I], R ₁ is a hydrogen atom, R ₂ is a hydroxyl group or a group represented by the above formula [III], or R ₁ is a group represented by the above formula [II]. And R ₂ is a hydroxyl group, and the amylin aggregation inhibitor according to claim 1. 3. In the above formula [I], R ₁ is a group represented by the above formula [II] and R ₂ is a hydroxyl group.
The amylin aggregation inhibitor described. 4. Amylin aggregation inhibitory activity, which comprises, as an active ingredient, rifamycins represented by the above formula [I] or the above formula [VI] or a pharmaceutically acceptable salt thereof. Or a therapeutic or prophylactic agent for a disease based on cytotoxicity resulting from amylin aggregation. 5. A rifamycin compound as an active ingredient,
Rifamycins represented by the above formula [I] or a pharmaceutically acceptable salt thereof, wherein R
_{The 1} is a hydrogen atom and R ₂ is a hydroxyl group or a group represented by the above formula [III], or R ₁ is a group represented by the above formula [II] and R ₂ is a hydroxyl group. A therapeutic or prophylactic agent. 6. A rifamycin compound as an active ingredient,
Rifamycins represented by the above formula [I] or a pharmaceutically acceptable salt thereof, wherein R
The therapeutic or prophylactic agent according to claim 4, wherein ₁ is a group represented by the formula [II] and R ₂ is a hydroxyl group. 7. The therapeutic or prophylactic agent according to any one of claims 4 to 6, wherein the disease is diabetes.