JPH08109138A - Cholesterol lowering agent - Google Patents

Abstract

PURPOSE: To obtain a cholesterol lowering agent based on principle lowering blood cholesterol concentration by decomposing enteric bile acid by oral administration of the agent, inhibiting reabsorption at the end of ileum and consuming cholesterol which is a raw material for bile acid. CONSTITUTION: This cholesterol lowering agent is obtained by immobilizing 3α-hydroxysteroid dehydrogenase anti/or 3α-hydroxycholanate dehydrogenase which is a bile acid hydrolase onto a polymer carrier. The polymer carrier is water-insoluble and a carrier capable of carrying out immobilization reaction with bile acid hydrolase is preferable as the polymer carrier and p- aminobenzylcellulose, polyaminopolystyrene, CNBr-activated cephalose, ethylene- maleic acid copolymer, agarose, etc., is exemplified as the polymer carrier and the particle diameter is preferably 1μm to 5mm, especially 5μm-1mm. Since the agent uses bile acid hydrolase, the absorbed part is not saturated as saturated in ion exchange resins and the agent effectively inhibits re-absorption of bile acid by using a small amount of the agent and readily can lower blood cholesterol value.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cholesterol lowering agent, and in particular, it is capable of decomposing intestinal bile acid by oral administration to inhibit reabsorption at the terminal ileum, and consuming cholesterol as a raw material of bile acid. Relates to a cholesterol lowering agent based on the principle of lowering blood cholesterol concentration.

[0002]

2. Description of the Related Art As a method for lowering blood cholesterol level, a method of orally administering an anion exchange resin to adsorb bile acid has been conventionally known (US Pat. No. 349).
No. 9960, US Pat. No. 3,780,171, Japanese Patent Publication No. 61
-54457).

The mechanism of lowering blood cholesterol level by orally administering an anion exchange resin is considered as follows. That is, the orally administered anion exchange resin adsorbs and fixes the bile acid in the intestine circulating in the enterohepatic system to prevent reabsorption of bile acid, and promotes the conversion of cholesterol into bile acid in the liver. As a result, blood cholesterol level is lowered.

Conventionally, cholestyramine has been used as a typical cholesterol lowering agent, which is a basic anion exchange resin in which an aliphatic quaternary ammonium salt is fixed as a functional group to a styrene resin ( U.S. Pat. No. 3,499,960, U.S. Pat. No. 3,780,171, and Japanese Patent Publication No. 61-54457). In addition to this, those using an imidazole base as a functional group (JP-A-60-209523).
No.), using vinyl pyridine (JP-A-2-214)
No. 711) and the like, and other than the styrene-based resin, an epoxy-based resin (JP-A-60-20
9523), saccharides such as cyclodextrin and polysaccharides, and polydiallylmethylamine derivatives (JP-A 64-22924).

However, the above-mentioned typical ion exchange resins were evaluated for their ability to adsorb bile acids in vitro in the presence of inorganic ions, but none of them was sufficient. This is a bile acid adsorbent based on the principle of ion exchange,
This is because only one molecule of bile acid can be adsorbed per unit of ion exchange group, and the adsorption site is saturated. Therefore, higher doses (eg, typical cholestyramine 8-1
(6 g / day), which was very difficult to drink.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above drawbacks, and an object thereof is to provide a novel cholesterol lowering agent having a small dose, that is, an excellent ability to inhibit bile acid absorption. It is to be.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the inventors of the present invention have found that the dose can be reduced by using a specific bile acid-degrading enzyme. In the present invention, 3α-hydroxysteroid dehydrogenase (3α
-Hydroxysteroid dehydrogenase) (EC 1.1.1.5
0) and / or 3α-Hydroxycholanate dehydrogenase (EC
1.1.1.12) is used.

The above-mentioned bile acid-degrading enzyme is immobilized on a polymer carrier in order to prevent degradation after administration and absorption into the body, and to improve retention of the enzyme in the intestine. As a fixing method, 3
α-Hydroxysteroid dehydrogenase (3α-Hydro
xysteroid dehydrogenase) and / or 3α-Hydroxycholanate dehydrogenase (3α-Hydroxycholanatedeh)
Ydrogenase) may be bound or encapsulated in a polymer carrier under neutral or alkaline conditions.

The above-mentioned polymer carrier is not limited as long as it is water-insoluble and can carry out an immobilization reaction with a bile acid-degrading enzyme. For example, p-aminobenzyl cellulose, polyaminopolystyrene, CNBr-activated sepharose ( Sepharose), CNBr-activated Sephadex (Sephadex), CNBr-activated cellulose, Sepharose CL (Sepharose C)
L), ethylene-maleic anhydride copolymer, agarose and the like. The particle size of the polymer carrier is not particularly limited and is preferably 1 μm to 5 mm, more preferably 5 μm to 1 mm.

The cholesterol-lowering agent of the present invention can be prepared into capsules, tablets, granules, powders and the like by a well-known technique in the technical field of pharmacy.
It may also be administered as a formulation mixed with one or more additives that are physically and chemically compatible.

Examples of the above additives include glucose,
Excipients such as lactose, crystalline cellulose, mannitol, cornstarch and sugar; binders such as hydroxycellulose, hydroxymethylpropylcellulose, carboxymethylcellulose, gelatin, gum arabic and polyvinyl alcohol; cornstarch, carboxymethylcellulose calcium, low substituted hydroxypropyl Disintegrators such as cellulose and potato starch; lubricants such as calcium stearate, magnesium stearate, talc, polyethylene glycol and hardened oil. Moreover, you may use a coloring agent, a flavoring agent, a flavoring agent, a stabilizer, etc. as needed.

Since the cholesterol lowering agent of the present invention has a very high bile acid adsorbing ability as compared with the ion exchange resin, the dosage is reduced as compared with the ion exchange resin-containing preparation regardless of which dosage form is selected. It is possible. In addition, the agent may be administered in a state of being dissolved or suspended in water or other liquid. Since this drug swells sufficiently in water, etc., it does not have the feeling of a rough foreign substance like conventional products of cholestyramine, and its dosage is small, making it easy to drink. The dose of the cholesterol lowering agent of the present invention is 0.1 to 5 g / day, preferably 0.2 to 3 g / day for adults, and it is usually taken twice a day in divided doses.

[0013]

Since the cholesterol-lowering agent of the present invention uses a bile acid-degrading enzyme, it does not saturate the adsorption site unlike an ion exchange resin, and effectively inhibits the reabsorption of bile acid even in a small amount, The blood cholesterol level can be easily lowered.

[0014]

[Preparation of cholesterol lowering agent]

(Example 1) CNBr-activated sepharose (CNBr-activ
ated Sepharose 4B, manufactured by Pharmacia) 10 g
After swelling in 0 ml of 1 mM hydrochloric acid for 15 minutes, the mixture was filtered through a fine-pore glass filter (G3 grade). 500
After washing 4 times with 1 ml of 1 mM hydrochloric acid, 50 ml of 0.5
It was washed with MNaCl / 0.1M NaHCO ₃ (coupling buffer, pH 8.3) to obtain a swollen gel.

3α-Hydroxysteroid dehydrogenase (SIGMA)
After dissolving 700 mg (350 mg protein) (manufactured by the company) in 70 ml of coupling buffer, the swelling gel was added and mixed by inversion at room temperature for 2 hours. After the reaction, the gel was isolated and 200 ml of 0.2 M glycine aqueous solution (pH 8.
0) was added to block the remaining active groups. Further, after washing with 200 ml of coupling buffer, 200 ml of 0.1M acetate buffer (pH 8.0), and 200 ml of coupling buffer in this order, they were suspended in physiological saline.
A cholesterol lowering agent containing 50 mg / ml gel was obtained.

(Example 2) High molecular weight agarose (Agarose LGT, manufactured by Nacalai Tesque) and low molecular weight agarose (Agarose L-200, manufactured by Shimizu Foods) Weight ratio 1:
A mixture of 1 was added to water to prepare a 10% by weight suspension,
This was heated at 121 ° C. for 20 minutes for dissolution to prepare an agarose solution. Next, 3α-hydroxysteroid dehydrogenase (3α-Hydroxysteroid dehydrogenase
, SIGMA) 700 mg (350 mg protei
n) was dissolved in 3.5 ml of water, added to 100 ml of the agarose solution heated to 40 ° C. and stirred. Further, about 700 ml of liquid paraffin heated to 40 ° C. was added and mixed, the agarose solution was dispersed in liquid paraffin, and this was ice-cooled with stirring to gel the agarose. Add 1000 ml of water and add 2000 rpm
It was centrifuged for 10 minutes. After collecting the remaining microcapsules, the microcapsules were suspended in physiological saline to obtain a cholesterol-lowering agent containing 50 mg / ml of gel.

(Comparative Example 1) Mu which is a resin component of Questran formulation (manufactured by Bristol-Myers Squibb)
romac 1x2 (particle size: 200-400 mesh,
Muromachi Chemical Co., Ltd.) was washed with water several times and then vacuum dried, and the obtained resin was suspended in physiological saline to obtain a cholesterol lowering agent containing 50 mg / ml of the resin.

[Bile Acid Adsorption Test in Human Bile] 8 μl of each cholesterol-lowering agent prepared in Examples 1 to 2 and Comparative Example 1 described above was immersed in 3 ml of human bile, and a rotary mixing device (R
OTATOR RT-50, made by Taitec) at 37 ° C
The mixture was mixed by tumbling for 3 hours. After that, 15,000 rp
Centrifugation was performed for 20 minutes at m, and the bile acid concentration in the supernatant was measured using EnzaVile 2 (Daiichi Pure Chemicals Co., Ltd.) to determine the adsorbed bile acid concentration. The results are shown in Table 1.

[0019]

[Table 1]

As is clear from Table 1, all the cholesterol-lowering agents of the present invention showed higher ability to eliminate bile acids than Comparative Example 1.

[Efficacy Evaluation Test] Sprague Daw
male rats (6 weeks old) were fed a basic diet (C
E-2, manufactured by CLEA Japan, was divided into the following groups 1 to 3 (6 animals / group), and the drug was administered by diet for one week.
One week later, after fasting for about 6 hours, blood was collected from the carotid artery of the rat, and the amount of cholesterol in the serum was determined as cholesterol E-.
It was measured using Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The serum cholesterol value is shown as an average value ± standard error of 6 animals in each group, and the inhibition rate was calculated by the following formula, and shown in Table 2. Group 1: Basic diet only Group 2: High cholesterol diet (basic diet + 1% cholesterol + 0.5% bile powder), plus 1 at 9:00 and 17:00
Forced administration of physiological saline 1 ml by Zonte twice a day (control group) Group 3: In addition to administration of high cholesterol diet, 9:00 and 17: 0
0 times twice a day, 1 ml of cholesterol lowering agents of Examples 1-2 and Comparative Example 1 was forcibly administered by Zonte

[0022] A: Serum cholesterol amount in high cholesterol diet group (second group) B: Serum cholesterol amount in basic diet group (first group) C: Serum cholesterol amount in each group

[0023]

[Table 2]

As is clear from Table 2, all of the cholesterol lowering agents of the present invention have a higher cholesterol lowering action than Comparative Example 1.

[0025]

INDUSTRIAL APPLICABILITY The cholesterol lowering agent of the present invention can reduce the dose and reduce the foreign body sensation as compared with the ion exchange resin, that is, it can provide a more easy-to-drink formulation and reduce the blood cholesterol level. It can be a novel drug for effectively reducing hyperlipidemia.

Claims (1)

[Claims] 1. A 3α-Hydroxysteroid dehydrogenase and / or 3
α-Hydroxycolanate dehydrogenase (3α-Hydro
xycholanate dehydrogenase) is a cholesterol lowering drug immobilized on a polymer carrier.