JPS634528B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel enzyme preparation, and more particularly to an easily absorbable enzyme preparation that is well absorbed from the intestinal tract.

In recent years, a large number of enzyme preparations have been commercially available, among which proteolytic enzymes such as seaprose, serratiopeptidase, lysozyme, and sphelicase are widely used clinically as anti-inflammatory agents.

Although the mechanism of action of this enzyme has not yet been fully elucidated, oral administration is generally used. However, as described below, according to research by the present inventors, the enzyme is degraded by trypsin, chymotrypsin, etc. in the intestinal tract, and the amount absorbed into the body is extremely small. Therefore, the present inventor conducted various studies to improve the absorption of the enzyme from the intestinal tract and increase its effect.
It has been discovered that when the enzyme is co-administered with an inhibitor such as trypsin or chymotrypsin described below, the absorption of the enzyme is significantly increased, and that the effect can be further improved by adding a surfactant to this, and the present invention completed.

That is, the present invention provides an easily absorbable enzyme preparation containing a protease and a trypsin and/or chymotrypsin inhibitor.

Trypsin and/or chymotrypsin inhibitors include the following formula (), () or () (In the formula, R ₁ represents a benzoyl group, an indole acetyl group, or a naphthylacetyl group that may have a substituent, R ₂ represents a lower alkyl group, and A represents a single bond or a linear or branched chain. represents an alkylene group, R represents a hydrogen atom, a lower alkyl group, an acylaminoalkyl group, or two R may be joined together to form a ring, and n
represents an integer from 1 to 3) Hydroxybenzoic acid derivative () represented by
This is an easily absorbable enzyme preparation containing an amidinophenol derivative () or gabexate ().

In the present invention, examples of the protease include seaprose, serratiopeptidase, lysozyme, and sphaericase.

In addition, the compounds of formulas () and (), which are inhibitors of trypsin, chymotrypsin, etc., are new compounds that have not yet been described in the literature, and can be produced, for example, as follows (Japanese Patent Application No. 54-56612; −
No. 63685).

Hydroxybenzoic acid derivative [] This is produced by reacting N,N-di-lower alkylethylenediamine () with hydroxybenzoic acid ester () according to the following reaction formula.
-di-lower alkylaminoethyl) hydroxybenzamide (), and then reacting this with carboxylic acid () or its reactive derivative.

(In the formula, R ₃ represents a lower alkyl group, R ₁ and
_R2 is the same as above) Amidinophenol derivative [] This is produced by reacting amidinophenol () with cyclohexanecarboxylic acid () according to the following reaction formula.

(In the formula, R, A and n are the same as above) Examples of the hydroxybenzoic acid derivative include N-(2-dimethylaminoethyl)-p-(benzoyloxy)benzamide (melting point: hydrochloride
178.5-181℃), N-(2-dimethylaminoethyl)-p-(β-indoleacetyloxy)benzamide (hydrochloride 175.5-178℃), N-(2-dimethylaminoethyl)-p-(1- naphthylacetyloxy)benzamide (hydrochloride 105.5-108℃),
N-(2-dimethylaminoethyl)-p-(toluyloxy)benzamide (hydrochloride 172-175°C) and N-(2-dimethylaminoethyl)-p-(2
-Methyl-5-methoxy-β-indoleacetyloxy)benzamide (hydrochloride 182-184°C), etc., and amidinophenol derivatives include:
For example, 4'-amidinophenyl cis-4-isopropylcyclohexane carboxylate (hydrochloride 175-179℃), 4'-amidinophenyl trans-4-isopropylcyclohexanecarboxylate (hydrochloride 188-189℃), 4'-amidinophenyl trans-4 -Formylaminomethylcyclohexanecarboxylate (hydrochloride 175.5-178
℃), 4'-amidinophenyl trans-4-acetylaminomethylcyclohexanecarboxylate (hydrochloride 198-201℃), 4'-amidinophenyl cis-4-acetylaminomethylcyclohexanecarboxylate (powder), 4'-amidinophenyl trans-4-n-butyroylaminomethylcyclohexanecarboxylate (viscous substance),
4'-amidinophenyl trans-4-isobutyroylaminomethylcyclohexanecarboxylate (hydrochloride decomposed at 174.5-177℃), 4'-amidinophenyl 3-cyclohexylpropionate (hydrochloride 170-172℃), 4'-amidinophenyl 2
- Cyclohexyl isobutyrate (hydrochloride 178~
179℃), 4'-amidinophenyl 1,2,3,4
-tetrahydro-1-naphthoate (methanesulfonate, 177-180°C) and 4'-amidinophenyl 1,2,3,4-tetrahydro-2-naphthoate (methanesulfonate, 212.5-214.2°C).

Furthermore, gabexate is a known compound that has trypsin inhibitory activity and is used as a therapeutic agent for pancreatic diseases.

The easily absorbable enzyme preparation of the present invention contains a protease as described above and a trypsin and/or chymotrypsin inhibitor represented by the formula (), (), or (). The compounding amount is preferably such that the weight ratio of the compound of formula (), (), or () is 0.1 to 20 to 1 of the proteolytic enzyme.

It is preferable to further include a surfactant in the enzyme preparation of the present invention, in which case absorption of the enzyme from the intestinal tract is further improved. As a surfactant,
For example, nonionic surfactants such as polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene hydrogenated castor oil derivative, sorbitan fatty acid ester, and sucrose fatty acid ester are used. The blending amount of this surfactant is based on the weight ratio of 1 part protease.
0.1-30 is preferred.

The above ingredients may include excipients, binders,
Together with a disintegrant and a lubricant, it is formulated into a preparation for oral administration, such as a powder, tablet, capsule, pill, etc., and enteric-coated tablets or enteric-coated capsules are particularly preferred.

The enzyme preparation of the present invention is administered in divided doses 3 to 4 times a day so that the amount of proteolytic enzyme is 5 to 15 mg each time.

As shown in the test examples described below, the easily absorbable enzyme preparation of the present invention prepared as described above is better absorbed from the intestinal tract and has better medicinal efficacy than when a proteolytic enzyme is administered alone. can be expected.

Next, test examples showing the effects of the enzyme preparation of the present invention, reference examples showing typical manufacturing methods of compounds () and (), and examples of the present invention will be given and explained.

Test example (1) Experimental animal Male domestic rabbit weighing 2.5-3 kg (Japanese native species, Kitayama Labes) (2) Proteolytic enzyme Seaprose protein content 0.94 mg/mg protein, casein hydrolysis activity: OD ₇₅₀ = 0.4/μg Protein / 30 minutes (3) Substrate N-acetyl-L-terocin-α-naphthyl ester (ATNE) (4) Test substance A: Seaprose 200mg/Kg B-1: Seaprose 200mg/Kg and N-(2
-dimethylaminoethyl)-p-(1-
naphthylacetyloxy)benzamide
Hydrochloride 100mg/Kg B-2: Seaprose 200mg/Kg and N-(2
-dimethylaminoethyl)-p-(β-
Indoleacetyloxy)benzamide hydrochloride 100mg/Kg B-3: Seaprose 200mg/Kg and 4'-amidinophenyl 1,2,3,4-tetrahydro-1-naphthoate methanesulfonate 100mg/Kg C: Seaprose 200mg/Kg Kg4'-amidinophenyl 1,2,3,4-tetrahydro-1
- Naphthoate methanesulfonate
100mg/Kg and sucrose palmitate 100
mg/Kg of the three components were homogenized under ice cooling and used as a homogeneous solution.

D: Seaprose 200mg/Kg and sucrose palmitate 100mg/Kg (5) Administration method Dissolve the test substance in 2ml/Kg of distilled water, and add pentobarbital (Dainippon Pharmaceutical Co., Ltd., Nembutal).
The abdomen of a rabbit to which 30 mg/Kg was intravenously administered was opened, and the drug was administered through a syringe needle while the 5 cm region below the pylorus was ligated. 15 minutes, 30 minutes, 1, 2, before and after administration
After 3, 4, 5, and 6 hours, blood was collected from the ear vein and centrifuged at 3000 rpm for 10 minutes to obtain serum.

6 Enzyme activity measurement method 50 μl of serum, 1% sodium dodecyl sulfate
100 μl and 550 μl of 50 mM Tris buffer (PH8.0) were mixed and incubated at 37° C. for 10 minutes. to this,
300 μl of a 10% ATNE dimethyl sulfoxide solution was added and incubated at 25° C. for 30 minutes. To this was added 100 μl of a 1% aqueous solution of Fast Violet B Salt (manufactured by Hoechst) and incubated at 0° C. for 10 minutes.

Add 1 ml of acetic acid to this and measure the absorbance (OD) at 515 nm.
was measured, and the absorption rate per ml of serum was calculated according to the following formula, setting the blood volume to 1/13 per kg of body weight.

Maximum OD value x 20 x 1000/13/Administered seaprose (Kg
total OD value x 100 = absorption rate (%) (7) Results Figure 1 shows the combined effect of proteolytic enzyme and compound () or (), and Figure 2 shows the effect when a surfactant is used in combination. As shown in the figure.

Reference example 1 (i) N-(2-dimethylaminoethyl)-p-hydroxybenzamide: 21.57 g (141.8 mmole) of p-hydroxybenzoic acid methyl ester and 25 g of N,N-dimethylethylenediamine
(283.6 m moie) was heated under reflux at 150°C for 20 hours. The reaction product was concentrated under reduced pressure, and ethyl acetate was added to the resulting residue to precipitate crystals. Further recrystallization from methanol-ether gave 14.2 g (yield 48.1%) of N-(2-dimethylaminoethyl)-p-hydroxybenzamide as colorless crystals.

Melting point: 117-122.5℃ Elemental analysis value: C ₁₁ H ₁₆ N ₂ O ₂ C H N Theoretical value (%) 63.44 7.74 13.45 Actual value (%) 63.29 7.69 13.28 (ii) N-(2-dimethylaminoethyl) -p-
(β-indoleacetyloxy)benzamide hydrochloride: N-(2-dimethylaminoethyl)-p-hydroxybenzamide 5.42g
(26m mole) and β-indoleacetic acid 5.26g
(30 m mole) of dicyclohexylcarbodiimide in 30 ml of dimethylformamide solution.
(30 mmole) was added and stirred at room temperature for 6 hours.
The resulting dicyclohexylurea was removed, water was added to the mother liquor, and the pH was adjusted to 2 to 3 with 1N hydrochloric acid under ice cooling. After washing with ethyl acetate, the aqueous layer was neutralized with saturated sodium bicarbonate solution under ice cooling, and extracted with ethyl acetate. The extract was washed with water, dried over sodium sulfate, and a methanol solution containing 0.47 g of hydrogen chloride was added to precipitate crystals. Recrystallization from methanol-ether yields 4.60 g of N-(2-dimethylaminoethyl)-p-(β-indoleacetyloxy)benzamide hydrochloride (yield
44.0%).

Melting point: 175.5-178℃ Elemental analysis value C ₂₁ H ₂₃ N ₃ O ₃・HCl C H N Theoretical value (%) 62.76 6.02 10.46 Actual value (%) 62.75 6.01 10.41 Reference example 2 4'-amidinophenyl 1,2, 3,4-tetrahydro-1-naphthoate: 1,2,3,4-
Tetrahydronaphthalene-1-carboxylic acid 2.64g
3.09 g of dicyclohexylcarbodiimide was added dropwise to a solution of 1.16 g of p-amidinophenol methanesulfonate in 10 ml of pyridine under ice cooling, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, dicyclohexylurea was removed and the mother liquor was concentrated to dryness.

Chloroform was added to the residue to remove insoluble materials, the solvent was distilled off, the solution was dissolved in methanol, and ether was added to give 4′-
Amidinophenyl 1,2,3,4-tetrahydro-1-naphthoate methanesulfonate 502
mg (yield 46.2%) was obtained.

Elemental analysis value _: C _{H N} S Calculated value (%) 58.45 _5.68 7.17 _8.21 Actual value (%) 58.49 5.58 7.18 8.21 Example 1 Enteric-coated capsule Seaprose 15 _mg 4′-amidinophenyl 1,
2,3,4-tetrahydro-1-naphthoate 15
mg and 150 mg of sucrose palmitate are mixed together, an appropriate amount of starch and crystalline cellulose are added thereto, the mixture is filled into gelatin capsules, and the capsules are enterically coated to produce the capsules.

Example 2 Enteric-coated granule-filled capsule N-(2-dimethylaminoethyl)-p-(β-indole acetyloxy) in 5 mg of Seaprose
Mix 10 mg of benzamide and 100 mg of polyoxyethylene hardened castor, add an appropriate amount of lactose,
Granules are prepared by adding crystalline cellulose and hydroxypropyl cellulose, and the granules are coated with enteric coating and filled into gelatin capsules.

Example 3 Enteric coated film tablet N-(2-dimethylaminoethyl)-p-(1-naphthylacetyloxy) in 5 mg of sphelicase
Mix 10 mg of benzamide and 50 mg of polyoxyethylene stearate, and add appropriate amounts of white sugar, carboxymethyl cellulose calcium,
It is manufactured by adding magnesium stearate and compressing it into tablets, and then applying an enteric coating to the tablets.

Example 4 Enteric-coated granules 10 mg of serratiopeptidase, 10 mg of 4'-amidinophenyl cis-4-isopropylcyclohexanecarboxylate and sucrose palmitate
Mix 200 mg of the product, add lactose, starch, and hydroxypropyl cellulose to make granules, and coat the granules with enteric coating.

Example 5 Enteric coated sugar-coated tablets 20 mg of 4'-amidinophenyltrans-4-isopropylcyclohexanecarboxylate and 50 mg of polyoxyethylene sorbitan monooleate were mixed with 10 mg of serratiopeptidase, and appropriate amounts of white sugar, starch, and hydroxypropyl were added to the mixture. It is manufactured by adding cellulose and magnesium stearate and compressing it into tablets, and applying enteric coating and sugar coating to the tablets.

[Brief explanation of the drawing]

Figure 1 shows the absorption rate when a protease and a hydroxybenzoic acid derivative or amidinophenol derivative are used together, and Figure 2 shows the absorption rate when a surfactant is used in addition to the two substances.

Claims (1)

[Scope of Claims] 1. An easily absorbable enzyme preparation characterized by containing a protease and a trypsin and/or chymotrypsin inhibitor. 2 Trypsin and/or chymotrypsin inhibitor has the following formula () or () (In the formula, R ₁ represents a benzoyl group, an indole acetyl group, or a naphthylacetyl group that may have a substituent, R ₂ represents a lower alkyl group, and A represents a single bond or a linear or branched chain. represents an alkylene group, R represents a hydrogen atom, a lower alkyl group, an acylaminoalkyl group, or two R may be joined together to form a ring, and n
represents an integer of 1 to 3) The easily absorbable enzyme preparation according to claim 1, which is a hydroxybenzoic acid derivative or an amidinophenol derivative represented by the following. 3. The easily absorbable enzyme preparation according to claim 1, wherein the trypsin inhibitor is gabexate. 4. The easily absorbable enzyme preparation according to claim 1, which further contains a surfactant. 5 Trypsin and/or chymotrypsin inhibitor to 1 protease by weight,
The easily absorbable enzyme preparation according to claim 1 or 4, which has a molecular weight of 0.1 to 10. 6. The easily absorbable enzyme preparation according to claim 4 or 5, wherein the weight ratio of surfactant to 1 part protease is 0.1 to 20.