JPH0441430A - Endothelin converting enzyme inhibitor - Google Patents

Abstract

PURPOSE:To obtain the subject inhibitor, containing phosphoramidon or salts thereof and useful as a treating and/or preventing agent for various diseases caused by excessive production of endothelin. CONSTITUTION:An inhibitor containing phosphoramidon, produced by actinomyces such as Streptomyces.tanashiensis, known to have powerful inhibitory action on a proteolytic enzyme thermolysin produced by Bacillus.thermoproteolyticus and expressed by the formula or salts thereof. The aforementioned compound is capable of suppressing excessive production of endothelin and antagonizing vascular and tracheal muscular contracting action in which the endothelin participates and useful as a remedy for human a hypertension, bronchial asthma, acute renal insufficiency, cardiac infarction, angina pectoris, cerebral infarction, cerebrovascular twitch and acute insufficiency after organ transplantation.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of the compound represented by the formula (phosphoramitone) or a pharmacologically acceptable salt thereof as an endothelin converting enzyme inhibitor.

Endothelin (hereinafter abbreviated as ET) is a polypeptide consisting of 21 amino acids, produced in vascular endothelial cells and various tissue cells of humans and various mammals, and has a strong smooth muscle contractile effect. . In particular, the vascular smooth muscle contraction effect is
It is the most powerful and long-lasting substance known. E.T.
The production mechanism is that preproendothelin is first biosynthesized and then processed within the cell to become big endothelin (
(hereinafter abbreviated as bET), and then Trp”-V of bET
It is said that ET is generated by hydrolyzing the "al" bond by endothelin converting enzyme (hereinafter abbreviated as ETCE). ET, which has a strong smooth muscle contraction effect, is widely distributed in various tissues in the body and plays a role in the homeostasis of the body. At the same time, overproduction of ET is associated with hypertension, ischemic heart failure, myocardial infarction, angina pectoris, acute renal failure, cerebral vasoconstriction after subarachnoid hemorrhage, cerebral infarction, bronchial asthma, and organ transplantation. It is considered to be one of the causes of subsequent acute failure, etc. Therefore, suppressing the overproduction of ET is considered to be essential for the prevention and treatment of the various diseases mentioned above, and the emergence of an ETCE inhibitor is eagerly awaited.

However, there are still no ETCE inhibitors, let alone ETCE inhibitors.
Its true nature is not clear.

The problem to be solved by the present invention is to find an inhibitor of ETCE.

Based on the above background, the present inventors first collected vascular endothelial cells from bovine carotid arteries, cultured them artificially, and discovered ETCE from the cultured cells. Next, we tested various compounds to find inhibitors for this ETCE, and found that
We found that phosphoramitone exhibits strong inhibitory activity.

Phosphoramiton is produced by Streptomyces tanashiensis.
[Tetrahedron Letters, Vol. 1, Sections 97-100 (1972)] and Bacillus thermoproteolyticus.
It is known to have a strong inhibitory effect on the protein enzyme thermolysin produced by P. moproteolyticus [Biochemical and Biophysical Research Communications (Biochemical andB.
iophysical Re5earch Commu
nications) Vol. 65, pp. 352-357 (
(1975)].

However, the E'TCE inhibitory effect of phosphoramitone has not been known at all.

As a result of extensive research, the present inventors have found that phosphoramitone or its pharmacologically acceptable salt has an excellent ETCE inhibitory effect, and has been found to be effective in various diseases caused by overproduction of ET, such as hypertension and ischemic heart failure. A pharmaceutical compound useful as a therapeutic and/or prophylactic agent for myocardial infarction, angina, acute renal failure, cerebral vascular zero line after subarachnoid hemorrhage, cerebral infarction, bronchial asthma, acute failure after organ transplantation, etc. They discovered something and completed the present invention.

Examples of biochemical and pharmacological experiments are described below to demonstrate that phosphoramitone is an excellent inhibitor of ETCE.

l ETCE's `` Endothelial cells were scraped from a freshly excised bovine carotid artery and dispersed using Tribun Digestion Method I. The cells were then cultured in the usual manner, and 2.6 x 101c of cultured cells were crushed using a glass homogenizer. The crude membrane fraction was collected by centrifugation at 10,000 × g and then sonicated at 105,00 × g.
A precipitate (microsome fraction) was obtained by centrifugation at 0xg. ETC
E activity is derived from synthetic human bET-1 (one of the three bETs)
The ET produced by reacting with the enzyme at neutral pH as a substrate
l (ET produced from bET-1) was measured by high performance liquid chromatography (hereinafter referred to as HPLC) and radioimmunoassay (hereinafter referred to as RIA). As a result, ETCE activity was found in the microsomal fraction mentioned above, and 0.5% Triton
-100 treatment solubilized ETCE from the microsomal fraction. This solubilized ETCE fraction contains 264 mg of protein and its specific activity is 3.7 nmol ET/m
g protein/hour. The optimum pH for the enzyme reaction is around 71, and the enzyme activity was significantly reduced at p) below 16.6 or above pH 7.6.

That is, this enzyme has the remarkable feature of being active only in an extremely narrow neutral pl region.Also, since it was completely inhibited by divalent metal chelating agents such as EDTA and phenanthroline, this enzyme This enzyme was found to be a type of metalloprotease with a metal coordinated to its active center.The molecular weight of the enzyme determined by gel filtration was approximately 100,000.

2. ETCE of Phosphoramine Based on the knowledge that ETCE obtained in Space Experiment Example 1 is a type of metalloprotease, we investigated ETCE using various metalloprotease inhibitors.
When the inhibitory effect was investigated, phosphoramitone inhibited ETCE.

That is, ETCEII μg and 1 μM bET-1 were added to 05M Tris-HCl buffer (
ET at 37°C for 190 minutes in pH 7.2).
The amount produced was quantified by RIA method, and ETC of phosphoramiton was determined.
E inhibitory activity was measured. From the results shown in Table 1, the 50% inhibitory concentration of phosphoramiton against ETCE is approximately 1 μM.
Met.

After extracting a pig's coronary artery, a spiral specimen with a width of 1 mm and a length of 10 mm was prepared. After removing endothelial cells, 95%02
．． It was suspended in a 5++1 Magnus tube filled with Krebs-Henseleit liquid saturated with a 5% CO2 gas mixture, and changes in tension were measured and recorded isometrically. 30nM bET-1
The effect of 1 mM of phosphoramitone on the contraction obtained by adding it into the Magnus tube was investigated. Note that phosphoramiton was added to the Magnus tube 60 minutes before the addition of bET-1.

As shown in Figure 1, phosphoramiton (1mM)
-1-induced vasoconstriction was almost completely suppressed.

A male Wistar Kyoto rat was anesthetized with bentoparbital sodium, and cannulae for blood pressure measurement and drug administration were inserted into the femoral artery and femoral vein, respectively. One day after the surgery, blood pressure and heart rate were measured using a pressure transducer without anesthesia or restraint. Note that the heart rate was measured using a heart rate monitor based on the pulse wave of blood pressure. b E T −12 nmol
The effects of 1-10 mg/kg of phosphoramitone on changes in blood pressure and heart rate induced by intravenous administration of 1-10 mg/kg of phosphoramitone were investigated. Note that phosphoramitone was administered intravenously 3 minutes before bET-1 administration.

As shown in Figure 2-3, phosphoramitone 1-10 mg/
kg dose-dependently suppressed the pressor response of bET-1 (Fig. 2) and the decrease in heart rate (Fig. 3). In particular, 10 mg/kg of phosphoramitone almost completely suppressed the above reaction.

A male guinea pig (bET-1) was anesthetized with urethane and alphafloralose, and artificial respiration was performed using a tracheal cannula. The animal's spontaneous respiration was abolished by succinylcholine.

Blood pressure was measured by connecting a cannula inserted into the common carotid artery to a pressure transducer. Changes in airway resistance were measured using the lateral pressure of the endotracheal cannula as an index according to the method of Conceto and Ressler. b The influence of 10 mg/kg of phosphoramitone on the pressor response and increase in airway resistance induced by intravenous administration of ET -14 nmol/kg was investigated. Note that phosphoramitone was administered intravenously 2 minutes before bET-1 administration.

Phosphoramiton 10mg/kg as shown in Figure 4-5
almost completely inhibited bET-1-induced pressor response (Fig. 4) and increase in airway resistance (Fig. 5).

As revealed by the above biochemical and pharmacological tests, phosphoramitone or its pharmacologically acceptable salts have excellent ETCE inhibitory effects, which can be used to treat various diseases caused by overproduction of ET, such as hypertension, Useful as a therapeutic and/or prophylactic agent for ischemic heart failure, myocardial infarction, angina pectoris, acute renal failure, cerebral vascular zero line after subarachnoid hemorrhage, cerebral infarction, bronchial asthma, acute failure after organ transplantation, etc. It is a pharmaceutical compound.

Phosphoramiton, which is the active ingredient of the present invention, can be administered as a free form or as a pharmacologically acceptable salt thereof. A pharmacologically acceptable salt can be easily produced by reacting phosphoramitone with a pharmacologically acceptable basic compound. Examples of the basic compound include sodium hydroxide, potassium hydroxide, carnoum hydroxide, sodium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, and the like.

Phosphoramiton or a pharmaceutically acceptable salt thereof can be administered either orally or parenterally.

The dosage form may be a solid preparation such as a tablet, gunpowder, powder, capsule, or granule, or a liquid preparation such as a solution, suspension, emulsion, or dispersion. Furthermore, when administered parenterally, it can also be used as an injection or a drip injection.

Furthermore, phosphoramitone or a pharmaceutically acceptable salt thereof can also be used in combination with a suitable pharmaceutical carrier. Examples of pharmaceutical carriers include gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, lactose, sugar,
Examples include corn starch, crystalline cellulose, potassium phosphate, glycine, magnesium stearate, talc, polyethylene glycol, potato starch, and sodium lauric sulfate.

The dosage of phosphoramitone or its pharmacologically acceptable salt varies depending on the type of disease, patient age, weight, severity of symptoms, route of administration, etc., but is usually 0.5 per day for adults, especially for oral administration. ~100n+g/kg, preferably 1-50+ng/kg is suitable.

(Left below) Example 1 (Tablet) Phosphoramiton 450g Corn starch 20.1g Lactose
82.4g polyvinylpyrrolidone 3.
0g crystalline cellulose 38.0g magnesium stearate 1.5g total
190 g of phosphoramitone, lactose, corn starch, and crystalline cellulose are mixed, an alcoholic solution of polyvinylpyrrolidone is added thereto, kneaded and granulated, and the mixture is sized after drying. Next, add magnesium stearate and use a tablet machine to make tablets with a diameter of 8 m.
It was made into a tablet with a weight of 190 mg.

Example 2 (injection) 10 g of phosphoramitone is dissolved in 2β of distilled water for injection.

After filtering this solution with a membrane flan filter with a pore size of 0.22 μM, the solution was filtered to 2 m7 using aseptic operation. Dispense into ampoules and seal them to make an injection.

Example 3 (powder) Phosphoramiton 10g lactose
90g0g total
Mix 100g of the above ingredients uniformly in a V-type mixer to make 1
Obtain 0x variance.

The endothelin converting enzyme inhibitor of the present invention containing phosphoramitone or a pharmacologically acceptable salt thereof can be used as a drug that antagonizes the vascular and tracheal muscle constriction effects associated with endothelin, and can also be used to treat hypertension in humans. It is useful as a therapeutic agent for bronchial asthma, acute renal failure, myocardial infarction, angina pectoris, cerebral infarction, cerebrovascular curvature, and acute failure after organ transplantation.

[Brief explanation of the drawing]

FIG. 1 shows the effect on big endothelin-1 contraction in porcine coronary artery specimens in the absence (•) and presence (0) of phosphoramitone. Figure 2 shows phosphoramitone 1 mg/kg (
(II) and 10 mg/kg (II). Figure 3 shows phosphoramitone 1 mg/kg on big endothelin 1-induced heart rate decrease (○) in rats.
(Mu) and 10 mg/kg (厘). Figure 4 shows changes in big endothelin-1-induced blood pressure in guinea pigs (○) of phosphoramitone 10n+g.
/kg (・). Figure 5 shows the change in airway resistance induced by ping endothelin-1 in guinea pigs (○) when 10 m of phosphoramitone was used.
Indicates the effect of g/kg (・).

Claims (1)

[Claims] (1) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ Endothelin converting enzyme inhibitor containing the compound represented by [I] (phosphoramidon) or its salt