JPS6151564B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an angiotensin converting enzyme inhibitor having the following structure. Thr-Thr-Met-Pro-Leu-Trp Conventionally, in vivo enzyme inhibitors found in actinomycete culture filtrates have various effects such as anti-inflammatory, anti-peptic ulcer, and anti-cancer effects, and are used as pharmaceutical agents. It has also been expected to be used as a research reagent. Regarding angiotensi converting enzyme inhibitors, several types of inhibitors produced by microorganisms have recently become known, including peptide inhibitors obtained from Brazilian snake venom and Japanese snake venom and gelatin inhibitors. It has been known for some time that this product was isolated from a solution treated with collagenase derived from a microorganism. In addition, the Squibb Company of the United States has synthesized captopril, a proline derivative, which has a strong inhibitory effect and is attracting attention as a new orally available drug. However, since all of these inhibitors are expensive, it is desired to develop inhibitors derived from natural products that can be obtained at low cost and have fewer side effects. On the other hand, the present inventors had previously succeeded in obtaining an angiotensin converting enzyme inhibitor by decomposing cow-derived casein with trypsin etc.
58-109425, JP-A-59-44323, JP-A-59-
44324). This time, the present inventors succeeded in preparing a new angiotensin converting enzyme inhibitor having the above structure by treating bovine casein with trypsin etc. as in the previous case. The inhibitor of the present invention exhibits an inhibitory effect on angiotensin converting enzyme. In this case, angiotensin converting enzyme converts angiotensinogen, which is secreted by the liver, into angiotensin (Asp-Arg-Val
－Tyr－Ile－His－Pro－Phe－His－Leu), which acts on angiotensin (Asp－Arg－Val－Tyr－Ile－His－Pro－Phe)
convert it to This angiotensin () causes the smooth muscle of the blood vessel wall to contract, increasing blood pressure, and also causes the smooth muscle of the gastrointestinal tract and uterus to contract in addition to the blood vessels, and also acts on the adrenal cortex to promote the secretion of aldosterone. It has the effect of causing Additionally, the enzyme kallikrein present in plasma degrades a protein called kininogen to produce pradikinin, which dilates blood vessels and lowers blood pressure, but this pradikinin is degraded and inactivated by the action of angiotensin converting enzyme. Thus, angiotensin converting enzyme produces, on the one hand, a pressor peptide (angiotensin) and, on the other hand, degrades an antihypertensive peptide (pradykinin), resulting in an increase in blood pressure. The inhibitor according to the present invention has an inhibitory effect on angiotensin converting enzyme that exhibits such an effect,
It is particularly effective as a hypotensive agent. To obtain an angiotensin converting enzyme inhibitor according to the present invention, bovine casein is decomposed with trypsin under conditions of pH 5.5 to 9.0, and the decomposed product is treated with hydrochloric acid to precipitate trypsin and undegraded casein. Remove with filter paper etc. The filtrate thus obtained is neutralized with an alkali such as sodium hydroxide and then purified to obtain a product. The present inhibitor can also be obtained using conventional peptide synthesis methods. That is, the amino group of one amino acid is protected with a benzyloxycarbonyl group or t-butoxycarbonyl group, the carboxyl group of the other amino acid or peptide is protected with a benzyl ester, etc., and DCC (N,N'-dicyclohexylcarbodiimide) is protected. ) etc. to couple. By repeating this operation, the protecting group is removed and the product can be purified and obtained. The property of the inhibitor according to the present invention at room temperature is a white powder, and the elution pattern of its aqueous solution by high performance liquid chromatography (reversed phase column, phosphate buffer - acetonitrile elution) is as shown in Figure 1 below. , by thin layer chromatography
The Rf values are shown in Table 1 below. Further, by dissolving it in 6M hydrochloric acid and performing hydrolysis at 110°C for 24 hours under vacuum, an amino acid mixture having the composition shown in Table 2 below can be obtained. The method of ingesting the angiotensin converting enzyme inhibitor of the present invention is generally performed by intravenous injection, for example,
An aqueous solution of the inhibitor is intravenously injected at a concentration of 0.01 to 1 mg of the inhibitor per 1 kg of animal. The angiotensin converting enzyme inhibitor according to the present invention can be applied to mammals that have the enzyme endogenously in their bodies, such as humans, rats, and dogs. Next, the present invention will be explained in more detail with reference to Examples. (Example) 80 g of bovine casein was suspended in 2 0.04 M phosphate buffer (PH7.4), 200 mg of trypsin (produced by PL Biochemicals, derived from pancreas) was added,
React for 15 hours at 37°C with stirring. After the reaction,
Add 100 ml of concentrated hydrochloric acid to the product to precipitate trypsin and undegraded casein. After removing this precipitate with filter paper, add sodium hydroxide to the filtrate and pH
Neutralize to 3.0 and remove the precipitate again with filter paper.
Further sodium hydroxide is added to this filtrate to neutralize it and adjust the pH to 7.0. Next, this solution is added to a Sephadex LH-20 column and purified by elution with distilled water. Then, the maximum active fraction at this time is collected and concentrated under reduced pressure. Note that the column processing conditions in this case are as follows. Column: Height 130 cm, inner diameter 5 cm Sample addition amount: 200 ml Flow rate: 120 ml/hr Elution: Distilled water Next, the active fraction fractionated with the Sephadex LH-20 was added to an ion retardation resin (AG11A8 manufactured by Bio-Rad). ) Add to the column and elute with distilled water. The active fractions are collected and concentrated under reduced pressure. Note that the column processing conditions in this case are as follows. Column: height 63 cm, inner diameter 4 cm Sample addition amount: 200 ml Flow rate: 100 ml/hr Elution: distilled water Next, the active fraction obtained above was added to the Sephadex LH-20 column again and rechromatized. The column processing conditions in this case are the same as above, and when the active fraction present at the elution volume of 1.8 is lyophilized, a white powder substance is obtained (approximately 85 mg is obtained from 80 g of casein). Next, the Rf value of this substance was determined by thin layer chromatography (silica gel plate, detection by UV absorption and ninhydrin color development), and it was as follows.

[Table] Next, we looked at the elution pattern of the substance of the present invention in liquid chromatography, and it was as shown in Figure 1.
The elution conditions are as follows. Column: Waters Radial Pack Cartridge C-18 for reversed phase Eluent: Phosphate buffer (10mMKH ₂ PO ₄ ,
50mM Na ₂ SO ₄ ) PH3.0: Acetonitrile (2:1, V/V) Flow rate: 1ml/min Detection: Ultraviolet absorption at 210nm In addition, the substance of the present invention was dissolved in 6M hydrochloric acid and heated at 110°C under vacuum for 24 hours. After heating for a period of time, analysis was performed using an amino acid analyzer, and the following results were obtained.

[Table] Next, when the substance of the present invention was treated with carboxypeptidase A, Trp (tryptophan) was further detected. Next, the primary amino acid sequence of the substance of the present invention was determined by an amino acid analyzer using the Edman degradation method and leucine aminopeptidase, carboxypeptidase A, and carboxypeptidase Y, and it was confirmed that it had the following structure. Thr-Thr-Met-Pro-Leu-Trp Next, the following experiment was conducted to measure the enzyme inhibitory activity of the substance of the present invention. First, 5g of Rabbittran Langacetone powder was mixed with 50ml of 0.1M sodium borate buffer (PH=
8.3) and centrifuged at 40000 g for 40 minutes, and the supernatant was further diluted 5 times with the above buffer to obtain an angiotensin converting enzyme solution. Put 0.03ml of the sample containing the substance of the present invention into a test tube,
To this was added 0.25 ml of hyperlylhistidylleucine (final concentration 5 mM, including 300 mM NaCl) as a substrate, and the mixture was allowed to react at 37°C for 30 minutes. Then 1N
After stopping the reaction by adding 0.25 ml of hydrochloric acid, 1.5
ml of ethyl acetate was added, and the absorption value at 228 nm of hyperlic acid extracted in ethyl acetate was measured, and this was taken as the enzyme activity. Note that under these conditions, the absorption value at 228 nm when the present inhibitor is not included is approximately 0.25. A plurality of such experiments were conducted, and the inhibition rate was calculated using the following formula. Inhibition rate = AB/A x 100 (%) A: 228nm absorption value without inhibitor (0.25) B: 228nm absorption value with inhibitor addition And inhibition when inhibition rate is 50% When the drug concentration ID ₅₀ was determined, it was 1.6×10 ⁻⁵ M for the inhibitor of the present invention.

[Brief explanation of the drawing]

FIG. 1 shows the analysis results of a purified angiotensin converting enzyme inhibitor by high performance liquid chromatography, with the vertical axis showing the intensity of ultraviolet absorption at 210 nm and the horizontal axis showing the elution time (minutes).

Claims (1)

[Claims] 1. An angiotensin converting enzyme inhibitor consisting of the following structure. Thr―Thr―Met―Pro―Leu―Trp