JPH0220263A - Composition for oral intake - Google Patents

Abstract

PURPOSE:To provide the title composition excellent in safety and vasodepressor activity, made up of, as an active ingredient, a peptide (or its acid-added salt) represented by Thr-Thr-Met-Pro-Leu-Trp. CONSTITUTION:The objective composition for oral intake (e.g. food, drug) for vasodepressor use, made up of, as an active ingredient, a peptide or its acid- added salt (e.g. hydrochloride) represented by Thr-Thr-Met-Pro-Leu-Trp. The above-mentioned peptide, being well-established, can be prepared, for example, by the following process: casein of bovine origin is degraded at a pH 5 - 9 by trypsin, and from the resulting degraded product undegraded casein is precipitated and removed by heating or acid treatment followed by concentration of the mother liquor and then purification; alternatively, it can be prepared by use of an organic chemical synthetic technique.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to an orally ingestible composition (food or medicine) for lowering blood pressure.

(Prior art) Hypertension is one of the diseases with the highest mortality rate in Japan today, and its treatment or prevention has become an urgent and important issue.

Hypertension includes secondary hypertension and essential hypertension, and the onset and pathology of the former, such as wild hypertension or endocrine hypertension, and the latter, essential hypertension, are both related to blood levels. It is well known that the active pbetide production system, especially the renin-angiotensin system, is deeply involved.
otensinConvert,irrg,Hnzy
me, hereinafter referred to as ACB) exists,
This enzyme produces an active peptide (angiotensin ■) that has a smooth muscle contraction effect on blood vessel walls, resulting in a strong increase in blood pressure.

Therefore, if this enzyme activity is inhibited, it may be possible to suppress the increase in blood pressure (lower blood pressure), and from this point of view, the search for inhibitors of various natural and synthetic substances is progressing, and synthetically synthesized substances have already been investigated. As for substances, certain types of proline derivative compounds have been recognized for their effectiveness and are put into practical use as antihypertensive agents.

On the other hand, ACE inhibitors derived from natural products have been found to have inhibitory activity in the decomposition products obtained by hydrolyzing gelatin with bacterial collagenase, and recently, ACE inhibitors have been found in the decomposition products of Ushiyama's casein with tribucin. It has been recognized that inhibitory peptides exist, and they have been isolated and purified (see JP-A-581094, No. 25). These natural ACE inhibitors are obtained from foods or food raw materials. , can be a low-toxic and highly safe antihypertensive agent, but in addition to the casein-derived inhibitory peptide F-4; Since it is expected that it can be mass-produced easily and at low cost, its practical use as an antihypertensive agent is being investigated.

However, bioactive peptides are generally hydrophilic and have a relatively high molecular weight, so they are poorly absorbed from the gastrointestinal tract, and in some cases, they may be degraded by gastrointestinal fluids and lose their activity. The route is often limited to intravenous administration by injection, and the casein-derived A
As is the case with CE-inhibiting peptides, the effectiveness of intravenous administration has heretofore been investigated, but the blood pressure-lowering effect was insufficient.

(Purpose of the Invention) The present inventors have developed a highly effective antihypertensive drug that has a high degree of safety.
As a result of intensive research to find an ingestible composition, we have completed the present invention, which is surprisingly effective when administered orally and is superior to intravenous administration.

(Structure of the Invention) That is, the present invention has the following structural formula (+) T h -Th r -M (!t -P r
The present invention relates to an orally ingested composition for lowering blood pressure, which contains a peptide represented by o -L e -T r p (I) or its acid salt as an active ingredient.

Preferred examples of the acid addition salt include pharmaceutically acceptable salts, such as hydrochloride, sulfate, succinate, citrate, and tartrate.

As mentioned above, it is considered extremely difficult for bioactive peptides to exhibit effective bioactivity by oral administration due to reasons such as hydrolysis and inactivation by gastrointestinal fluids or poor absorption from the gastrointestinal tract. ing.

Considering these facts, it is completely unexpected that the peptide represented by the structural formula (I) used in the present invention exhibits a strong antihypertensive effect even when administered (ingested) orally, as shown in test examples later. This has only become clear through the present invention. Furthermore, the previously attempted intravenous administration was not able to be put to practical use as a treatment for hypertension due to its insufficient blood pressure lowering effect.
The present invention has great significance in that it has become possible to put it into practical use for the first time.

Peptyl" used in the present invention is already known and is a partial structure of bovine casein, and can be obtained by tryptic hydrolysis of the protein. Alternatively, an Iqa chemical synthesis method can be used.

Peptide (I) is prepared, for example, as follows. Ushiyama casein is decomposed with trypsin under conditions of pH 5.0 to 9.0, and the decomposed product is subjected to heat treatment or acid treatment to stop the reaction and to precipitate and remove undecomposed casein. Next, if necessary, the mother liquor is neutralized with an alkali, concentrated 2 to 3 times under reduced pressure, and 1. , H
-v7A, etc., and eluted with distilled water.
Both fractions showing Cr3 inhibitory activity are collected, and if necessary, the same purification is repeated to obtain peptide (I). Further, an example of a method for synthesizing peptide (I) is shown below.

(C, Chemical Synthesis Method) An example of the synthesis method is shown below. Here, peptide synthesis is performed using the in-phase symmetric acid anhydride method using polystyrene resin as an insoluble carrier. Note that all amino acids herein refer to the L-configuration, and the abbreviations of the protecting groups of amino acids represent the following residues.

Boc: tert, l1utyloxy-ca
rbonyl group PAM: p-methox
y phenyl acetamide
l resinBzl: Henzyl group Z: Bundyloxycarbonyl group Tos: p-tolylsulfonyl group AA": nth amino acid AA'(AA'PA) cross-linked to polystyrene resin
H-AAI by deprotecting M) with TFA
-PAM is synthesized, BocAA''-01-1 is condensed with DCC in dichloromethane and dimethylformamide, BocAA2-AA' -PAM is synthesized, and PAM is converted to unreacted A using acetic anhydride. Capping.

The obtained BoC-AA''-AA'-PAM was again subjected to deprotection reaction using TFA, and B
o c-AA”-OH is condensed, and in the same manner, AA
The condensation reaction is carried out up to 6. Note that the functional groups of the amino acid side chains used are blocked as shown below.

Thr(Bzzri, Met(SuIlfoxide)
), Pro (none) Trp (Formy Il)
+ Leu (none) After the condensation reaction, a deprotection reaction was performed using HF to remove 13oc, PAM, and side chain protecting groups, and H-Thr-Thr-Met-Pro-L
Obtaining eu-Trp When the orally ingested composition of the present invention is used as a medicine for the treatment or prevention of hypotension,
The peptide of structural formula (I) is prepared in the form of a preparation for oral administration together with a pharmaceutically acceptable carrier (excipient, lubricant, binder, coloring agent, flavoring agent, excipient, etc.) according to a conventional method. For example, it is administered orally in the form of tablets, capsules, troches, powders, fine granules, granules, etc.

On the other hand, when used as a health food, it may be in the form of an oral preparation similar to the above, or in the form of a solid or liquid food or luxury item (for example, sweets, powdered tea, alcoholic drinks, sports drinks, etc.). And it is sufficient. The blending amount varies depending on the dosage form, but generally O, 1 to 30%.
is preferred.

Doses generally range from 0.1 mg to 50 mg per day for adult males.
The range is 0 mg/kg body weight, and administration from a range of
An appropriate amount is selected depending on the purpose of intake (intake). In addition, the intake may be done at once or divided into several doses.

Examples of the production of peptide (I), which is the active ingredient of the oral composition of the present invention, and the results of animal experiments (hypertensive lowering test and acute toxicity test) are listed below.

In addition, the activity (I D s.) of the ACE inhibitory peptide is
It was measured by the following method.

(Measurement of ACE inhibitory activity of ACE inhibitory peptide) i) Preparation of angiotensin converting enzyme solution (ACE solution) 5 g of rabbit anguanand powder (manufactured by Sigma)
50 m It of 0.1'M borate buffer (+) 1
18.3), centrifuged at 40,000xg for 40 minutes, and the supernatant was further added with the above buffer.
It was diluted 10 times to obtain an angiotensin converting enzyme solution.

ii) Place 0.03 ml of the activity measurement sample into a test tube, and add 250 μl of Hyplylu-1-Histidyl-Leucine [manufactured by Sigma, Co., Ltd., final concentration 5] as a substrate.
Contains 300 mM NaC#. ] was added, and after incubation at 37°C for 10 minutes, 0.1 ml of the above enzyme solution was added and reacted at 37°C for 30 minutes. Then IN hydrochloric acid 0.25 m j! After stopping the reaction by adding 1
．． 5 m 11 of ethyl acetate was added and stirred vigorously for 15 seconds. Then, centrifuge at 3.50 rpm for 15 minutes to form a 1 m7 layer of ethyl acetate. was collected. The ethyl acetate layer was heated at 120° C. for 30 minutes to remove the solvent. After removing the solvent, distilled water 1mA! was added, and the absorption of the extracted hyperlic acid (absorbance at 228 nm) was measured, which was taken as the enzyme activity.

The inhibition rate was calculated using the following formula.

Inhibition rate - (A-B)/AX 100% A: Absorbance at 2'28 nm when no inhibitor is included B: Absorbance at 228 nm when inhibitor is added and the enzyme reaction solution when the inhibition rate is 50% ID, the inhibitor concentration in. shall be.

<Production Example 1> 320 g of milk casein and L-lipsin (PLBioch
Made by em, EC, 3, 4, 21, 4) 800m
g of 0.04 M phosphate buffer (p I-17,4)
81 and reacted at 37°C for 15 hours. concentrated hydrochloric acid 4
Add 00m+2 to stop the reaction and eliminate the formed precipitate. f) Neutralize the furnace liquor to H7 using N a OIf.

200 mj2 of the tr=solution was applied to a Sephadex L H~2o column (5 x 130 cm), both fractions having A' CB inhibitory activity were collected, and this was repeated three times. This was applied to a Bio-Rad AC-11A8 column (4 x 63 cm), fractionated and concentrated, and then applied to a Sephadex L H-20 column (5 x 130 cm and 3 x 105 cm).
A CE inhibitory activity fraction peptide (I) is obtained.

The ■D5o of peptide (I) was determined to be 16 μM.

Production Example 2 Next, a test example regarding the antihypertensive effect of the synthetically obtained peptide of structural formula (I) will be shown.

Test Example 1 Laso I/Metra 1-cho of the peptide of structural formula (I)
Antihypertensive effect after 11 doses (I) Test method 12-week-old male spontaneously hypertensive rats (Japanese Charles
River Inc.) 15 animals were placed at a temperature of 23±2°C and a humidity of 55±5.
The mice were housed in a small animal room and allowed free access to water and feed (MF, manufactured by Oriental Yeast Co., Ltd.). After the rats were acclimatized for two weeks while having their blood pressure measured once a week,
The animals were subjected to experiments when they developed hypertension.

The test specimen was administered in L1 by dissolving the sample in distilled water (prepared immediately before administration) and adjusting the dose to 130 mg/kg body weight.

Blood pressure was measured using non-heated, non-invasive rat blood pressure a1 (manufactured by Toiden, DSR80) before and 3 hours after administration.
1A), the systolic blood pressure value of each rat was continuously measured 10 times by the tail-cuff method, and the average value was determined. The results are shown as the average value of 5 animals per group.

(2) Test results The results are shown in Table 1.

Table 1 Blood pressure (mmHg) Immediately before administration (204) 2 hours after administration (I81) 〃4' (I99) As is clear from the results in Table 1, the peptide of structural formula (I) showed significant It is found that it has a hypotensive effect.

Test Example 2 Acute toxicity test (I) Sample A 10% aqueous solution of Vepti F of structural formula (I) was used as a sample.

(2) Experimental animals Animals: ICR mice (Kuchimoto Yura) Number of samples: 20 males and 20 females Weight at start of test: Males 24-26g Females 22-24g Rearing conditions during the period: ? ! A degree 22±2℃ Humidity 50±5
% Solid sample (CB-2, Nippon Talea) Ad libitum tap water (3) Test method After preliminarily rearing the animals for one week, they were subjected to experiments in groups of 10 animals. After fasting for 16 hours before administration, the experimental group received the maximum possible dose of 3 g of sample (30 ml/kg of I0% aqueous solution, and the control group received 30 mff/kg of tap water, each forcibly administered orally using a gastric probe. .

For 7 days after administration, the animals were observed daily for survival and general symptoms.

(4) Test results The LDso of the peptide of structural formula (I) was 3 g/kg or more, and there was no death of any animals. General symptoms such as weakness, wasting, collapse, crouching, crawling on stomach, lying on the side, change in body hair color, change in skin temperature, sweating, piloerection, hair loss, contamination of hair, increase/decrease in respiratory rate, irregular breathing, wheezing, etc. were also observed, but nothing was observed. There was no change.

From this result and the fact that it is derived from casein, a food ingredient, it is clear that the peptide of structural formula (I) of the present invention is extremely safe.

(Effects of the Invention) The present invention has made it possible to provide an orally ingestible composition (food or medicine) for lowering blood pressure that is highly safe and highly effective.

Examples will be described in detail below.

In addition, all parts in the examples mean parts by weight.

Example 1 Tablet [Composition] Peptide of structural formula (I) 20.0 parts Hydroxypropyl methyl cellulose 5.0 milk'r
*
6 1. 5 Sucrose
0.05 Carboxymethyl cellulose 7.5 Magnesium stearate 1.0 Starch glycolic acid sorter 5.0 Peptide of structural formula (I) 20.0 parts, milk F' 61.5
1 part, 0.05 part of sucrose, and 7.5 parts of carboxymethylcellulose were suspended in 25 parts of a 70% ethanol aqueous solution containing 5 parts of hydroxypropylmethylcellulose, kneaded, and vacuum-dried to obtain a dried product. To this dried product, 1.0 part of magnesium stearate and 5.0 parts of sodium starch glycolate were added, and tablets (I tablets 40
0mg) was prepared.

Example 2 Lozenge [Composition] Peptide of structural formula (I) 5.0 parts milk
sugar
50.0 蔗 @
3 9. 8 Tragacanth powder 5.0 Peppermint oil 0.2 Lactose 50.0 parts, Potato 17! 39.8 parts of powdered tragacanth, 5.0 parts of powdered tragacanth, and 0.2 parts of pevermin l-0.
A solution prepared by dissolving 5.0 parts of Peptide A in 3.5 parts of distilled water was added and mixed well.

Next, the above-mentioned mixture was spread with a rolling pin onto a glass plate sprinkled with starch to form a sheet with a thickness of about 5 mm, and then
It was punched out with a mold and dried to give a lozenge (I, 0 g/piece).

Example 3 Ice cream skimmed milk powder 8.0% vegetable fat 10.0 sand I!
13.0 stabilizer
0.3 Emulsifier
0.3 Vanilla flavor - 0.1 The peptide egg yolk water of Production Example 1 was prepared by the usual production method.

Example 4 Yogurt milk whole milk skimmed milk powder Granulated sugar water Peptide of Production Example 1 egg yolk It was created using a normal manufacturing method.

0, 1 12.4 55.8 (I0.0g/cup) 64.0 4, 0 5.0 7.0 1 0, 0 0, 1 9.9 (I0,0g/cup) =17

Claims (1)

[Claims] An orally ingestible composition for antihypertensive use comprising a peptide represented by the following structural formula (I) [Gene sequence is available] (I) or an acid addition salt thereof as an active ingredient.