JPS5877892A - Renin inhibitor - Google Patents

Abstract

PURPOSE:The titled substance, recoverable from a human placenta, having a specific molecular weight and isoelectric point and specific inhibitory action on renin without inhibiting trypsin, plasmin, etc., and useful as a remedy for renin- angiotensin exasperation, e.g. hypertension. CONSTITUTION:A precipitate fraction obtained by fractionating an extract with an aqueous solvent of a human placenta with 40-50% ammonium sulfate is adsorbed in an anionic exchanger, and the adsorbed substance is then subjected to the gel filtration to recover the aimed substance, having 65,000-75,000 molecular weight measured by the disk electrophoretic method with sodium dodecyl sulfate (SDS)-polyacrylamide gel and 4.6-5.2pH isoelectric point, and capable of specifically inhibiting renin without inhibiting trypsin, plasmin, callicrein and urokinase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a renin inhibitor that has a specific inhibitory effect on renin from human placenta, and a method for producing the same.

Renin is proteolytic oxygen, +1[I plasma α, -
It acts on angiotensinoken in the 1β-globulin fraction to form angiotensin 1. This angiotensin 1 is converted to angiotensin 2, which has physiological activity, by a converting enzyme present mainly in the lungs. Asigiotensin tomb is the most powerful stimulant negative that exists in the living body, and it also has an influence on the aldosterone system and sodium ion regulatory system, and can prevent obesity and pregnancy syndrome.
It is considered to be a causative agent of hemophilia.

Therefore, it is thought that substances that suppress the renin-angiotensin system have the potential to be therapeutic for the diseases associated with the photopropagation of the biological 1-segment system, and much research and development has been carried out to date. .

Conventionally, biologically derived renin inhibitors include * and plasma white rice phospholipids [s, Yang (S, 5en) et al., Biochemistry (Biochemistry) Vol. 6]
Vol. 1572 (1967): Bacchio, A.
Baggio) et al., Kurini Riki, Kimi Riki, Acta (Oli)
nica chimica Acta) Volume 45% No. 6
7 (1978)], Deoxy l1li in bile? ″
Acid derivatives [Kokufu Tatsube, Modern Medicine, Vol. 26, No. 209
8 (1971)], mW from α- and β-hemoglobin chains! M specific fragment (H.

R,J, Workman et al., Biochemistry Vol. 18,
No. 3029 (1974)], pepstatin isolated from Actinomycetes strain [U Umezawa, Journal of Antibiotics (Journal
of Antibiotics) No. 23i! 11
p. 259 (1970)] and] Group II group compounds CK and Poulsen (K
II Poulsen et al., Biochemistry, Vol. 12, No. 3
8 7 7i (1978)].

However, these renin inhibitors have weak inhibitory activity against renin, require extremely large doses to exert their effects, are questionable about their physiological role, or are toxic and therefore have no applicability to living organisms. Although it is possible, it has not yet reached the point of practical application.

Therefore, the present inventors have carried out research to obtain a better renin inhibitor, and have discovered that a renin inhibitor that is non-toxic and has excellent activity exists in the human placenta. The present invention was completed by establishing a method for efficiently recovering substances.

The present invention is characterized in that (1) it can be recovered from human placenta, and the molecular type measured by disk electrophoresis of SDS-polyacrylamide gel is 65,000 to 75,000, and the equimolar point is p.
H4,6-5.2, trypsin, plasmin,
Kallikrein, a renin inhibitor that does not harm urokinase and has a specific inhibitory effect on renin, and (2)
The aqueous solvent nf+ product from the human placenta is precipitated with 40-50% ammonium sulfate, and the above renin inhibition is achieved by adsorbing it to an anion exchanger and then passing through the gel mouth. to the manufacturing method.

The human placenta used in the present invention is preferably one after blood components have been removed, and is preferably fresh or freshly frozen and preserved.

Note that blood components are usually removed by washing with steamed water, physiological saline, etc.

Examples of the aqueous solvent used for extraction from the placenta in the present invention include steamed stomach water, Naikonshokutankyu, and Yumachiso.

Extraction from the placenta with an aqueous solvent is carried out, for example, as follows. The placenta is minced, homogenized with aqueous +lIl, pH adjusted to 40-50, centrifuged, and the supernatant is collected as an extract.

The extract thus obtained is subjected to salting out fractionation using ammonium sulfate.

The salting-out fraction is obtained, for example, by first adding ammonium sulfate to the extract to make it 20 to 80% saturated, then collecting the supernatant, and adding ammonium acid to this to make it 40 to 50% saturated. It is struck by collecting the sinking chestnut. More precisely, for example, 20 to 80
Dissolve ammonium sulfate in such a manner that the mixture will be dissolved in ammonium sulfate, leave to stand, and centrifuge to separate the sunken chicken and L oil, collect the supernatant, and readjust the pH to 6 to 8. After that, ammonium sulfate is added again to saturation of 40 to 50%, and the mixture is separated to obtain a precipitate.

After the precipitate thus obtained is brought into contact with an anion exchanger, the absorbent is eluted and a bath product is recovered.

The precipitate is generally 2 to 10mM intoxicant solution (
A mildly bulky vinegar such as pH 7.5-85) is brought into contact with an ion exchanger. In addition, the ion exchanger is equilibrated in advance with the same sand sludge used to dissolve the precipitate.

As anion exchangers, weak to medium basic anion exchangers,
Examples include DEAE-Cellulose, DEAE-Sephatex, DOWEX (■), and Amberlite (■).

The adsorbent preferably contains the same W used to dissolve the precipitate.
After washing well with i-[san], the sample is subjected to the next elution operation.

Elution from the anion exchanger adsorbate is carried out, for example, by a direct swelling concentration gradient method with a final concentration of 50 to 100 mM, and both fractions eluted from about 20 to 40 mM may be fractionated. The elution can also be carried out by increasing the salt concentration of each yuzu salt, such as sodium chloride.

Q) +I Φ The eluate is usually concentrated, dialyzed, desalted and lyophilized, and then subjected to molecular sieving by gel filtration to recover the renin inhibitor activity.

For example, the carrier used for gel filtration has a molecular weight of 5.
0,000 to 500,000 compounds passed through a molecular sieve, a supercarrier such as Sephadex, agarose,
Examples include Biogel, Sephacryl 0, and the like.

The renin inhibitor obtained by the method of the present invention is #IA+
The specific activity is 200 to 800 times higher than that of the ammonium acid precipitated fraction, and the [gi yield is 70 to 80%].
It is.

The properties of the renin light-hazardous substance thus obtained are as follows.

Molecular weight determined by disk electrophoresis of 1.8DS-polyacrylamide gel: 65,000 to 75,000 2, isoelectric point pH: 4,6 to 52, 8, trypsin, plasmin, kallikrein.

It does not inhibit urokinase, but has a specific inhibitory effect on renin.

The renin inhibitor of the present invention is a new substance that exhibits a specific and strong inhibitory effect on renin, and since this substance is derived from humans, it is similar to other animal-derived protein preparations when administered to humans. There is no need to worry about immunological side effects. Moreover, this substance is not only useful as a therapeutic agent for various disorders associated with the renin-angiotensin system, but also extremely useful in elucidating the physiological and pathological problems of the renin-angiotensin system.

After that, heat treatment, sterilization, sterilization, lyophilization, dispensing, and formulation are carried out according to conventional pharmaceutical manufacturing techniques to provide a pharmaceutical containing a novel placental renin inhibitor. be. However, this does not apply when used as a reagent for biochemistry or pharmacology.

Examples are given below to explain the present invention in more detail. In the present invention, the activity of the renin inhibitor was measured using as an index the effect of asgiotensin on violet, which is formed when renin and a renin substrate are reacted. Renin is extracted from human amniotic fluid (E,
Lum-ber, Enzymologia
zymologia).

Vol. 40, p. 829 (1971)], with some modifications, to obtain a renin substrate.

The F/-4 fraction by Cohn's fractionation method was used. [K.

Arakawau, Krinika, Kimika, Acta (Olin.

Chim, Acta), Volume 22, Fee 809 (19
1968)] Reactions include renin, renin substrate, and aprotinin.

Furthermore, add the test solution, and after 16 hours of bending at 87°C,
The reaction was stopped by heat treatment, and angiotensin-1 in the supernatant obtained by centrifugation was measured by radioimmunoassay or biological measurement using the degree of increase in blood pressure of anesthetized rats as an index.

Example A placenta delivered after a normal delivery was minced and washed repeatedly with distilled water to remove mixed blood components. After homogenizing this placental tissue with physiological saline, the pH was adjusted to 4.
Adjust to 8. After standing, it was centrifuged to obtain a supernatant. Ammonium sulfate was added to the supernatant to achieve 25% saturation, stirred, and allowed to stand still. After standing, the pH of the resulting supernatant was adjusted to 7.0 by centrifugation, and ammonium sulfate was dissolved again to achieve 45% saturation. After centrifuging this, the resulting precipitate was collected and used as a raw material for renin inhibitor purification.

This purified raw material was added to 5mM phosphoric acid &P solution (pH 8,0).
After dissolving in the DEAR, which had been flattened in advance due to its looseness,
After loading the cellulose and washing thoroughly with the same 11 Toshin, it was bathed by a linear density gradient bathing method using phosphorus@*'flJ with a final concentration of 50 mM.

Renin I is eluted from a concentration of about 25mM! A fraction of the total amount was collected. At this time, it was also possible to elute the renin inhibitory activity by increasing the degree of sodium chloride. The eluted renin inhibitory activity was dialyzed, desalted, and lyophilized.

Next, Sephacryl S-200 superfine (Da 2.5X) equilibrated with phosphate-added physiological saline & (pH 7,0)
The 11F4 renin silk-damaging substance whose renin inhibitory activity was recovered by gel filtration at 100 cm) was purified 288 times compared to ammonium sulfate precipitation, and the recovery rate was 76%.

(1) Negative No. 7 of the specification, "Sephatex" procedural amendment δ on the first line (voluntary) December 23, 1982 Commissioner of the Patent Office 1 Indication of the case 1982 Patent #177446 2 Invention 0 Title Lane obstruction Substance 3: Relationship with the case of the person making the amendment Mr. Patent Applicant? '+ (?', f+,) Midori Juji Co., Ltd. 4, Agent 〒541 6 Number of inventions to be increased by amendment None も 1 " 7. Subject of amendment

Claims (2)

[Claims] (1) One harvest from a human placenta has a molecular weight of 65,000 to 75,000 as measured by disk electrophoresis iAB method on 8D8-polyacrylamide gel. The isoelectric point is pH4,
6 to 5.2, does not inhibit trypsin, plasmin, kallikrein, or urokinase, and has a v4i action specifically on renin. (2) E) 40-50% i of aqueous solvent extract from placenta
After adsorbing the E# fraction obtained by distilling K# ammonium onto an anion exchanger, the 8D8
- Molecular wrinkles measured by disk electrophoresis of polyacrylamide gel are 65,000 to 75,000. Isoelectric point or p
H4.6 to 6.2, and produces trypsin, plasmin, kallikrein, and urokinase [4t]. A patented method for producing a renin inhibitor that has a specific inhibitory effect on renin.