JPS6041051B2 - Peptide for regulating intestinal intrinsic motility - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to compounds isolated from the posterior lobe of the bovine pituitary gland that have active activity against smooth muscle of the gastrointestinal tract and other tissues.

More particularly, the present invention relates to certain low molecular weight peptides, which stabilize intestinal proper motility in animals, thereby preventing ileitis, colitis, spasmodic constipation, etc.
It is effective in the treatment of conditions associated with spasmodic diarrhea, diverticulitis, peptic obliquus and irregularity of beristartin action.
The products of this invention are certain low molecular weight peptides that can be isolated from the posterior lobe of the bovine pituitary gland and also include pharmaceutically acceptable salts of the peptides.

The invention also relates to physiologically effective compositions containing the above-mentioned compounds as the main active ingredient together with a pharmaceutically acceptable carrier. In addition to their utility for the control of positive proper motility (motmty), certain of the peptides of this invention have a high degree of vasoconstriction (vagmoconstriction).
It also exhibits oconstric ■r) activity.

Accordingly, the products and pharmaceutically acceptable salts are useful in the treatment of shock and other conditions in which constriction of capillaries and arteries is not desired. For convenience, the products produced singly by the process of this invention are designated as Coherjn A, B and C.

Coherin A and B are the special subject of this invention. Both of these are effective for controlling explicit proper motion. Furthermore, although both of them exhibit vasoconstrictor nerve activity, the activity of coherin A is rather low. It is currently believed that coherin C acts as a carrier. The isolation procedure for these products is as follows.

In Step 1, acetone-dried bovine posterior pituitary gland powder (50 g) (Mills Laboratories) was added to the ice bath for 2 hours.
×125' ammonium acetate (0.2M, pH 5.
6) was homogenized for 5 minutes. 18,000 evening, 4 at 5℃
After centrifuging for 0 minutes, the supernatant solution was treated with a lotus algae furnace additive (Celite, Jones, Masovil, Analytical) for 3 days.
of diethylaminoethyl cellulose (DEAE) (Whatman, DE23), filtered and pre-equilibrated with 0.2 ammonium acetate (50 mL).
×61 Cape Qi) (Step-2).

DEAE column with 0.09 M ammonium acetate pH 5
．． 6, the eluate is separated and collected, and its fractionation (
fraction) for the control of intestinal contractions and roux. En・
Contraction adhesion (c) of the large intestinal adjacent segment with the fistula (fist mountain a)
Tests were conducted for activity as assessed by (orherence). The main biological activity is 1825 to 396
5 secretion (total volume 2140', 2A280/skin = 4608
) was eluted from DEAE cellulose in the fractionation between

DEAE cell.

The active fraction obtained from the base column was freeze dried in vacuum. 10' ammonium acetate (0.0
2M. pH 5.6) and cross-linked dextran.
The gel was transferred to a column (50 x 61 columns) of gel (Pharmashin Corporation, Sephadex G-5 fine powder). In step-3, ammonium acetate PH of 0.02
Elution was performed using 5.6.

Active fractionation is 1460 and 2580 (Ve/Vo=3
．． 08). The fractions eluted from Sephadex G-50 were lyophilized in vacuo and the product (2.1 min.) was dissolved in 0.02 M ammonium acetate (5 min.). The solution was applied to a column (50 x 109 columns) of cross-linked dextran gel (Pharmacia Cephadex G-2 ultrafine powder) as described above (Step-4) and separated as described above. Active ingredients range from 1460 to 2580 (Ve
/Vo=2.33). In a similar manner, the active fraction obtained from Sephadex G-25 was freeze-vacuum dried and dissolved in 4' acetic acid 0.1 M, and a highly cross-linked dextran gel (pre-equilibrated in the same solvent) ( Sephadex G-10) column (
10×105 (comparison ship)) (Step-5). The coherin isolated in step 15 is a compl which is bound by either electrostatic or van der Waals forces.
ex).

The substance is the product of step-4, Sephadex G-1.
0 to 248 enemies and 363' (Ve/Vol.4
7) by elution at a rate of 0.5' per minute during the fractionation. The precision of the bound coherin was determined by dissolving the product 78 of step-5 in 0.2 M' of 19 units of acetic acid. This solution was mixed with butano-acetic acid-water (4:1).
:5) Extracted with the upper phase (non-aqueous phase) (3×10 volumes). The non-aqueous extracts were combined and concentrated in vacuo to a white solid. When the solid was dissolved in 10 g of 95% ethanol and left at 5° C. for 4 hours, fine white crystals were formed. The coherin rust remains bound after repeated recycling with Cephadex G-10 in 0.1M acetic acid.

The coherin bell body isolated in step 15 was dissociated, and the PH2
．． Continuous flow electrophoresis (contin
When the flow electrophores are repeated, coherins A, B, and C are generated. Coherin rust body (Step 15) (ZA280 = 175
) in acetic acid 0.2M, pH 2.75 to obtain A280=
A concentrate of 4.0 was obtained. This solution is applied at a rate of 0.42 M per hour (18 V/arc; 0.83 hA/arc) at a point 175 bars from the vertical centerline. The electrolyte was 0.2M acetic acid, and the elution rate was 1.2 tubes per hour. Coherin A was present in fractions 13 and 19, corresponding to the drip tip of the 95-leg cathode at the point of application.

The bodies obtained after Bouling and freeze-vacuum drying of these tubes were extracted with isopropanol (3 x 3 tubes) and collected by centrifugation. Under these conditions (pH2
．． In 8), coherin B is isolated in fractions 5 to 9, which are 15 times more cathode at the point of application.

Coherin C elutes in fractions 17 to 19 (6 times the cathode at the point of application). Tubes containing Coherin A, B and C, respectively, were pooled, freeze-vacuum dried, and separately p.
Cycled to pneumophoretic homogeneity in a continuous flow electrophoresis machine at H 2.8 and 3.5.

For pH 3.5 the electrolyte was 0.02M ammonium acetate in 0.2M acetic acid. Circulating on axis 3.5, coherin A is present in fractions 19-21, B in fractions 15-19, and C in fractions 23-25. The fractional numbers of the Beckmann-Spinco model CP remain unchanged, thus indicating their position relative to the anode and cathode.
The numbers are from 1 to 32 from cathode to anode. Coryrin A, B and C are isolated after being cycled through continuous flow electrophoresis at pH 2.8 and 3.5, followed by ethanol extraction to remove other peptides and traces of free amino acids. Coherin A is a peptide containing at least glycine, and coherin B is also a peptide, but with a somewhat higher molecular weight.

Moreover, coherin B contains glycine, and further contains at least glutamic acid and serine. Both peptides are soluble in water, slightly soluble in absolute ethanol, but insoluble in acetoso and ether. The physical and chemical properties of the product are shown in Table 1. Table 1. Notes on Table 1: 'a - UV measurements were performed using a Curly Model 11 spectrophotometer.

The solvent was 0.1N hydrochloric acid for pHI and 0.1N sodium hydroxide for pH11.

{b} Electrophoresis
) was carried out using Whatman M paper on a Research Substances Company instrument, and the electrolyte buffer was as follows. pH2 Formic acid, Acetic acid (14.2V
/ Fairy tale. 85mA/en) PH3.5 pyridine, acetic acid (1
4.6V/arc. 8 pine/inhibition) spots 8.5 Rubital buffer (.0 shoes) (8,13V/arc 1,25hA/skin) Two MS y=bowl; In the formula, △MS is bromophenol full one and This is the difference in migration from the sample (skin), and MBPB is the migration of bromophenol blue.

[c} The isoelectric point was taken as the pH at which zero migration occurs from the origin using the lightning electrophoresis device described in {b} above.

{d} Chromatography is performed using silica gel on glass (
Merck, Silica Gel G) was used.

Solvent tl} N-butanol-pyridine-acetic acid-water (60
:40:12:48), [2-N-butanol-acetic acid-
Water (4:1:5). te} Melting points are not corrected.

Coffret micro melting point Determined using a hot stage. {f' molecular weight is dextran (cephadex G-
50) Biological tests were routinely performed on the main fractions isolated in all the steps described above.

Inu Hashi-en-Yfist (Ro-like-en-Yfist)
ulae) so that intestinal absorption could be monitored by a pressure transducer in the lumen of the ileal segment.

Coherin activity was determined by mechanical as well as electrical means. Fractional activity is demonstrated by a pronounced inhibitory effect on intestinal contraction within 3 days after intravenous injection of coherin. Fractions for evaluation analysis were routinely dissolved in one normal saline solution. Another Isolation Procedure Example Coherins A, B and C can also be isolated by the following procedure.

The first steps in this operation are steps 1 to 3 of the procedure described above.
is the same as

The eluate from step 3 is first lyophilized and then extracted with isopropanol. The residue is dissolved in acetic acid, equilibrated with the acid and passed through a Sephadex G-10 column. Melt it with 0.1M acetic acid. The eluate is then subjected to continuous flow electrophoresis with 8-12 side passes at pH 2.8 and 3.5 to isolate the desired subunits. Following the above operations, step 5 can be performed on the same machine.

However, the eluates from step 5 containing coherin A, B and C may be pooled, lyophilized and each cycled and repeated for continuous flow electrophoresis. Each pool is large enough to accommodate pH 2.0, 2.8 and 3.
．． It is possible to cycle 8 to 12 times in each of 5. Under these conditions, coherins A, B, and C each maintain homogeneity. However, coherin A dissociates to produce two fractions, A and A2. Methods for isolating individual subfractions may be used as follows.

The initial extraction operation is identical to that previously described for step 1 of the previous example.

However, in step 2, the DEAE cellulose column is washed with water and scaled with 0.08M ammonium acetate. Step 3 remains the same, but in step 4:
Isobu if necessary. Panol extraction may be used to convert the syrup into a powder. Step 5 is the same as Examples 1 and 2. The main new attempt in this operation is in step 6.
This is generally done as follows. Self-adex G
Swell 150 super fines and 100 mg in water, 0
．． Wash in the column (6xlood) with 5 liters of 2M acetic acid.

230 washed gels were placed in a polyacrylic tray (19x
20.2 x 2.4 arc) to form a thick layer on the depth 6 side. Place the tray in the appropriate location on a water-cooled platform using an electric agitation device (Research Substances, Inc.). And Whatman Elementary M
A wick of oven paper (18.5 x 27 togs) is inserted into the gel at each end. Insert the free ends of the wick into the anode and cathode containers, respectively. Apply 50 samples of 0.1 M acetic acid (A280 = 21) along the middle line of the gel with a micropipette.

Bromophenol (10-4 in 0.1 MHAc)
Apply the 10 Buddhas of M) to the line in the middle for reference. Glass plate (20 x 20) to retard evaporation
Cover the tray with Electrophoresis was continued for 6 hours at 10.30V/G, 0.58rA/skin.

In order to determine the peptide component, a test piece of Wattmaso MM furnace (loosely 1×20) is inserted into the gel parallel to the direction of current passage, quickly removed, dried and treated with fluorescamine. The gel slab was cut into 5 strips, transferred to a glass chromatography column (2 x 30 columns), and lysed with 0.1M acetic acid (150 ml per fraction). Freeze-dry the port liquid. Fractions are identified by evaluation for biological activity. The homogeneity of their fractionation can be tested by paper electrophoresis and also by chromatography on thin layers of Sephadex G-50 and silica gel using the procedure described above. The operation involves different pH,
For example, you can repeat with 3.5. In order to analyze the remarkable hakama properties of coherin peptides, the following operations were performed.

The results are shown in Table 3. Exposure migration was carried out using Whatman 3MM oven paper and the following buffers using equipment manufactured by Research Substances.

Buffer: mother 2, formic acid, acetic acid (14.shio/c, .8 shoes a/ga): pH 3.5, pyridine, acetic acid (14.5v
/arc,. 85 hA/skin) or ammonium acetate 0.0
5 molar or ammonium triethyl acetate 0.05
Molar concentration: pH 8.5, Parbital buffer, 0.
08M (8.1 circles/arc, 1,20 A skin), or diethylaminoethanol 0.05 molar concentration, pHil. 0
Na2C030.08M (8.1W/skin, 1,2 shoes A
/skin). MS V=arm; In the formula, Ms is the difference in migration between bromophenol flu and the sample (skin), and NZPB is the migration of bromophenol flu.

The constant dew point is the fence when the migration from the origin is zero using the electrophoresis described above.

Chromatography was performed using silica gel on glass (Merck & Co., Silica Gel G) and Eastman Chromatogram.
It was done with a thin layer of the silica gel on a sheet.

The solvents in each case were [1}n-butanol-pyridine-acetic acid-water (60:40:12:48) and [2}
The mixture was n-butanol-acetic acid-water (4:1:1).
The migration speed of the peptides was compared to that of bacitracin A.

RBAc was divided by the migration distance of bacitracin A. Equal to the migration distance (nmxloo) of the sample peptide. Molecular weight was evaluated based on migration ratio on a thin layer of DemoStranger (Sephadex G-50) along with amino acid analysis.

The results are shown in Table 2. The biological activity of each of the peptides was tested to determine the level of control of positive intrinsic motility.

Dogs were prepared with Leh-en-Wy such that intestinal contractions could be monitored with a pressure transducer in the ileal segment. Coherin activity was measured by mechanical as well as electrical means. Fractional activity was demonstrated by significant inhibition of intestinal contractions within 3 days after intravenous injection of coherin peptide.
Assay fractions were dissolved in standard saline solution as is customary.
The results of the bioassay are shown in Table 3. Table 2 *Molar ratios expressed to the nearest integer Table 3 Because the compounds of this invention are amphoteric, both pharmaceutically acceptable metal salts and acid addition salts can be prepared and are effective.

Typical useful salts include alkali and alkali metal salts, especially ammonium and amine salts such as calcium, sodium and potassium salts, cyclohexylamine and piveridine salts, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, lactic acid, Organic and inorganic salts are included, such as citric acid, tartaric acid, oxalic acid, succinic acid, maleic acid, gluconic acid, and the like. The compounds may be prepared by conventional operations commonly utilized for the preparation of the above-mentioned salts, for example by titration of an aqueous solution with an acid or base and removal of water by lyophilization. Alternatively, the salt may be precipitated by dissolving the compound in water, adding a suitable acid or base, and adding a water-miscible non-solvent such as acetone. Although the products of this invention may be administered alone, they will generally be administered with a non-occlusive pharmaceutically acceptable carrier.

In this case, these proportions will be determined by the compatibility and chemical nature of the particular carrier, the chosen mode of administration and standard pharmaceutical practice. It can also be administered orally in the form of tablets or capsules containing excipients such as, for example, starch, lactose, certain clays, and the like. Usually these will be enteriocoated (enteriocoa d) to make them more resistant to stomach acids and digestive enzymes. However, because of their relatively low molecular weight nature, they can be absorbed before being hydrolyzed by the proteolytic enzymes of the stomach. For intravenous, intramuscular, or subcutaneous administration, other solutes may also be used in the form of sterile solutions containing salines or glucose sufficient to render the aqueous solution isotonic. A wide variety of dosage unit forms are possible. The attending physician or veterinarian will determine the medication regimen that will be effective.

Claims (1)

[Scope of Claims] 1 Contains aspartic acid, serine, glutamic acid, proline, glycine, alanine, leucosine, phenylalanine, lysine and araginine in a molar ratio of 1:1,
Soluble in water, slightly soluble in absolute ethanol and insoluble in acetone, with a molecular weight of 1000-1500, with a maximum of 276 nm and a corresponding minimum of 249 nm at pH 3.5 in the ultraviolet region of the spectrum.
A peptide coherin B and a pharmaceutically acceptable salt thereof, characterized in that it has m. 2 Contains aspartic acid, serine, glutamic acid, proline, glycine, alanine, leucosine, phenylalanine, lysine and araginine in a molar ratio of 1:1,
Soluble in water, slightly soluble in absolute ethanol and insoluble in acetone, with a molecular weight between 1000 and 1500, with a maximum of 276 nm and a corresponding minimum of 249 nm at pH 3.5 in the ultraviolet region of the spectrum.
1. A composition for controlling intrinsic motility of the intestine, characterized in that the active main ingredient is the peptide coherin B having the following properties: m or a pharmaceutically acceptable salt thereof. 3. The composition for controlling intestinal intrinsic motility according to claim 2, characterized in that a carrier is present.