CA1160624A - Psp and salts thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The invention relates to a novel purified polypeptide which is recoverable from porcine pancreas glands. The amino acid composition of the said polypeptide has been determined to be as follows:
Trp (2), Lys (4), His (1), Arg (5), Asx (10), Thr (3), Ser (9), Glx Cl2), Pro (12), Gly (6), Ala (6), Cys 1/2 (14), Val (7), Met (2), Ile (3), Leu (1), Tyr (2), Phe (7).
The purified polypeptide is of potential utility as a medicament, for example as a spasmolytic agent or a diagnostic aid.

Description

WA/TON/UF
80~722 PANCREATIC SPASMOLYTIC POLYPEPTIDE

This invention is directed to a novel purified polypeptide or a physiologically acceptable salt thereof, as well as to a method for recovery and purification thereof and to the use thereof as a spa ~ lytic agent. The purified poly-peptide of this invention, wHich is recoverable from porcine pancreas, has surprisingly been shown to possess smooth muscle relaxing or spasmolytic effects. It has, therefore, been accorded the trivial name of Pancreatic Spasmolytic Polypep-tide, hereinafter for the sake of convenience abbreviated to PSP. PSP shows interesting pharmacological properties.

Spasmolytic agents or antispasmodics, such as atropine, congeners thereof and synthetic drugs having an atropine-like effect, are widely used for the treatment of a variety of ailments, in particular of smooth muscle spasms and hypermotility states~
However, the intended action of such drugs is usually accompanied by a number of side effects attributable to their general character of being anticholinergics.

As a diagnostic aid in gastrointestinal radiology, particularly in conjunction with an X-ray visualizati`on medium for improving visualization of the gastrointestinal, biliary and urinary tracts, atropine-like anticholinergic drugs have also been commonly used. Such a drug is usually administered parenteral-ly and, owing to the size of the dose needed to induce relaxation, the side effects classical to those agents are usually encountered.

Recently, parenteral administration of the peptide hormone glucagon consisting of 29 amino acids was introduced as an alternative means of reducing gastrointestinal motility in conjunctiOn with radiographic examinations (vide U.S. Patent No. 3,8~2,301). However, glucagon exerts a plurality of actions in the human body incluaing a strong influence on metabolic regulatory functions, the most conspicuous effects being the induction of hyperglycemia and lipolysis. Thus, although the introduction of glucagon in endoscopy accomplished certain advantages, undesirable side effects were not completely abolished. It is an object of this invention to provide a spasmolytic agent which, whilst possessing antispasmodic and smooth muscle relaxing effects comparable to those of known agents, exhibits substantially reduced side effects.

According to one aspect of the present invention there is provided a novel purified polypeptide exhibiting the following amino acid composition:

Trp (2), Lys (4), His (l~, Arg (5), Asx (lO), Thr (3), Ser (9), Glx (12), Pro (12), Gly (6), Ala (6), Cys~ tl4!) , Val (7), Met (2), Ile (3), Leu (l), Tyr (2), Phe (7), wherein the determinations are subjected to the usual error of + lO per cent of the indicated figures. The partial amino acid sequence ccmprising a total of ~5 amino acids frcm the N-terminal, is believed to be:

pyrGlu-Lys-Pro-Ala-Ala-Cys-Arg-Cys-Ser-Arg-Glx-Asx~Pro-~ys-Asx-. ~0 15 -Arg-Val-Asx-Cys-Gly-Phe-Pro-Gly-Ile-Thr-Ser-Asx-Glx-Cy~-Phe-Thr-Ser--Gly-Cys-Cys-~he-Asx-Ser-Glx-Val-Pro-Gly-Val-Pro-Trp-, wherein pyrGlu (residue l) stands for pyroglutamic acid.

The a~breviat~ons for the amino acids appear from J.Biol.Chem. 243 (1968), 3558.

The present invention also provides a method for preparing purified PSP, whic~ method comprises isolating PSP
from porcine pancreatic tissue prefera~ly from the insulin salt cake by a com~ination of chromatograph~ and precipitation processes. --The insulin salt cake may ~e prepared as follows:whole, neatly defatted porcine pancreas glands are finely comminuted under frozen cond~t~ons and then su~jected to the conventional extraction process for recovery of insulin, that is extracted with a mixture of water and an organic water-misci~le solvent, such as a lower aliphatic alkanol, for example ethanol or isopropanol, in an acid medium, for example, a medium having a pH in the range of ~rom about 1.5 to 5 when measured with a pH meter in the mixture. The acid pH is o~tained ~y the addition of an acid. In the mixture, the organic solvent is present in a concentration in the range of from a~out 40 to 80% ~v/v) when all the components are mixed.
The resulting slurry is stirred at a temperature in the range of from a~out 5C to am~ient, followed by removal of the gland residue, for example ~y centrifugation. The extract is then neutralized to a pH in the range of from about 5 ko 9, and clarified, for example by centrifugation. The extract is acidified to a pH in the range of from about 3 to 4, where-after the extract is freed of any organic solvent, for example by evaporation at reduced pressure, followed by removal of lipid compounds, for example by centrifugation. Insulin admixed with other proteins and polypeptides, such as PSP, is salted out from the concentrated extract so o~tained, for example ~y the addition of sodium chloride to a concentration in the range of from a~out 10 to 30% ~w/v) r and the precipitate formed is isolated, for example by centrifugation, thus affording the salt cake.

The salt cake thus obtained may then be dissolved in water and crude insulin isolated by isoelectric precipitation at a pH in the range of from about 4.~ to 5.7, for example about 5.3, optionally in the presence of metal ions, for example zinc ions, and recovered, usually by centrifugation. The supernatant is given a pH in the range of from about 5.7 to 7, preferably about 6.5. The precipitate formed, containing some insulin, i5 centrifuged off. In order to remove ancillary substances, such as salts, excess of EDTA is added to the above second supernatant, followed by the addition of a water-miscible organic solvent, preferably ethanol (usually, from 5 to 20 volumes). The mixture is left to precipitate overnight at about 4C and then centrifuged. The precipitate is dried in vacuo, yielding a dry powder, hereinafter referred to as "superna-tant protein". By this procedure, practically all protein material of the "supernatant protein" is recovered.
PSP can be obtained in a crude crystalline form from a solution of "supernatant protein" in water (about 10 parts).
The solution is stirred gently while acid, for example acetic acid, is added in the course of about 3 hours until a pH in the range of from about 3.8 to 4.8, preferably about 4.3, is attained. The mixture is then chilled and the stirring is continued for 3 days, preferably at about 4C. A crop of relatively large, bar-shaped, birefringent crystals is isolated, for example by centrifugation, and dried in vacuo.
The material so obtained may be further purified, preferably by applying consecutive steps of anion and cation exchange chromatography.
I'o illustrate the procedure, anion exchange chromatography may be performed on a column of "QAE-Sephadex A-25" (supplied by Pharmacia AB, Sweden), using the eluent stated on Fig 1 of the accompanying drawings (TRIS being tris (hydroxymethyl) aminomethane).

P~ f~

The chromatogram obtained by monitoring the optical density of Eractions at 276 nm shows one main peak. The pool corresponding to the main peak is adjusted to p~ 7.4 and then mixed with a water-miscible organic solvent, for example ethanol ~4 volumes). ~pon standing at ~C for 2 days a precipitate is recovered by centrifugation and dried in vacuo.

The material so obtained can be further purified by cation exchange chromatography, for example on a column of "SP-Sephadex C-25`' (supplied by Pharmacia). Elution may be effected with the eluent stated on Fig 2 of the accompanying drawings. The chromatogram, obtained in the same manner as above, shows a main peak. Pooled fractions corresponding thereto are evaporated to dryness, the residue is dissolved in water at a pH
in the range of from about 6 to 8, for example about 7, mixed with an excess (about 12 volumes) of a water miscible organic solvent, for example ethanol, and left overnight under similar conditions as described above. Purified PSP , which precipi-tates from the solution, is isolated by centrifugation, washed with ethanol, and dried in vacuo.

Alternatively , PSP containing protein may be obtained from the mother liquor arising when isolating the salt cake using sodium chloride in a concentration of from 10 to 20~ (w/v)- by an additional salting out process. The precipitate is recovered, for example by centrifugation. Purified PSP can be obtained from the precipitate by the use of anion and/or cation chromatography in any order.

By a further method, PSP containing protein may be isolated from the above extract of pancreas glands obtained using a mixture of water and an organic water-miscible sol~ent by adsorption to a cation or anion exchanger, for example alginic acid, sulphonated polystyren or aminoethylcellulose. Thereafter, the ion exchanger is washed and the protein is eluted with an aqueous medium. The isolation by the use of an ion exchanger is performed by methods which are analogous to known methods.
:~' PSP obtained ~y any of th~ a~ove methods has the following characteristics:
Molecular weight, calculated from the amino acid composit~on: aBout 11,700.
Molecular we~ght, determined ~y sodium dodecyl sulp~ate ~ gel electrop~ores~s ~Nevil~e: J. ~iological C~emistry 246 C1~711 6328~: a~out 10,700.
Electrophorectic characteristics:
~ asic DISC electrop~oresis (basic DEl in polyacryl-amide gel as descri~ed ~y J. Schlichtkrull et al. (Horm.
Meta~ol.Researc~, Suppl. Series 5 (1974l 1341 shows essenkially a single ~and with Rf 0.65 - O.75. A similar pattern is obtained in analytical electro-focusing in polyacrylamide gel ~y which method the pI is determined to a~out 4.4. ~ry~5i~
Products o~tained upon treatment of PSP with~tr ~r ~-chymotrypsin, CNBr, acid, or pyroglutamate aminopeptidase as descri~ed ~elow, have a spasmolytic activ~ty of the same order as that of PSP.
''Tryp's'in''treatment:
Twenty mg of PSP was dissolved in 20 mQ of 0.01 M
NH4HCO3 (pH: 7.8) and preincu~ated for 5 minutes at 37C.
After addition of 100 ~1 of 0.001 M HCl containing 0.4 mg TPCK-trypsin ~o~tained from Worthington Biochem. Corp.), the mixture was incubated at 37C for 15 minutes and then lyophiliz-ed.
a-Chymotrypsin trea*ment:
Twenty mg of PSP was dissol~ed in 2ao ~1 of 0.1 M
NaOH and 1800 ~1 of 0.05 M NH4HCO3 (pH: 8.0) was added. The solution was preincubated for 5 minutes at 37C and 50 ~1 of 0.001 M HCl containing 100 ~g a-chymotrypsin (obtained from Sigma Chemical Company) was added. The incubation was contin-ued for one hour at 75C and the reaction was stopped by the addition if 50 ~1 concentrated acetic acid, whereafter the solution was lyophilized.

~ 2 CNBr treatment Twenty mg of PSP was dissolved ~n ~ ml of 70% (v/v) formic acid containing 72 mg CNBr. T~e m~xture was stored at room temperature for 40 hours and then lyop~ilized. The lyophilizat~on was then repeated after add~tion of 2 ml of water.
Acid treatment Samples of 1 mg PDP, dissolved in 100 ~Q of 0.5 N
hydrochloric acid, were incubated at 37C for 2, 10 and 21 days.
After incu~ation t~e protein of each sample ~as precipitated quantitativel~ ~y the addition of 2 ml of acetone. The pre-cipitate was isolated ~y centrifugation, washed with 2 ml of acetone and dried in vacuo. The samples so obtained and a sample of untreated PSP were analysed by basic DE, vide supra, with the proviso t~at the time of electrophoresis was reduced to give Rf = 0.53 for PSP. In the sample ;ncubated for 2 days a series of ~ands were observed with Rf ranging from 0.53 to 0.86.
In the samples incu~ated for 10 and 21 days only a single band with Rf 0.86 appeared. The results indicated that a partial deamidation of PSP had occurred after 2 days and a complete deamidation after 10 days of incubation.
Pyroglutamase aminopeptidase treatment A sample of 6 mg PSP was dissolved in 2 ml of 50 mM, sodium monohvdrogen phos~hate, 3~ ~ p-mercaptoethanol, 1 m~
EDTA buffer with a pH of 7.8. A so~ution of 2.5 mg p~roglut-amate aminopeptidase (obtained from oehm~gcr ~lannhcl~) in 0.5 ml of the above buffer was added. The mixture was incu~ated for 16 hours at 39C and thereby lyophilized.
(2.5 mg pyroglutamate aminopeptidase used contained about 10 mu enzymatic activity.

The purity of the final PSP product may be checke~ by analytical isoelectric focusing (IEF) and basic DISC
electrophoresis (basic DE, ~ ). The product migrates essentially as a single band in both systemsO IEF is performed according to the instructions of LKB brochure I-1804-E02: "LKB
Ampholine PAG" plates for analytical electrofocusing on polyacrylamide gels (LKB-Produkter AB, Bromma, Sweden).
Likewise, gel filtration of the polypeptide on "Bio-Gel P-3O" (supplied by Biorad Laboratories, Richmond, California, V.S.A.) using 1 molar acetic acid as the eluent, reveals only a single peak.
PSP has been analysed for a number of immunoreactivities according to methods known in the art. The results obtained are presented in Table 1:
Table 1 Immunoreactant Contents (Ppm2 Insulin (lRl) 3 - 6 ~'otal glucagon (total GLI) 0.02 Pancreatic glucagon (pancreatic GLI) 0.02 Vasoactive intentinal peptide (VIP) 0.02 Pancreatic polypeptide (porcine) 0.08 C-Peptide (porcine) 0.1 Somatostatin 0.002 The immunoreactivity of PSP is measured by a highly specific radioimmunoassay which is developed to detect down to 250 pg per ml.
Antibodies were prepared by immunizing rabbits with "supernatant protein" (0.5 ml of a solution contining approximately 4 mg protein per ml) mixed with Freund's adjuvant (0.5 ml) twice weekly for a period of 20 weeks. Beginning from the 13th day after the first immunization, a total of 10 blood samples (10 ml) ~rom each animal, taken at regular intervals over a period of 172 days, were collected. The antisera obtained were tested for affinity and capacity and a suitable antiserum was selected for use in the radioimmunoassay.

125I-PSPw~s prepared by the lactoperoXidase method developed by Thorell and Johansson (Biochim.Biophys.Acta 251 ~1971) 363). The radioiodinated PSP ~s purified by anion exchange chromatography as known in the art and used for polypeptide radioimmunoassay according to the procedure developed by L.G. Heding (Diabetologica 7 (i971), 10).

.
Furthermore, the present invention relates to salts of PSP and, as examples of such salts, salts with cations such as sodium, potassium, magnesium, calcium and zinc and acid addition salts with organic or inorganic acids such as formic, methansulfonic, hydrochloric and sulphuri.c acid, can be mentioned.
For the sake of brevity, the designation PSP Compounds is used to cover PSP and physiologically acceptable salts thereof.

PSP Compounds and glucagon were found to be about equipotent in their inhibition of the amplitude of the contractions of electrically stimulated guinea pig ileum ln vitro, vide Table 2.
PSP and glucagon were dissolved in 0.9% sodium chloride with 0.1%
human serum albumin Table 2 Inhibitory effect in per cent Concentration in the organ bat~, M PSP Glucagon ,-lo~6 - 49 51 This effect of PSP Compounds was blocked by phentolamine but not by naloxone. The spontaneous motility of the isolated ileum from reserpine-treated guinea pigs was inhibited by PSP.

Likewise, PSP Compounds were found to be about as potent as glucagon with respect to its inhibitin in vivo of .the peristalsis in mice, vide Table 3, an effect which again could 6~6Z~
be blocked b~ phentolamine.

Table 3 Drug Per cent of intestine traversed by ( 50 mg/kg sub- charcoal compared to a control cutaneously) , Glucagon 66 Atropinsulphate 64 .. . . . .. . .. . . ..
PSP reduces intestinal motility in rabbits in vivo after administration intravenously or intraluminally in the intestine. The motility was recorded by means of a balloon catheter in the intestine connected to a pressure transducer. In 5 out of 5 rabbits (fr~m 2.5 to 3.0 kg body weight)400 ~g PSP
administered intravenously or 5 cm ~Xom the balloon into the lumen of the intestine caused a marked reduction of the intestinal motility, almost to atonia. 200 ~g ~ad a clear effect in 3 out of 5 rabbits.
Glucagon had the same effect, but only when administered intravenously.

PSP was found to delay the absorption of [U-14CJ protein hydrolysate in pigs and in pancreatectomised dogs and of [U-14C~
ovalbumin in panareatectomised doges, when the compound was administered perorally in a capsule with 3 mg PSP. The pigs and the dogs weighed about 30 kg. 100 ~Ci [U-14C] protein hydrolysate or 5 ~Ci lU-14C] ovalbumin was mixed with a suspension of 1 g/kg Idon and administered through stomach tubes. Maximum plasma dpm values were reached from 30 to 40 minutes later after administration of PSP
as compared to placebo. This delay in absorption caused by about 100 ~g/lg pf PSP orally probably reflects a reduced gastro-intestinal motility.

`` PSP comp~unds were found to be devoid of an~ In vitro ~ 'ect on the release of glucagon or insulin or on lipolysis and of any in vivo effect on blood glucose. Nor did an intravenously injected dose of up to 1 mg/kg exert any significant efect on the blood pressure of the anest~etized rat.

~ The above p~armacological data indicate the potential value of PSP compounds for t~e prevention and treatment of smooth muscle spastîc conditions, ~r example in the intestine. Due to the lack of metabolic effects, PSP compounds may prove advantageous as a substitute for gluaagon in endoscopy and in radiological procedures.

PSP compounds may ~e administered intravenously as a bolus or as an infusion. When an effect of prolo~ged nature, slower in onset, is desired, PSP compounds may be administered as a depot from which it is slo~ly mobilized by the blood stream such as intra~
muscularly or su~cutaneously in a region of good peripheral circulation supply. The fact that the biological activity and the immunoreactivity is maintained after exposure of PSP to gastric juice, trypsin, and chymotrysin and the experiments described above showing delayed a~sorption after oral administration of PSP points to the oral route as a possible way of administration. Therefore, PSP
may be administered through an endoscope during the endoscopy procedures or PSP~may be mixed with the contrast media, e.g. barium sulphate, during the radiology procedure.

The dosage rates of PSP Compounds can be adjusted according to the magnitude of desired response and other factors routinely taken into consideration in establishing the dosage.
As an example of a dosage ra~ge, from 10 to ~Oo ~ g per kg body weight can be mentioned, although a lower or igher dosage may be administered.

The present invention also relates to a pharmaceutical composition comprising PSP Compounds and one or more pharmaceutically acceptable carrier(s) . As examples of such carriers,preservatives and sodium chloride can be mentioned.

6~
" I

In an attempt to secure that the desired result is obtained after administration of a PSP Compound it is advisable to use a starting material for preparing PSP preparations which has a purity of at least 50%, preferably a purity of at least 90~ of a PSP Compound.
According to hitherto unpublished data pancreatin pills contain PSP (for example about 1 per thousand).
Because of its content of enzymes pancreatin pills have been used for pancreatectomized patients and patients with chronic pancreatitis. Commercial insulin has now been found to contain about 30 ppm PSP.

Any novel feature or combination of features described herein is considered essential.

The following Examples, which, however, are not con-sidered to be limiting, are presented to illustrate the process for preparing PSP. Highly purified PSP is PSP which essentially migrates as a single band in the above IEF
and basic DE systems.

Example 1.
A salt cake originating from 94 kg of porcine pancreas glands was dissol~ed in water to a volume of 3.2 1 . The pH
of the solution was adjusted to 5.3, whereafter the precipi-tate was removed by centrifugation. The pH of the supernatant was adjusted to 6~5 and the suspension thus formed was centri-fuged. The solution was mixed with 32 ml of 0.5 M Na4EDTA and 35 1 of ethanol. The mixture was left overnight at 4C and then centrifuged. The precipitate was dried in vacuo yielding 50 g of dry supernatant protein powder.

A solution of the powder in 500 ml of water was stirred gently while 1 M acetic acid was added slowly by means of a peristaltic pump until a pH of 4.30 was attained (af~er about 3 hours of pumping). Stirring was then continued for 3 days at 4C whereby crystallization occured. The crop of crystals (bar-shaped by appearance,possibly orthorhombic and showing birefrinye~ce) wére harvested by centrifugation, suspended in 500 ml of water at 4C with stirring overnight, centrifuged and dried in vacuo. The yield was 5.2 g.

4 g of this material was dissolved in 50 ml of 50 per cent (v/v) ethanol and 50 ml of eluent (vide Fig 1) at pH 8.6. The solution was subjected to anion exchange chroma-tography as shown in Fig 1. The pool from the main peak was given a pH of 7.4, mixed with 4 volumes of 96 per cent (v/v) ethanol and then stored at 4C for 2 days. The precipitate was isolated by centrifugation, washed twice with 150 ml of 96 per cent (v/v) ethanol and dried in vacuo. The yield was 2.6 g.

2.5 g of this material was dissolved in 125 ml of 50 per cent (v/v) ethanol and 125 ml eluent (vide Fig 2) ~t pH 4.7 and then subjected to a cation exchange chromatography as shown in Fig 2. The pool from the main (only visible) peak was evaporated to dryness. The residue was dissolved in . ~

water and the pH of the solution was adjusted to 7.1 (the final volume was about 90 ml). The solution was mixed with 1200 ml of 96 per cent (v/v) ethanol and the mixture was stored at 4 C overnight. The precipitate was isolated by centrifugation, washed twice with 150 ml of 96 per cent (v/v) ethanol, and dried ln vacuo. The yield was 1.~ g of highly purified PSP fulfilling the purity requirements stated in Table 1. -Example 2.
20 g of supernatant protein powder, produced asdescribed in Example 1, was dissolved in 200 ml of water.
208 ml of 96 per cent (v/v) ethanol was added, followed by adjustment of pH to 4.6 with acetic acid. A small precipitate was removed by centrifugation. The supernatant, which slowly became turbid, was subjected to cation exchange chromatography on a 2.5 x 80 cm column of "SP-Sephadex C-25", equilibrated in Eluent 1 (0.4 M acetic acid, 0.05 M sodium acetate, 50 per cent (v/v) ethanol, pH: 4.6). Linear gradient elut:ion was performed between 3 1 of Eluent 1 and 3 1 of Eluent 2 (0.3 M sodium acetate/50Fer cent;(v/v) ethanol, pH: 8.7). Fractions of 10 ml were collected at an elution rate of 40 ml/h. The fractions corresponding to the large peak appearing from fractions 100 to 130 were pooled. The pool was given a pH of 8 and then mixed with 1.8 1 of 96 per cent (v/v) ethanol. The mixture was stored at 4C for 24 hours. The precipitated protein was isolated by centrifugation, washed twice with 150 ml of 96 per cent (v/v~ ethanol and dried in vacuo. Yield:
2.8 g. 2.5 g of this material was dissolved in 250 ml of a TRIS bu~fer (0.0575 M TRIS, 0.05 N HCl, pH: 7.~ ). The solu-tion was subjected to anion exchange chromatography on a 2.5 x 50 crn column of "QAE-Sephadex A-25", equilibrated in a TRIS buffer (0.115-M TRIS, 0.1 N HCl, pH: 7.4). The column was eluted with the equlibration buffer at a rate of 30 ml/h.
Fractions of 10 ml were collected. The fractions corresponding to the central major part of the peak showing a maximum at fraction 225 were pooled. The pool (620 ml) was mixed with 60 ml of 5 M sodium chloride an~ 12 1 of 96 per cent ~v/v) ethanol.

~ _ . _ . . . _ . _ _ . ... _ . ~ _ i - 15 -~ 6;~1 The mixtu~-e was stored at 4~C for 24 hours. ~he precipitated protein was isolaked by centrifugation, washed twice with 150 ml of 96 per cent (v/v) ethanol and dried in vacuo.
Yield: 1.7 y of highly purified PSP.

Example 3.
To 150 1 of an a~ueous solution which was obtained b~,~ evaporation of an extract from 250 kg of porcine pancreas gl.~nds and which was freed from insoluble material, 22.5 kg of sodium chloride were added. The mixture was stirred to dissolve the salt added and the resulting precipitate was removed by centrifugation thus affording the salt cake. To the mother liquor (162 1) was added 34 kg of ammonium sulphate with continued stirring for 2 hours at room temperature affording a precipitate which was isolated by centrifugation 223 g of the wet product were dissolved by addition of 50~ ml of a buffer (0.05 M formic acid, 0.01 M sodium hydroxide buffer, pH: 3.2). The conductivity of the solution was reduced to 4 mS by dialysis against water. The solution was applied on a 5 x 50 cm column of "SP-Sephadex C-25"
equilibrated with Buffer I (0.1 M formic acid, 0.02 M sodium hydroxide, pH: 3.2). After application of the solution, the column was ~uted with a linear gradient of sodium chloride from 0 to 0.27 M in Buffer I. The total volume of the eluent was 5.5 1. The column was then further eluted with Buffer I
containing 0.27 M sodium chloride. The flow during the appli-cation al~ elutioh was 100 ml per hour and fractions of 15 ml were collected. The chromatogram obtained by monitoring the optical density of the fractions at 276 nm showed one main peak from fraction 420 to 530. The pool corresponding to the main peak was adjusted to a pH of 7.4 and then mixed with 20 volumes of 96 per cent (v/v) ethanol. Upon standing at 4C for 48 hours, a precipitate was recovered by centrifugation and dried in vacuo. Yield: 6 g. The material so obtained was further purified by anion exchange chromatography on a column of "Q~E-Sephadex A-25", as described in Example 2. Yield: 3.4 g of highly purified PSP.

EXH I B I T A

SUPPLEMENTARY DI SCLOSURE
Among the interesting pharmacological properties of PSP referred to herein, in addition to the spasmolytic effect, PSP also possesses an inhibitory effect on gastric acid secretion. Patients with duodenal ulcers benefit from treatment with agents which inhibit gastric acid secretion.
However, the same patients suffer from an increased gastro-intestinal motility. PSP combines two effects which are highly desirable in the treatment of patients with duodenal ulcers, an inhibitory effect on the gastrointestinal motility, and an inhibitory effect on gastric acid secretion.
As an example of a known medicament which is used to inhibit gastric acid secretion, cimetidin may be mentioned~
However, cimetidin possesses frequent adverse effects such as diarrhoea, exanthema, elevation of liver enzymes, and gynecomastia. As PSP is a polypeptide which is to be dosed orally and as it is not absorbed in substantial amounts in the gastrointestinal tract, it is not likely to have systemic adverse effects.
PSP was found to inhibit pentagastrin stimulated gastric acid secretion in rats and cats with chronic gastric fistulas. 10 ~g PSP infused over 1 hour to rats was found to be as effective in inhibiting the acid secretion after 5 ~g pentagastrin s.c. as 1 ~g somatostatin, i.e. the peptides are equipotent on a molar bas s. 10 ~g/kg PSP s.c. and 250 ~g PSP
orally in a capsule were effective in cats.
The data indicate the value of PSP in the treatment of gastroduodenal ulcers. Therefore, PSP may be administered orally in capsules to patients with gastroduodenal ulcers.

Claims (16)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for isolating a purified polypeptide PSP
exhibiting the following amino acid composition:

Trp (2), Lys (4), His (1), Arg (5), Asx (10), Thr (3), Ser (9), Glx (12), Pro (12), Gly (6), Ala (5), Cysl/2 (14), Val (7), Met (2), Ile (3), Leu (1), Tyr (2), Phe (7), wherein the determinations are subiect to the usual error of ? 10 percent of the indicated figures, and having a partial amino acid sequence which from the N-terminal is:

pyrGlu-Lys-Pro-Ala-Cys-Arg-Cys-Ser-Arg-Glx-Asx-Pro-Lys-Asx-Arg-Val-Asx-Cys-Gly-Phe-Pro-Gly-Ile-Thr-Ser-Asx-Glx-Cys-Phe-Thr-Ser-Gly-Cys-Cys-Phe-Asx Ser-Glx-Val-Pro-Gly-Val-Pro-Trp-, wherein pyrGlu (residue 1) stand for pyroqlutamic acid, or a physiologically acceptable salt thereof, comprising i) forming an extract of porcine pancreas qlands ii) causing precipitation of said polypeptide from the extract, in the semi-pure form, by appropriate adjustment of extract conditions, iii) redissolving the semi-pure form of said polypeptide so obtained, to form a solution thereof, iv) and subjectinq said solution to chromatographic separation so as to recover therefrom said polypeptide in purified form.

2. A method according to claim 1, wherein the extract of porcine pancreas glands is obtained by extracting the glands with an acidic mixture of water and water-miscible organic solvent, removing the precipitate, appropriately adjusting the pH of the liquid extract and removing residual organic solvent, thereby obtaining said extract of porcine pancreas glands in the form of an insulin salt cake.

3. A method according to claim 2, wherein the process of obtaining the insulin salt cake includes the step of salting out from the liquid extract, after removing residual organic solvent.

4. A method accroding to claim 2, wherein crude insulin is removed from the salt cake by forming a solution of said salt cake with a pH in the range from about 4.9 to 5.7, subjecting the solution to isoelectric precipitation, and separating the precipitate from the resulting supernatent liquid.

5. A method according to claim 4, wherein a further insulin containing precipitate is deposited from said resulting supernatant liquid by adjusting the pH of said resulting supernatant to the range from about 5.7 to 7, and wherein the precipitate so formed is removed to leave a residual second supernatant liquid containing dissolved supernatant protein which includes said polypeptide PSP.

6. A method according to claim 5, wherein said polypeptide is isolated from the supernatant protein by recovering solid supernatant protein from said second supernatant liquid, redissolving the solid supernatant in aqueous solution under acidic conditions, chilling the solution and recovering crystals of said polypeptide from the acidic solution.

7. A method according to claim 6, wherein said polypeptide so formed is further purified by a chromatographic method.

8. A process according to claim 7, wherein said chromatographic method includes use of a cation and/or an anion exchanger.

9. A process according to claim 8, wherein the chromatography is carried out with the collection of the major part of the main PSP peak.

10. A method according to claim 1, which comprises extracting the porcine pancreas glands with an acidic mixture of water and water-miscible organic solvent, removing the precipitate of gland residue, appropriately adjusting the pH of the liquid extract to an acid range and removing residual organic solvent, salting out an insulin salt cake by addition of a suitable salt thereto under conditions favouring the solubility of said polypeptide in the mother liquor, separating the insulin salt cake precipitate from the mother liquor, and subsequently extracting said polypeptide from the mother liquor by salting out therefrom, followed by purification thereof.

11. A method according to claim 1, wherein porcine pancreas glands are extracted with a mixture of water and an organic water-miscilbe solvent to form said extract, precipitation from said extract takes place by absorption on an ion exchanger, and the polypeptide is recovered from the ion exchanger by redissolving in an appropriate solvent.

12. The polypeptide PSP whenever prepared or produced by a process according to claim 1, claim 2 or claim 3 or an obvious chemical equivalent thereof.

13. The polypeptide PSP whenever prepared or produced by a process according to claim 4, claim 5 or claim 6 or an obvious chemical equivalent thereof.

14. The polypeptide PSP whenever prepared or produced by a process according to claim 7, claim 8 or claim 9 or an obvious chemical equivalent thereof.

15. The polypeptide PSP whenever prepared or produced by a process according to claim 10, or an obvious chemical equivalent thereof.

16. The polypeptide PSP whenever prepared or produced by a process according to claim 11, or an obvious chemical equivalent thereof.