IE50966B1 - Spasmolytic polypeptide - Google Patents

Description

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 spasmolytic agent. The purified polypeptide 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 Polypeptide, hereinafter for the sake of convenience abbreviated to PSP. Besides, PSP has surprisingly been found to possess an inhibitory effect on gastric acid secretion.

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 visualization 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 parenterally and, owing to the size of the dose needed to induce relaxation, the side effects classical to those agents are usually encountered. 50ΘΘ6 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. 3,862,301). However, glucagon exerts a plurality of actions in the human body including 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 provided certain advantages, undesirable side effects were not completely abolished.

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. it is an object of this invention to provide a spasmolytic agent which, whilst possessing antispasmodio and smooth muscle relaxing effects comparable to those of known agents, exhibits substantially reduced side effects.

Apart from the spasmolytic effects of PSP, the peptide 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 gastrointestinal 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.

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 (1), Arg (5), Asx (10), Thr (3), Ser (9), Glx (12), Pro (12), Gly (6), Ala (6), Cys J (14), Val (7), Met (2), lie (3), Leu (1), Tyr (2), Phe (7), wherein the determinations are subjected to an error of + 10 percent 10 of the indicated figures, having a molecular weight, calculated from the amino acid composition, of about 11,700, having a molecular weight, determined by sodium dodecyl sulphate gel electrophoresis of about 10,700 and having a basic DISC electrophoresis in polyacrylamide gel showing essentially a single band with 0.65-0.75, with a similar pattern being obtained in analytical electrofocusing in polyacrylamide gel, by which method the pH is determined to about 4.4, or a physiologically acceptable salt thereof.

The amino acid sequence is selieved to be: pyrGly-Lys-Pro-Ala-Ala-Cys-Arg-Cys-Ser-Arg-Gln-Asp-Pro-Lys-Asn20 1 5 10 15 Arg-Val-Asn-Cys-Gly-Phe-Pro-Gly-Ile-Thr-Ser-Asp-Gln-Cys-Phe-Thr20 25 30 Ser-Gly-Cys-Cys-Phe-Asp-Ser-Gln-Val-Pro-Gly-Val-Pro-Trp-Cys-Phe35 40 45 Ser-Pro-Leu-Pro-Ala-Gln-Glu-Ser-Glu-Glu-Cys-Val-Met-Gln-Val-Lys50 55 60 Ala-Arg-Lys-Asn-Ser-Gly-Tyr-Pro-Gly-Ile-Cys-Pro-Glu-Asp-Cys-Ala65 70 75 Ala-Arg-Asn-Cys-Cys-Phe-Ser-Asp-Thr-Ile-Pro-Glu-Val-Pro-Trp-Cys30 80 85 90 95 Phe-Phe-Pro-Met-Ser-Val-G1u-Asp-Cys-Hi s-Tyr, 100 105 wherein pyrGlu (residue 1) stands for pyroglutamyl. 50986 The aooreviations for uib ammo acids are taken from J. Biol. Chem. 243 (1968), 3558.

The present invention also provides a method for preparing purified PSP, which method comprises isolating PSP from porcine pancreatic tissue preferably from the insulin salt cake by a combination of chromatography and precipitation processes, as well as a method which comprises extracting the glands with a mixture of water and a water-miscible organic solvent in an acid medium, removing the precipitate, neutralising the extract, and isolating polypeptiue from the extract thus obtained whereafter the polypeptide, if desired, is converted into a salt thereof.

The insulin salt cake may be prepared as follows: Whole, neatly defatted porcine pancreas glands are finely comminuted under frozen conditions and then subjected to the conventional extraction process for recovery of insulin, that is extracted with a mixture of water and an organic water-miscible solvent, such as a lower (that is containing less than 5 carbon atoms) aliphatic alkanol, for example ethanol or isopropanol, in an acid medium, for example a medium having a pH in the range of from about 1.5 to 5 when measured with a pH meter in the mixture. The acid pH. is obtained by the addition of an acid.

In the mixture, the organic solvent is present in a concentration in the range of from about 49% to 80% (v/v) when all the components are mixed. The resulting slurry is stirred at a temperature in the 50866 range of about 5’ to ambient followed by removal of the gland residues, e.g., by centrifugation. The extract pH is adjusted to the range of from about 5 to 9. Then, after being clarified, for example by centrifugation, the extract is acidified to a pH in the range of from about 3 to 4, whereafter 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 including PSP, is salted out from the concentrated extract so obtained, for example by the addition of sodium chloride to a concentration in the range of from about 10 to 30% (w/v); the precipitate which forms js isolated, for example by centrifugation, thus affording tbs insulin salt cake.

The salt cake thus obtained is dissolved in water and crude insulin isolated by isoelectric precipitation at a pH in the range of from about 4.9 to 5.7, for example about 5.3, optionally in the presence of metal ions, for example zinc ions. The insulin precipitate is recovered, usually by centrifugation. The supernatant is then given a pH in the range of from about 5.7 to 7, preferably about 6.5. The precipitate that forms, containing some insulin, is also centrifuged off. In order to remove ancillary substances, such as salts, an excess of EDTA is added i° . 50866 above second supernatant, followed by the addition of a watermiscible organic solvent, preferably ethanol {usually from to 20 volumes). The mixture is left to precipitate overnight at about 4°C. and then centrifuged to recover precipitated material. The precipitate is dried in vacuo, yielding a dry powder, hereinafter referred to as supernatant protein By this procedure practically all protein material of the supernatant protein is recovered.

PSP can be isolated in a crude crystalline form a solution of the supernatant protein in water {about 10 per cent by weight). 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 pH 4.3,is attained. The mixture is then chilled and the stirring is continued for 3 days, preferably at about 4"C. A crop of relatively large, bar-shaped, birefringent crystals is isolated. for example, by centrifugation, and then dried in vacuo.

The material so obtained may be further purified, preferably by applying consecutive steps of anion and cation exchange chromatography.

Referring now to the drawings.

Figure 1 illustrates the details of an exemplary anion exchange chromatographic purification of crude PSP; and, Figure 2 illustrates the’details of cation exchange chromatographic purification of the PSP fraction from the anion exchange purification of Figure 1.

To 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 Figure 1 of the accompanying drawings (TRIS being tris[hydroxymethyl]aminomethane).

The chromatogram obtained by monitoring the optical density of fractions at 276 nm shows one main peak. The pool corresponding to the main peak is adjusted to pH 7.4 and then mixed with a water-miscible organic solvent, for example ethanol (4 volumes). Upon standing at 4°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 U of SP-Sephadex C-25 (supplied by Pharmacia). Elution may be effected with the eluent stated on Figure 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 precipitates from the solution, is isolated by centrifugation, washed with ethanol, and dried in vacuo.

'Sephadex is a trade mark 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). The mother liquor is subjected to an additional salting out process. is recovered, for example by centrifugation, obtained from the precipitate by the use of anion and/or cation chromatography in any order.

The precipitate Purified PSP can be 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 solvent 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 preformed by methods which are analogous to known methods.

PSP obtained by any of the above methods has the following characteristics: Molecular weight, calculated from the amino acid composition: about 11,700.

Molecular weight, determined by sodium dodecyl sulphate gel electrophoresis (Neville: J. Biological Chemistry 246 (1971) 6328): about 10,700.

Electrophoretic characteristics: Basic DISC electrophoresis (basic DE) in polyIQ acrylamide gel as described by J. Schlichtkrull et al.

(Horm. Metabol. Research, Suppl. Series 5_ (1974) 134) shows essentially a single band with Rf 0.65-0.75. A similar pattern is obtained in analytical electrofocusing in polyacrylamide gel by which method the pi is determined to about 4.4 Products obtained upon treatment of PSP with trypsin, a chymotrypsin, CNBr, acid, or pyroglutamate aminopeptidase as described bela·;, have a spasmolytic activity of the same order as that of PSP.

Trypsin treatment: Twenty mg of PSP was dissolved in 20 ml of 0.01 M NH^HCO (pH : 7.8) and preincubated for 5 minutes at 37°C. After addition of 100 μΐ of 0.001 M HCl containing 0.4 mg TPCK-trypsin (obtained from Worthington Biochem.Corp.), the mixture was incubate: at 37°C for 15 minutes and then lyophilized. α-Chymotrypsin treatment: Twenty mg of PSP was dissolved in 200 μΐ of 0.1 M NaOH and 1800 μΐ of 0.05 M NH4HCO3 (pH: 8.0) was added. The 50986 solution was preincubated for 5 minutes at 37°C and 50.gl of 0.001 M HCl.containing 100 gg α-chymotrypsin (obtained from Sigma-Chemical Company)was added. The incubation was continued for one hour at 37°C and the reaction was stopped by the addition of 50 gl concentrated acetic acid, whereafter the solution was lyophilized.

CNBr treatment Twenty mg of PSP was dissolved in 2 ml of 70% (v/v) formic acid containing 72 mg CNBr. The mixture was stored at room temperature for 40 hours and then lyophilized. The lyophilization was then repeated after addition of 2 ml of water.

Acid treatment Samples of 1 mg PSP, dissolved in 100 gl of 0.5 N hydrochloric acid, were incubated at 37°C for 2, 10 and 21 days. After incubation the protein of each sample was precipitated quantitatively by the addition of 2 ml of acetone. The precipitate was isolated by 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 that the time of electrcphores was reduced to give R^ = 0.53 for PSP. In the sample incubated for 2 days a series of bands were observed with R^ ranging from 0.53 to 0.86. In the samples incubated for 10 and 21 days only a single band with Rf 0.86 appeared. The results indicate that a partial deamidation of PSP had occurred after 2 days and a complete deamidation after 10 days of incubation.

Pyroglutamate aminopeptidase treatment A sample of 6 mg of PSP was dissolved in 2 ml of 50 mM sodium monohvdrogen phosphate, 30 mM p-mercaptoethanol, mM EDTA buffer with a pH of 7.8. A solution of 2.5 mg of pyroglutamate aminopeptidase (obtained from 3oehringer Mannheim) in 0,5 ml of the above buffer was added. The mixture was incubated for 16 hours at 37 °C and thereby lyophilized. (2.5 mg of pyroglutamate aminopeptidase used contained about 10 mU enzymatic activity.) 50968 The purity of the final PSP product may be checked by analytical isoelectric focusing (IEF) and basic DISC electrophoresis (basic DE, vide supra).The product migrates essentially as a sifigle band in both systems. 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).

*Ampholine is a Trade Mark Likewise, gel filtration of the polypeptide on Bio-Gel P-30 (supplied by Biorad Laboratories, Richmond, Califormia, U.S.A.) using 1 molar acetic acid as the eluent, reveals only a single peak.

PSP has been analyzed for a number of immunoreactivities according to methods known in the art. The results obtained are presented in Table 1: Table 1 Immunoreactant Contents (ppm) Insulin (IRI) 3-6 Total glucagon (total GLI) <0.02 Pancreatic glucagon (pancreatic GLI) <0.02 Vasoactive intestinal peptide (VIP) <0.02 Pancreatic polypeptide (porcine) ^0.08 C-Peptide (porcine) <0.1 Somatostatin λ, 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 containing approximately . 4 mg protein per ml) mixed with Freund's adjuvant (0.5 ml) twice weekly for a period of 26 weeks. Beginning from the 13th day after 30 the first immunization, a total of 10 blood samples (10 ml) from 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. 125 I-PSP was prepared by tht. lactoperoxidase method developed by Thorell and Johansson (Biochim.Biophys.Acta 251 (1971) 363). The radioiodinated PSP was purified by anion exchange chromatography as known in the art aiid used for polypeptide radioimmunoassay according to the procedure developed by L.G. Heding (Diabetologica T_ (1971), 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 sulphuric acid, can be mentioned.

For the sake of brevity, the designation PSP Compounds is used to cover PSP and physioloqically acceptab1 salts thereof.

PSP and glucagon were found to be about 15 equipotent in their inhibition of the amplitude of the contractions of electrically stimulated guinea pig ileum in vitro, vide Table 2. PSP and glucagon were dissolved in 0.9% sodium chloride with 0.1% human serum albumin, (percentages by weight).

Table 2 Concentration in the organ bath, M Inhibitory effect in per cent PSP Glucagon 10~5 89 89 io"6 49 51 107 21 24 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. 30968 Likewise, PSP Compounds were found to be about as potent as glucagon with respect to its' inhibition jn vivo of the peristalsis in mice, vide Table 3, an effect which again could be blocked by phentolamine.

Table 3 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 Out of 5 rabbits (from 2.5 to 3.0 kg body weight) 400 pg PSP administered intravenously or 5 cm from the balloon into the lumen of the intestine caused a marked reduction of the intestinal motility almost to atonia. 200 μg PSP had 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-'^c] protein hydrolysate in pigs and in pancreatectomised dogs and of (U-14Cl ovalbumin in pancreatectomised dogs, when the compound was administered perorally in a capsule with 3 mg PSP. The pigs and the dogs weighed about 30 kg.. 100 gCi [O-^4C] protein hydrolysate or 5 pCi [U-^4C] 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/kg of PSP orally probably reflects a reduced gastrointestinal motility.

PSP was found to inhibit .pentagastrih stimulated gastric acid secretion in rats and cats with chronic gastric fistulas. 10 pg PSP infused over 1 hour to rats was found to TO be as effective in inhibiting the acid secretion after 5 pg pentagastrin s.c. as 1 pg somatostatin, i.e. the peptides are almost eguipotent on a molar basis. 10 jig/kg PSP s.c. and 250 pg PSP orally in a capsule were effective in cats.

PSP Compounds were found to be devoid of any in vitro effect 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 effect on the blood pressure of the anesthetized rat.

The above pharmacological data indicate the 20 value of PSP Compounds Cor the treatment and prevention of smooth muscle spastic conditions, for example in theintestin.e. Due to the lack of metabolic effects,PSP Compounds may prove advantageous as a substitute'for glucagon in endoscopy and in radiological procedures.

Besides the data indicate the value of PSP in the treatment of increased gastrointestinal motility and gastroduodenal ulcers.

PSP compounds may be administered intravenously as a bolus or as an infusion. When an effect of prolonged nature, slower xn onset, is desired, PSP Compounds may be administered as a depot *rom which it is slowly mobilized by the blood stream such as intramuscularly ο»· subcutaneously in a region of good peripheral *Idon is a Trade Mark circulation supply. The fact that the biological activity and the immunoreactivity is maintained after exposure of PSP to gastric juice, trypsin, and chymotrypsin and the experiments described above showing delayed absorption and inhibition of gastric acid secretion 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. PSP may be administered orally in capsules to patients with gastroduodenal ulcers.

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 range, from 10 to 200 pg per kg body weight can be mentioned, although a lower or higher 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.

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 dry weight of a PSP Compound (percentages by weight).

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. I per thousand" designates that 1 kg of pancreatin pills contain about 1 g of PSP.

The following Examples, which, however, are not considered 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 dissolved in water to a volume of 3.2 1 . The pH of the solution was adjusted to 5.3, whereafter the precipitate was removed by centrifugation. The pH of the supernatant was adjusted to 6.5 and the suspension thus formed was centrifuged. The solution was mixed with 32 ml of 0.5 M Na^EDTA and 35 1 of ethanol. The mixture was left overnight at 4°C 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 (after about 3 hours of pumping). Stirring was then continued for 3 days at 4°C. whereby crystallization occured. The crop of crystals (bar-shaped by appearance, possibly orthorhombic and showing birefringence) were harvested by centrifugation, suspended in 500 ml of water at 4°C with stirring overnight, centrifuged and dried in vacuo. The yield was 5.2 g. 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 chromatography as shown in Fig 1. The pool frcm the main peak was given a pH of 7.4, mixed with 4 volumes of 96 per cent (v/vi ethanol and then stored at 4°C for 2 days. The precipitate was isolated by centrifugation, washed twice with 150 ml of 96 per cent fv/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) at 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 bycentrifugation, washed twice with 150 ml of 96 per cent (v/v) ethanol, and dried in vacuo. The yield was 1.8 g of highly purified PSP fulfilling the purity requirements stated in Table 1.

Example 2. g of supernatant protein powder, produced as described 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, per cent (v/v) ethanol, pH: 4.6). Linear gradient elution was performed between 3 1 of Eluent 1 and 3 1 of Eluent 2 (0.3 M sodium acetate,50 per 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 4°C 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 buffer (0.0575 M TRIS, 0.05 N HCl, pH: 7.4). The solution was subjected to anion exchange chromatography on a 2.5 x 50 cm 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 and 12 1 of 96 per cent (v/v) ethanol.

The mixture was stored at 4°C 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: 1.7 g of highly purified PSP.

Example 3.

To 150 1 of an aqueous solution which was obtained by evaporation of an extract from 250 kg of porcine pancreas glands 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 500 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 eluted 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 application and elution 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 4°C 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 QAE-Sephadex A-25, as described in Example 2. Yield: 3.4 g Of highly purified PSP.

EXAMPLE 4 A preparation for parenteral administration containing 1 mg of PSP per ml may be prepared as follows: lg of PSP and 99 g of lactose are dissolved in 5 1 liter of distilled water and the pH is adjusted to 7.0. The solution is thereafter steril filtered. The sterile solution is filled in 10 cc vials in such a way that each vial contains 10 ml of the solution. Thereafter, the solutions are lyophilised and the vials are sealed at aseptic conditions. 1q The preparation in any of the vials is to be dissolved in 10 ml of sterile water before administration.

EXAMPLE 5 Oral preparations may be prepared as follows: 100 mg of PSP is admixed with 9 g of maize starch, 8 g of lactose, and 180 mg magnesium stearate until a homogeneous mixture is obtained. The mixture is filled in hard gelatine capsules No. 3 in such a way that each capsule contains 1 mg of PSP.

Claims (27)

1. A purified polypeptide 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 (6), Cys| (14), Val (7), Met (2), He (3), Leu (1), Tyr (2), Phe (7), wherein the determinations are subjected to an error of + 10 per cent of the indicated figures, having a molecular weight, calculated from the amino acid composition, of about 11,700, having a molecular weight, determined by sodium dodecyl sulphate gel electrophoresis of about 10,700 and having a basic DISC electrophoresis in polyacrylamide gel showing essentially a single band with R f 0.65-0.75, with a similar pattern being obtained in analytical electrofocusing in polyacrylamide gel, by which method the pH is determined to about 4.4 and having an amino acid sequence which so far elucidated is believed to be: pyrGlu-Lys-Pro-Ala-Ala-Cys-Arg-Cys-Ser-Arg-Gln-Asp-Pro-Lys-Asn15 10 15 Arg-Val-Asn-Cys-Gly-Phe-Pro-Gly-Ile-Thr-Ser-Asp-Gln-Cys-Phe-Thr20 25 30 Ser-Gly-Cys-Cys-Phe-Asp-Ser-Gln-Val-Pro-Gly-Val-Pro-Trp-Cys-Phe35 40 45 Ser-Pro-Leu-Pro-Ala-Gln-Glu-Ser-Glu-Glu-Cys-Val-Met-Gln-Val-Lys50 55 60 Ala-Arg-Lys-Asn-Ser-Gly-Tyr-Pro-Gly-Ile-Cys-Pro-Glu-Asp-Cys-Ala65 70 75 Ala-Arg-Asn-Cys-Cys-Phe-Ser-Asp-Thr-Ile-Pro-Glu-Val-Pro-Trp-Cys80 85 90 Phe-Phe-Pro-Met-Ser-Val-Glu-Asp-Cys-His-Tyr, 100 105 wherein pyrGlu (residue 1) stands for pyroglutamic acid, or a physiologically acceptable salts thereof.

2. A polypeptide according to Claim 1, which has a purity of at least 50 per cent by weight, preferably higher than 90 per cent by weight.

3. A polypeptide according to Claim 2, which has a purity of at least 90 per cent by weight.

4. A polypeptide according to Claim 1, which is in crystalline form.

5. A method for isolating a purified polypeptide according to Claim 1, which comprises isolating the same from porcine pancreas glands by a combination of chromatography and precipitation.

6. A method according to Claim 5, comprising extracting the glands with a mixture of water and a water-miscible organic solvent in an acid medium, removing the precipitate, neutralising the extract, and isolating PSP from the extract thus obtained using separation methods known per se, whereafter PSP, if desired, is converted into a salt thereof.

7. A method according to Claim 5, which comprises isolating the same from an insulin salt cake.

8. A method according to Claim 6, wherein the isolation is performed by chromatography by the use of a cation and/or an anion exchanger.

9. A method according to Claim 8, which includes a crystallization process.

10. A method according to Claim 8, wherein the chromatography is carried out by the collection of a major part of the main PSP peak.

11. A method according to Claim 6, which comprises isolating PSP from the mother liquor arising when preparing an insulin salt cake, by a further salting out procedure followed by chromatography using an anion and/or a cation exchanger.

12. A method according to any cne of Claims 7-10, wherein the salt cake is substantially freed of insulin by removing the precipitate formed in a solution of tne salt cake given a pH in the range of from 4.9 to 5,7, and optionally removing the precipitate formed in the supernatant at a pH in the range of from 5.7 to 7.0. before the chromatography is carried out.

13. A method according ‘-.o Claim 9, wherein the crystallisation process is carried out by adjusting the pH of the solution to a value in the range of from about 3.8 to 4.8.

14. A polypeptide according to Claim 1 whenever purified by a method in accordance with any one of Claims 5-13, 2?

15. A pharmaceutical composition which comprises an effective amount of a purified polypeptide in accordance with any one of Claims 1-4 and 14, in association with a suitable physiologically acceptable carrier or excipient.

16. An aqueous sterile solution of a purified polypeptide in accordance 5 with any one of Claims 1-4 and 14.

17. An aqueous sterile solution according to Claim 16, which contains about 0.9 per cent by weight sodium chloride.

18. An aqueous sterile solution according to Claim 16 or 17, which contains preservatives such as parabene or ohenol. 10

19. A purified polypeptide in accordance with any one of Claims 1-4 and 14, for use as a medicament.

20. A purified polypeptide according to Claim 19, for use as a spasmolytic agent.

21. A purified polypeptide according to Claim 20, for use as a 15 diagnostic aid.

22. A purified polypeptide according to Claim 20, for use as a therapeutic agent.

23. A purified polypeptide according to Claim 22, for use as a prophylactic agent. 20

24. A solution according to Claim 16, 17 or 18, wherein the purified polypeptide is present in a concentration of from 0.1 to 200 mg per ml.

25. A solution according to Claim 24, wherein the purified polypeptide is present in concentration of from 0.5 to 25 mg per ml.

26. A method for preparing a purified polypeptide substantially as ?5 hereinbefore described with reference to any of Examples 1 to 5.

27. A purified polypeptide as defined in claim 1 whenever prepared by a method as claimed in any one of Claims 5-13 and Claim 26.