NO802683L - PROCEDURE FOR PREPARING POLYPEPTID PSP - Google Patents

Description

The present invention relates to a hitherto unknown

purified polypeptide or physiologically tolerable salts thereof, a method for producing and purifying the polypeptide and the use of the polypeptide as a spasmolytic agent. It has surprisingly been shown that the purified polypeptide according to the invention, which can be extracted from porcine pancreatic glands, exhibits a relaxing effect on smooth muscle or spasmolytic

effect. The compound has therefore been assigned the trivial designation Pancreatic Spasmolytic Polypeptide, which is abbreviated here for convenience to PSP. PSP exhibits interesting pharmacological properties.

Spasmolytic agents or antispasmodics such as atropine, similar compounds and synthetic drugs with atropine-like activity are widely used to treat various diseases, especially spasms in the smooth muscles, and conditions with hypermotility. However, the desired effect of such drugs is usually accompanied by several side effects due to their general properties as anticholinergic substances.

Atropine-like anticholinergic drugs have also largely found use as a diagnostic aid in gastrointestinal radiology, especially in conjunction with an X-ray imaging medium to improve the imaging of the gastrointestinal area, the bile duct area and the urinary tract. Such a drug is usually administered parenterally, and the side effects that are classic for the agents in question usually occur due to the dose amount necessary to achieve a relaxing effect.

Parenteral administration of the peptide hormone glucagon consisting of 29 amino acids was recently introduced as an alternative means of reducing gastrointestinal motility in the context of radiographic examinations (cf. U.S. Patent No. 3,862,301). However, glucagon produces several effects in the body, including a strong impact on metabolic functions, among which the most obvious effects are the induction of hyper-glycaemia and lipolysis. Thus, although the introduction of glucagon to endoscopy brought certain advantages, the undesirable side effects were not completely eliminated. An object of the present invention is the provision of a spasmolytic agent which, while exhibiting antispasmodic effects and relaxing effects on smooth muscles, which effects are comparable to the effects of the known agents, exhibits substantially reduced side effects.

According to one aspect of the present invention, a hitherto unknown, purified polypeptide with the following amino acid composition is provided:

Trp (2), Lys (4), His (1), Arg (5), Asx (10), Thr (3), Ser (9), Glx (12), Pro (12), Gly (6), Ala (6), Cys 1/2 (14), Val (7), Met (2), Ile (3), Leu (1), Tyr (2), Phe (7), where the determinations are affected by the usual error of ±10% of the stated values. The partial amino acid sequence comprising a total of 45 amino acids from the N-terminal end is believed to be the following:

where pyrGlu (amino acid residue 1) denotes pyroglutamic acid.

The amino acid sequence for PSP is believed to be as follows:

The abbreviations for the amino acids appear in J.Biol.Chem., 243,

(1968), 3558.

The present invention also relates to a method for producing purified PSP, which method is characterized in that PSP is isolated from pancreatic tissue from pigs, preferably from the insulin salt cake, by a combination of chromatography and precipitation.

The insulin salt cake can be prepared in the following way: Whole, carefully defatted porcine pancreatic glands are crushed in the frozen state and then extracted in the usual way to extract insulin, i.e. extracted with a mixture of water and an organic, water-miscible solvent such as a lower aliphatic alkanol, e.g. ethanol or isopropanol, in an acidic medium, for example a medium with a pH value in the range from approx. 1.5 to approx. 5, when measured with a pH meter in the mixture. The acidic pH value is achieved by adding an acid. in the mixture, the organic solvent is present in a concentration in the range from approx. 40 to approx. 80% (volume/volume) when all ingredients are mixed. The resulting slurry is stirred at a temperature in the range from approx. 5°C to room temperature,• after which the rest of the glands are removed, for example by centrifugation. The extract is then neutralized to a pH value in the range from approx. 5 to approx. 9 and is managed, e.g. by centrifugation. The extract is acidified to a pH value in the range of approx. 3 to approx. 4, after which the extract is freed from organic solvent, e.g. by evaporation at reduced pressure, followed by removal of fatty compounds, e.g. by centrifugation. Insulin, which is mixed with other proteins and polypeptides such as PSP, is salted out from the concentrated extract obtained in this way, for example by adding sodium chloride to achieve a concentration in the range from

about. 10 to approx. 30% (weight/volume), and the precipitate formed is isolated, e.g. by centrifugation, whereby the salt cake is obtained.

The salt cake obtained in this way can then be dissolved in water, and crude insulin can be isolated by isoelectric precipitation at a pH value in the range from approx. 4.9 to approx. 5.7, e.g. at approx. 5.3, optionally in the presence of metal ions, for example zinc ions, and the crude insulin is recovered, usually by centrifugation. The supernatant is adjusted to a pH value in the range from approx. 5.7 to approx. 7, preferably at approx. 6.5. The formed precipitate containing some insulin is filtered off. For the removal of excipients such as salts. an excess of EDTA is added to the above-mentioned 2. supernatant, followed by the addition of a water-miscible organic solvent, preferably ethanol (usually from 5 to 20 volumes). The mixture is allowed to stand overnight at approx.

4<Q>C for precipitation and subsequent centrifugation. The precipitate is dried in a vacuum, whereby a dry powder is obtained which is hereinafter referred to as "supernatant protein". At this point, practically all the protein in "supernatant protein" is recovered.

PSP can be obtained in crude, crystalline form from a solution

of "supernatant protein" in water (about 10 parts). The solution is stirred carefully, while during approx. 3 hours are added

acid, e.g. acetic acid, until a pH value in the range of approx. 3.8 to approx. 4.8, preferably approx. 4>3. The mixture is then cooled, and stirring is continued for 3 days, preferably at approx. 4°C. One extracts, for example by centrifugation,

a product consisting of relatively large, rod-shaped, birefringent crystals that are dried in a vacuum.

The product thus obtained can be further purified, preferably using anion and cation chromatography in logical order.

To clarify the method, anion exchange chromatography can be performed on a column with "QAE-Sephadex A-25" (sold by Pharmacia AB, Sweden) using the one in fig. 1

specified eluent.

The chromatogram obtained by reading the absorption of fractions at 276 nm gives one main peak. What is collected and which corresponds to the main peak is adjusted to a pH value of 7.4 and then mixed with a water-miscible organic solvent, for example ethanol (4 volumes). After standing at 4°C

for 2 days, a precipitate is recovered by centrifugation which is dried in a vacuum.

The product obtained in this way can be further purified by cation chromatography, for example on a column with "SP-Sephadex C-25" (sold by Pharmacia). The elution can be carried out with the one in fig. 2 specified eluent. The chromatogram obtained in an analogous manner as described above shows one main peak. Collected fractions corresponding to the main peak are evaporated to dryness, and the residue is dissolved in water at a pH value. in the area from approx. 6 to approx. 8, for example approx. 7, is mixed with an excess (approx. 12 volumes) of a water-miscible organic solvent, for example ethanol, and allowed to stand overnight under similar conditions as described above. Purified PSP that precipitates from the solution is isolated by centrifugation, washed with ethanol and dried in vacuum.

Alternatively, PSP-containing protein can be obtained by further salting out the mother liquor obtained, when the salt cake is isolated using sodium chloride in a concentration of from 10 to 20%

(weight/volume). The precipitate is recovered, for example by centrifugation. Purified PSP can be obtained from the precipitate using anionic and/or

or cation chromatography in any order.

In a further method, PSP-containing protein can be recovered by adsorption on a cation or anion exchanger, for example alginic acid, sulphonated polystyrene or aminoethyl cellulose, from the above-mentioned extract of pancreatic glands obtained using a mixture of water and an organic with water miscible solvent. The ion exchanger is then washed, and the protein is eluted with an aqueous medium. The recovery using an ion exchanger is carried out analogously to known methods.

PSP which is extracted by any of the above

specified methods, have the following characteristics:

Molecular weight (calculated on the basis of the amino acid composition): approx. 11,700.

Molecular weight (determined by sodium dodecyl sulfate gel electrophoresis (Nevillé: J.Biol.Chem. 246, 6328)): approx. 10,700.

Electrophoretic characteristics:

By basic DISC electrophoresis (basic DE) in polyacrylamide gel (Horm. Metabol. Research, Suppl. Series 5_, 134) essentially a single band with an R^ value of 0.65-0.75 is obtained. An analogous pattern is obtained by analytical electrofocusing in polyacrylamide gel, and the PI value is determined in this way to approx. 4.4.

Products obtained by treating PSP with .trypsin, α-qhymo-trypsin, CNBr, acid or pyroglutamate aminopeptidase as described below have spasmolytic activity of the same order of magnitude as

PSP.

Trypsin treatment:

20 mg of PSP are dissolved in 20 ml of 0.01 M ammonium bicarbonate solution (pH value: 7.8), and the mixture is incubated for 5 minutes at 37°C. The mixture is incubated at 31°C for 15 minutes after the addition of 100 µl of 0.001 M hydrochloric acid containing 0.4 mg of TPCK-trypsin (available from Worthington Biochem. Corp.) and then lyophilized. g- Chymbrypsin treatment: 20 mg of PSP is dissolved in 200 µl of 0.1 M sodium hydroxide solution, and 1800 µl of 0.05 M ammonium bicarbonate solution (pH value: 8.0) is added. The solution is incubated for 5 minutes at 37°C, and 50 µl of 0.0 µl M hydrochloric acid containing 100 µg of α-chymotrypsin (available from Sigma Chemical Company) is added. The incubation is continued for 1 hour at 37°C, and the reaction is stopped by the addition of 50 µl of concentrated acetic acid, after which the solution is freeze-dried. CNB r— treatment; 20 mg of PSP is dissolved in 2 ml of 70% (vol/vol) formic acid inside containing 72 mg of CNBr. The mixture is stored at room temperature for 40 hours and then freeze-dried. After adding 2 ml of water freeze-drying is carried out again.

Acid treatment:

Samples containing 1 mg of PSP dissolved in 100 µl

0.5 N hydrochloric acid, incubated at 37°C for 2, 10 and 21 days. After incubation, the protein in each sample is precipitated quantitatively by adding 2 ml of acetone. The precipitate is recovered by centrifugation, washed with 2 ml of acetone and dried in a vacuum. The samples obtained in this way and a sample of untreated PSP are analyzed by basic DE (cf. above) with the caveat that the electrophoresis time is reduced, so that an R^ value of 0.53 is obtained for PSP.

In the sample that had been incubated for 2 days, a series of bands with R^ values between 0.53 and 0.86 is obtained. In the samples that were incubated

for 10 and 21 days, only a single band with an Rf value of 0.86 is obtained. It appears from the results that after 2 days a partial deamidation of PSP has occurred and a complete deamidation after 10 days of incubation.

Pyroglutamate aminopeptidase treatment:

Dissolve 6 mg of PSP in 2 ml of 50 mM sodium monohydrogen phosphate,

30 mM p-mercaptoethanol, 1 mM EDTA buffer with a pH value of 7.8. A solution of 2.5 is added. mg of pyroglutamate aminopeptidase (available from Boehringer, Mannheim) in 0.5 ml of the above buffer. The mixture is incubated for 16 hours at 37°C and then freeze-dried.

(2.5 mg of the pyroglutamate aminopeptidase used contained

about. 10 mU enzyme activity).

The purity of the PSP final product can be checked by analytical isoelectric focusing (IEF) and basic DISC electrophoresis (basic DE, cf. above). The product travels mainly as a single band in both systems. IEF is carried out as indicated in the instructions in LKB brochure I-1804-E02: "LKB Ampholine PAG" plates for analytical electrofpcusing on polyacrylamide gels (LKB-Produkter AB, Bromma, Sweden)..

In the case of gel filtration of PSP on "Bio-Gel P-30" (sold by Bio-rad Laboratories, Richmond, California, U.S.A.) only a single peak is likewise obtained, since IM acetic acid is used as eluent.

Using known methods, PSP is analyzed for a number of immunoreactivities. The results obtained are shown in table 1:

PSP's immunoreactivity is determined by an extremely specific "radioimmunoassay" that has been developed, so that it is sensitive down to 250 pg/ml.

Antibodies are produced by immunizing rabbits with "supernatant protein" (0.5 ml of a solution containing about 4 mg of protein per ml) which is mixed with 0.5 ml of Freund's adjuvant,

Twice a week during 26 weeks. Starting from the 13th day

after the first immunization, a total of 10 blood samples of 10 ml are collected at regular intervals during 172 days from each animal. The antisera obtained are examined for affinity and capacity, and a suitable antiserum is selected for use in the radioimmunoassay.

125

I-PSP is produced by the lactoperoxidase method (Biochim. Biophys. Acta 251, 363). Radioiodinated PSP is purified in a known manner by anion exchange chromatography and used for polypeptide radioimmunodetermination (Diabetologica 7, 10). -

The present invention also relates to salts of PSP, and

Examples of such salts include salts with cations such as sodium, potassium, magnesium, calcium and zinc and acid addition salts with organic or inorganic acids such as formic acid, methanesulphonic acid, hydrochloric acid and sulfuric acid. For the sake of brevity, the designation PSP compounds is used for PSP and physiologically tolerable salts thereof.

It has been shown that PSP compounds and glucagon are approximately equipotent with respect to inhibition of the amplitude of the contractlons of electrically stimulated guinea pigs in vitro, cf. Table 2. PSP and glucagon were dissolved in a 0.9% sodium chloride solution with 0.1% human serum albumin.

This effect of PSP compounds is blocked by phentolamine, but not by rialoxone. The spontaneous motility of the insulating ileum from reserpine-treated guinea pigs is inhibited with PSP.

It has similarly been shown that PSP compounds are almost as powerful as glucagon with regard to inhibition of peristalsis in mice in vivo, cf. table . 3, an effect that can be blocked with phentolamine.

PSP reduces intestinal motility in rabbits in vivo after intravenous or intraluminal administration in the intestine. The motility is determined using a balloon catheter in the intestine which is connected to a pressure transducer. In 5 out of 5 rabbits (from 2.5 to 3.0 kg body weight) 400 µg of PSP administered intravenously or 5 cm from the balloon into the intestinal cavity produced a marked decrease in intestinal motility, almost to atony. 200 ug PSP had a clear effect in 3 out of 5 rabbits. Glucagon had the same effect, but only when administered intravenously.

PSP has been shown to delay the absorption of [U-^C]-protein hydrolyzate in pigs and in pancreatectomized dogs and of [U-^C]-ovalbumin in pancreatectomized dogs when the compound is administered orally in a 3 mg PSP capsule. The pigs and dogs weighed approx. 30 kg. 100 uCi [U-"^C]-protein hydrolyzate or 5 uCi [U-^C]-ovalbumin is mixed with a suspension of 1 g "Idon"/kg. and administered through a gastric tube. Compared to placebo, maximum plasma dpm was achieved -values from 30' to 40 minutes later after administration of PSP This delay in absorption due to about 100 µg PSP/kg orally,

is probably an expression of reduced gastrointestinal motility.

It has been shown that PSP compounds in vitro do not have any effect on the release of glucagon or insulin or on lipolysis, and the compounds have no effect on blood sugar levels in vivo. Furthermore, an intravenously injected dose of up to 1 mg/kg produced no significant effect on blood pressure in anesthetized rats.

The above-mentioned pharmacological data indicate that PSP compounds are valuable for the prevention and treatment of spastic conditions in smooth muscle, for example in intestinal VdT~ qr\ mn~ due to their lack of metabolic effects, PSP compounds can advantageously be used instead of glucagon for endoscopy and for radiological determinations.

PSP compounds can be administered intravenously as a bolus or as an infusion. When an effect with extended duration is desired - slower at the beginning - PSP compounds can be administered as a depot from which they are slowly mobilized by the blood flow such as intramuscularly or subcutaneously in an area with good peripheral circulation. The fact that the biological activity and immunoreactivity are maintained after PSP has been treated with gastric juice, trypsin and chymotrypsin, and the above-mentioned experiments showing delayed absorption after oral administration of PSP, indicate that it is possible to administer PSP orally. PSP can thus be administered via an endoscope during the endoscopy, or PSP can be mixed with the contrast medium, for example barium sulfate, during the radiological examination.

The dosage amount of PSP compounds can be adjusted depending on the magnitude of the desired effect and other factors, which are usually taken into account when determining the dosage. As an example of a dose range, from 10 to 200 ug/kg body weight can be stated, although lower or higher doses can be administered.

It is possible to prepare pharmaceutical preparations containing PSP compounds and one or more pharmaceutically tolerable excipients. As examples of such excipients can

preservatives and sodium chloride are indicated.

A suitable preparation contains an effective amount of the purified polypeptide PSP together with a suitable physiologically tolerable carrier.

A suitable preparation form is an aqueous sterile solution of the purified polypeptide PSP containing 0.9% sodium chloride and possibly preservatives such as phenol or a paraben.

The purified polypeptide is preferably present in a

amount of from 0.1 to 200 mg/ml, in particular from 0.5 to 25 mg/ml.

As an attempt to ensure that after administering a PSP connection one gets the desired result, it is advisable . for the production of PSP preparations to use a starting material that has a purity of at least 50%, preferably a purity of at least 90% of a PSP compound.

According to previously unpublished data, pancreatin pills contain PSP (e.g. approx. 1/1000). Due to their content of enzymes, pancreatin pills have been used for pancreatectomy patients and patients with chronic pancreatitis. It has now been shown that commercial insulin contains 30 ppm PSP.

The procedure for producing PSP is explained in more detail in

the following. The term "right-purified PSP" denotes PSP that moves essentially as a single band in the above-mentioned IEF system and basic DE^ system.

Example 1.

A salt cake originating from 94 kg of pig pancreatic glands is dissolved in water to a volume of 3.2 1. The pH value of the solution is set to 5.3, after which the precipitate is removed by centrifugation. The pH value of the supernatant is adjusted to 6.5, and the the formed suspension is centrifuged. The solution is mixed with 32 ml of 0.5 M Na 2 EDTA and 35 1 of ethanol. The mixture is allowed to stand

overnight at 4°C and then centrifuged. The precipitate is dried in vacuum, whereby 50 g of dry supernatant protein powder is obtained.

A solution of the powder in 500 ml of water is stirred carefully, while 1 M acetic acid is slowly added using a peristaltic pump, until a pH value of 4.30 is achieved (after approx. 3 hours of pumping). Stirring is continued for 3 days at 4°C, whereby crystallization occurs. The crystals (rod-shaped appearance, possibly orthorhombic and birefringent) are collected by centrifugation, suspended in 500 1 water at 4°C with stirring overnight, centrifuged and dried in vacuum.

The yield is 5.2 g.

4 g of this product is dissolved in 50 ml of 50% (volume/volume) ethanol and 50 ml of the eluent used in connection with Figure 1 with a pH value of 8.6. The solution is subjected to anion exchange chromatography as indicated in Figure 1. What is collected from the main peak is adjusted to a pH value of 7.4, mixed with 4 vol.96% (vol/vol) ethanol and then allowed to stand at 4°C

for 2 days. The precipitate is isolated by centrifugation, washed twice with 150 ml of 96% (volume/volume) ethanol and dried in vacuum. The yield is 2.6 g.

2.5 g of this product is dissolved in 125 ml of 50% (volume/volume) ethanol and 125 ml of the eluent used in figure 2 with. a pH value of 4.7, and cation exchange chromatography is then carried out as indicated in Figure 2. What is collected from the main peak (the only one indicated in the figure) is evaporated to dryness. The residue is dissolved in water, and the pH value of the solution is set to 7.1 (the final volume is approx. 90 ml). The solution is mixed with 1200 ml of 96% (volume/volume) ethanol, and the mixture is allowed to stand overnight at 4°C. The precipitate is collected by centrifugation, washed 2 times with 150 ml 96% (vol/vol) ethanol and dried

in vacuum. The yield is 1.8 g of highly purified PSP which meets the purity criteria specified in Table 1.

Example 2.

20 g of supernatant protein powder, which has been prepared as described in example 1, is dissolved in 200 ml of water. 208 ml of 96% (volume/volume) ethanol is added, after which the pH value is set to 4.6 with acetic acid. During centrifugation, a small precipitate is removed. The supernatant, which slowly becomes cloudy, is subjected to cation exchange chromatography on a 2.5 x 80 cm column of "SP-Sephadex C-25" equilibrated in eluent 1 (consisting of 0.4 M acetic acid, 0.05 M sodium acetate and 50 % (vol/vol) ethanol, pH value 4.6).

Elution with a linear gradient is performed between 3 1 eluent 1

and 3 1 eluent 2 (consisting of 0.3 M sodium acetate and 50% (vol/vol) ethanol, pH value: 8.7). Fractions of 10 ml are collected at an elution rate of 40 ml/hour. The fractions corresponding to the large peak that appears from fraction no. 100 to no. 130 are collected. The collected is adjusted to a pH value of 8 and then mixed with 1.8 1 96% (volume/volume) ethanol. The mixture is stored at 4°C for 24 hours. The precipitated protein is collected by centrifugation, washed twice with 150 ml of 96% (volume/volume) ethanol and dried in vacuum. The yield is 2.8 g. 2.5 g of this product is dissolved in 250 ml of a TRIS buffer (consisting of 0.0575 M TRIS, 0.05 N hydrochloric acid, pH value: 7.4). The solution is subjected to anion exchange chromatography on a 2.5 x 50 cm column with "QAE-Sephadex A-25" equilibrated in a TRIS buffer

(0.115 M TRIS, 0.1 N hydrochloric acid, pH value: 7.4). The column is eluted with the equilibration buffer at a rate of 30 ml/hour. Fractions of 10 ml are collected. The fractions that correspond to

the central predominant part of the peak which gives the maximum at fraction No. 225 is collected. The collected (620 ml) is mixed with 60 ml of 5 M sodium chloride solution and 12 1 of 96% (vol/vol) ethanol. The mixture is stored at 4°C for 24 hours. The precipitated protein is collected by centrifugation, washed twice with 150 ml of 96% (volume/volume) ethanol and dried in vacuum. The yield is 1.7 g of highly purified PSP.

Example 3.

22.5 kg of sodium chloride are added to 150 1 of an aqueous solution which is obtained by evaporating an extract from 250 kg of pig pancreatic glands and which has been freed from undissolved components. The mixture is stirred until the added salt dissolves, and the resulting precipitate is removed by centrifugation, whereby the salt cake is obtained. 34 kg of ammonium sulphate is added to the mother liquor (162 1) with stirring for 2 hours at room temperature, whereby a precipitate is obtained, which is isolated by centrifugation. 223 g of wet product is dissolved by adding 500 ml of a buffer (consisting of 0.05 M formic acid, 0.01 M sodium hydroxide, pH value: 3.2). The solution's conductivity is reduced to 4 mS by dialysis against water. The solution is put on a 5 x 50 cm column with "SP-Sephadex C-25" which is equilibrated with buffer I (consisting of 0.1 M formic acid, 0.02 M sodium hydroxide, pH value: 3.2). After application of the solution, the column is eluted with a linear gradient of sodium chloride from 0 to 0.27 M in buffer I. The total volume of eluent is 5.5 1. The column is then further eluted with buffer I containing 0.27 M sodium chloride. The flow rate during application and elution is 100 ml/hour, and fractions of 15 ml are collected. The chromatogram obtained by determining the absorption of the fractions at 276 nm shows a main peak from fraction no. 420 to no. 530. What is collected corresponding to the main peak is adjusted to a pH value of 7.4 and then mixed with 20 volumes of 96 % (volume/volume/ ethanol. After standing at 4°C for 48 hours, a precipitate can be recovered by centrifugation which is dried in vacuum. The yield is 6 g. The product obtained in this way is further purified by anion exchange chromatography on

one column with "QAE-Sephadex A-25" as described in Example 2. The yield is 3.4 g of highly purified PSP.

. Example 4.

A preparation for parenteral administration containing

1 mg PSP per ml can be prepared in the following way:

1 g of PSP and 99 g of lactose are dissolved in distilled water, and the pH is adjusted to 7.0. The solution is then sterilized. The sterile solution is filled into 10 ml vials

such a way that each vial contains 10 ml of solution. The solutions are then freeze-dried, and the vials are closed under aseptic conditions.

The product in the vials must be dissolved in 10 ml of sterile water before administration.

Example 5.

Oral preparations can be prepared in the following way:

100 mg PSP is mixed with 9 g corn starch, 8 g lactose and

180 mg of magnesium stearate, until a homogeneous mixture is obtained. The mixture is filled in hard gelatin capsules (no. 3) on such

way that each capsule contains 1 mg of PSP.

Processes for the production of PSP and salts thereof

can be set up in the following way:

1) Method for extracting a purified polypeptide

PSP as defined above, in that the polypeptide is recovered from porcine pancreatic glands by a combination of chromatography and precipitation. 2) Process according to point 1), where the glands are extracted with a mixture of water and a water-miscible organic solvent in an acidic medium, the precipitate is removed, the extract is neutralized, and PSP is recovered from the thus obtained extract using separation methods which are in and known, after which PSP, if desired, is converted into a salt thereof. 3) Method according to point 1, where the product is extracted from an insulin salt cake. 4) Method according to point 2, where the recovery is carried out by chromatography using a. cation and/or anion exchangers. 5) Method according to point 4, where crystallization is carried out. 6) Method according to point 4, wherein the chromatography is carried out while collecting the main part of the main PSP peak. 7) Method according to point 2, where PSP is recovered from the mother liquor which is formed when an insulin salt cake is produced, by a further salting out followed by chromatography using an anion and/or cation exchanger.. 8) Method according to any one of points 3 -6, where the salt cake is essentially freed from insulin by removing the precipitate that forms in a solution of the salt cake which is adjusted to a pH value in the range from approx. 4.9 to approx. 5.7, preferably approx. 5.3, and that the precipitate that forms in the supernatant at a pH value in the range from approx. 5.7 to approx. 7.0, preferably approx. 6.5, possibly removed before the chromatography is carried out. 9) Method according to point 5, where the crystallization is carried out by setting the pH value of the solution to a value in the range from approx. 3.8 to approx. 4.8, preferably approx. 4.3.

Claims (9)

1. Purified polypeptide with 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 1/2 (14), Val (7), Met (2), Ile (3), Leu (1), Tyr (2), Phe (7 ), where the determinations are subject to the usual error of 1 10% of the stated values, and with a partial amino acid sequence which from the N-terminal end is supposed to be: pyrGlu-Lys-Pro-Ala-Ala-Cys-Arg- Cys-Ser-Arg-Glx-Asx-Pro-5 10 Lys-Asx-Arg-Val-Asx-Cys-Gly-Phe-Pro-Gly-Ile-Thr-Ser-Asx-Glx-Cys-15 20 25 Phe -Thr-Ser-Gly-Cys-Cys-^Phe-Asx-Ser-Glx-Val-Pro-Gly-Val-Pro-Trp-30 35 40 45 where pyrGlu (residue 1) denotes pyroglutamic acid or a physiologically tolerable salt thereof . 2. Polypeptide according to claim 1, characterized, in that it has a purity of over 50% by weight, preferably over 90% by weight. 3. Polypeptide according to claim 1, characterized in that it exists in crystalline form. 4. Use of a purified polypeptide according to any one of claims 1-3 as a spasmolytic agent, preferably as a diagnostic agent or as a prophylactic agent. 5. Method for extracting a purified polypeptide according to claim 1, characterized in that pig pancreatic glands are extracted with a mixture of water and a water-miscible solvent under extraction conditions that are adapted to the extraction of insulin from the glands, and that the polypeptide (PSP) is then extracted from the extracted extract. 6. Method according to claim 5, characterized in that the extracted extract is freed from the water-miscible solvent by evaporation, after which the fatty substances are removed. 7. Method according to claim 6, characterized in that the insulin and accompanying proteins are salted out after removal of the relevant fatty substances, that the salt cake is dissolved in water, that the resulting solution is adjusted to a pH value between 4.9 and 5.7, preferably 5.3 , and that the sediment formed is removed. 8. Method according to claim 6, characterized in that the insulin and accompanying proteins are salted out after removal of fatty substances. and that the formed salt cake is removed. 9. Method according to claim 5, characterized in that the recovery is carried out by separation into a fraction that is more enriched with regard to the polypeptide in question, followed by chromatographic purification and concentration of the fraction enriched with the polypeptide in question, until a fraction is obtained that contains at least 50%, calculated on dry weight, of the relevant polypeptide.