NL8005084A - PURIFIED POLYPEPTIDE, METHODS FOR PREPARING IT, AND PHARMACEUTICAL PREPARATIONS CONTAINING THE PURIFIED POLYPEPTIDE. - Google Patents

Description

ΐ -, Ν.Ο. 29,397

Purified polypeptide, methods of preparation thereof, as well as pharmaceutical compositions containing the purified polypeptide.

The present invention relates to a newly purified polypeptide or a physiologically acceptable salt thereof, as well as to a method of recovering and purifying it and to pharmaceutical preparations, particularly suitable as spasmolytic agents, containing the new purified polypeptide.

The purified polypeptide of the present invention, which can be recovered from porcine pancreas, has surprisingly been found to have smooth muscle relaxing or spasmolytic effects. Therefore, the trivial name has been given of pancreatic spasmolytic polypeptide, hereinafter abbreviated PSP for convenience.

PSP exhibits interesting pharmacological properties.

Antispasmodics or antispasmodics, such as atropine, related products thereof and synthetic drugs with an atropine-like effect, are widely used for the treatment of various ailments, in particular smooth muscle cramps and hypermotility states. However, the intended effect of such drugs is usually accompanied by a number of side effects due to their general character of anticholinergics.

As a diagnostic tool in gastrointestinal radiology, in particular in conjunction with an X-ray visualization media for improving the visualization of the gastrointestinal, bile and urinary tracts, atropine-like anticholinergic medicines commonly used. Such a drug is usually administered parenterally and, due to the size of the dosage necessary to induce relaxation, the known side effects for these agents are encountered.

Recently, the parenteral administration of the peptide hor-moon glucagon, consisting of 29 amino acids, was introduced as an alternative means of reducing gastrointestinal motility in conjunction with radiographic studies (see U.S. Patent 3 * 862,301). However, glucagon exerts a multitude of actions in the human body, including a strong influence on metabolic regulation functions, the most prominent effects of which are hyperglycemia induction and lipolysis. Thus, while the introduction of glucagon has certain benefits in endoscopy, unwanted side effects have not been completely eliminated. It is an object of the present invention to provide a spasmolytic agent which, while possessing antispasmodic and smooth muscle relaxing effects similar to those of known agents, exhibits significantly reduced side effects.

In one aspect of the present invention, there is provided a new purified polypeptide showing the following amino acid composition: 10 Trp (2), Lys (if), His (1), Arg (5), Asx (10), Thr ( 3), Ser (9) »

Glx (12), Pro (12), Gly (é), Ala (6), Cys * (1lf), Val (7), Met (2), Ile (3), Leu (1), Tyr (2) Phe (7), where the determinations are subject to the usual error of - 10% of the indicated numbers. The partial amino acid sequence, comprising a total of 45 amino-15 acids from the nitrogen terminal, is believed to be as follows: pyrGlu-Lys-Pro-Ala-Ala-Cys-Arg-Cys-Ser-Arg-Glx- Asx-Pro-Lys-Asx- 5 10 15 -Arg-Val-Asx-Gys-Gly-Hie-Pro-Gly-Ile-Thr-Ser-Asx-Glx-Gys-PheJrhr-Ser- 20 25 30 - -Gly -Cys-Cys-Phex-Ser-Glx-Val-Pro-Gly-Val-Pro-Trp-, 35 40 45 20 where pyrGlu (residue 1) stands for pyroglutamic acid.

The abbreviations for the amino acids are evident from J.Biol.Chem.

243 O908), 3558.

The present invention also provides a method for the preparation of purified PSP, which method comprises isolating PSP from porcine pancreatic tissue, preferably from the insulin salt cake, by a combination of chromatographic and precipitation methods.

The insulin salt cake can be prepared as follows: Whole, neatly defatted pig pancreas glands are divided into small pieces under freezing conditions and then subjected to a conventional extraction process for extracting insulin, which is extracted with a mixture of water and an organic, water-miscible solvent, such as an aliphatic alkanol with a small number of carbon atoms, for example ethanol or isopropanol, in an acid medium, preferably an environment with a pH in the range of about 1.5 to 5, when measured with a pH- meter in the mixture. The acidic pH is obtained by the addition of an acid. In the mixture, the organic solvent is present in an 8005084 44 concentration in the range of about 40 to 80% by volume when all components are mixed. The resulting suspension is stirred at a temperature in the range from about 5 ° C to ambient temperature, followed by removal of the gland residue, for example, by centrifugation. The extract is then neutralized to a pH in the range of about 5 to 9, and clarified, for example, by centrifugation. The extract is acidified to a pH in the range of about 3 to 4, after which the extract is freed from any organic solvent, for example, by evaporation under reduced pressure, followed by removal of lipid compounds, for example by centrifugation. Insulin, mixed with other proteins and polypeptides, such as PSP, is salted out from the concentrated extract thus obtained, for example, by the addition of sodium chloride to a concentration in the range of about 10 to 30% w / v, and the precipitate formed is isolated, for example, by centrifugation, whereby the salt cake is obtained.

The salt cake thus obtained can then be dissolved in water and impure insulin can be isolated by isoelectric precipitation at a pH in the range of about 4.9 to 5.7, for example about 5.3, optionally in the presence of metal ions, for example zinc ions, and are usually recovered by centrifugation. The supernatant is adjusted to a pH in the range of about 5-7 to 7 He preferably about 6.5. The precipitate formed, which contains some insulin, is centrifuged. To remove minor products, such as salts, excess EDTA is added to the aforementioned second above layer, followed by the addition of a water-miscible organic solvent, preferably ethanol (usually 5 to 30 parts by volume). The mixture is left to precipitate overnight at about 4 ° C and then centrifuged. The precipitate is dried under reduced pressure to obtain a dry powder, hereinafter referred to as "supernatant protein". By this method, almost all protein material is recovered from the "supernatant protein".

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 over about 40 hours until a pH in the range of about 3.8 to 4.8, preferably about 1.3 +, is reached. The mixture is then cooled and the stirring treatment is continued for 3 days, preferably at about / f ° C. An amount of relatively large, rod-shaped, birefringent crystals are isolated, for example, by centrifugation, and dried under reduced pressure.

The material thus obtained can be further purified, preferably by using successive steps of anion and cation exchange chromatography.

To illustrate this method, an anion exchange chromatography can be performed on a column of "QAE-Sephadex A-25" (supplied by Pharmacia AB, Sweden), using the one used in Figure 1 of the accompanying drawings eluent (TRIS is tris (hydroxymethyl) aminomethane).

The chromatogram obtained by recording the optical density of fractions at 276 nm shows a major peak. The base product corresponding to the main peak is adjusted to a pH of 7Λ and then mixed with a water-miscible organic solvent, for example, ethanol (/ vol parts by volume). After 2 days at 2 ° C, a precipitate is collected by centrifugation and dried under reduced pressure.

The material thus obtained can be further purified by cation exchange chromatography, for example on a column of HSP-Sephadex. C.-25 "(supplied by Pharmacia). Elution can be accomplished with the eluant shown in Figure 2 of the accompanying drawings. The chromatogram obtained in the same manner as described above shows a major peak. Corresponding pooled fractions evaporated to dryness, the residue is dissolved in water at a pH in the range of about 6 to 8, e.g. about 7 *, mixed with an excess of 30 (about 12 parts by volume of water) of a water-miscible organic solvent, e.g. ethanol and stored overnight under similar conditions as described above Purified PSP precipitated from solution is obtained by centrifugation, washed with ethanol and dried under reduced pressure.

Protein-containing PSP can also be obtained from the mother liquor, which results when the salt cake is isolated using sodium chloride at a concentration of 10 to 20% w / v by an additional salting process. The pre-ifO citrate is collected, for example by centrifugation. Purified 8005084 5 PSP can be obtained from the precipitate using an anion and / or cation chromatography in any order.

According to a further method, protein-containing PSP can be isolated from the aforementioned pancreatic extract obtained by using a mixture of water and an organic water-miscible solvent by adsorption on a cation or anion exchanger, for example alginic acid, sulfonated styrene or aminoethyl cellulose. The ion exchanger is then washed and the protein is eluted with an aqueous medium. The isolation using an ion exchanger is carried out according to methods analogous to known methods.

PSP obtained by any of the aforementioned methods has the following properties:

Molecular weight, calculated from the amino acid composition: about 11,700.

Molecular weight determined by sodium dodecyl sulfate disc gel electrophoresis (Neville: J. Biological Chemistry 2IF6 (1971) 6328): about 10,700. ...

Electrophoretic Properties: Basic disc electrophoresis (base DE) in polyacrylamide 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 by analytical electrofocusing in polyacrylamide gel, according to which method the pi is determined to be approximately 4 , 4

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

Trypsin treatment 20 mg PSP were dissolved in 20 ml 0.01 M NH 4 HCO 4 (pH 7, δ) and preincubated at 37 ° C for 5 minutes. After addition of 100 µl of 0.001 M HCl containing 0.1 mg TPCK trypsin (obtained from Worthington Biochem. Corp.), the mixture was incubated at 37 ° C for 15 minutes and then lyophilized.

q-Chymotrypsin treatment 20 mg PSP were dissolved in 200 µl 0.1 M NaOH and 1800 µl 0.05 M NH 4 HCO 3 (pH 8.0) were added. The solution / Pre-incubated at 37 ° C for 5 minutes and 50 µl of 0.001 M HCl containing 100 µg of 40 α-chymotrypsin (obtained from Sigma Chemical Company) were added. The incubation was continued at 37 ° C for one hour and the reaction was stopped by the addition of 50 µl concentrated acetic acid, after which the solution was lyophilized.

CNBr treatment 5 20 mg PSP were dissolved in 2 ml 70 vol. # Formic acid containing 72 mg CNBr. The mixture was stored at ambient temperature for hours and then lyophilized. Lyophilization was then repeated after adding 2 ml of water.

Soak 10 samples of 1mg PSP, dissolved in 100µl 0.5N hydrochloric acid, were incubated at 37 ° C for 2, 10 and 21 days. After incubation, the protein of each sample was quantitatively precipitated by adding 2 ml of acetone. The precipitate was isolated by centrifugation, washed with 2 ml of acetone and dried under reduced pressure.

The samples thus obtained and a sample of untreated PSP were analyzed by base DE, see above, provided that the electrophoresis time was reduced to R 1. = 0.53 for PSP. In the sample incubated for 2 days, a series of bands were observed with R 1, ranging from 0.53 to 0.86. In the 10 and 21 da-20 gene incubated samples, only a single band with R ^ 0.86 appeared. The results indicate that partial deamidation of PSP had occurred after 2 days and complete deamidation after 10 days of incubation.

Pyroglutamate aminopeptidase treatment. A sample of 6 mg PSP was dissolved in 2 ml 50 mM sodium monohydrogen phosphate, 30 mM p-mercaptoethanol, 1 mM EDTA buffer with a pH of 7-8 * A solution of 2.5 mg pyroglutamate amino- peptidase (obtained from Boehringer Mannheim) in 0.5 ml of the aforementioned buffer was added. The mixture was incubated at 37 ° C for 16 hours and lyophilized thereby. (The 2.5 mg pyroglutamate aminopeptidase used contained about 10 mu of enzymatic activity).

The purity of the final PSP product can be checked by analytical isoelectric focusing (IEF) and base disc electrophoresis (base DE, see above). The product migrates mainly as a single band in both systems. IEF is performed according to the instructions of the LKB brochure I-180 * f-E02: "LKB Ampholine PAG '* plates for analytical electro-focusing on polyacrylamide gels (LKB-Produkter AB, Bromma, Sweden). 40 Also shows gel filtration of the polypeptide at 8005084 i * 7 "Bio-Gel P-30" (supplied by Biorad Laboratories »Eichmond,

California, U.S.A.) using 1 molar acetic acid as an eluent, only a single peak.

PSP has been analyzed for a number of immunoractivities by methods known per se. The results obtained are given in Table A.

Table A

Immunoreagent Assay (ppm)

Insulin (IEI) 3-6 10 Total glucagon (total GLI) <0.02

Pancreatic glucagon (pancreatic GLI) <0.02

Vasoactive visceral peptide (VIP) <0.02

Pancreatic polypeptide (pig-like) 0.08 C-Peptide (pig-like) <0.1 15 Somatostatin / -v 0.002

The immunoreactivity of PSP is measured according to a very specific radioimmunoassay, which is designed to be down to 250 µg per ml.

Antibodies were prepared by immunizing rabbits with 20 "supernatant protein" (0.5 ml of a solution containing approximately if mg protein per ml) mixed with the Freund additive (0.5 ml) twice a week for a period of time from 26 weeks. Beginning from the third day after the first immunization, a total of 10 blood samples (10 ml) from each animal, taken at regular intervals over a 172-day period, were collected.

The anti-sera obtained were tested for affinity and potency and an appropriate antiserum was selected for use in the radioimmunoassay.

125 J-PSP was prepared according to the lactoperoxidase method developed by Thorell and Johansson (Biochim.Biophys.Acta 251 (1971) 363). The radioiodine-containing PSP was purified by anion exchange chromatography, as known, and used for polypeptide radioimmunoassay according to the method developed by L.G. Heding (Diabetologica £ (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 acid, methanesulfonic acid, hydrochloric acid and sulfuric acid can be mentioned. Briefly, if appropriate, the designation PSP connections is used to describe PSP

0 Λ Λ E Λ Q /.

and include physiologically acceptable salts thereof.

PSP compounds and glucagon were found to be approximately equipotent in their inhibition of the amplitude of the contractions of electrically stimulated guinea pig ileum in vitro, see Table B. PSP and glucagon were dissolved in 0.9% sodium chloride with 0.1% human serum albumin.

Table B

Concentration in the Respiratory effect in% organ bath, M PSP Glucagon 10 10 "5 89 89 10" 6 49 51 10 "7 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.

Also, PSP compounds were found to be about as potent as glucagon in their in vivo inhibition of the peristalsis in mice, see Table C, an effect which can be blocked again by phentolamine.

Table C

Drug% intestine, which passed charcoal (50 mg / kg subcutaneously) _ compared to a control_ PSP 78 25 Glucagon 66

Atropine sulfate 64 PSP decreases visceral motility in rabbits in vivo after intravenous or intraluminal gut administration. The motility was registered by means of a balloon catheter 30 in the viscera connected to a pressure transducer. In 5 out of 5 rabbits (with a body weight of 2.5 to 3 * 0 kg) 400 / Ug PSP administered intravenously or 5 cm of the balloon in the intestinal lumen caused a marked decrease in intestinal motility, almost to muscle weakness. 200 µg had a clear effect in 3 out of 5 rabbits. Glucagon had the same effect, only when administered intravenously.

1 ii PSP was found to delay the absorption of [α-x!] Protein hydrolyzate in pigs and dogs with pancreas removed and [-** C] ovalbumin in dogs with pancreas removed, 8005084 3 «9 when the compound is peroral was administered in a capsule containing 3 mg PSP. The pigs and dogs weighed about 30 kg. 10OCyuCi [U- ^ Cj protein hydrolyzate or 5 / uCi [U-1ifC] ovalbumin were mixed with a suspension of 1 g / kg Idon® and administered through gastric tubes. Maximum plasma ppm values were reached from 30 to ifO minutes after PSP administration compared to a placebo. This delay in absorption caused by about 100 µg / kg / kg of PSP orally probably reflects reduced gastrointestinal motility.

PSP compounds were found to be devoid of any in vitro effect on glucagon or insulin release or on lipolysis and any in vivo effect on blood glucose. Nor did an intravenously injected dose up to 1 mg / kg have any significant effect on the blood pressure of the anesthetized rat.

The foregoing pharmacological data indicate the potential value of PSP compounds for the prevention and treatment of spastic smooth muscle conditions, for example, in the viscera. Due to the lack of metabolic effects, PSP compounds may prove to be effective as a substitute for glucagon in endoscopy and radiological methods.

PSP compounds can be administered intravenously as a large pill or as an infusion. When an effect of a continued nature, slower in the beginning, is desired, PSP compounds can be administered as a depot, from which they are slowly mobilized by the bloodstream, such as intramuscularly or subcutaneously 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 chymotrypsin and the tests described above, which show delayed absorption after oral administration of PSP, indicates the oral route as a possible route of administration. Therefore, PSP can be administered by an endoscope during the endoscopy methods or PSP can be mixed with the contrast media, for example barium sulfate, during the radiology method.

The dosing rates of PSP compounds can be adjusted according to the size of the desired response and other factors that are routinely taken into account when determining the dosage. As an example of a dosage range, 10 to 200 µg per kg of body weight although an i * 0 lower or higher dose can be administered.

The present invention also relates to a pharmaceutical composition containing PSP compounds and one or more pharmaceutically acceptable carriers. Preservatives and sodium chloride can be mentioned as examples of such carriers.

In an effort to ensure that the desired result is obtained after administration of a PSP compound, it is recommended to use a starting material for the preparation of PSP preparations having a purity of at least 50% * 10, preferably a purity of at least at least 90% of a PSP connection.

According to hitherto unpublished data, pancreatin pills contain PSP (e.g., about 1 per 1000). Due to its enzymes content, pancreatin pills have been used for patients with removed pancreas and patients with chronic pancreatitis. Commercial insulin appears to contain about 30 ppm PSP.

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

However, the following examples, which are not considered to be limiting, are given for the method of preparing PSP .... to explain. Highly purified PSP is PSP, which practically migrates as a single band in the aforementioned IEF and base DE systems.

Example I

A salt cake from 94 kg of pig pancreatic glands was dissolved in water to a volume of 3 2 liters. The pH of the solution was adjusted to 5-3 and 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 2 EDTA and 35 liters of ethanol. The mixture was stored at 4 ° C overnight and then centrifuged. The precipitate was dried under reduced pressure to obtain 50 g of a dry "supernatant protein" powder.

A solution of the powder in 500 ml of water was stirred gently, while 1 M acetic acid was slowly administered by a peristaltic pump until a pH of 4.30 was reached (after about 3 hours of pumping). Stirring was then continued for 40 days at 4 ° C, with crystallization taking place.

8005084 11

The yield of crystals (rod-shaped by appearance, possibly showing orthorhombic and birefringence) was collected by centrifugation, suspended in 500 ml water at 5 ° C with stirring overnight, centrifuged and dried under reduced pressure. The yield was 5 * 2 g.

k g of this material were dissolved in 50 ml of 50 vol. # ethanol and 50 ml of eluent (see Fig. 1) at a pH of 8.6. The solution was subjected to anion exchange chromatography as shown in Fig. 1. The main peak base product was adjusted to a pH of 7tk, mixed with if parts by volume of 96 vol. Ethanol and then stored at if ° C for 2 days. The precipitate was isolated by centrifugation, washed twice with 150 ml of 96% ethanol and dried under reduced pressure. The yield was 2.6 g.

2.5 g of this material were dissolved in 125 ml of 50 vol. # Ethanol and 125 ml of eluent (see Fig. 2) at a pH of if 7 and then subjected to a cation exchange chromatography as shown in Fig. 2 The main peak stock (visible only) was evaporated to dryness. The residue was dissolved in water and the pH of the solution was adjusted to 7 µl (the final volume was about 90 ml). The solution was mixed with 1200 ml of 96% ethanol by volume and the mixture was stored at if ° C overnight. The precipitate was isolated by centrifugation, washed twice with 150 ml of 96% ethanol and dried under reduced pressure. The yield was 1.8 g of highly purified PSP, which meets the purity requirements listed in Table A.

Example II

20 g of "supernatant protein" powder, prepared as described in Example 1, were dissolved in 200 ml of water. 208 ml of 96% ethanol by volume were added, followed by adjusting the pH to if 6 with acetic acid. A small amount of precipitate was removed by centrifugation. The supernatant layer, which slowly became cloudy, was subjected to cation exchange chromatography on a 2.5 x 80 cm column of "SP-Sephadex G-25n, equilibrated in eluent 1 (0, if M acetic acid, 0.05 M sodium acetate, 50 vol. # ethanol, pH: if, 6), Linear gradient elution was performed between 3 liters of eluent 1 and 3 liters of eluent 2 (0.3 M sodium acetate, 5 ° vol. # Ethanol, pH: 8.7) fractions of ifO 10 ml were collected at an elution rate of ifO ml / h The 8005084 fractions corresponding to the large peak, appearing from fraction 100 to 130 were collected The collected product was brought to a pH of 8 and then mixed with 1.8 liters of 96 volume percent 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% ethanol and dried under reduced pressure. Yield 2.8 g. 2.5 g of this material were 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 exchange chromatography on a 2.5 x 50 cm column of MQUA-

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 equilibrium buffer at a rate of 30 ml / h. Fractions of 10 ml were collected The fractions corresponding to the central major portion of the peak, which show a maximum at fraction 225, were combined This combined product (base product, 620 ml) was mixed with 60 ml of 5 M sodium chloride and 12 liters of 96% ethanol by volume The mixture was stored for 24 hours at 4 ° C. The precipitated protein was isolated by centrifugation, washed twice with 150 ml of 96% ethanol by volume and dried under reduced pressure. 7 g highly purified PSP.

Example III

To 150 liters of an aqueous solution, which was obtained by evaporation of an extract from 250 kg of pig pan-25 creas glands and which was free from insoluble material, was added 22.5 kg of sodium chloride. The mixture was stirred to dissolve the added salt and the resulting precipitate was removed by centrifugation, thus providing the salt cake. To the mother liquor (162 liters), 34 kg of ammonium sulfate was added with continued stirring for 2 hours at ambient temperature to obtain a precipitate, which was isolated by centrifugation. 223 g of the wet product were dissolved by adding 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 fed to a 5 x 50 cm column of HSP-Sephadex C-25% equilibrated with buffer I (0.1 M formic acid, 0.02 M sodium hydroxide, pH 3.2). After applying the solution, the column was eluted with a linear gradient of sodium chloride from 0 to 0.27 M in buffer I.

8005084 13

The total eluent volume was 5 * 5 liters. The column was then further eluted with buffer I containing 0.27 M sodium chloride. Flow during application and elution was 100 ml per hour and 15 ml fractions were collected. The chromatogram obtained by recording the optical density of the fractions at 276 nm showed a major peak from fraction i + 20 to 530. The base product, corresponding to the major peak, was adjusted to a pH of 7 * 4 and then mixed with 20 96 parts by volume of ethanol. After storing at 1 ° C for hours, a precipitate was collected by centrifugation and dried under reduced pressure. Yield: 6 g. The material thus obtained was further purified by anion exchange chromatography on a column "QAE-Sephadex A-25" as described in Example II. Yield: 3 * 4 g of highly purified PSP.

15 Image 17

A preparation for parenteral administration containing 1 mg PSP per ml can be prepared as follows: 1 g PSP and 99 g lactose are dissolved in 1 liter of distilled water and the pH is adjusted to 7.0. The solution is then sterile filtered. The sterile solution is filled into 10 cm bottles in such a way that each bottle contains 10 ml of solution. The solutions are then lyophilized and the vials are closed under aseptic conditions.

The formulation in each of the vials should be dissolved in 10 ml of sterile water prior to administration.

Image 7

Oral preparations can be prepared as follows: 100 mg PSP are mixed with 9 S corn starch, 8 g lactose and 180 mg magnesium stearate until a homogeneous mixture is obtained.

The mixture is filled into hard gelatin capsules No. 3 ° P in such a way that each capsule contains 1 mg of PSP.

8005084

Claims (15)

1. Purified polypeptide having 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), 5 Ile (3), Leu (1), Tyr (2), Phe (7) j where the determinations are subject to the usual error of + 10% of the indicated values and with a partial amino acid sequence, which, if clarified , from the terminal N it is supposed to be: pyrGlu-Lys-Pro-Ala-Ala-Cys-Arg-Cys-Ser-Arg-Glx-Asx-Pro-Lys-Asx- 10 5 10 15 Arg-Yal- Asx-Cys-Gly-Ibe-Pro-Gly-Ile-Thr-Ser-Asx-Glx-Cys- & e-Thr- 20 25 30 Ser-Gly-Cys-Cys-Shex-Ser-Glx-Yal-Pro -Gly-Val-Pro-Trp-, 35 40 45 wherein pyrGlu (residue 1) stands for pyroglutamic acid, or a physiologically acceptable salt thereof. Polypeptide according to claim 1, characterized in. k, that the polypeptide has a purity of greater than 50% by weight, preferably greater than 90% by weight. Polypeptide according to claim 1 or 2, characterized in that the polypeptide is in crystalline form. 4. A method of isolating a purified polypeptide according to claim 1, characterized in that the polypeptide is isolated from swine pancreatic glands by a combination of chromatography and precipitation. 5. Process according to claim 4, characterized in that the glands are extracted with a mixture of water and a water-miscible organic solvent in an acidic medium, the precipitant is removed, the extract is neutralized and PSP is isolated from the extract thus obtained under application of separation methods known per se, after which PSP is optionally converted into a salt thereof. 6. A method according to claim 4, characterized in that the polypeptide is isolated from an insulin salt cake. 7. Process according to claim 5, characterized in that the isolation is carried out by chromatography using a cation and / or an anion exchanger. 8. Method according to claim 7, characterized in that the method comprises a crystallization process. 9. Process according to claim 7, characterized in that the chromatographic treatment is carried out with the collection 80 0508 4 15 of the main part of the main ESP peak. 10. Process according to claim 5, characterized in that PSP is isolated from the mother liquor, which results when an insulin salt cake is prepared, by a further salting-out method, followed by chromatography using an anion and / or a cation exchanger. 11. Process according to claims 6 to 3, characterized in that the salt cake is practically freed from insulin by removing the precipitate formed in a solution of the salt cake, which has been brought to a pH in the range of about 4f9 to 5 > 7j, preferably about 5j3, and optionally removing the precipitate formed in the above layer at a pH in the range of about 5.7 to 7.0, preferably about 6.5, before the chromatography is performed. Process according to claim 8, characterized in that the crystallization process is carried out by adjusting the pH of the solution to a value in the range from about 5.8 to 4.8, preferably about 4.3 · 13 Pharmaceutical composition characterized by the presence of an effective amount of a purified polypeptide according to claims 1 to 3, respectively obtained by using one or more of the methods according to claims 4 to 12 together with a suitable physiologically acceptable carrier or the like cutting agent. 14. An aqueous sterile solution of a purified polypeptide according to claims 1 to 3 obtained respectively by one or more of the methods according to claims 4 to 12, preferably containing about 0.9% sodium chloride and optionally preservatives such as paraben or phenol. Solution according to claim 14, 1 characterized in that the purified polypeptide is present in a concentration of 0.1 to 200 mg per ml, preferably 0.5 to 25 mg per ml. Process for the preparation of a pharmaceutical preparation, characterized in that a purified polypeptide according to / T according to / T is brought into a form suitable for such use. 8005084