FR2464942A1 - POLYPEPTIDE, METHOD FOR ISOLATION, AND THERAPEUTIC USES - Google Patents

Abstract

The novel purified polypeptide has the following composition of amino acids: 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). These determinations are subject to the usual margin of error of +/- 10% of the number, given in brackets, of the relevant amino acids. Part of the amino acid sequence, determined from the N-terminal end of the peptide, has the following composition: In this sequence, pyrGlu at amino acid residue 1 represents pyroglutamic acid. The novel polypeptide may also be present in the form of its physiologically acceptable salts. It has a purity which is higher than 50% by weight, preferably higher than 90% by weight, and is in crystalline form. The polypeptide is isolated from porcine pancreas by a combination of chromatography and precipitation. The polypeptide is stored in aqueous sterile solutions containing about 0.9% by weight of sodium chloride and, if desired, preservative. The novel polypeptide is used in pharmaceutical compositions together with a suitable, physiologically acceptable carrier or excipient, as a spasmolytic, diagnosis aid, therapeutic agent or prophylactic agent.

Description

Polypeptide, process for its isolation,
and its therapeutic uses.

 The present invention relates to a novel purified polypeptide, in the free state or in the form of a physiologically acceptable salt, to a process for isolating and purifying it and to its therapeutic uses, in particular as a spasmolytic agent. .

The purified polypeptide according to the invention, which can be isolated from the porcine pancreas, was unexpectedly found to have smooth muscle relaxation effects or spasmolytic effects. It has therefore been given a common name spasmolytic pancreatic polypeptide, hereby repealed for the sake of simplicity and simplicity PPS.The PPSpossed "interesting pharmacological properties.Spasmolytic or antispasmodic agents such as atropine, related drugs and synthetic drugs having effects similar to those of atropine, are very often used for the treatment of various disorders, and in particular for the treatment of smooth muscle spasms and states of hypermotility. The beneficial effect of these drugs is usually accompanied by a number of side effects attributable to their general anti-cholinergic agents.

 Atropine-like anti-cholinergic drugs are also commonly used as diagnostic aids in gastrointestinal radiology, particularly in conjunction with an X-ray visualization product to improve visualization of the gastrointestinal tract. , biliary and urinary. Such a drug is usually administered parenterally, and because of the importance of the dose required to induce relaxation, the usual side effects to these agents are generally encountered.

 Recently, the parenteral administration of the glucagon peptide hormone, consisting of 29 amino acids, has been proposed instead to reduce gastrointestinal motility on the occasion of radiographic examinations (see US Pat. 3862301). However, glucagon has multiple effects in the body, including a strong influence on metabolic regulatory functions, the most obvious effects being the induction of hyperglycemia and lipolysis. Thus, if the introduction of glucagon endoscopy brings certain benefits, it has not eliminated completely undesirable side effects.

 The present invention aims at the development of a spasmolytic agent which, having anti-spasmodic and smooth muscle relaxation effects comparable to those of known agents, also has considerably reduced side effects.

In one of its aspects, the invention therefore relates to a novel purified polypeptide having the following composition in amino acids
Trp (2), Lys (4), His (1), Arg (5), Asx (10), Thr (3),
Ser (9), Glx (12), Pro (12), Gly (6), Ala (6) Cysl / 2 (14), Val (7),
Met (2), Ile (3), Leu (I), Tyr (2), Phe (7).

The determinations are subject to the usual error of + 10% of the figures indicated. It is believed that the partial amino acid sequence comprising a total of 45 amino acids from the terminal nitrogen is pyrGlu-Lys-Pro-Ala-Ala-Cys-Arg-Cys-Ser-Arg-Glx-Asx. -Pro-
5 10
Lys-Asx-Arg-Val-Asx-CysT Ly-Phe-Pro-Gly-Ile-Thr-Ser-Asx -
15 20 25
Glx-Cys-Phe-Thr-Ser 4 ly-Cys-Cys-Phe-Asx -Ser-Glx-Val-Pro-
30 35 40
Gly-Pro-Val-Trp.

45
In the above scheme, pyrGlu (residue 1) represents pyroglutamic acid.

 The abbreviations used for the amino acids are those appearing in J.Biol.Chem. 243 (1968), 3558.

The invention also includes a process for preparing the purified PPS, which method comprises isolating the
PPS of the pancreatic tissue of the pork, preferably of the insulin release cake, by a combination of chromatography and precipitation operations.

 The insulin release cake may be prepared as follows: Carefully degreased pork pancreas glands are finely ground finely and subjected to the conventional extraction procedure for isolation of insulin. that is to say, it is extracted with a mixture of water and a water-miscible organic solvent, for example a lower aliphatic alkanol such as methanol or isopropanol, in an acidic medium, for example a medium having a pH in the range of 1.5 to 5, the measurement being made using a pH meter in the mixture.The acidic pH is obtained by adding an acid, in the mixture, the organic solvent is present in a concentration in the range of about 40 to 80% by volume after mixing all the components. The resulting dispersion is stirred at a temperature in the range of about 50 ° C. to room temperature, after which residues of the glands are removed, for example by centrifugation. The extract is then neutralized to a pH in the range of about 5 to 9, then clarified, for example by centrifugation. The extract is then acidified to a pH of approximately 3 to 4, then all the organic solvents are removed, for example by evaporation under reduced pressure, and then the lipid compounds, for example by centrifugation. Insulin, mixed with d Other proteins and polypeptides, such as PPS, are salted out of the concentrated extract obtained under these conditions, for example by the addition of sodium chloride at a concentration in the range of about 10 to 30% (w / v); the precipitate is isolated for example by centrifugation; it is this precipitate which constitutes the release cake.

 The release cake, thus obtained, is then dissolved in water; the crude insulin is then 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 is isolated, usually by centrifugation. The supernatant liquid is adjusted to a pH of about 5.7 to 7, preferably about 6.5. The formed precipitate which contains a little insulin is centrifuged. To remove the accompanying products such as salts, an excess of ethylene diamine tetraacetic acid is added to the second supernatant above, followed by a water-miscible organic solvent, preferably ethanal (usually 5% by weight). to 20 volumes). The mixture is allowed to precipitate overnight at about 40 ° C. and then centrifuged. The precipitate is dried under vacuum; a dry powder is obtained, hereinafter referred to as "protein of the supernatant liquid". This procedure makes it possible to substantially recover the proteins contained in the "proteins of the supernatant liquid".

 PPS can be obtained in a crude crystalline form from a solution of "supernatant protein" in water (about 10 parts). The solution is stirred gently and an acid, for example acetic acid, is added in about three hours to a pH in the range of about 3.8 to 4.8, preferably about 4.3. . The mixture is then refrigerated and stirring continued for three days, preferably at about 40 ° C. A birefringent crystal crop in the form of relatively large tablets is isolated, for example by centrifugation, and dried under vacuum.

 The product obtained under these conditions can be purified more deeply, preferably by subsequent operations of anion exchange chromatography and cation exchange chromatography.

 By way of example, anion-exchange chromatography can be carried out under the following conditions: a column of "QAE-Sephadex A-25" (product of the firm Pharmacia AB, Sweden) is used, to a dimension of bed of 2.5 x 80 cm; the eluent consists of Tris (it is tris (hydroxymethyl) aminomethane) 0.08 M, 0.02 N HCl, 0.08 M NaCl, 50% by volume ethanol, pH 8.5. The operation is carried out on the crystals obtained from the proteins of the supernatant liquid. The elution rate is about 40 ml / hour and the size of each of the fractions is about 13 ml. The chromatogram obtained by continuous measurement of the optical density of the fractions at 276 nm shows a principal peak. It is shown in Figure 1 of the accompanying drawings. The collected portion, corresponding to the main peak, is adjusted to pH 7.4, then mixed with a water-miscible organic solvent, for example ethanol (4 volumes). After standing for two days at 40 ° C., a precipitate is collected by centrifugation and dried under vacuum.

 The product thus obtained can be further purified by cation exchange chromatography, for example on a column of "SP-Sephadex C-25" from Pharmacia, Sweden. The size of the bed is 2.5 x 25 cm and the eluent consists of 0. 4 M CH3COOH, 0.05 M CH3COONa, 50% by volume ethanol, pH 4.7. The elution rate is about 50 ml / hour and the size of each of the fractions about 15 ml. The chromatogram obtained as described above has a main peak. It is represented in FIG. 2 of the appended drawings. The collected part, corresponding to the main peak, is evaporated to dryness; the residue is redissolved in water at a pH in the range of 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 conditions similar to those above. The purified PPS which precipitates from the solution is isolated by centrifugation, washed with ethanol and dried under vacuum.

 Proteins containing PPS can also be obtained from the mother liquors obtained during the isolation of the release cake by using sodium chloride at a concentration of 10 to 20% (weight / volume) by a new salting operation. . The precipitate is collected, for example by centrifugation. The purified PPS can be obtained from the precipitate by anionic and / or cationic chromatography in any order.

 In another method, the PPS-containing proteins of the pancreatic extract described above can be isolated using a mixture of water and a water-miscible organic solvent by adsorption on a cation or anion exchanger, for example alginic acid, sulphonated polystyrene or aminoethylcellulose. The ion exchanger is then washed and the proteins eluted by an aqueous medium. The isolation using an ion exchanger is carried out according to similar procedures to known procedures.

The PPS 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 electrophoresis in a sodium dodecyl sulfate gel (Neville: J. Biological Chemistry 246 (1971) 6328): about 10,700.

Characteristics of electrophoresis
Basic discontinuous electrophoresis ("basic DE") in a polyacrylamide gel, performed as described by J. Schlichtkrull et al. (Horm.Metabol.Research,
Suppl. Series 5 (1974) 134) has essentially a single band at R f 0.65-0.75. A similar result is obtained by the analytical electrofocusing in a polyacrylamide gel, this method making it possible to determine the pI which is about 4.4.

The products obtained by treatment of the PPS with trypsin, a-chymotrypsine CNBr, an acid or pyroglutamate aminopentidase, coinire written below, have a spasmolytic activity of the same order of magnitude as that of
PPS.

Trypsin treatment
20 mg of PPS are dissolved in 20 ml of 0.01 M NH4HCO3 (pH 7.8) and preincubated for 5 minutes at 370 ° C.

After addition of 100 μl 0.001 M HCl containing 0.4 mg TPCK-trypsin (from Worthington Biochem Corp.), the incubation mixture was incubated for 15 minutes at 370 ° C. and lyophilized.

Treatment with a-chymotrypsin
20 mg of PPS are dissolved in 200 μl of 0.1 M NaOH and 1800 μl of 0.05 M NH 4 HCO 3 (pH 8.0) are added. The solution was preincubated for 5 minutes at 370 ° C and 50 μl of 0.001 M HCl containing 100 μg of α-chymotrypsin (from Sigma Chemical Company) was added. The incubation is continued for one hour at 370 ° C., then the reaction is stopped by addition of 50 μl of concentrated acetic acid and the solution is then lyophilized.

Treatment with CNBr
20 mg of PPS are dissolved in 2 ml of 70% by volume formic acid containing 72 mg of CNBr. The mixture is kept at room temperature for 40 hours and then lyophilized. Freeze-drying is then repeated after addition of 2 ml of water.

Acid treatment
Samples of 1 mg of PPS dissolved in 100 ml of 0.5 N hydrochloric acid are incubated at 370 ° C. for 2, 10 and 21 days. After incubation, the protein of each of the samples is precipitated quantitatively by the addition of 2 ml of acetone. The precipitate is isolated by centrifugation, washed with 2 ml of acetone and dried under vacuum. The samples obtained are analyzed with a sample of PPS not treated with basic DE (see above), but the electrophoresis duration is reduced so as to obtain a value of Rf equal to 0.53 for the
PPS. In the sample incubated for 2 days, a series of bands with
Rf ranging from 0.53 to 0.86. In samples incubated for 10 and 21 days, only one band with a Rf value of 0.86 was observed. These results show that partial deamidation of the PPS occurred after 2 days, and complete deamidation after 10 days of incubation.

Pyroglutamate aminopeptidase treatment
A sample of 6 mg of PPS was dissolved in 2 ml of a solution of 50 mM sodium hydrogen phosphate 30 mM p-mercaptoethanol, 1 mM EDTA buffer pH 7.8. A solution of 2.5mg of Boehringer's pyroglutamate aminopeptidase is added
Mannheim in 0.5 ml of the buffer above. The mixture is incubated for 16 hours at 37 ° C., then lyophylized (2.6 mg.dc.-the pyroglutamate aminopeptidase used contains about 10 mU of enzyme activity).

The purity of the final PPS can be controlled by
analytical isoelectric focusing ("IEF") and electro
pork is discontinued basic (DE basic,
see above). The product essentially migrates under
form of a single band in both systems. IEF
is done according to the instructions in the brochure
I-1804-E02 from LKB-Produkter AB, Bromma, Sweden
LKB Ampholine PAG, flat for electrofocusing
polyacrylamide gels ".

Similarly, the filtration of the gel polypeptide, the
"Biosel P-30" from Biorad Laboratories, Richmond,
California, USA, with 1M acetic acid as
eluent, reveals only one peak.

 PPS has been analyzed for a number of immunoreactivities according to known procedures.

The results obtained are reported in the ta
I below
Table I.

Immunoreactive Content (ppm)
Insulin (IRI) 3 to 6
Total Glucagon (Total GLI) <0.02
Pancreatic Glucagon (Pancreatic GLI) <0.02
Vasoactive intestinal peptide (VIP) <0.02
Pancreatic (pork) polypeptide X 0.08
C-peptide (pork) <0.1
Somatostatin X 0.002
The immunoreactivity of PPS was measured by a high specificity radioimmunotest developed to detect amounts that can fall to 250 mg per ml.

 Antibodies were prepared by rabbit immunization with the "supernatant protein" (0.5 ml of a solution containing about 4 mg of protein per ml) mixed with 0.5 ml of Freund's adjuvant, twice per hour. week for 26 weeks. Beginning on the 13th day after the first immunization, a total of 10 blood samples (10 ml) of each of the animals taken at regular intervals were collected over a period of 172 days. The obtained antisera were tested for affinity and capacity and an antiserum appropriate for use in the radioimmunoassay was selected.

125
125I-PPS was prepared by the lactoperoxidase procedure developed by Thorell and Johansson (Biochim Biophys. Acta 251 (1971) 363). The radio-iodinated PPS was purified by ion exchange chromatography according to a known procedure and used for the radioimmunoassay of the polypeptides according to the procedure developed by LG Heding (Diabeto) ogica 7 (1971). ).

 The invention also comprises the salts of PPS and for example salts of cations such as sodium, potassium, magnesium, calcium and zinc cations and salts formed by addition with organic or inorganic acids such as formic, methanesulfonic acids. , hydrochloric and sulfuric.

 The term "PPS compounds" covers PPS and its physiologically acceptable salts.

 It has been found that PPS compounds and glucagon have about equal potency in inhibiting the contraction amplitude of electrically stimulated guinea pig ileum in vitro; cf. Table II below.

PPS and glucagon were dissolved in 0.9% sodium chloride with 0.1% human serum albumin.

Table II.

Concentration in% inhibition effect on organ bath, M
PPS Glucagon
10-5 89
1o-6 49 51 21 21 24
This effect of the PPS compounds is blocked by phentolamine but not by naloxone. Spontaneous motility of ileum isolated from reserpine-treated guinea pigs is inhibited by PPS.

 Likewise, it has been found that PPS and its salts have an activity approximately equal to that of glucagon with respect to the in vivo inhibition of peristalsis in mice; cf. Table III below; this effect can also be blocked by phentolamine.

Table III.

Drug (50 mg / kg per Percentage of intestine subcutaneously) crossed by charcoal,
compared to a witness
PPS 78
Glucagon 66
Atropine sulfate 64
PPS decreases intestinal motility in rabbits in vivo after intravenous or intraluminal administration in the intestine. Motility is recorded by a balloon catheter placed in the intestine and connected to a pressure transducer. For 5 out of 5 rabbits (weighing 2.5 to 3.0 kg each), 400 μg of intravenously administered PPS or 5 cm of the balloon in the lumen
of the intestine cause a marked decrease in
intestinal motility, almost to atony. 200 g have a net effect on 3 out of 5 rabbits. The glucagon has the same
effect, but only when administered
intravenous itself.

PPS has been found to delay the absorption of
protein hydrolyzate [U-14Cj in pigs and at
Pancreatectomized dogs and ovalbumin [U-14C]
in pancreatectomized dogs, after administration
Peroral in a capsule containing 3 mg of PPS. The
pigs and dogs weighed about 30 kg. We mixed
100 ci -4 'protein hydrolyzate tU-14Cg or 5 Ci oval
butin [U ~ 14Css with a suspension of 1g / kg of a protein concentrate such as that sold by Ferrosan under the name Idon and administered by gastric tube.The maximum plasma dpm values were reached 30 to 40 minutes after the administration of the PPS, oeTpartively to a plaoebo administration.This delay of abc
Sorption caused by approximately 100 p / kg of oral PPS probably reflects reduced gastrointestinal mobility.

 It has been found that PPS compounds lack any in vitro effect on the release of glucagon or insulin or on lipolysis and any in vivo effect on blood glucose. A dose of up to 1 mg / kg intravenously injected also has no marked effect on the blood pressure of the anesthetized rat.

The results of pharmacological tests reported
above highlight the potential value of PPS compounds for the prevention and treatment of smooth muscle spasm conditions, for example in the intestine. Because of
they do not cause metabolic effects, the compounds
of PPS can be used advantageously as
glucagon substitutes in endoscopy and in operas
radiological findings. They advantageously possess important safety.

The acute toxicity study did not show any adverse effects after intraperitoneal injection of 200 mg PPS / kg
mouse.

PPS compounds can be administered intravenously
venous, as a food bolus or as an infusion.When
If there is a slower prolonged effect to be triggered, the
composed of PPS ----------------------------------------------- in the state of deposition slowly mobilized by the blood stream, for example the subcutaneous or intramuscular deposit in a region with good peripheral circulation. The fact that the biological activity and the immunoreactivity can be conserved after exposure of PPS to gastric juice, trypsin and chymotrypsin, and the results of the tests described above demonstrating the delayed absorption after oral administration of PPS indicates that the oral route is a route of administration. possible. Therefore, PPS can be administered by means of an endoscope during endoscopic operations; it may be mixed with the contrast medium, for example barium sulfate, for radiological operations.

 The doses of the PPS compounds can be adjusted according to the importance of the desired response and the other factors usually taken into consideration.

For example, doses of 10 to 200 μg per kg of body weight may be indicated, but lower or higher doses may be given.

 In the case of administering PPS compounds according to the invention in the form of sterile aqueous solutions, solutions containing them in a concentration of 0.1 to 200 mg / ml, preferably 0, will be advantageously used. 5 to 25 mg / ml.

 The invention also includes therapeutic compositions containing PPS compounds with one or more pharmaceutically acceptable carriers. Such carriers include preservatives and sodium chloride.

 To be sure to achieve the desired result after administration of the PPS compounds, it is recommended to prepare them from a starting product to arrive in the final product at a purity of at least 50S preferably from minus 90% of the PPS compound.

 According to previously unpublished research findings, pancreatin pills contain PPS (e.g., about b / cc). Because of their enzyme content, pancreatin pills have been used in pancreatectomized patients and in patients with chronic pancreatitis. It has also been found that commercial insulin contains about 30 ppm of PPS.

 Any new feature or combination of features described in this application is considered essential.

 The examples which follow illustrate the invention without, however, limiting its scope; in these examples, the indications of parts and percentages are by weight, unless otherwise indicated. On the other hand, when talking about a high-purity PPS, it will be a PPS that essentially migrates as a single band in the basic IEF and DE systems described above.

 Exemption 1.

 A release cake obtained from 94 kg of porcine pancreatic glands is dissolved in water at a volume of 3.2. The pH of the solution is adjusted to 5.3 and the precipitate is separated by centrifugation. The pH of the supernatant is adjusted to 6.5 and the suspension is centrifuged. The solution is mixed with 32 ml of 0.5 M tetrasodium ethylenediaminetetraacetate and 35 l of ethanol.

The mixture is left overnight at 40.degree. C. and then centrifuged. The precipitate is dried under vacuum; 50 g of dry protein powder of supernatant are obtained.

 A solution of the powder in 500 ml of water was gently stirred by slowly adding acetic acid using a peristaltic pump, up to pH 4.3 (after about 3 hours of pumping). Stirring is continued for 3 days at 40C; crystallization occurs. The crystals (in the form of tablets apparently, perhaps orthorhombic and having the birefringence phenomenon) are collected by centrifugation, suspended in 500 ml of water at 40 ° C. and stirred overnight; centrifuged and dried under vacuum. Yield: 5.2 g.

 4 g of this product are dissolved in 50 ml of 50% by volume ethanol and 50 ml of the eluent consisting of 0.08 M Tris, 0.02 N HCl, 0.08 M NaCl, 50% ethanol. volume at pH 8.6 (see Figure 1) .The solution is subjected to anion exchange chromatography as described above and illustrated in Figure 1 of the accompanying drawings. The collected portion corresponding to the main peak is adjusted to pH 7.4, mixed with 4 volumes of ethanol at 96% by volume, and then maintained for 2 days at 40C. The precipitate is isolated by centrifugation, washed twice with 150 ml of 96% ethanol and dried under vacuum. Yield 2.6 g.

2.5 g of this product are dissolved in 125 ml of 50% strength ethanol and 125 ml of the eluent consisting of
0.4M CH3COOH, 0.05M CH3COONa, 50% by volume ethanol, pH 4.7, and cation exchange chromatography as described above and illustrated in Figure 2 of the accompanying drawings. The collected portion corresponding to the main piz (the only visible one) is evaporated to dryness.

The residue is dissolved in water and the pH of the solution is adjusted to 7.1 (final volume about 90 ml). The solution is mixed with 1200 ml of 96% ethanol and the mixture is kept overnight at 40 ° C. The precipitate is isolated by centrifugation, washed twice with 150 ml of 96% ethanol and dried under vacuum.

Yield: 1.8 g of high purity PPS meeting the purity requirements given in Table I below
Example 2

 20 g of the supernatant protein powder obtained as described in Example 1 are dissolved in 200 ml of water. 208 ml of ethanol at 96% by volume are added and adjusted to pH 4.6 with acetic acid. A small precipitate is removed by centrifugation. The slowly turbid supernatant liquid is subjected to cation exchange chromatography on a 2.5 x 80 cm column of "SP-Sephadex C-25", equilibrated with eluent 1 (0.4 M acetic acid, 0.05 M sodium acetate, 50% by volume ethanol, pH 4.6). The linear gradient elution is carried out with 3 l of the eluent 1 to 3 l of the eluent 2 (0.3 M sodium acetate, 50% by volume ethanol, pH 8.7).

Fractions of 10 ml are collected at the elution rate of 40 ml / h. Fractions corresponding to the major peak appearing fractions 100 to 130 are collected.

The collected portion is adjusted to pH 8 and then mixed with 1.8 l of 96% by volume ethanol. The mixture is kept at 40C for 24 hours. The precipitated protein is isolated by centrifugation, washed twice with 150 ml of 96% ethanol and dried under vacuum. Yield: 2.8 g. 2.5 g of this product are dissolved in 250 ml of a Tris buffer (0.0755 M Tris,
0.05 N HCl, pH 7.4). The anion exchange chromatography solution is subjected to a 2.5 x 50 cm column of "QAE-Sephadex A-25", equilibrated with a Tris buffer (0.117 M Tris, 0.1 N HCl, pH 7.4). The column is eluted with the equilibration buffer at a rate of 30 ml / h. Fractions of 10 ml are collected. Fractions corresponding to the main central part of the peak, the maximum of which is at fraction 225, are pooled. This collected portion (620 ml) is mixed with 60 ml of 5 M sodium chloride and 12 ml. 1 of ethanol at 96% by volume.

 The mixture is kept at 40C for 24 hours. The precipitated protein is isolated by centrifugation, washed twice with 150 ml of 96% ethanol and dried in vacuo. Yield: 1.7 g of high purity PPS.

Example 3

 To 150 liters of an aqueous solution obtained by evaporation of an extract of 250 kg of pancreatic pig glands freed of insoluble matter, 22.5 kg of sodium chloride are added. It is stirred to dissolve the added salt, the precipitate is separated by centrifugation and the release cake is thus obtained. To the liqueursmers (162 l) was added 34 kg of ammonium sulphate stirring continuously for 2 hours at room temperature; a precipitate is obtained which is isolated by centrifugation. 223 g of the wet product are dissolved again by adding 500 ml of a buffer (0.05 M buffer of formic acid, 0.01 M of sodium hydroxide, pH 3.2). The conductivity of the solution is reduced to 4 mS by dialysis against water. The solution is placed on a 5 x 50 cm column of "SP-Sephadex C-25" equilibrated with buffer I (0.1 X formic acid). 0.02 M sodium hydroxide, pH 3.2).

The column is then eluted with a linear gradient of sodium chloride from 0 to 0.27 M in buffer I. The total eluent volume is 5.5 l. The column is further eluted by buffer I containing the chloride. The flow rate, during column feeding and elution, is 100 ml / h and the collected fractions are 15 ml. The chromatogram obtained by continuous control of the optical density of the fractions at 276 nm has a main peak ranging from fraction 420 to fraction 530. The portion corresponding to the main peak is brought together and adjusted to pH 7.4, then mixture with 20 volumes of ethanol at 96% by volume. After standing for 48 hours at 40 ° C., a precipitate is separated by centrifugation and dried under vacuum. Yield: 6 g. The product is further purified by anion exchange chromatography on a column of "QAE-Sephadex A-25", as described in Example 2. Yield: 3.4 g of high purity PPS .

EXAMPLE 4
A preparation for parenteral administration containing 1 mg PPS / ml is prepared as follows:
Dissolve 1 g. of PPS and 99 g of lactose in 1 liter of distilled water and the pH was adjusted to 7.0. The solution is then sterile filtered. 10 ml vials are filled with the sterile solution such that each vial contains 10 ml of the solution. Then, the solutions are lyophilized and the flasks are sealed under aseptic conditions.

The preparation of each of these vials will be dissolved in 10 ml of sterile water before administration.

EXAMPLE 5
Oral preparations can be prepared as follows
100 mg of PPS are mixed with 9 g of malus starch, 9-g of lactone and 180 mg of magnesium stearate until a homogeneous mixture is obtained. No. 3 gelatin capsules are filled with the mixture in such a manner that each capsule contains 1 mg of PPS.

Claims (17)

 1. purified polypeptide characterized in that it has the composition of amino acids ciapres Trp (2) Lys (4), His (1), Arg (5), Asx (10), Thr (3), Ser (9), Glx (12), Pro (12), Gly (6), Ala (6), Cysl / 2 (14), Val (7), Met (2), Ile (3), Leu (1), Tyr (2), Phe (7), the determinations being subject to the usual error of + 10% of the figures indicated, and in that that it probably presents, according to the current knowledge, the partial amino acid sequence, from the nitrogen hereinafter pyrGlu -Lys-Pro-Ala-Ala-Cys-Arg-Cys-Ser -Arg- Glx-Asx -Pro  5 10 Lys-Asx-Arg-Val-Asx-Cys Wly-Phe-ProWly-Ile-Thr -Ser-Asx-  15 20 25 GIx Cys-PheThr-Ser-Gly-Cys-Cys-Phe-Asx-Ser-Glx-VaI-Pro-  30 35 40 Gly-Val-Pro-Trp,  Wherein pyrGlu (residue 1) represents pyroglutamic acid, and its salts. physiologically acceptable.  2. Polypeptide according to claim 1, characterized in that it has a purity greater than 50% by weight, and preferably greater than 90% by weight.  3. Polypeptide according to claim 1, characterized in that it is in the crystallized state.  4. Process for isolating a purified polypeptide according to claim 1, characterized in that this polypeptide is isolated from the porcine pancreatic glands by a combination of chromatographies and precipitates.  5. Method according to claim 4, characterized in that the glands are extracted with a mixture of water and a water-miscible organic solvent in an acid medium, the precipitate is removed, the extract is neutralized. and isolating the polypeptide from the extract thus obtained using known separation methods per se, after which; if desired, it is converted into a salt.  6. Process according to claim 4, characterized in that the polypeptide is isolated from an insulin release cake.  7. Process according to claim 5, characterized in that the isolation is carried out by chromatography by means of a cation exchanger and / or anion exchanger.  8. Process according to claim 7, characterized in that a crystallization operation is also carried out.  9. The method of claim 7, characterized in that the chromatography is carried out with harvest of most of the product corresponding to the main peak of the polypeptide.  10. Process according to claim 5, characterized in that the polypeptide is isolated from the mother liquors obtained during the preparation of an insulin release cake by carrying out a new release followed by chromatography with the aid of anion exchangers and / or cation exchangers.  11. Method according to any one of claims 6 to 9, characterized in that the release cake is substantially free of insulin by separating the precipitate formed in a solution of the release cake with a pH in the range of about 4.9 to 5.7, preferably about 5.3, and optionally separating the precipitate formed in said solution of the release cake at a pH in the range of about 5.7 to 7.0, of preferably about 6.5, before the chromatography operations.  Process according to claim 8, characterized in that the crystallization step is carried out by adjusting the pH of the solution to a value in the range of about 3.8 to 4.8, preferably about 4.3. .  13. A polypeptide purified by a process according to claim 1 purified by a process according to any one of claims 4 to 12.  14. A pharmaceutical composition characterized in that it comprises an effective amount of a purified polypeptide according to any one of claims 1 to 3 and 13 in association with a pharmaceutically acceptable purified vehicle or excipient.  15. A composition according to claim 14, characterized in that it consists of a sterile aqueous solution of the purified polypeptide, preferably containing about 0.9S sodium chloride and optionally preservatives such as a parahydroxybenzoate or phenol .  16. The pharmaceutical composition as claimed in claim 15, characterized in that the purified polypeptide is present in the solution at a concentration of 0.1 to 200 mg / ml, preferably of 0.5 to 25 mg / ml.  17.- Application of a purified polypeptide according to any one of claims 1 to 3 and 13 as a drug in particular as a spasmolytic agent, diagnostic auxiliary product, therapeutic agent and prophylactic agent.