CA1176193A

CA1176193A - Peptide, process for their preparation and pharmaceutical composition containing them

Info

Publication number: CA1176193A
Application number: CA000402283A
Authority: CA
Inventors: Hildegard C. Nee Kornmayer Schaller; Heinz Bodenmuller
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften
Priority date: 1981-05-06
Filing date: 1982-05-05
Publication date: 1984-10-16
Also published as: JPS57185244A; JPS6043080B2; EP0064302B1; EP0064302A1; ATE9994T1; DE3117934A1; DK200082A; US4457917A; DE3261044D1

Abstract

ABSTRACT OF THE DISCLOSURE
The peptide pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe is a neuropeptide of interest as a research substance and in pharmaceutical preparations; it is produced synthetically according to known peptide-synthesizing methods, or by extracting animal tissue with an organic solvent and purifying the extract by chromatography particularly through ion-exchangers, through weakly cross-linked molecular-sieve columns and by high pressure liquid chromatography.

Description

~76~93 The invention relates to a hitherto unknown peptide consisting of eleven (11) amino acids, to a method for obtaining it, and to the use thereof in pharmaceutical preparations.
The peptide according to the invention comprises 11 amino acids and exhibits the following sequence:
pGlu-pro-pro-Gly-Gly-ser-Lys-val-Ile-Leu-phe~
The sequence has been confirme~ by synthesis.
The peptide according to the invention exhibits the following retention times in a reverse phase~octyl-silane column (LiChrosob RP-8*, particle size 7l~m, column size 250-4 mm) at a flow rate of 1 ml/min.;
i) in 50% methanol in 5 mM of ammoniumbicarbonate isocratic 7.6 min.
ii) with gradient (10 min.) from 40 to 60% methanol in 5 mM of ammoniumbicarbonate 10.5 min.;
iii) in 30% acetonitrile in 0.1% TFA (trifluoroacetic acid) isocratic 8 min.;
iv) with gradient ~10 min.) from 20 to 40% acetonitrile in 0.1% of TFA 11.2 min.
The peptide according to the invention is the active substance in the so-called "cephalic activator" (or head-activator). In the case of hydra, the compound is also responsible for cephalo-specific determination, i.e. an interstitial stem-cell is determined by the connection to the nerve cell. These effects are obtained at very low concen-trations, of the order to 10 13 M. In the case of mammals, the compound has a growth stimulating effect upon embryonal brain cells, and it has a hypertensive ~pressor) effect with intraventricular application. By reason of its biological properties, the peptide is an interestin~ research chemical *trademark 11761~3 and also opens up interesting possibilities as a pharmaceutical preparation.
It has been found that the peptide of the invention may be obtained from animal tissues, more particularly from the hypothalamus region of the brain, from the intestines of mammals, or from whole coelenterata.
According to an aspect of the invention, there is provided a process for obtaining the peptide of the inventicn comprising:
i~ extracting an animal tissue with an organic solvent, centrifuging,chromatographically purifying a concentrated extract and subjecting a peptide-containing fraction to high pressure liquid chromatography, or ii) synthesizing said peptide by a conventional peptide synthesis,or iii) cloning of coded nucleic acid.
In a particular embodiment the method of the invention is characterized in that the animal tissue is extracted with an organic solvent, for example acetone, acetic acid or methanol, is centrifuged, and the concentrated extract which suitably has been concentrated by ultrafiltra-tion is purified by chromatography employing ion-exchangers, for example weakly acid anion-exchangers or weakly basic cation-exchangers, and through weakly cross-linked molecular sieve columns, for example dextran or polyacrylamide, the fractions containing the peptide sought being then subjected to high pressure liquid chromatography, for example, through reverse phase octyl- or octadeca-silyl columns.
It is possible to obtain by this method from 20 to 30 nanomols of the peptide in pure form from 200 ky of sea anemone and 3000 rat intestines, respectively. From a - 13 7~3 human hypothalamus, the method produces about 1 nanomol of the peptide. If the peptide is produced by conventional methods of peptide synthesis from amino acids, it is readily available in any desired quantities.
The peptide acccording to the invention may be synthesized by any of the peptide-synthesizing processes, for example by the Merryfield solid phase synthesis with symmetrical anhydrides (Hoppe Seyler Z.Physiol.Chem.353, 1973-1976 (1972)) or in the liquid phase with mixed anhydrides (Liebigs Ann.Chem.572, 190-194 (1951)), with or without the use of automaticpeptide synthesis.
Synthesis by cloning of coded nucleic acid is also possible.
As shown by the formula, the peptide contains two molecules each of proline and glycine and 7 other amino acids once each. A peculiarity characteristic of many peptide hormones is the presence of a pyroglutamic acid at the amino end. The molecule is characterized by considerable hydrophobicity in the carboxy end part.
When animal tissues are used to obtain the peptide, the tissues are suitably comminuted as finely as possible in the organic solvent, for example methanol, at a temperature not exceeding 10C. After centrifuging, the concentrated left over is washed with petroleum ether and chloroform in order to separate any lipids. Subsequent chromatography is preferably carried with an approximately neutral pH. This may, if desired, be preceded by treatment with à weakly basic anion exchanger, for example a cross-linked dextran carrying diethylaminoethyl groups, the peptide being combined at 5 mM of ammonium acetate at a pH of less th-n ~L176193 7.5 and being again eluted at a pH of less than 3.5.
Because of its atypical absorption properties, a product obtainable commerically under the name of Sephadex G-10* has been found particularly satisfac*ory as a weakly cross-linked dextran gel, while the product commerically obtainable under the name of Biogel P 2* has been found particularly satis-factory as a polyacrylamide gel. However, it is also possible to use other dextran gel or polyacrylamide gel preparations having comparable properties, especially as regards the degree of cross-linking.
Elution is carried out with a dilute salt solution, for example 0.1 M of NaCl. The desired pH ~alue in the given range may be adjusted with any desired buffer sub-stances, preferably tris-buffers.
For the high pressure liquid chromatography, the preferred carrier material is reverse phase octyl silane, obtainable commerically under the name of `'LiChrosorb RP-8*".
The peptide of the invention is held back on this carrier material under polar conditions (a little organic component, for example methanol or acetonitrile in the mobile phase), and it is eluted under apolar conditions (a lot of organic component in the mobile phase).
As already mentioned hereinbefore, synthetic production of the peptide may be carried out by conventional protein synthesizing methods. The pyroglutamic acid group at the amino end may be formed by the incorporation, for example, of glutamine and subsequent cyclizing by heating in an acid, for example TFA (trifluoroacetic acid).
Due to the properties described hereinbefore of the new peptide, this neuropeptide, which first occurs in the animal world and has`maintained its structure strictly *trademark 1~7~93 up to man, constitutes an interesting research substance and may be used as the active substance in pharmaceutical preparations for regulating growth and/or as a transmitter substances.
The invention is further illustrated in particular embodiments by reference to the accompanying examples.

Between 10 and 13 kg of deep frozen Anthopleura elegantissima have Pacific Biomarine Laboratories, California, are chopped into pieces measuring between 1 and 3 cm, and cold methanol (-20C) is added until the pieces are well covered (about 1 part of the chopped pieces to 2 parts of methanol).
The mixture thus obtained is homogenized (Ultraturax*). The temperature must not exceed 10C (cool with a bath of ice, industrial ~aCl or cattle salt). The homogenized products thus obtained are centrifuged for 10 minutes at 2000 r.p.m. (pre-cool the rotor and the centrifuge and allow to run at 4~C).
The supernatant is collected and stored at -20C.
The sediments are dissolved in the centrifuge vessel with cold (-20C) methanol and homogenizing and centrifuging is repeated twice. The combined supernatants are concentrated in a rotary evaporator to between 10 and 15 litres and are shaken with cold petroleum-ether. The petroleum-ether phase is discarded.
Washing is repeated until the green colour is in the ether. The washed solution is extracted thrice with cold chloroform and the first chlorofGrm-phase once. The collected water-phases are concentrated into the smallest possible volume (in a rotary evaporator) and are frozen and dried.
The freeze-dried residue is dissolved in a minimum of methanol, is centrifuged and the supernatant is *trademark , - 5 -~76~93 recovered. The sediment is again extracted with methanol and centrifuged. The collected supernatants are evaporated, dissolved in distilled water (total volume about 500 ml), are adjusted to a pH of 7.6 and 250 ml at a time, are charged into a Sephadex G-10* column (total volume 5.3 litres, 10 x 73.5 cm).
Alternatively, 100 g of Sephadex DEAE-A-25* per 500 ml of extract are equilibrated overnight with 5 mM of ammonium acetate pH 7.6, are shaken for 30 min. with the supernatant and are centrifuged for 30 min. at 1500 r.p.m. The supernatant is added to new DEAE (100 g), and this is repeated until colour no linger combines with the DEAE-Sephadex* (usually 3 to 4 times). The DEAE-Sephadex*
is eluted with 5 mM of ammonium acetate until there is no longer any colour in the left over material. The eluate is neutralized, concentrated, 25 ml at a time are charged into a Sephadex-G-10* column (total volume 250 ml, 5.5 x 13 cm) and are chromatographed with distilled water.
The 9.5 to 14.5 litre fractions of the 5.3 litre G-10 column, or the 500 to lOOOml fractions of the 250 ml G-10 column, are now concentrated to 20 ml, are charged into an acrylamide-gel column (Biogel P-2*, 660 ml, 4.5 x 50 cm) and are eluted with 5 mM of ammoniumbicarbonate solution.
The 300 to 450 ml fractions are combined, are concentrated to 1 ml, are charged into a further G-10 column (volume 25 ml, 1.5 x 16 cm), and are eluted with 0.1 M of NaCl and 0.01 M
of tris-HCl at a pH of 7.6. The combined 29 to 42 fractions are concentrated to 1 ml, are charged into an acrylamide gel column (Biogel P-2*, 40 ml, 1.3 x 47 cm), and are eluted with the same elution agent as in the preceding step.
The active fractions are combined, concentrated in'o 0.5 ml and placed in a Pasteur capillary pipette containing 0.5 ml of LiChrosorb RP-8*, particle size 10 um~
*trademark 117~ 3 which is equilibrated with 20% of methanol in 5 mM of ammonium carbonate. After washing with the equilibrating agent, elution is carried out with 3 ml of 80% methanol in 5 mM of ammoniumbicarbonate solution, the eluate is dried, is taken up again in 0.1% trifluoroacetic acid and the treatment in the Pasteur capillary pipette is repeated, but with 3 ml of 40% acetonitrile in 0.1 TFA as the elution agent. The eluate is concentrated and charged into a high pressure liquid chromatography column (LiChrosorb RP-8*, particle size 7 ~m 250 x 4 mm), which is equilibrated with 20% of acetonitrile 0 in 0.1% of trifluoroacetic acid and, with a gradient of up to 40% of acetonitrile, is eluted in 0.1% of trifluoroacetic acid for 10 min. at a flow rate of 1 ml/min. The retention time of the peptide of the invention amounts to 10.6 min.
The eluate is concentrated to 50 ~1 and is again charged into a similar high pressure liquid chromatography column which is equilibrated with 40% methanol in 5 mM of ammoniumbicarbonate and is eluted with a gradient of up to 60% of methanol for 10 min. Flow rate 1 ml/min. The retention of the peptide of the invention amounts to 10.5 min. This produces 1 nanomolof pure compound.

1000 Rats (between 2 and 3 months old) were narcot-ized with carbon dioxide, the abdominal wall was opened and the intestine, without the pancreas was taken out from the pylorus to the last thickening of the intestine before the anus. The intestines were collected on 20 litres o~ cold methanol, were mixed with 0.05% of phenylmethylsulphonyl fluoride, and were then processes as described in Example 1 This produced 10 nanomols of the peptide of the invention.

*trademark 1176~3 A human hypothalamus (20 hours after death) was homogenized at 4C with an excess of methanol and was then treated twice for 2 minutes ultrasonically. A supernatant was produced by centrifuging, and this was concentrated with petroleum-ether and washed with chloroform. After re-extrac-tion with water, as described in Example 1, the aqueous phase was concentrated to 10 ml, was charged into a Sephadex G-10*
column 250 ml in volume as described in Example 1, and was eluted. The 500 to 1000 ml fraction was concentrated and charged directly into a 0~5 ml LiChrosorb RP-8* column.
Further purification was carried out as in the latter stages of Example 1. This produced one nanomol of the peptide of the invention.

*trademark

Claims

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as follows:

1. A method for obtaining a peptide exhibiting the amino acid sequence:
pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe comprising:
i) extracting an animal tissue with an organic solvent, centrifuging, chromatographically purifying a concentrated extract and subjecting a peptide-containing fraction to high pressure liquid chromatography, or ii) synthesizing said peptide by a conventional peptide synthesis, or iii) cloning of coded nucleic acid.

2. A method according to claim 1 i) comprising:
a) extracting an animal tissue with an organic solvent, b) centrifuging the solvent extract, c) purifying the thus obtained concentrated extract by chromatography through an ion-exchanger and through a weakly cross-linked molecular sieve, and subjecting the peptide-containing fraction to high pressure liquid chromatography.

3. A method according to claim 1 ii) comprising a Merryfield solid phase synthesis with symmetrical anhydrides.

4. A method according to claim 1 ii) comprising a liquid phase synthesis with mixed anhydrides.

5. A method according to claim 2, which comprises repeating steps a) and b) before proceeding to step c).

6. A method according to claim 1 i) wherein said animal tissue is from the hypothalamus region of the brain.

7. A method according to claim 1 i) wherein said animal tissue is from the intestines of animals.

8. A method according to claim 1 i), wherein said animal tissue is from whole coelenterata.

9. A method according to claim 1 i) wherein said animal tissue is Anthopleura elegantissima.

10. A method according to claim 1, including a step of recovering a peptide of formula -pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe.

11. A method according to claim 2, including a step of recovering a peptide of formula -pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe.

12. A method for obtaining a peptide exhibiting the amino acid sequence:
pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe comprising:
i) extracting an animal tissue with an organic solvent, centrifuging, chromatographically purifying a concentrated extract and subjecting a peptide-containing fraction to high pressure liquid chromatography.

13. A method according to claim 12, including a step of recovering a peptide of formula -pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe.

14. A peptide exhibiting an amino acid sequence:
pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe whenever prepared by the method of claim 1, or by an obvious chemical equivalent.

15. A peptide exhibiting an amino acid sequence:
pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe whenever prepared by the method of claim 2, or by an obvious chemical equivalent.

16. A peptide exhibiting an amino acid sequence:
pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe whenever prepared by the method of claim 12, or by an obvious chemical equivalent.

17. A peptide of formula:
pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe whenever prepared by the method of claim 10, or by an obvious chemical equivalent.

18. A peptide of formula:
pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe whenever prepared by the method of claim 11, or by an obvious chemical equivalent.

19. A peptide of formula:
pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe whenever prepared by the method of claim 13, or by an obvious chemical equivalent.