MXPA96003991A - Peptides to inhibit the release of peps - Google Patents

Abstract

The present invention relates to peptides derived from the C-terminal sequence of sorbin, but containing at least one amino acid residue of configuration D, which inhibits the release of pepsin and, therefore, are particularly useful in the treatment of ulcers or of esophagitis The present invention also relates to substitute salts and derivatives, as well as to pharmaceutical compositions containing said peptide.

Description

PEPTIDES TO INHIBIT THE RELEASE OF PEPSINA BACKGROUND OF THE INVENTION The present invention aims at peptides capable of inhibiting the release of pepsin, substitution derivatives and salts of such peptides, as well as pharmaceutical compositions containing them. The peptides can be used in the treatment of diseases related to the release of pepsin and, more particularly, for the treatment of ulcers or esophagitis. Recently a new peptide was isolated from the intestine of the pig; this one called sorbin, has 153 natural amino acids (WO 89/06241). The sorbin, as well as the peptide fragments of the C-terminal end of the sorbin (containing when more 40 amino acids), are capable of causing an increase in the process of absorption by the mucosa. However, the modification of these peptide fragments by insertion of at least one amino acid residue of configuration D confers, in an unexpected manner, another biological activity on the modified peptide analogs: it inhibits the release of pepsin, biological activity that the peptides not modified do not present. This activity is particularly interesting in certain circumstances. Indeed »the general REF: 23019 mechanisms of gastric secretion in mammals are well known at present. Gastric digestion, then, is the result of the action of enzymes, hydrochloric acid and pepsin. Pepsin is a protein; It is also, together with gastrin, one of the main components of gastric juice. Its main physiological function is to start the digestion of proteins. In contrast, numerous studies have shown the significant role of pepsin in the formation of ulcers. Consequently, in certain circumstances, it may be adequate to at least partially inhibit the release of pepsin. DESCRIPTION OF THE INVENTION The present invention aims at a peptide of the general formula I Al A2 A3 X wherein A. represents the residue L-Thr or D-Thr; or one of the following sequences, in which at least one amino acid residue can have the D configuration: Val-Thr, Pro-Val-Thr, Arg-Pro-Val-Thr, Glu-Arg-Pro-Val-Thr, His-Glu-Arg-Pro-Val-Thr, Gln-His-Glu-Arg-Pro- Val-Thr, Leu-Gln-His-Glu-Arg-Pro-Val-Thr, Ile-Leu-Gln-His-Glu-Arg-Pro-Val-Thr, Ser-Ile-Leu-Gln-His-Glu- Arg- Pro-Val-Thr, Se-Ser-Ile-Leu-Gln-His-Glu-Arg-Pro-Val-Thr, and s-Ser-Ser-I le- Leu -Gln- His-Glu-Arg- Pro -Val-Thr, Gly-Lys-Ser-Ser-Ile-Leu-Gln-His-Glu-Arg-Pro-Val-Thr, Pro-Gly-Lys-Ser-Ser-I le-Leu-Gln-His- Glu-Arg- Pro-Val -Thr or Glu-Pro-Gly-Lys-Ser-Ser-I le-Leu-Gln-His-Glu-Arg-Pro-Val-Thr; A_ represents the sequence Lys-Pro-G ln-Al a in which at least one amino acid residue can have the D configuration; A "represents a covalent bond or the peptide sequence -Gly-A4-Ag, wherein A. and A_ represent, each independently, a basic amino acid residue; and X represents a hydroxy group; Not me; alkylamino, characterized in said peptide of formula I because it contains at least one amino acid residue of configuration D.

The present invention also aims at substitution derivatives of the peptides of the general formula I, wherein one or more amino acid residues are substituted with one or more of the peptide protecting groups commonly used in the chemistry of the peptides for use. biological; when several protective groups are used, these are not necessarily the same. Preferably, the protecting groups are selected from the group consisting of lower alkyl groups such as methyl or tert-butyl; phenyl; benzyl or substituted benzyl such as trimethoxybenzyl; 2-chlorobenzyloxycarboni lo; 9-fluorenyl-methyloxycarboni lo; tert-buti loxicarboni lo; acetyl; sulfonyl and phosphoryl. The present invention also aims at peptides containing the amino acid sequence Aj-A ^ -A-, where A., A_ and A_ are as previously defined. The present invention also relates to pharmaceutically acceptable salts of peptides such as those defined above. These salts can be obtained with organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, benzoic, salicylic, succinic, methylsulfonic acids. toluenesulfonic, mineral acids such as hydrochloric, sulfuric or phosphoric acid, or polymeric acids such as tannic acid or carboxymethylcellulose. A. and 5 represent. each independently, when present in the peptides according to the present invention, are basic amino acid residues of configuration D or L. Preferably, A. and A5 represent, each independently, an amino acid residue Lys, D -Lys, Arg or D-Arg. As mentioned above, the porpoids according to the present invention contain one or more amino acid residues of configuration D. The present invention particularly considers peptides that contain an amino acid residue of configuration D, which peptides may have such protective groups co or those defined in the present invention. They can be cited, for example. the following peptides.

Thr-Lys-Pro-Gln-D-Ala-NH2. Thr-Lys-Pro-Gln-D-Ala-Gly-Lys-Lys-NH2, Thr- (acetyl) Lys-Pro-Gln-D-Ala-NH2, Pro-Val-Thr-Lys-Pro-GIn-D -Ala-NH2, Pro-D-Val-Thr-Lys-Pro-Gln-Ala-NH2, Prc-Val-Thr-Lys-Prc-Gln-Ala-Gly-Arg-D-Arg. Pro-Val-Thr- (acétyl) Lys-Pro-Gln-D-Ala-NH2, D-Pro-Val-Thr-Lys-PrrGln-Ala-NH2, His-GIu-Arg-Pro-Val-Thr-Lys -Pro-Gln-D-Ala-NH2, pe-Ieu-Gln-His-Glu-Arg-Pro-Val-Thr-Lys-Prc-Gln-D-Ala-NH2et Glu-Pro-Gly-Lys-Ser- Ser-Ile-Leu-Gln-His-Glu-Arg-Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH2.

The amino acid residue of configuration D is preferably located at the C-terminal or N-terminal end. Preferred peptides having an amino acid residue of configuration D at the C-terminal position are peptides in which A. represents Lys-Pro-Gln-D-Ala and A3 represents a covalent bond. Preferred peptides having an amino acid residue of configuration D at the N-terminal position, are the peptides in which A. represents D-Thr or a sequence such as that defined above, in which the amino acid residue in N position -terminal of this sequence is of configuration D. More particularly, similarly. the present invention considers the peptides having two amino acid residues of configuration D, said peptides being able to be substituted with protective groups such as those defined in the present invention. We can mention, for example, the following peptideOB, D-Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH2, D-Pro-Val-Thr-Lys-Pro-Gln-Ala-Gly-D-Lys-Lys-NH2, Pro-D- Val-Thr-Lys-Pro-Gln-D-Ala-NH2et D-Pro-Val-Thr- (acetyl) Lys-Pro-Gln-D-Ala-NH2.

Preferably. the peptides contain two amino acid residues of configuration D. presenting an amino acid residue of configuration D at the C-terminal position. The second amino acid residue may be located at any site of the peptide chain, but preferably at the N-terminal position of the peptide. Preferred peptides are peptides in which A represents D-Thr or a sequence such as those defined above. wherein the amino acid residue in the N-terminal position of this sequence has a D configuration; A_ represents Lys-Pro-Gln-D-Ala and A "represents a covalent bond. Such peptides are preferably substituted with a protecting group, and more particularly with an acetyl protecting group on the Lys residue.

The peptides according to the present invention can be prepared according to any of the classical methods known in the synthesis of peptides. For example, the synthesis can advantageously be carried out in solid phase according to the following scheme: the formation of the peptide chain begins with the fixation of a first C-terminal amino acid of the chain, on a resin, by means of its carboxylic group; its amine function is protected with a protective group such as t-buti loxycarboni lo (Boc). After fixing the first C-terminal amino acid, the amine function is deprotected by washing the resin with an acid. In the case of protection with the Boc group, the deprotection can be carried out by washing with trifluoroacetic acid. The second amino acid, whose amino function is protected, is coupled, via its carboxyl function, to the unprotected amino function of the first C-terminal amino acid of the chain. The coupling is preferably carried out in the presence of a coupling agent, such as dicyclohexylcarbodiimide or diisopropylcarbodiimide. The peptide chain thus formed comprises two amino acids whose amino terminal function is protected. As described above, this amino terminal function is deprotected and subsequently a third amino acid can be fixed. The desired peptide chain is thus obtained by fixing the amino acids, one after the other. After removal of all protecting groups, the peptide is released from the resin. The synthesis of a peptide according to the present invention »namely, Pro-Va1-Thr-Lys-Pro-Gln-D-Ala-NH2 >; it was briefly described in the previous paragraph. Other peptides according to the present invention can be prepared by appropriate modifications of the above peptide synthesis, which are known to those skilled in the art. The synthesis is carried out in solid phase, at room temperature. The mode of operation used comprises the following steps: Deprotection, neutralization and coupling. The resin used is of the polystyrene type with 1% divinylbenzene (Merrifield resin). The fixation of the Boc-D-Ala on the Merrifield resin is carried out in the presence of cesium carbonate, in toluene and dimethylformamide (DMF). The amino terminal function of the amino acids used is protected by the Boc group. These Boc groups are displaced by trifluoroacetic acid, followed by several washes with methylene chloride and isopropanol. The aminated groups are neutralized with trieti lamina, which is followed by several washes. Threonine and Valine are converted after coupling into a hydroxybenzotriazole ester in the presence of diisopropyl carbodiimide (DIPCDI), with glutamine; The hydroxybenzotriazole ester is prepared directly in the reactor. The plant and the two prolines are transformed into symmetrical anhydride after coupling. In any case, the coupling is carried out in the presence of diisopropylethylamine. The side chain of lysine is protected by an Fmoc group, but that of threonine is not protected. The last terminal coupling, the displacement of the Fmoc group. is obtained with piperidine in dimethylformamide after displacing the Boc group from the N-terminal amino function of Proline. The peptide is obtained by detaching it from the resin, after a treatment with ammonia in methanol / DMF. Subsequently, the crude product thus obtained is purified. The present invention also considers pharmaceutical compositions containing, as an active ingredient, an effective amount of at least one peptide of formula I as defined above, a substitution derivative of said peptide as defined above, or a peptide containing the amino acid sequence A, -A-A, as defined above, in association with a pharmaceutically acceptable diluent or carrier.

The peptides according to the present invention can be administered orally, intravenously, parenterally, subcutaneously, intraperitoneally or intramuscularly. The pharmaceutical composition can be presented in the form of a gel, a tablet, a lyophilized or a liquid, according to the selected administration route. The pharmaceutical composition can also be in the form of a sustained release formulation. Orally, the peptide according to the present invention can be administered, in humans, at a dose of 5 to 100 jug / kg per day. Intravenous or subcutaneously, a compound according to the present invention can be administered, in humans, at a dose of 1 to 12 and kg / kg. one to three times a day. In animals. the compounds according to the present invention are retained in a significant amount in the organism several days after an acute administration and more particularly the Pro-Va1-Thr-Lys-Pro-Gln-D-Ala-NH ", which is retained in an amount greater than 10%. Toxicity Subacute toxicity was studied in rats and dogs. After administrations of doses that were up to 4000 jug / kg / d, no signs of toxicity and no mutagenesis-inducing signal were observed four weeks after administration. In humans, a subcutaneous or intravenous injection, with a dose of 200 Ltg / kg, did not induce any biological, clinical or pathological abnormality. Pharmacology The therapeutic interest of the compositions according to the present invention was determined by the following experiments. The intensity of the gastric response was measured by determining the volume of gastric secretion induced. Cats were operated under general anesthesia, in which the intervention allows to observe the stomach in two parts: the Heidenheim pouch and the gastric fistula. These two bags are derived to the outside, in order to recover the secretions of hydrochloric acid, pepsin and gastric juice, both during the basal phase and after the stimulus. Cats suffer from chronic fistulas, then, these can undergo a certain number of tests every week and be their own controls. The stimulation of pepsin secretion is obtained by administration in live animals. of pentagastrin (PG) and PIV (vasoactive intestinal peptide). by perfusion for two hours at a ratio of 2 and 4 g / kg / h.

One hour after stimulation with pentagastrin and PIV, these peptides are added to the infusion, at a dose of 100 pmol / kg / h. The volume of gastric juice is collected from 30 minutes before perfusion, until the end of it. The dose of pepsin in the gastric juice (as homogeneous as possible) was evaluated by a proteolytic spectrophotometric method. The results obtained in 9-12 experiments are reported in the tables presented below: pepsin secretion is expressed in mg / 15 min. average of two periods of 15 min per test during basal secretion and average of 6 periods of 15 min during stimulated secretion. Certain peptides according to the present invention, which have at least one amino acid residue of configuration D, were compared with their analogs whose amino acid residues are all of L configuration.

Table 1 Comparison of the activity of a peptide according to the present invention PD: Pro-Va1-Thr-Lys-Pro-G1n-D-A1a-NH2 with its analog P, Pro-Val-Thr-Lys-Pro-Gln-Ala -NH ^.

Table 2 Activity of peptides according to the present invention PD2: D-Pro-Val-Thr-Lys-Pro-Gln-Ala-NH2. PD3: Pro-Val-Thr- (acetyl) Lys-Pro-Gln-D-Ala-NH2 and PD. : D-Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH ".

Table 3 Activity of peptides according to the present invention PD5: Pro-D-Val-Thr-Lys-Pro-Gln-Ala-NH2, PD £ or: Pro-D-Val-Thr-Lys-Pro-Gln-D -Ala-NH and PD7: D-Pro-Val-Thr- (acetyl) Lys-Pro-Gln-D-Ala-NH2.

Table 4 Activity of peptides according to the present invention PDQ: His-Glu-Arg-Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH PDg: pe-lL / Hi- ^ ln-His-Glu -Arg-PrO-Val-ThrH. ^ And .D 10 G1u-Pro-Gly-Lys-Ser-Ser-11e-Leu-G1n-His-G1u-Arg-Pro-V 1-Thr-Lys-Pro-G1 n-D-A1a-NH2 Table 5 Comparison of the activity of the peptide according to the present invention P 8 His-G1u-Arg-Pro-Va 1-Thr-Lys-Pro-Gl nD-A1-NH, with its analog PQ His-Glu-Arg- Prrr-Val-Thr-Lys-Pro-Gln-Ala-NH ,, Table 6 Peptide activity according to the present invention 11 Thr-Lys-Pro-G1n-D-A1-NH. 12 Thr- (cetyl) Lys-Pro-Gln-D-A1a-NH, It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates. Having described the invention as above, the content of the following is claimed as property.

Claims (23)

1. CLAIMS 1. A peptide of the general formula I Al A2 A3" wherein, A ,, represents the residue L-Thr or D-Thr; or one of the following sequences in which at least one amino acid residue can be of D configuration; Val-Thr, Pro-Val-Thr, Arg-Pro-Val-Thr, Glu-Arg-Pro-Val-Thr, His-Glu-Arg-Pro-Val-Thr, Gln- • His-Glu-Arg-Pro -Val-Thr, Leu- • Gln- • His-Glu-Arg-Pro-Val-Thr, Ile- • Leu- Gln-His-Glu-Arg-Pro-Val-Thr, Ser- -Ile-Leu- Gln-His-Glu-Arg-Pro-Val-Thr, Ser- • Ser- • Ile-Leu-Gln-His-Glu-Arg-Pro-Val-Thr, Lys- • Ser- • Ser- 11e-Leu- Gln-His-Glu-Arg-Pro-Val-Thr, Gly- • Lys- • Ser-Ser-1le-Leu-Gln-His-Glu-Arg-Pro-V l-Thr, Pro- -Gly- • Lys -Ser-Ser-Ile-Leu-Gln-His-Glu-Arg-Pro-Val-Thr or Glu- -Pro-Gly-Lys-Ser-Ser-Ile-Leu-Gln-His-Glu-Arg-Pro -Val-Thr; A represents the Lys-Pro-Gln-Ala residue wherein at least one amino acid residue can be of D configuration; A "represents a covalent bond or the peptide sequence -Gly-A.-A ... in which A- and A_ represent, each independently, a basic amino acid residue; X represents a hydroxy group; Not me; ali lamino; said peptide of the formula I characterized in that it contains at least one amino acid residue of configuration D.
2. 2. A peptide containing the amino acid sequence A1-A2 ~ A3 in which A-, A2 and A3 are as defined in the claim 1, characterized in that it contains at least one amino acid residue of configuration D.
3. 3. A substitution derivative of a peptide according to any of claims 1 or 2, characterized in that one or more amino acid residues is substituted with one of the groups protectors normally used in the chemistry of peptides for biological use; when two or more are present, these are not necessarily identical.
4. 4. A peptide according to claim 3, characterized in that it contains at least one lysine residue protected by an acetyl group.
5. 5. A peptide according to any of the preceding claims, characterized in that it is in the form of its pharmaceutically acceptable salt.
6. 6. A peptide according to any of claims 1 to 5, characterized in that it contains an amino acid residue of configuration D.
7. 7. A peptide according to claim 6, characterized in that its amino acid residue of configuration D is located in the C-terminal or N-terminal end.
8. 8. A peptide according to claim 7, characterized in that its amino acid residue of configuration D is located at the C-terminal end.
9. 9. A peptide according to any of claims 6 to 8, characterized in that A "represents Lys-Pro-Gln-D-Ala and A- represents a covalent bond.
10. 10. A peptide according to claim 9, characterized in that it has the formula Thr-Lys-Pro-G1n-D-Ala-NH2 or Thr- (aceti 1) Lys-Pro-Gln-D-Ala-NH2.
11. 11. A peptide according to claim 9, characterized in that it has the formula Pro-Val-Thr-Lys-Pro-Gln-D-Al -NH or Pro-Val-Thr- (aceti 1) Lys-Pro-G1n- D-A1a-NH-,.
12. 12. A peptide according to claim 9, characterized in that it has the formula His-G1u-Arg-Pro-V 1-Thr-Lys-Pro-G1n-D-A1a-NH2.
13. 13. A polypeptide according to claim 9, characterized in that it has the formula 11e-Leu-G1n-His-Glu-Arg-Pro-Va1-Thr-Lys-Pro-G1n-D-A1a-NH2.
14. 14. A peptide according to claim 9, characterized in that it has the formula G1u-Pro-Gly-Lys-Ser-Ser-11e-Leu-G1n-His-G1u-Arg-Pro-Va1-Thr-Lys-Pro-G1n-D-A1a-NH ,,.
15. 15. A peptide according to claim 7, characterized in that its amino acid residue of configuration D is located at the N-terminus.
16. 16. A peptide according to claim 15 »characterized in that A. represents D-Thr or a sequence as defined above, in which the N-terminal amino acid residue of this sequence has a D.
17. 17 configuration. A peptide according to claim 16. characterized in that it has the formula D-Pro-Va1-Thr-Lys-Pro-G1n-A1a-NH ,,.
18. 18. A peptide according to any of claims 1 to 5, characterized in that it contains two amino acid residues of configuration D.
19. 19. A peptide according to claim 18 »characterized in that its amino acid residue of configuration D is located in the C position -terminal.
20. 20. A peptide according to any of claims 18 or 19, characterized in that the second amino acid residue of configuration D is located at the N-terminal position of the peptide.
21. 21. A peptide according to claim 20, characterized in that it has the formula D-Pro-V 1-Thr-Lys-Pro-G1n-D-A1-NH2.
22. 22. A peptide according to claim 20, characterized in that it has the formula D-Pro-Va1-Thr- (aceti 1) Lys-Pro-G1n-D-A1-NH2.
23. 23. Pharmaceutical compositions characterized in that they contain, as an active ingredient, an effective amount of at least one peptide according to any of claims 1 to 22, in association with a pharmaceutically acceptable diluent or carrier.