Peptides for inhibition of the release of pepsin, the pharmaceutical composition

 

(57) Abstract:

Describes a new synthetic peptides of the General formula I A1-A2-A3-X where A1= L - or D-Thr, or one of the following sequences, in which at least one radical of the amino acids may be D-configuration at the end of the claims; A2-Lys-Pro-Gln-Ala, in which at least one radical of the amino acids may be D-configuration; A3means a covalent bond; X = hydroxy, amino, alkylamino group and named the peptide of formula I contains at least one radical of the amino acid of D-configuration, and may contain one or several identical or different protective groups, or their pharmaceutically acceptable salts. Also described pharmaceutical composition, inhibiting the release of pepsin, based on the compounds of formula I. 3 C. and 18 h.p. f-crystals, 6 PL.

The object of the invention are peptides capable of inhibiting the release of pepsin, their substituted derivatives and their salts, and pharmaceutical compositions containing these peptides. The peptides can be used in the treatment of diseases associated with the release of pepsin, and, in particular, in the treatment of ulcers or esophagitis.

This activity is of particular interest. General mechanisms of gastric secretion in mammals is now well known. Gastric digestion is carried out under the action of enzymes, hydrochloric acid and pepsin. Pepsin is a protein that, along with gastrinom, is one of the main components of gastric juice. Its main physiological function is to initiate decomposition of proteins. However, numerous studies have shown that it promotes the formation of ulcers. Therefore, in certain situations it is desirable to slow down, at least partially, the release of pepsin.

The object of the invention is a peptide of General formula I

A1-A2-A3-X

where A1means a radical of L-Thr or is et D-configuration, submitted after the claims;

A2means the sequence Lys-Pro-Gln-Ala, in which at least one radical of the amino acids may be D-configuration;

A3means a covalent bond or a peptide sequence Gly-A4-A5where A4and A5denote each, independently of one another, a radical of the basic amino acids; and

X is hydroxy, amino, alkylamino group and named the peptide of formula I characterized in that it contains at least one radical of the amino acid of D-configuration.

The invention also relates to substituted derivatives of the peptides of General formula I, in which one or more amino radicals substituted by one or more protective groups of peptides, which are widely used in chemistry and biology, in the case of substitution of more protective groups, their identity is not required. Preferably, the protective group selected from the group lower alkyl, such as methyl or tertiary butyl; phenyl; benzyl or substituted benzyl, such as trimethoxybenzyl; 2-chlorobenzenesulfonyl, 9-fertilityscore, tert-butyloxycarbonyl; acetyl; sulfonyl and phosphoryl.

The object of izobreteny the 1, A2and A3are as defined above.

The invention concerns also pharmaceutically acceptable salts of the peptides defined above. These salts can be obtained with organic acids such as acetic acid, lactic, maleic, citric, malic, ascorbic, benzoic, salicylic, succinic, methansulfonate, toluensulfonate, or mineral acids such as hydrochloric acid, sulfuric acid or phosphoric, or with polymeric acids such as tannin or carboxymethyl cellulose.

A4and A5signify, independently from each other, when they are present in the peptides according to the invention, the radicals of the primary amino acid configuration D or L. Mostly A4and A5represent, independently, a radical amino acid Lys, D-Lys, Arg or D-Arg.

The above peptides according to the invention contain one or more radicals of amino acid of D-configuration. The object of the invention, in particular, are peptides containing the radical of the amino acid of D-configuration, while these peptides can be substituted with protective groups specified in the present invention. We can give the following example peptides, which are represented by m or N-terminal segment. Preferred peptides that have radical amino acid of D-configuration at the C-end, are peptides in which A2means Lys-Pro-Gln-D-Ala and A3means covalent bond. Preferably, the peptides, which have a radical amino acid of D-configuration at N-end, represented peptides, in which A1mean D-Thr or above the sequence, and the radical of the amino acids at the N-end of this sequence has the D-configuration.

The object of the invention also include peptides that have two radical amino acid of D-configuration, and these peptides can be substituted with protective groups, defined by the present invention. We can give the following example of peptides.

D-Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH2,

D-Pro-Val-Thr-Lys-Pro-Gln-Ala-Gly-D-Lys-NH2,

Pro-D-Val-Thr-Lys-Pro-Gln-D-Ala-NH2and

D-Pro-Val-Thr-(acetyl)Lys-Pro-Gln-D-Ala-NH2.

Mainly, peptides containing two radical amino acid of D-configuration, have one radical at the C-end, and the second can be located anywhere on the peptide chain, but mainly it is on the N end of the peptide. Preferred peptides are those in which A1represents the D-Thr or configuration, A2means Lys-Pro-Gln-D-Ala, and A3means covalent bond. Such peptides, predominantly, are peptides having one or more protective groups, and, in particular, acutiloba protective group on the radical Lys.

The peptides according to the invention can be obtained in accordance with one of the known classical methods in the field of peptide synthesis, for example, using solid-phase synthesis, which is carried out according to the following scheme: education peptide chain starts with the fixation of the first amino acid from the C-terminal end of the chain on the resin via its carboxyl group; the amino group is protected by a joining tert-butyloxycarbonyl (Boc). After joining the COOH-group of the first amino acid is removed protection with NH2group by washing the resin with acid. In case the protective group Boc, unlocks can be done using triperoxonane acid. The second amino acid with a protected amino group is attached through its carboxyl group on the released amino group of the first amino acid. The binding occurs mainly in the presence of a binding agent, such as dicyclohexylcarbodiimide or diisopropylcarbodiimide. The peptide chain is formed so clicks the group and then carry out the joining of the third amino acid. Thus, there is a serial connection of amino acids one by one and build the desired peptide chain. After removing all the protective groups of the peptide otscheplaut from resin.

Synthesis of a peptide according to the invention, namely Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH2briefly described next. The rest of the peptides according to the invention can be obtained by appropriate modifications described below peptide synthesis, which are well known to the specialists in this field.

The synthesis is carried out in solid phase at room temperature. Used operating mode includes the following steps: removing protection, neutralization and binding. Used resin such as polystyrene, structured on a 1% divinylbenzene (resin Merifield). Fixation of Boc-D-Ala on the resin Merifield is performed in the presence of cesium carbonate in toluene and dimethylformamide (DMF). Terminal amino group of amino acids used is protected by the Boc group.

These groups are replaced by Boc triperoxonane acid, and then produce several washes with methylene chloride and isopropanol. Amino groups are neutralized with triethylamine, followed by several washes. Threonine and valine before linking into ester of Girasole is produced directly in the reactor. The both lysine and Proline are transformed into the symmetric anhydride before binding. In all cases, the binding is carried out in the presence of diisopropylethylamine. The side chain of lysine is protected by a group of Fmoc, while the side chain of threonine is not protected. When the last binding, group Fmocremoved using piperidine in dimethylformamide before substitution group Boc N-terminal amino group of Proline. The peptide is produced by cleavage from the resin after treatment with ammonia in methanol/DMF. Thus obtained, the original product is then cleared.

The aim of the invention are also pharmaceutical compositions containing as active ingredient at least one of the aforementioned peptide of formula I, one of the above substituted derivative of such a peptide or a peptide containing the above-mentioned amino acid sequence of the A1-A2-A3, together with a pharmaceutically acceptable carrier or diluent.

The peptides of the present invention can be administered orally, intravenously, parenterally, subcutaneously, intraperitoneally or intramuscularly.

The pharmaceutical composition may be in the form of a gelatinous cap is azizia can also be in the form of drug delayed allocation.

The peptide of the present invention can be assigned to a person for oral administration in a dose of from 5 to 100 mcg/kg / day.

Intravenously or subcutaneously connection according to the invention can be assigned to a person in a dose of from 1 to 12 mcg/kg from one to three times a day. In animals, the compounds of the present invention found in large numbers in a few days after the in-depth application, in particular, the peptide Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH2detected in excess of 10%.

Toxicity

Toxicity was studied in rats and dogs. Four weeks after injection of doses up to 4000 mg/kg/day, there was no evidence of toxicity and no signs telling mutagenic abilities. For a person subcutaneous or intravenous injection of a dose of 200 µg/kg does not entail any biological, clinical or pathological abnormalities.

Pharmacology

therapeutic interest of the compounds of the present invention is defined by the following experiment.

The intensity of gastric reaction is measured by determining the amount of induced gastric secretion.

The cats had surgery under General anesthesia. Stomach b is aruru to collect the secretion of hydrochloric acid, pepsin and gastric juice as during the main phase, and after stimulation. Since these animals had chronic fistula, they were subjected to a specific number of tests each week and served as both the reference samples. Stimulation of the secretion of pepsin was achieved by perfusion of the introduction of animals pentagastrin (PG) and VIP (vasoactive gastric peptide) for 2 hours at the rate of 2 and 4 μg/kg/h

One hour after stimulation pentagastrinom and VIP perfusion peptides were added at a dose of 100 pmol/kg/hour, Volume of gastric juice was measured within 30 minutes before and after perfusion. The amount of pepsin in the gastric juice is determined by proteolytic spectrophotometric method. The results obtained in 9-12 experiments presented in the following tables, pepsin secretion is expressed in mg/15 minutes, the average of two periods of 15 minutes during basal secretion and the average value of the 6 periods of 15 minutes during stimulated secretion.

Some of the peptides of the invention having at least one radical of D-amino acids, compared with their counterparts, in which all the radicals of amino acids have L-configuration.

Example 1

Specific mg

Mannitol - 25,00 mg

Sodium chloride - 4,50 mg

In the form of a solution:

The peptide according to the invention is 0.30 mg

Alcohol - 250,00 mg

Water - 0.75 mg

Emulsion:

The peptide according to the invention: - 0.30 mg

Polysorbate - 5,00 mg

Neutral oil - 500,00 mg

Water - 0.50 mg

In the form of tablets:

The peptide according to the invention is 0.45 mg

Hydroxypropylcellulose - 22,00 mg

Magnesium stearate - 0.10 mg

Example 2

Amino acids peptides PD2-PD7connect with each other as indicated in the description of the classical method using butyloxycarbonyl (Boc), which was used to obtain peptide PD1and also using as a starting resin 4-methylbenzhydrylamine.

The binding of different amino acids produced in dimethylformamide (DMF) in the presence of benzotriazol-1-yl-oxides(dimethylamino)phosphonium-hexaphosphate (BOP), 1-hydroxybenzotriazole (HOBt) and N, N-diisopropylethylamine (DIEA).

The process of binding and removing the protection stages are listed below:

1 - TFA - (1 × 1 min)

2 - TFA - (1x3 min)

3 - DCM - Fast laundering

4 - isopropyl alcohol - (1 × 1 min)

5 - DMF - (1x3 min)

6 - binding/DMF - (1h12 min)
At each stage we used 10 ml of solvent per 1 g of resin.

In the process of synthesis of peptides used the following protective groups of the side chains:

- benzyl for threonine

- 2-CIZ for lysine

- for acetyl lysine in the peptide PD7and PD3.

To abort the process by using the resin to the mixture was added 10 ml of hydrofluoric acid in 1 gram of peptide resin and incubated for 45 minutes at 0oC in the presence of paracresol as a cleaner.

After evaporation of the hydrofluoric acid, the product of the synthesis was washed in ether, was dissolved in TFA, precipitated with ether, and dried.

Faction, sufficiently purified, separated and liofilizirovanny. The final products were examined using analytical methods high-performance liquid chromatography (HPLC), mass spectroscopy and amino acid analysis.

1. The peptide of General formula I

A1-A2-A3-X

where A1means a radical of L-Thr or D-Thr; or one of the following sequences, in which at least one radical of the amino acids may be D-configuration, shown in the graphical part;

A2is a sequence Lys-Pro-Gln-Ala, in which tou communication;

X represents a hydroxy, amino, alkylamino and named the peptide of formula I contains at least 1 radical D-configuration, and may contain one or several identical or different protective groups, or their pharmaceutically acceptable salts.

2. The peptide containing the amino acid sequence of A1-A2-A3where A1, A2and A3determined in accordance with paragraph 1, the peptide contains at least one radical of the amino acid of D-configuration.

3. The peptide under item 1 or 2, containing at least one radical of the lysine protected acutiloba group.

4. The peptide according to any one of paragraphs.1 to 3, containing one radical amino acid of D-configuration.

5. The peptide under item 4, in which the radical of the amino acid of D-configuration is-the end or at the N-end.

6. The peptide under item 5, in which the radical of the amino acid of D-configuration is-the end.

7. The peptide according to any one of paragraphs.4 = 6, where A2denotes Lys-Pro-Gln-D-Ala and A3means covalent bond.

8. The peptide under item 7, having the formula Thr-Lys-Pro-Gln-D-Ala-NH2or Thr-(acetyl)Lys-Pro-Gln-D-Ala-NH2.

9. The peptide under item 7, having the formula Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH2.

11. The peptide under item 7, having the formula Ile-Leu-Gln-His-Glu-Arg-Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH2.

12. The peptide under item 7, having the formula Glu-Pro-Gly-Lys-Ser-Ser-Ile-Leu-Gln-His-Glu-Arg-Pro-Val-Thr-Lys-Pro-Gln-D-Ala-NH2.

13. The peptide under item 5, in which the radical of the amino acid of D-configuration is on the N end.

14. The peptide under item 13, in which A1denotes a D-Thr or above the sequence in which the radical of the amino acids at the N-end of this sequence has the D-configuration.

15. The peptide according to p. 14, having the formula D-Pro-Val-Thr-Lys-Pro-Gln-Ala-NH2.

16. The peptide according to any one of paragraphs.1 to 3, which contains two radical amino acid of D-configuration.

17. The peptide according to p. 16, in which the radical of the amino acid of D-configuration is located on the C-end.

18. The peptide according to any one of paragraphs.16 - 17, whose second radical amino acid of D-configuration is located on the N-end of the peptide.

19. The peptide under item 18, having the formula D-Pro-Val-Thr-Lys-Pro-Gln-Ala-NH2.

20. The peptide under item 18, having the formula D-Pro-Val-Thr-(acetyl)Lys-Pro-Gln-D-Ala-NH2.

21. The pharmaceutical composition inhibiting the release of pepsin, containing the active ingredient, associated with a pharmaceutically acceptable carrier or diluent is e, one peptide according to any one of paragraphs.1 - 20.

 

Same patents:

The invention relates to medicine, namely to the field of preparative biochemistry, and for onlinebingo, method of its production and pharmaceutical compositions containing onlineby

The invention relates to biotechnology, genetic engineering

The invention relates to medicine, namely to the chemical-pharmaceutical industry and relates to a composition for stimulating the growth of osteoblasts containing FRPT and vitamin D
The invention relates to medicine, Allergology
The invention relates to medicine, in particular to the treatment of diseases characterized by autoimmune aggression

The invention relates to medicine, more specifically to the Neuroimmunology, namely to test for antigens associated with multiple sclerosis

The invention relates to medicine, in particular to Oncology

The invention relates to pharmaceutical industry and the receipt of liofilizirovannogo preparation of alpha-fetoprotein (AFP), belonging to the group of immunomodulators and used as a treatment for cancer

The invention relates to the field of medicine and are expected to be used in the treatment of malignant tumors
The invention relates to the food industry

FIELD: medicine, pediatrics.

SUBSTANCE: the present innovation deals with treating motor-autonomic disorders in children associated with affected function of central nervous system. For this purpose one should puncture perineural areas in the region of the main nervous trunks with alfetin dissolved in cerebrolysine. Additionally, one should puncture in projection area of cervical and lumbar spinal thickenings and areas that correspond to segmentary innervation of organs with affected function and, also, in scalp areas depending upon the character of patient's disorders. The method suggested provides improved autonomic-trophic impact of nervous system.

EFFECT: higher efficiency of therapy.

2 ex

FIELD: medicine, cardiology, gastroenterology.

SUBSTANCE: invention relates to a method for treatment of ulcer-erosion injures in gastroduodenal region in patients with arterial hypertension. Method involves detection of immune disturbances and carrying out the combined immunomodulating therapy and hypotensive therapy. Immunocorrecting complex consists of licopide, cortexinum, vetoronum TK in arterial hypertension of I-II degree and comprises superlymph additionally in arterial hypertension of III degree. Method provides attaining optimal results in treatment for relatively short time due to adequate immunocorrection in such patients.

EFFECT: improved method for treatment.

5 cl, 6 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: vaccine is high molecular weight protein conjugate with angiotensine II taken in high molecular weight protein : angiotensine II proportion of 1:12-55 in % by weight. The conjugate is modified with equilibrium quantity of immunocompetent polyelectrolyte like polyoxydonium.

EFFECT: stable physiological response within prolonged period of 6-12 months.

3 tbl

FIELD: chemistry of peptides, medicine.

SUBSTANCE: invention relates to compound of the structure: N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-leu)-NH2 designated for using as an active component in manufacturing a medicinal agent used for inhibition of natural β-amyloid peptides aggregation in patients suffering with disorder associated with β-amyloidosis and in treatment of Alzheimer's disease. Also, invention relates to a pharmaceutical composition, a method for inhibition of natural β-amyloid peptides, a method for detection of natural β-amyloid, a method for detection for the presence or absence of natural β-amyloid peptides in biological sample and to a method for treatment of patient with disorder associated with β-amyloidosis.

EFFECT: improved method for detecting, valuable medicinal properties of agent.

14 cl, 5 tbl, 5 dwg, 9 ex

FIELD: genetic engineering, pharmaceutical and medical-biological industry.

SUBSTANCE: invention proposes a chimeric sequence of nucleic acid encoding a fused polypeptide able to bind with the vessel endothelium growth factor (VEGF) and to inhibit its specific mitogenic effect. The fused polypeptide molecule comprises immunoglobulin-like domain 2 of VEGF-receptor Flt1, immunoglobulin-like domain 3 of VEGF-receptor Flk 1 or Flt4 and multimerizing component represented by either domain Fc IgG or heave chain IgG. By expression of the proposed chimeric sequence or its two successively joined copies in a host-cell a monomer or dimer of the fused polypeptide are prepared, respectively, that can be used for suppression of VEGF activity in mammals, in particular, in humans. New VEGF inhibitors differ from the known one by the improved pharmacokinetics.

EFFECT: improved preparing method, valuable biological properties of polypeptide.

23 cl, 67 dwg, 1 tbl, 35 ex

FIELD: medicine, pharmacology, biochemistry, pharmacy.

SUBSTANCE: invention relates to a pharmaceutical composition and industrial product comprising proteins Src and Yes of tyrosine kinase type together with a pharmaceutically acceptable carrier wherein at least one among protein Src and protein Yes represents active kinase and at least one among protein Scr and protein Yes represents inactive kinase, and to their amino acid sequences also. Invention provides the selective inhibition of tyrosine kinases activity, reduces damages or traumas associated with enhanced permeability of tissue vessels.

EFFECT: valuable medicinal properties of modulators.

16 cl, 16 dwg, 14 ex

Up!