Peptide with immunomodulatory activity

 

(57) Abstract:

The invention relates to medicine, namely to compounds with immunomodulatory properties. As immunomodulator proposed peptide patterns

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where R is H, lower acyl or lower alkyl, and X is an aromatic or heteroaromatic amino acid or its derivative. 3 table.

The invention relates to medicine, namely to new peptide structures with immunomodulatory properties.

Known medical use of a large number of Immunostimulants, such as drugs ginseng and schisandra, derivatives of nicotinic acid, thymalin and so on (Mashkovsky M. D. Medicines, T. 11, M, Medicine, 1988, S. 169).

The disadvantage of most natural products is low activity due to the low content of active principle, the presence of such effects.

We know a significant number of Immunostimulants peptide, for example, taktivin (French patent N b, class A 61 K 39/41), thymosin (Goldstein A. L. et al, Proc. Nat. Acad. Sci. USA, 1972, v. 69, p. 1856-1863), obtained from natural raw materials. These drugs constitute a set of polypeptides of different lengths, affecting nulinio unwanted side effects. In addition, the practical use of such drugs is difficult due to the complexity of the methods of their extraction from natural raw materials, low output, significant variability in their physical and chemical properties.

Search synthetic peptides with selective activity on account of changes in the structure would be more appropriate. Synthetic peptide Immunostimulants are the peptide Glu-Asp-Ser-Ser-Thr-Gly-Trp-Asp-OH (US patent N 4478828, class A 61 K 37/00, C 07 C 103/52, 1984), analogues thymopentin (European patent class. C 07 K 7/64, 1985, US patent N 5013723, class A 61 K 37/02, 1991), etc.

Even more interesting is the creation of a peptide of Immunostimulants on the basis of short selectively active highly active peptides, the synthesis of which is economically advantageous.

The prototype of the claimed invention was the peptide H-L-Glu-L-Trp-OH, the lack of which is less high immunomodulating activity.

The problem faced by the authors in creating the present invention was to develop more effective and safe immune modulators on the basis of short peptides.

It was found that this problem can be solved by using at least one of the peptides aminosilica or its derivative, for example ester, amide, hydrazide, etc.

Peptides receive standard technology, peptide synthesis on solid phase (J. M. Steward and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. San Francisco, 1969), or by synthesis in solution (E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press (New York), 1965) followed by removal of protective groups and purification of the final product.

As an interim protection-amino group of amino acids for solid-phase method of synthesis using tert-butyloxycarbonyl group. For protection of side radicals of amino acids using the following groups: for histidine benzyloxyethanol; tyrosine 2,6-dichlorobenzidine; formyl tryptophan, etc. For condensation of use dicyclohexylcarbodiimide or dicyclohexylcarbodiimide with the addition of 1-hydroxybenzotriazole. Release are 50% solution triperoxonane acid in methylene chloride and neutralizing with 5% solution of diisopropylethylamine in methylene chloride.

Cleavage of these peptides from the resin and release the side of the protective groups are using anhydrous hydrogen fluoride, and the cleansing - gel - and obremenitve chromatography.

Example 1. Synthesis of peptide g-L-glutamyl-L-tryptophan (1)

0,6 teleformula, add 0.2 g (0,0018 mol) N-oxysuccinimide, cooled to minus 5oC with vigorous stirring poured to the reaction mixture a solution of 0.37 g (0,0018 mol) of N,N-dicyclohexylcarbodiimide 2.0 cm3of dimethylformamide. The mixture was stirred at 0oC 1 h and left for 12 h at room temperature. Filtered dicyclohexylamine, added to the filtrate to 0.72 g (0,0022 mol) of the hydrochloride benzyl ester of L-tryptophan and 0.3 cm3(0,0023 mol) of triethylamine and the reaction mixture is stirred 16 h at room temperature. The solution is filtered, diluted with 50.0 cm3water and extracted three times with 40 cm3ethyl acetate. The combined organic extracts are washed sequentially 20.0 cm3water, twice 20.0 cm32 n sulfuric acid, twice 20.0 cm3a saturated solution of sodium chloride and dried with anhydrous sodium sulfate.

Evaporated the organic solution under reduced pressure, the residue is treated 16,0 cm350%-aqueous solution triperoxonane acid in methylene chloride for 45 min, again evaporated under reduced pressure and added 25,0 cm3isopropyl alcohol.

To the obtained solution of 0.3 g (0,00048 mol) of fluoroacetate demonia. The reaction mixture is heated to 50oC, is added in small portions 5.0 g of 10% palladium on coal, suspended in 25,0 cm3water, and stirred for 30 minutes, Filtered off the catalyst, evaporated isopropyl alcohol under reduced pressure, an aqueous solution lyophilized, was added to the residue 20.0 cm3water and again lyophilized.

The obtained freeze-dried purified by reversed-phase chromatography on a column of Ultraprep C18, gradient (10-30%) of acetonitrile in 0.1% triperoxonane acid. The content of the basic substance according to the optical density not less than 97% Structure confirmed by amino acid analysis of hydrolyzed peptide obtained by hydrolysis of 3 N. paratoluenesulfonyl. The analysis revealed the following relationship amino acid residues: Glu 1,0(1); Trp 0,9(1).

Example 2. Synthesis of peptide g-L-glutamyl-Nin-formyl-L-tryptophan (II)

For the synthesis of peptide g-L-glutamyl-Nin-formyl-L-tryptophan is loaded into the reaction vessel 0.6 g of tert-butyloxycarbonyl-Nin-formyl-L-tryptophyl-polymer. The content of tryptophan 0.50 mmol/g of polymer. Peptide chain further increase according to the following program (see tab.1).

If the ninhydrin test is positive, pogo residue use a-benzyl ester N-carbobenzoxy-L-glutamic acid.

Upon completion of the synthesis, the peptidyl-polymer dried in a vacuum desiccator over pjatiokisi phosphorus to constant weight. 1 g of peptidyl-polymer is transferred to the digester to work with liquid hydrogen fluoride, add 1 ml of thioanisole and 1 ml of meta-cresol, cooled to a temperature of -78oC and for 10 min distilled 10 ml of anhydrous hydrogen fluoride. The reaction mixture is heated to 0oC and stirred for 60 min, then distilled hydrogen fluoride. The residue is transferred to the filter SCHOTT and washed with ethyl acetate and ether. The peptide extracted with 50 ml of 25% acetic acid, diluted with water and lyophilizers.

After desalting on a column with Sephadex G-10 obtained lyophilized purified by reversed-phase chromatography on a column of Ultraprep C18, gradient (10-30%) of acetonitrile in 0.1% triptocaine acid. The content of the basic substance, according to the optical density, at least 96% Amino acid composition of the peptide corresponds to theoretical.

Example 3. Synthesis of peptide g-L-glutamyl-b-thienyl-D-alanyl-amide (III).

Synthesis, blocking, purification and characterization of the peptide are carried out in the same way that the peptide of example 2, except that the reaction vessel sagrei the release to the environment of anhydrous hydrogen fluoride for 2 hours The content of b-thienyl-D-alanine 0.5 mmol/g of polymer.

The content of the basic substance, according to the optical density not less than 97%

Example 4. Synthesis of peptide N-methyl-g-L-glutamyl-L-tryptophan (IV).

Synthesis of peptide (IV) is carried out in the same way that the peptide (II) of example 2, except that at the stage of condensation as the joining of amino acid residue use a-benzyl ester N-tertbutyloxycarbonyl N-methyl-L-glutamic acid.

Upon completion of the synthesis, the peptidyl-polymer dried in a vacuum desiccator over pjatiokisi phosphorus to constant weight. 1 g of peptidyl-polymer is transferred to the digester to work with liquid hydrogen fluoride, added to 6.5 ml of dimethyl sulfide, 0.8 ml of meta-cresol and 0.4 ml identicial, cooled to a temperature of -78oC for 10 min, 2.5 ml distilled anhydrous hydrogen fluoride. The reaction mixture is heated to 0oC and stirred for 120 min, then distilled hydrogen fluoride and dimethyl sulfide. The residue is transferred to the filter SCHOTT and washed with ethyl acetate and ether. The peptide extracted with 50 ml of 25% acetic acid, diluted with water and lyophilizers.

After desalting on a column with Sephadex G-10 Rila 0.1% triptocaine acid. The content of the basic substance, according to the optical density not less than 97% of the Structure of matter was substantiated by the conditions of synthesis and amino acid composition. The amino acid composition of the peptide corresponds to theoretical.

Example 5. Synthesis of peptide N-acetyl - g-L-glutamyl-L-tryptophan (V).

Synthesis of peptide (IV) is carried out in the same way that the peptide (II) of example 2, except at the stage of condensation as the joining of amino acid residue use a-benzyl ester N-tertbutyloxycarbonyl L-glutamic acid. Dipeptidyl-polymer handle 50% triperoxonane acid in methylene chloride (2 times, 2 and 30 min), washed with methylene chloride (4 times, 2 min) and incubated in a mixture containing of 0.11 ml of triethylamine, and 0.28 ml of acetic anhydride and 5 ml of methylene chloride.

Upon completion of the peptidyl-polymer was washed with methylene chloride (6 times for 2 min), dried in vacuum, will unlock and clear, as shown in example 4 (peptide IV). The content of the basic substance, according to the optical density, at least 96% Amino acid composition of the peptide corresponds to theoretical.

Immunomodulating activity of the claimed peptides were evaluated in tests of their wlia the main growth factor of T-cell interleukin-2.

Example 6. The effect of peptides on the differentiation of precursor T-cell bone marrow of mice.

A suspension of bone marrow cells of mice succumbing dislocation sewing vertebrae, obtained by flushing femurs with saline. Peptides at various concentrations is added to the received cells in the environment of the Needle and incubated for one hour at 37oC. the Level of expression of a marker of differentiation of T-lymphocytes q-antigen define a method complementation cytolysis using antibodies to q-antigen (Terasabi P. I. et al. in Manual of Tissue Typing Techniques, Nathional Institute of Health, Bethesda, 1972, p. 50-55). The data are given in table.2.

Example 7. The effect of peptides on the production of interleukin-2 by mouse splenocytes.

Data on the influence of peptides on the induction of synthesis of the main growth factor T-cell interleukin-2 (Gillis, S. et. al. J. Immunol. 1978, v. 120, p. 2027-2032) are given in table. 3.

Drugs are not toxic, as 1000-fold excess of therapeutic dose (1 mg/kg) death was not detected in any one animal.

The peptide of General formula

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where R is H, lower acyl or lower alkyl;

and X is an aromatic or heteroaromatic amino acid or its proizvodi the

 

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