Derivatives of oligopeptides containing d-alkyltrimethyl and are able to activate the release of growth hormone

 

(57) Abstract:

In the present invention are described peptides of General formula A-D-X-Mrp-B, as well as their additive salts with pharmaceutically acceptable organic or inorganic acids where the substituents a, D, X or have specified in the description of the values. The compounds are able to activate the release of growth hormone. Their introduction into the body through the mouth increases this activity. 2 c. and 11 C.p. f-crystals, 1 table.

The invention relates to oligopeptides derivative containing amino acid D-2-alkyltrimethyl, which is capable of releasing growth hormone (GH) from the somatotropic cells and active when administered orally.

Increased levels of growth hormone (GH) in mammals after administration of compounds that induce the release of GH can lead to the acceleration of growth and increased muscle mass and increased productivity of milk, if after the introduction of reach sufficiently high concentrations of GR. In addition, it is known that increased levels of growth hormone in mammals can be achieved by the introduction of well-known means for release such as hormones release of growth hormone (GHRH).

Increased levels of the hormone kotoryj previously described, for example, in U.S. patents 4 223 019, USA 4 223 020, USA 4 223 021, USA 4 224 316, USA 4 226 857, USA 4 228 155, USA 4 228 156, USA 4 228 157, USA 4 228 158, USA 4 410 512, USA 4 410 513, USA 4 411 890, USA 4 839 344.

Therefore, at the present time requires a very simple oligopeptides with a short circuit, capable of stimulating the release of growth hormone, provided an easy and convenient method of obtaining them, as well as easy cleaning and manufacturing formulations, which is active when administered orally. Deghenghi et al. (Journal of Pediatric Endocrinology and Metabolism, 8, 311-313 (1995) described aminoacylation of tetrapeptides that are active in vivo in the release of growth hormone. In WO 96/10040 described tetrapeptide containing D-2-alkyltrimethyl and can stimulate the release of growth hormone when administered orally. Deghenghi et al. (Life Sciences, vol.54, 18, 1321-1328, 1994) discussed GR-releasing activity of the Hexapeptide too.

It was found that the oligopeptides with a very short chain having at least one residue of D-2-alkylthiophene (2-Mg), have activity release of growth hormone (GH) from the somatotropic cells.

Another distinguishing feature of the present invention is very high activity and a favorable ratio of activity at Petey this series.

The oligopeptides of the present invention have the formula:

A-D-X-D-Mrp-B

where a represents hydrogen, 2-aminoisobutyric, 4-aminobutyryl, D refers to dextro-isomer, X is Mg, where Mg denotes the 2-alkyltrimethyl formula:

< / BR>
where R is hydrogen, Cho, SO2CH3mesitylene-2-sulfonyl, RHO3(CH3)2, RHO3H2; R1is1-C3is an alkyl group, or X represents a residue of a protected serine, Ser(Y), where Y can be a benzyl, n-chlorbenzyl, 4-methoxybenzyl, 2,4,6-trimethoxybenzyl, tert-bootrom; represents NR2R3where R2and R3that may be the same or different, represent hydrogen or C1-C3is an alkyl group; a group OR4where R4represents hydrogen or C1-C3is an alkyl group; or represents a group C-Lys-NH2where is Phe or MGy.

The invention is based on the fact that different oligopeptides with a short circuit, which actuates the release and increase the levels of HGH in the blood of animals, characterized by the fact that all of them contain the peptide chain of the D-isomer of 2-alkylthiophene (D-2-Me-Trp or D-Mrp).

Oligopeptides, Eva, 2-aminoisobutyric, 4-aminobutyryl, D refers to dextro-isomer, X is Mg, where Mg denotes the 2-alkyltrimethyl formula:

< / BR>
where R is hydrogen, Cho,SO2CH3mesitylene-2-sulfonyl, RHO3(CH3)2, RHO3H2; R1represents C1-C3is an alkyl group, or X represents a residue of a protected serine, Ser(Y), where Y can be a benzyl, p-chlorbenzyl, 4-methoxybenzyl, 2,4,6-trimethoxybenzyl, tert-bootrom; represents NR2R3where R2and R3that may be the same or different, represent hydrogen or C1-C3is an alkyl group; OR4where R4represents hydrogen or C1-C3is an alkyl group; or represents a group C-Lys-NH2where is Phe or MGy, and additive salts with pharmaceutically acceptable organic or inorganic acids of any one of these polypeptides.

Used here, the abbreviation of amino acid residues is in accordance with standard nomenclature for peptide: Lys = lysine.

In addition,

Aib = 2-aminoisobutyric;

GAB = 4-aminobutyryl;

Mrp = 2-alkyltrimethyl;

Bzl = benzyl;

p-Cl-Bzl = BR>
Mts = mesitylene-2-sulfonyl.

In accordance with the present invention the alkyl denotes a lower alkyl containing from 1 to 3 carbon atoms. Examples of lower alkyl are methyl, ethyl, propyl, isopropyl. Among them, the methyl group is greatly preferred.

All three-letter abbreviations of amino acids, preceded by "D" mean amino acid residue with a D-configuration. When the amino acid is represented by only three-letter abbreviation, it has the L-configuration.

Preferred compounds of the present invention, releasing growth hormone are:

(a) GAB-D-Mrp-D-Mrp-Phe-Lys-NH2;

(b) GAB-D-Mrp-D-Mrp-Mrp-Lys-NH2;

(c) Aib-D-Mrp-D-Mrp-NH2;

(d) Aib-D-Mrp-Mrp-NH2;

(e) Aib-D-Ser(Bzl)-D-Mrp-NH2;

where Mg is 2-methyltryptophan, GAB and Aib such as defined above, and additive salts with pharmaceutically acceptable organic or inorganic acids of any one of these oligopeptides.

These compounds are preferably administered orally method but can also enter intranasal or parenteral, or you can get them in the form of effective forms for controlled release, such as biodegradable cap which you can synthesize by conventional methods of peptide chemistry in the solid phase, and the solution, or by classical methods known in this field. The synthesis in solid phase begins with the C-terminal part of the peptide. Suitable starting material can be obtained, for example, attach the required protected alpha-amino acids to charmitalian resin, hydroxymatairesinol resin, benzhydrylamine resin (BHA) or pair-methylbenzhydrylamine resin (p-Me-BHA). As an example chlorotoluenes resin is a solid material with a trademark BIOBEADSSX1 supplied by BioRad Laboratories, Richmond, California. Getting hydroxymercuri described by Bodansky et al., Chem. Ind. (London) 38,15997, (1966). The BHA resin described Pietta and Marshall, Chem. Comm. , 650 (1970), and released to the market by the firm Peninsula Laboratories Inc., Belmont, California.

After the initial joining of the protective group of alpha-amino acids can be removed using a variety of acidic reagents, including triperoxonane acid (TFA) or hydrochloric acid (model HC1), dissolved in organic solvents at room temperature. After removal of the protective group of the alpha-amino acids, the remaining protected amino acids can be linked in the desired order. Each protected amino acid is usually injected into the reaction in a threefold excess with isopropylcarbodiimide (DIC), dissolved, for example, in methylene chloride (CH2Cl2or dimethylformamide (DMF) and their mixtures. After the formation of the target amino acid sequence of the target peptide can be split from the media-resin by treatment with a reagent such as hydrogen fluoride (HF), which it is not only the peptide from the resin but also the usual protective groups of the side chains. When used klimatisierung resin or hydroxymatairesinol resin, treatment with HF leads to the formation of acid peptide in the free form. When using a BHA resin or p-Me-BHA, treatment with HF leads directly to the formation of amide peptide in the free form.

Discussed above method in the solid phase are known in this area and was described by Atherton and Sheppard, Solid Phase Peptide Synthesis (IRL Press, Oxford, 1989).

Some of the methods of synthesis in solution, which can be used for the synthesis of peptide parts of the invention are described in detail Bodansky et al., Peptide Synthesis, 2ndedition, John Wiley and Sons, New York, n.y., 1976, and jones, The Chemical Synthesis of Peptides (Clarendon Press, Oxford, 1994).

These compounds are administered to animals and people in an effective dose, which can be easily determined by the person skilled in the art and may vary depending what I dose is in the range from about 0.1 to 10 μg total peptide per 1 kg of body weight. When the compound is administered orally, usually need a higher number. For example, for oral administration to a human level dose is usually from about 30 to about 1000 micrograms of polypeptide per 1 kg of body weight. The exact level is easily determined empirically on the basis of the above description.

Compositions which contain as active ingredient an organic or inorganic salt additive described above oligopeptides, and combinations thereof, optionally in a mixture with filler, diluent, matrix, or coating for sustained release (active ingredient), also included in the scope of the present invention. Especially interesting pharmaceutical composition with delayed release containing biorstwami matrix suitable for subcutaneous implantation. Examples of these matrices are described in W09222600 and W09512629.

The activity of these compounds in vivo was determined on ten rats, which were subcutaneously injected dose of 300 µg/kg) or different doses in the study of the ratio of the dose-response according to the method described in detail by the authors R. Deghenghi et al. Life Sciences, 54, 1321 (1994). The results are tabulated and released growth hormone (GH) was measured 15 min after we introduce the methods of synthesis in the solid phase, as described in "Solid phase peptid synthesis technique by E. Atherton and R. C. Sheppard, IRL Press, Oxford University Press, 1984, and using fluorenylmethoxycarbonyl (Fmoc) as a protective group was derived peptide:

GAB-D-2-Mrp-D-2-Mrp-Phe-Lys-NH2< / BR>
where Mg is 2-methyltryptophan, mol.weight 779,9 found 778,4; purity (HPLC) 98,0%.

Example 2

Analogously to example 1 was obtained following peptide:

GAB-D-Mrp-D-Mrp-Mrp-Lys-NH2,

where Mg is 2-methyltryptophan, mol.weight 830,8 found 831,3; purity (HPLC) 98,0%.

Example 3

Analogously to example 1 was obtained following peptide:

Aib-D-2-Mrp-D-2-Mrp-NH2,

where Mg is 2-methyltryptophan, mol.weight 502,6 found 503,3; purity (HPLC) 99,0%.

Example 3

Analogously to example 1 was obtained following peptide:

Aib-D-2-Mrp-D-2-Mrp-NH2,

where Mg is 2-methyltryptophan, mol.weight 502,6 found 503,3; purity (HPLC) 99,0%.

Example 4

Analogously to example 1 was obtained following peptide:

Aib-D-2-Mrp-2-Mrp-NH2,

where Mg is 2-methyltryptophan, mol.weight 502,6 found 503,3; purity (HPLC) 99,0%.

Example 5

Analogously to example 1 was obtained following peptide:

Aib-D-Ser(Bzl)-D-Mrp-NH2,

where MGy D-X-D-Mrp-B

where a represents 2-aminoisobutyric, 4-aminobutyryl;

D denotes dextro-isomer;

X represents Mrp, where Mrp denotes 2-alkyltrimethyl formula

< / BR>
where R represents hydrogen;

R1is1-C3is an alkyl group;

or X represents a residue of a protected serine, Ser(Y), where Y can be a benzyl, p-chlorbenzyl, 4-methoxybenzyl, 2,4,6-trimethoxybenzyl, tert-bootrom;

In represents NR2R3where R2and R3each individually represent hydrogen;

or represents a group C-Lys-NH2where is Phe or Mrp, and additive salts with pharmaceutically acceptable organic or inorganic acids of any of these polypeptides.

2. The peptide under item 1, where Mrp is 2-methyltryptophan.

3. The peptide under item 1, where In is C-LysNH2where defined above.

4. The peptide under item 1, having the formula

GAB-D-Mrp-D-Mrp-Phe-Lys-NH2.

5. The peptide under item 1, having the formula

GAB-D-Mrp-D-Mrp-Mrp-Lys-NH2.

6. The peptide under item 1, having the formula

Aib-D-Mrp-D-Mrp-NH2.

7. The peptide under item 1, having the formula

Aib-D-Ser(Bzl)-D-Mrp-NH2.

8. The peptide according to PP. 4-7, where Mrp is 2-is but an animal.

10. Peptides under item 9, where the specified animal is a mammal.

11. The pharmaceutical composition exhibiting activity against activation of the release of growth hormone in animals comprising as an active ingredient the peptide on PP. 1-10 in an effective quantity, and the specified peptide may be optionally mixed with the carriers and fillers.

12. The pharmaceutical composition according to p. 11, in the form for parenteral, oral administration, injection with adjustable release subcutaneous implants.

13. The composition according to p. 11, in the form for oral administration.

 

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