The polypeptide compounds containing d-2-alkyltrimethyl, stimulating the release of growth hormone

 

(57) Abstract:

Describes a new peptide having the formula (I) AD-Mrp(Al)n-B -, where A-D-Thr-His, imidazolate, isonipecotic, 4-aminobutyryl, D-refers to destroytimer, MGy is a 2-(C1-C3-alkyl)tryptophan, n is 0 or 1, B-D-Trp, D-Nal, C-NH2, pheLysNH2H additive salts of pharmaceutically acceptable organic or inorganic acids. The peptide includes the amino acid D-2-alkyltrimethyl, which has the ability to release somatropin growth hormone. 3 c. and 6 C.p. f-crystals, 1 table.

The present invention relates to oligopeptides compounds that have activity by oral administration comprising the amino acid D-2-alkyltrimethyl, which has the ability to release somatropin growth hormone (GH).

Prior art

Increased levels of growth hormone (GH) in mammals after administration of compounds that induce the secretion of GH can lead to accelerated growth, increased muscle mass and increased milk production. In addition, it is known that increased levels of growth hormone in mammals can be obtained due to the introduction of factors, releasing growth hormone, capitalship can be obtained by introducing peptides releasing growth hormone, some of them have already been described, for example in US 4223019, US 4223020, US 4223021, US 4224316, US 4226857, US 4228155, US 4228156, US 4228157, US 44228158, US 4440512, US 4410513, US 4411890 and US 4839344.

For this reason, there is a need for a fairly simple short-chain polypeptides, promoting the release of growth hormone, provided that they are simple and easy to obtain, easy cleaning and the formation of compositions that are active when administered orally.

One of the most studied peptides growth hormone (GHRP), was known for many years as GHRP-6 (C. Y. Bowers et al., Endocrinology 114:1537 (1984)), a peptide with the formula His-D-Trp-Ala - Trp-D-Phe-Lys-NH2, GHRP-6 releases growth hormone in vitro and in vivo and has oral activity in animals and humans. Studied its molecular mechanism and one of its analogues - heptapeptide GHRP-1 (Cheng et al., Endocrinology 124: 2791 (1989); M. S. Akman et al., Endocrinology 132:1286 (1993)). Found that in contrast to naturally occurring GHRH, these peptides to release GH, act through different receptors, using a mechanism that is independent of camp and which acts through other intracellular pathways such as the mobilization of supply calcium and process-dependent protein kinase C (SW) go, the introduction of D-2-alkylthiophene (2-Mrp) in Oligopeptide group GHRP, known modifies the intracellular mechanism of release of GH. In some cases, it was observed a significant increase in adenylate cyclase activity in the anterior pituitary of the mouse and human, but the specific mechanism remained unclear.

Thus, modification of tryptophan (D-conformation), retrieved by using a single alkyl group in position 2, positive impact on the stability of amino acid residue Trp (R. Deghenghi WO 91/18016 published 28.11.1991 and R. Deghenghi et al., Life Sciences 54, 1321, (1994)), except only responsible for a sudden change in the intracellular mechanisms that are now not dependent on calcium, partly dependent adenylate cyclase, and more similar to the mechanisms GHRH, like other peptide hormones. (James D. Watson et al., Molecular Biology of the Gene, 4th ed. , The Benjamin/Cummings Publishing Company, Inc., Menlo Park, California, 1987, p. 60). Another distinguishing feature of the present invention is a very high activity of some Penta-, hexa-, and heptapeptides, as well as a very favorable ratio activity/force in the group of smaller tetrapeptides in oral introduction.

The oligopeptides of the present Izaberite L-amino acid or its D-isomer, provided that A is not His, if B represents Trp, C is D-Phe-Lys-NH2or A represents imidazolyl, isonipecotic, 4-aminobutyryl, 4-(aminomethyl)cyclohexanecarbonyl, Glu-Tyr-Ala-His, Tyr-Ala-His, Tyr-His, D-Thr-His, and more preferably A is D-Ala, D-Thr, Tyr; D refers to destroytimer, Mrp is 2-alkyltrimethyl, preferably 2-methyltryptophan; n represents 0 or 1; B represents L - or D-Trp, Phe or D-Nal; C represents NH2D-Phe-Lys-NH2, Phe-LysNH2D-Trp-LysNH2D-Phe-Lys-ThrNH2D-Phe-Lys-D-ThrNH2, OR, in which R represents an alkyl group, a C1-C3more preferably, when B represents Trp, then C is Phe-LysNH2or D-Phe-LysNH2and when B is Phe, then C is D-Trp-LysNH2.

Detailed description of the invention

The present invention relates to short-chain polypeptides that contribute to the release and increase levels of growth hormone in the blood of animals, each of which includes a peptide chain of the D-isomer of 2-alkylthiophene (D-2-Me-Trp or D-Mrp).

These polypeptides included in the scope of the present invention, expressed by the following formula

A-D-Mrp-(Ala)n-B-C

in which A present is no Trp, C is D-Phe-LysNH2or A represents imidazolyl, isonipecotic, 4-aminobutyryl, 4-(aminomethyl)cyclohexanecarbonyl, Glu-Tyr-Ala-His, Tyr-Ala-His, Tyr-His, D-Thr-His, and more preferably, A is D-Ala, D-Thr, Tyr; D refers to destroytimer, Mrp is 2-alkyltrimethyl, preferably 2-methyltryptophan; n represents 0 or 1; B represents L-or D-Trp, or D-Nal; C represents NH2D-Phe-LysNH2, Phe-LysNH2D-Trp-LysNH2D-Phe-Lys-ThrNH2D - Phe-Lys-D-ThrNH2, OR, in which R represents an alkyl group, a C1-C3more preferably, when B represents Trp, then C is Phe-LysNH2or D-Phe-LysNH2and when B is Phe, then C is D-Trp-LysNH2and additive salts of pharmaceutically acceptable organic or inorganic acids of any of these polypeptides.

The used abbreviations amino acid residues are used here according to the standard nomenclature of peptides:

Gly = Glycine

Tyr = L-Tyrosine

Ile = L-Isoleucine

Glu = L-Glutamic acid

Thr = L-Threonine

Phe = L-Phenylalanine

Ala = L-Alanine

Lys = Lysine

Asp = L-Aspartic acid

Cys = L-Cysteine

Arg = L-Arginine

Gln = L-Glutamine

Pro = L-Polyptoton

Val = L-Valine.

In addition,

D -- Nal = D- -Nafcillin;

INIP = Isonipecotic;

IMA = Imidazolylalkyl;

GAB = 4-aminobutyryl;

Mrp = 2-alkyltrimethyl.

In accordance with the present invention under the alkyl is meant a lower alkyl comprising from 1 to 3 carbon atoms. Examples of lower alkyl are methyl, ethyl, propyl, isopropyl. Among them, a methyl group is most preferable.

The letter "D" preceding a three letter abbreviation indicates the D-configuration amino acid residue.

The preferred connection, releasing growth hormone in the embodiment of the present invention are:

INIP-D-Mrp-D-Trp-Phe-Lys-NH2;

INIP-D-Mrp-D-Nal-Phe-Lys-NH2;

IMA-D-Mrp-D-Trp-Phe-Lys-NH2;

IMA-D-Mrp-D-Nal-Phe-Lys-NH2;

GAB-D-Mrp-D-Trp-Phe-Lys-NH2;

GAB-D-Mrp-D-Nal-Phe-Lys-NH2;

GAB-D-Mrp-D -- Nal-NH2;

GAB-D-Mrp-D -- Nal-OC2H5;

4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-Trp-Phe-Lys-NH2;

4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-Nal-Phe-Lys-NH2;

D-Ala-D-Mrp-Ala-Trp-D-Phe-LysNH2;

D-Thr-D-Mrp-Ala-Trp-D-Phe-LysNH2;

His-D-Mrp-Ala-Phe-D-Trp-LysNH2;

Tyr-His-D-Mrp-Ala-Trp-D-Phe-LysNH2;

and additive salts of pharmaceutically acceptable organic and inorganic acids of any of these polypeptides.

These compounds can enter parenteral, intranasal and oral administration, or you can make the recipe with controlled release in the form of biodegradable microcapsules, microspheres, subcutaneous implants, etc.

Data polypeptide compounds in accordance with the present invention can be obtained by conventional chemical means, as by solid-phase synthesis or synthesis in solution, or by classical methods known in the art. Solid-phase synthesis starting from the C-terminal end of the peptide. Prepare an appropriate source material, for example, attaching the required protected alpha-amino acid to chlorotoluenes resin, hydroxymatairesinol resin, benzhydrylamine resin (BHA) or parametermessagefile resin (p-Me-BHA). For example, chlorotoluene resin sold under the trademark BIOBEDSSX 1 Laboratory BioRad, Richmond, California. Getting hydroxymethylene resin described in Bodansky et al., Chem. Ind. (London) 38, 15997, (1966). Getting BHA-resin described Pietta and Marshal, Chem. Comm., 650 (1970) and sold Peninsula Laboratorie who are using a variety of acidic reagents, includes triperoxonane acid (TFA) or hydrochloric acid (HCl) dissolved in organic solvents at room temperature. After removal of the protective group of the alpha-amino acids, the remaining protected amino acids are sequentially added in a specific order. Each protected amino acid can usually respond approximately more than three times, using an appropriate carboxyl activating group, such as dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC), dissolved, for example, in methylene chloride (CH2Cl2) or dimethyl-formamide (DMF) and mixtures thereof. After completion of the construction of the necessary amino acid sequence, peptide otscheplaut from resin carrier by treatment with a reagent such as hydrogen fluoride (HF), which it is not only the peptide from the resin, but also, more generally, the protective groups of the side chains. When used klimatisierung resin or hydroxymatairesinol the resin by treatment with HF leads to the formation of acidic peptide in the free form. When using a BHA resin or resin p-Me-BHA treatment with HF leads directly to the formation of amide peptide in the free form.

Consider what the Chida (IRL Press, Oxford, 1989).

Some ways carried out in solution, which can be used for the synthesis of peptide components of the present invention, described in Bodansky et al., Peptide synthesis, 2nd edition, John Wiley & Sons, new York, N. Y. 1976 and Jones, the Chemical synthesis of peptides, (Clarendon Press, Oxford, 1994).

These compounds can enter the animal and human in an effective dose, which can be easily determined by the person skilled in the art and which can be modified in accordance with the type, age, sex and weight of a treated subject. For example, when intravenously preferred dose is in the limit from about 0.1 μg to 10 μg of total peptide per kg of body weight. When oral administration is usually required large quantities. For example, at a dose level of dosing usually varies from about 30 μg to about 1000 μg polypeptide per kg of body weight. The exact dose can be easily determined empirically on the basis of the above description.

Compositions which include as an active ingredient of organic and inorganic salt additive of the above polypeptides and combinations thereof, optionally in a mixture with a carrier, diluent, SV is. farmacevticheskie forms with delayed release, including decaying foundations, suitable for subcutaneous implantation, are particularly interesting. Examples of these bonding agents are described in WO 9222600 and WO 9512629.

Biological activity

The activity of these compounds in vivo was determined in ten days on rats, which were injected subcutaneously (s.c.) a dose of 300 µg/kg) or different doses in the study of dose-response relationships, in accordance with the described studies in R. Deghenghi et al., Life Sciences 54, 1321, (1994). The results are summarized in the Table below, the number of released GH was measured 15 minutes after injection.

Standard sample GHRP-2 had the structure of the D-Ala-D-Nal-Ala-Trp-D-Phe-Lys-NH2(Chen and Clarke, J. Neuroend. 7, 179 (1995)).

Effect in vitro on adenylate cyclase activity was measured in cells of the anterior pituitary of rats weighing 150 g and showed a 30% increase compared to the base line when EC50=0,23 nm for peptide D-Ala-D-Mrp-Ala-Trp-D-Phe-LysNH2whereas GHRP-6 (His-D-Trp-Ala-Trp-D-Phe-LysNH2) led to iactiveaware.

The following examples illustrate in more detail the present invention.

EXAMPLE 1

Synthesis of GAB-D-Mrp-D-Trp-Phe/Lys-NH2(Mrp = 2-methyltri is preparing the resin for packaging column reactor in accordance with one of several ways, well-known experts in this field, explained the example in "Solid-phase synthesis of peptides", edited by A. Atherton and R. C. Sheppard, IRL press at Oxford University press, 1989. Protected amino acids are Fmoc-Lys(Fmoc)-Opfp (Opfp = pentafluorophenyl ester), Fmoc-Phe-Opfp, Fmoc-D-Trp-Opfp, Fmoc-D-2-Me-Trp-Opfp and Fmoc-GAB-Opfp (GAB = gamma-aminobutyric acid). Alternatively, the reagents Castro, THIEF and Rumor (compare Le Nguen and Castro (1988), B: Chemistry of peptides 1987, S. 231-238. Fund for protein research, Osaka; and Tetragedron Letters 31, 205 (1990)) can also be used mainly as directly connecting reagents.

After removal and separation, called peptide was isolated by purification in the form of acetate salt. The degree of purification (ghvd): 98%, M. C. (M+N+)=764,3 (M. C. calculation=763,9).

EXAMPLE 2

In accordance with the procedure described in Example 1, was synthesized and obtained in the form of the acetate salts of the following peptides:

INIP-D-Mrp-D-Trp-Phe-Lys-NH2; the degree of purification (ghvd)=99,0%,

M. C.(M+H+=790,4; calculation = 790,0)

INIP-D-Mrp-D-Nal-Phe-Lys-NH2; the degree of purification (ghvd)=96,5%,

M. C.(M+H+=801,4; calculation = 801,0)

IMA-D-Mrp-D-Trp-Phe-Lys-NH2; the degree of purification (ghvd)=99,2%, M. C.(M+H+= 786,5; calculation = 786,8)

IMA-D-Mrp-D-Nal-Phe-Lys-NH2; the degree of purification (ghvd)=97,3%, M. C. (M+H+=79

4-(aminomethyl)cyclohexanecarbonyl-D-Mrp-D-Nal-Phe-Lys-NH2;

D-Ala - D-Mrp-Ala-Trp-D-Phe-LysNH2;

D-Thr-D-Mrp-Ala-Trp-D-Phe-LysNH2;

His-D-Mrp-Ala-Phe-D-Trp-LysNH2;

Tyr-His-D-Mrp-Ala-Trp-D-Phe-LysNH2;

His-D-Mrp-Ala-TrpNH2;

D-Thr-D-Mrp-Ala-TrpNH2;

in which Mrp is 2-methyltryptophan, a INIP, IMA and GAB presented above.

1. The peptide having the formula I

A-D-Mrp-(Ala)n-B-S,

A - D - Thr - is, imidazolate, isonipecotic, 4-aminobutyryl;

D - refers to dextrosol;

Mrp is a 2-(C1-C3-alkyl)tryptophan;

n is 0 or 1;

In is D-Trp, D-Nal;

With represents NH2, he Lys NH2,

and their additive salts of pharmaceutically acceptable organic or inorganic acids.

2. The peptide under item 1, in which Mrp is chosen from 2-methyltryptophan, 2-ethyltryptamine, 2-propylthiophene, 2-isopropylnaphthalene.

3. The peptide under item 1, in which Mrp is 2-methyltryptophan.

4. The peptide under item 1, in which a represents imidazolyl, isonipecotic, 4-aminobutyryl.

5. The peptide under item 1, selected from the group including

imidazolate-D-Mrp-D-Trp-he-Lys-NH2,

imidazolate-D-Mrp-D--Nal-he-Lys-NH2,

isonipecotic-D-Mr 4-aminobutyryl-D-Mrp-D--Nal-he-Lys-NH2,

4-aminobutyryl-D-Mrp-D--Nal-NH2.

6. The peptide under item 4, in which Mrp is 2-methyltryptophan.

7. The peptides of General formula I under item 1, with the ability to stimulate the release of growth hormone in animals.

8. The pharmaceutical composition active against stimulating the release of growth hormone in animals comprising as an active ingredient the peptide on PP.1 to 5 in an effective amount, and this peptide may optionally be mixed with carriers or fillers.

9. The composition according to p. 8 in the form of a parenteral, intranasal, oral administration with controlled release for subcutaneous implantation.

Priority points:

27.09.94 - p. 1: a - imidazolyl, isonipecotic, 4-aminobutyryl, D-Thr is; n = 0; In = D-Trp, D-Nal; p. 5;

16.06.95 - p. 1: n = 1, C = NH2, he-Lys-NH2; PP.2 and 3; p. 4: a - imidazolate; PP.7 and 8.

 

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