MXPA97002275A

MXPA97002275A - Polypeptidic compounds containing d-2 alkyltriptophan capable of promoting the hormone release of growth

Info

Publication number: MXPA97002275A
Application number: MXPA/A/1997/002275A
Authority: MX
Inventors: Deghenghi Romano
Original assignee: Deghenghi Romano
Priority date: 1994-09-27
Filing date: 1997-03-26
Publication date: 1997-06-01

Abstract

The present invention relates to a peptide of the formula: AD-Mrp (Ala) nBC, wherein A is any natural L-amino acid or its D-isomer, as long as A is not His if B is Trp and C is D -Phe-Lys-NH2; or A is imidazolacetyl, isonipecotyl, 4-aminobutyryl, 4- (aminomethyl) cyclohexylcarbonyl, Glu-Try-Ala-His, Try-Ala-His, Try-His, D-Thr-His; refers to the dextrorotatory isomer, Mrp is 2-alkyltriptofan, n is 0 or 1, B is L- or D-Trp, Php or D-Beta-naphthylalanine, C is NH2, D-Phe-LysNH2, D-Trp-LysNH2, D-Phe-LysNH2, Phe-LysNH2, D-Phe-Lys-ThrNH2, D-Phe-Lys-D-ThrNH2, OR, where R is an alkyl group of 1 to 3 carbon atoms, and the acid addition salts organic or inorganic pharmaceutically acceptable ingredients of any of said polypeptides, wherein these compounds are capable of promoting the release of growth hormone and are active by the or

Description

POLYPETHYDES THAT CONTAIN D-2- ALKYLTRYPTOPHAN CAPABLE OF PROMOTING THE RELEASE OF GROWTH HORMONE BACKGROUND OF THE INVENTION The present invention relates to odopeptide compounds containing an amino acid D-2-alkytriptofan and which are capable of releasing growth hormone (GH) from atotropic substances and which are active by the oral route. The increase of the levels of growth hormone (GH) in mammals after the administration of compounds that induce the release of GH. it can cause an acceleration of the growth and an increase of the muscular mass and an increase of the production of milk. if high enough GH levels are obtained after administration. In addition, it is known that the increase in growth hormone levels in mammals can be achieved by the administration of agents known to be growth hormone releasers, as growth hormone releasing hojrones (GHRH). The increase of growth hormone levels in mammals. it can also be obtained by administering growth hormone releasing peptides, some of which have already been REF: 24246 described, for example, in US Patent Nos. 4,223,019. 4,223,020. 4,223,021, 4,224,316, 4,226,857. 4,228,155, 4,228,156. 4,228,157, 4,228,158. 4,410,512. 4,410,513. 4,411,890 and 4,839,344. Thus. At present, simple short chain polypeptides are desirable, capable of promoting the release of growth hormone under conditions in which they can be prepared easily and conveniently, as well as being easy to purify and formulate. and that are active when administered orally. One of the most studied growth hormone releasing peptides (GHRP) has been known for many years as GHRP-6 (CY Bowers et al., Endocrinclogy 114: 1537 (1984) and has the formula His-D-Trp- Ala-Trp-D-Phe-Lys-NH- GHRP-6 releases growth hormone in vi tro and in vivo and is active orally in animals and humans, its molecular mechanism has been studied, as well as that of its heptapeptide analog GHRP-1 (Chepg et al. Endocrinology 124: 2791 (1989): M.S. Ak an et al. Endocrinology 132: 1286 (1993)). It has been found that, continously, natural GHRHs. these peptides act & through different receptors for the release of GH and through a different mechanism, which is independent of cAMP and which operates through other intracellular routes. as the mobilization of calcium supplies and a protein kinase C dependent process (PKC-dependent process) (L. Bresson-Bepoldin and L. Dufy-Barbe, Cell.Calcium 15, 247, (1994).) BRIEF DESCRIPTION OF THE INVENTION Surprisingly, it has now been found that the introduction of a D-2-alkyltriptofan (2-Mrp) in the oligopeptide of the GHRP series. modifies the known intracellular mechanism of GH release. In some cases, there has been a substantial increase in the activity of adenyl cyclase in the anterior pituitary glands, both of murine origin and of human origin, but the specific mechanism remains to be clarified. Thus, the modification given by a single alkyl group in the 2-position of tryptophan (in its conformation D), further favorably increases the stability of the Trp residue (R. Deghenghi WO 91/18016 published on 11.28.1991 and R. Deghenghi et al. al., Life Sciences 54. 1321, (1994)). which is responsible for a totally unexpected change of intracellular mechanisms. which are now independent of calcium, sometimes dependent on adenyl cyclase and more similar to those of GHRH. as well as other peptide hormones (James D. Watson et al .. Molecular Biology of the Gene 4Q edition, The Ben amin / Cummings Publishing Company, Inc., Menlo Park, California, 1987. p.60). Another unexpected feature of the present invention. is the very high power of some penta-, hexa- and hepta-peptides and also the favorable relation actividac./potencia by oral route of the tetrapeptides of the series. The oligopeptides of the present invention have the following formula: A-D-Mrp- (A1a) -B-C. wherein A is a natural L-amino acid or its D- isomer. as long as A is not His if B is Trp and C is D-Phe-Lys-NH ,. or A is an imidazolaceti group, isonipecotyl. 4-aminobutiri lo. 4- (aminometi 1) cyclohexanecarboni lo. 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 the dextrorotatory isomer. Mrp is 2-alkyltriptofan. preferably 2-methyltriptofan; n is 0 or 1; B is L- or D-Trp. Phe or D-ß-Nal: C is NH2, D-Phe-Lys-NH2, Phß-Lys-NH2. D-Trp-Lyi? -NH D-Phe-Lys-Thr-NH2. D-Phe-Lys-D-Thr-NH2. OR, wherein R is an alkyl group of 1 to 3 carbon atoms, preferably, when B is Trp, then C is Phe-Lys-NH, D-Phe-Lys-NH, and when B is Phe. then C is D-Trp-Ly: 5-NH2.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the discovery that different short chain polypeptides promote the release and increase of the levels of growth hormone in the blood of animals. they are characterized because they all comprise, in the peptide chain. a D- isomer of 2-alkyltriptofan (D-2-Me-Trp or D-Mrp). The polypeptides included in the scope of the present invention are defined by the following formula: wherein A is a natural L-amino acid or its D- isomer. as long as A is not His if B is Trp and C is D-Phe-Lys-NH ", or A is an imidazolacßti lo group. isonipecotilo. 4- aminocarbonyl, 4- (aminomethyl) cyclohexanecarbonyl. Glu-Tyr-Ala-His. Tyr-Ala-His, Tyr-His. D-Thr-Hia. and more preferably A is D-Ala. D-Thr, Tyr; D refers to the dextrorotatory isomer, Mrp is 2-alkyltriptofan. of preferer.cia 2-methyltriptofan: n is 0 or 1; B is L- or D- Trp. Phe or D-ß-Nal; C is NH2. D-Phe-Lys-NH2. Phe-Lys-NH2.

D-Trp-Lys-NH2, D-Phe-Lys-Thr-NH2, D-Phe-Lys-D-Thr-NH2. OR. where R is an alkyl group of 1 to 3 carbon atoms. more preferably. when B is Trp. then C is Phe-Lys-NH., or D-Phe-Lys-NH_ and when B is Phe. then C is D-Trp-Lys-NH_ and the addition salts with pharmaceutically acceptable organic or inorganic acids of any of said polypeptides. The abbreviations for the amino acid residues used herein, are in accordance with the standard nomenclature for peptides: G1 to licina Tyr »LT.irosina He-L-Isoleucina Glu - Acid L-Glut? Mico Thr - L - Treonina Phe - L -Finyl alanine Ala • L-Alanine Lys - L-Lysine Asp - L-Asp arctic acid Cys - L - Cystine Arg - L - Arginine Gln = L - Glutamine Pro »L - Proline Leu - L - Leucine Met - L - Methionine Ser - L - Serine Asn = L -? Sparag - ina His - L - Histidine Trp «L - Tryptophan Val = LV lina. Further. D-ß-Nal-D-β-Naphthialanine, INIP = Isonipecotyl; IMA - Imldazoi i lacet lo: GAB ß 4- < _minobutiri it; Mrp = 2- < iqui itriptofano. In accordance with the present invention, the term "alkyl" means a lower alkyl, which comprises} from 1 to 3 carbon atoms. Examples of lower alkyls are methyl. ethyl. propyl »isopropi Lo. Among these, the methyl group is the most preferred. All abbreviations of three letters of the amino acids preceded by a letter "D" indicate the configuration D of the amino acid residue. Preferred growth hormone releasing compounds in the embodiment of the present invention are: INIP-D-Mrp-D-Trp-Phe-Lys-NH2; INIP-D-Mrp-D-ß-Nal-Phe-Lys-NH2. IMA-D-Mrp-D-Trp-Phe-Lys-NH2; IMA-D-Mrp-D-β-N 1-Phe-Lys-NH 2; GAB-D-Mrp-D-Trp-Phe-Lys-NH2; GAB-D-Mrp-D-ß-Nal-Phe-Lys-NH2: GAB-D-Mrp-D-β-Nai-NH 2; GAB-D-Mrp-D-0-Nal-OC2H5; 4- (amino? Ret 1) cic lohexanocarboni 1-D-Mrp-D-Trp-Phe-Lys-NH; 4- (aminormeth 1) cyclohexanecarboni 1-D-Mrp-D-β-Nal-Phe-Lys-NH ^; D-A 1 a-D-Krp-A-Trp-D-Phe-Lys-NH2; D-Thr-D-Mrp-Ala-Trp-D-Phe-Lys-NH 2; Hi s-D-Mr -A 1 a-Phe-D-Trp-Lys-NH.,; Tyr-His-D-Mrp-Al -Trp-D-Phe-Lys-NH-; His-D-Mrp-Ala-Trp-NH2; D-Thr-D-Mrp-Ala-Trp-NH2; where Mrp is 2-methyltriptofan. INIP, IMA and GAB are as previously defined. and addition salts with pharmaceutically acceptable organic or inorganic acids of any of said polypeptides. These compounds can be administered parenterally. but they are administered more conveniently by intramuscular and oral route, or they can be formulated in controlled release systems. such as biodegrable microc? caps, microspheres. subcutaneous and similar implants. The polypeptide compounds according to the present invention can be synthesized according to the normal methods of peptide chemistry. both in solid phase and in solution, or by means of the classical methods known in the art. Solid phase synthesis begins at the C-terminal end of the polypeptide. You can prepare a suitable starting material, for example, by binding the necessary protected a-amino acid to a chloro-resin. to a hydroxymethyl resin. to a resin enzhidri lamina (BHA) or to a resin para-meti lben hidri lamina (p-Me-BHA). For example, a chloromethyl resin is sold, under the trademark BIOBEADS (R) SX 3 .. by BioRad Laboratories, Richmond. California. The preparation of the hydroxymethyl resin is described by Boda sky et al. Chem. Ing. (London) 38. 15997, (1966). The BHA resin is described by Pietta and Marxhall. Chem. Comm. , 650 (1970) and commercially available at Peninsuld Laboratories Inc .. Belmont, California. After the initial binding, the protective group of the α-amino acid can be removed by means of different acid reagents. including trifluoroacetic acid (TFA) or hydrochloric acid (HCl) dissolved in organic solvents at room temperature. After removing the protective group of the a-amino acid. Protected amino acid resins can be coupled, step-by-step, in the desired order. Each protected amino acid can usually be reacted with an excess of about three times, using a suitable carboxyl activating group. such as dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIO dissolved, for example in methylene chloride (CH-C1) or in dimethylformamide (DMF) and mixtures thereof After the desired amino acid sequence has been completed , the desired peptide can be released from the support resin by a treatment with a reagent such as hydrogen fluoride (HF), which not only releases the peptide from the resin, but also breaks down the most common protective groups of the chains When using a chloromethyl resin or a hydroxymethyl resin, treatment with HFα causes the formation of the acid peptide in free form.When a BHA or p-Me-BHA resin is used, the HF treatment directly produces the formation of the amide peptide in free form The solid phase process described above is known in the art and was described by Atherton and Sheppard, Sun, Phase Peptide Synthesis (IRL Press, Oxford, 1989). Some solution methods that can be used to synthesize the peptide portions of the present invention are detailed in Bodansky ßt al., Peptide Synthesis. 23. edition. John Wiley &; Sons. New York N.Y. 1976 and in Jones, The Chemical Synthesis of Peptides, (Clarendon Press, Oxford, 1994). These compounds can be administered to animals and humans, at an effective dose that can be easily determined by those skilled in the art, and which can vary according to the species, age, sex and weight of the treated subject. For example, in humans. when administered intravenously. the preferred dose. falls in the range of about 0.1 to 10 ug of total peptide per kg of body weight. When administered orally, typically larger amounts are needed. For example, in humans for oral administration, the dose level is typically from about 30 to about 1000 μg of the polypeptide per kg of body weight. The exact level can be easily determined, empirically, based on the above description. The compositions comprising as an active ingredient the inorganic addition salts of the above-described polypeptides and combinations thereof, optionally. mixed with a vehicle, diluent. sustained release matrix or coating. they also fall within the scope of the present invention. The pharmaceutical forms of sustained release. which comprise bioerodible matrices.

Suitable for subcutaneous implantation, they are particularly interesting. Examples of these matrices are described in patents WO9222600 and W09512629. Biological activity The viral activity of these compounds was determined in ten-day-old rats, which were injected subcutaneously (sc) with a dose of 300 ug / kg or with different doses in dose-response studies, according to what is described in more detail by R. Deghenghi et al., Life Sciences 54. 1321. (1994). The results are summarized in the Table presented below. GH was measured after 15 minutes of treatment. The GHRP-2 (reference standard) has the structure D-Ala-D-β-N l-Ala-Trp-D-Phe-Lys-NH 2 (Chen and Clarke, J. Neuroend, 7, 179 (1995).

TABLE Peptide Dosage wg / kg Control of GH 6H l iberated 3 c (ng / tl) (og / il) Hi s-D-Mrp-A 1 a-Trp-D-Phe-Lys-300 31 ± 8 176 ± 20 Thr-NH2; Hi s-D-Mr p-A 1 a-Trp-D-Phe-Lys-300 31 ± 8 169 ± 27 D-Thr-NH2; D-Thr-D- «r p-A 1 a-Tr p-D-Phß-300 31 ± 8 266 ± 20 Lys-D-Thr-NH2; D-Thr-Hi s-D-Mrp-A 1 a-Trp-D-300 31 ± 8 86 ± 19 Phe-Lys-NH 2; D-A l a-D- «rp-A l a-Trp-D-Phß- 40 34 ± 1 200 ± 20 Lys-NH2: D-A l a-D-ttrp-A la-Trp-D-Phe-320 34 ± 1 251 ± 32 Lys-NH2; His-D-Mrp-Ala-Trp-NH2; 5000 69 ± 14 124 ± 37 imidazole aceti 1-D-Mrp-Ala-300 20 ± 3 159 ± 27 Trp-D-Phe-Lys-NH2 INIP-D-Mrp-D-Trp-Phe-Lys-NH2 300 15 155 INIP-D-Mrp-D-ß-N 1-Phe-Lys-300 15 150 NH2 GAB-D-Mrp-D-Trp-Phe-Lys-NH2 300 10 110 (GHRP-2) 300 10 98.6 The activity of the adenyl cyclase was measured in cells of the anterior pituitary gland of rats weighing 150 g and showed an increase of 30% compared to the baseline. with an EC_n = 0.23 nm for the peptide D-Ala-D-Mrp-Ala-Trp-D-Phe-Lys-NH-, while the GHRP-6 (His-D-Trp-Ala-Trp-D-Phe -Lys-NH_) was naive. The following examples further illustrate the present invention. EXAMPLE 1 Synthesis of GAB-D-Mrp-D-Trp-Phe-Lyß-NH. (Mrp »2-methyltri tofan). The synthesis was carried out in solid phase with 9-f luorenilmeti loxicarbóni l- (Fmoc-amino acids), which included the preparation of the resin, the assembly in a reactor column according to one of several methods available to the technicians in the subject, as exemplified in "Solid phase peptide synthesies" by E. Atherton and RC Sheppard. IRL Press at Oxford University press. 1989. Protected amino acids are Fmoc-Lys (Fmoc) -Opfp (Opfp »pentafluorophenester), Fmoc-Phe-Opfp, Fmoc-D-Trp-Opfp, Fmoc-D-2-MeTrp-Opfp and Fmoc-GAB-Opfp (GAB = gamma-aminobutyric acid). Alternatively, Castro, BOP and PyBOP reagents (see Le Nguyen and Castro (1988) in Peptide Chemistry 1987, p.231-238, Protein Research Foundation Osaka; and Tetrahedron Letters 31, 205 (1990)). they can also be used advantageously as direct coupling reagents. After being detached and isolated, the title peptide was purified in the form of an acetate salt. Purity (CLAP): 98%. PM (M + H +) = 764.3 (theoretical - 763.9). EXAMPLE 2 In accordance with the procedures described in Example 1, the following polypeptides were prepared and obtained in the form of acetate salts: INIP-D-Mrp-D-Trp-Phe-Lys-NH2; purity (HPLC) -99.0%, MW (M-rH + -790.4, theoretical 0 790.0) INIP-D-Mrp-D-ß-Nal-Phe-Lys-NH2; purity (HPLC) - 96.5% MW (M + H + - 801.4, theoretical »801.0) IMA-D-Mrp-D-Trp-Phß-Lys-NH2; purity (HPLC) - 99.2%. MW (M + H + - 786.5; theoretical - 786.8) IMA-D-Mrp-D-ß-Nal-Phe-Lys-NH2; purity (HPLC) - 97.3%, MW (M + H + - 798.3, theory - 797.9) GAB-D-Mrp-D-ß-Nal-Phe-Lys-NH2; 4- (aminomethylcyclohexanecarboni lD-Mrp-D-0-Nal-Phe-Lys-NH2; 4- (aminor? Eti 1) cyclohexanecarbonyl-D-Mrp-D-ß-Nal-Phe-Lys-NH7; D-A1a -D- ^ rp-A1a-Trp-D-Phe-Lys-NH2; D-Thr-D-Mrp-Ala-Trp-D-Phe-Lys-NH,; His-D-Mrp-1a-Phe-D-Trp-Lys-NH2: Tyr-His-DM? -p-Ala-Trp-D-Phe-Lys-NH2: His-D-Mrp-A1a-Trp-NH2 D-Thr-D-Mrp-Ala-Trp-NH2; where Wrp is 2-methyltriptofan. INIP, IMA and GAB are as previously defined. It is stated that in relation to this date. The best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention. Having described the invention as an antecedent, what is contained in the following is claimed as property.

Claims

CLAIMS 1. A peptide of the formula: AD-Mrp- (Ala) -BC where A is any natural L-amino acid or its D- isomer, as long as A is not His if B is Trp and C is D -Lys-NH_; or A is an imidazolaceti group. isonipecot i lo, 4-aminobutyri lo, 4- (aminomet i 1) cyclohexanecarboni, Glu-Tyr-Aia-His, Tyr-Ala-His, Tyr-His. D-Thr-His; D refers to the dextrorotatory isomer. Mrp is 2- (alkyl of 1 to 3 carbon atoms) tryptophan; n is 0 or 1; B is L- or D-Trp. Phe or D-ß-Nal; C is H2 > D-Trp-Lys-NH2. D-Phe-Lys-NH2 # Phe-Lys-NH2. D-Phe-Lys-Thr-NH2. D-Phe-Lys-D-Thr-NH2. OR. wherein R is an alkyl group of 1 to 3 carbon atoms, and the addition salts with pharmaceutically acceptable organic or inorganic acids of any of said polypeptides.
2. A peptide according to claim 1, characterized in that Mrp is selected from the group consisting of 2-methyltriptofan, 2-ethyltriptofan, 2-propyltriptofan. 2-isopropyltriptofan.
3. A peptide according to claim 1, characterized in that Mrp is 2-methyltriptofan.
4. A peptide according to claim 1, characterized in that A is amidazoleceti lo. isonipecotí lo, 4-aminobutiri lo. D-Ala. D-Thr. Tyr.
5. - A peptide according to claim 1. characterized in that C is Phe-Lys-NH D-Trp-Ly? -NH_. NH_. or. wherein R is an alkyl group of 1 to 3? toms of carbon.
6. A peptide according to claim 1, characterized in that it is selected from the group consisting of IMA-D-Mrp-D-Trp-Phe-Lys-NH2; IMA-D-Mrp-D-ß-Nal-Phe-Lys-NH2; INIP-D-Mrp-D-Trp-Phe-Lys-NH2; INIP-D-Mrp-D-ß-Nal-Phe-Lys-NH2; GAB-D-Mrp-D-Trp-Phe-Lys-NH2; GAB-D-Mrp-D-ß-Nal-Phe-Lys-NH2; GAB-D-Mrp-D-β-N 1-NH 2; GAB-D-Mrp-D-ß-Nal-OC2H5; 4- (aminometi 1) cyclohexanecarboni l-D-Mrp-D-Trp-Phe-Lys-NH- ,; 4- (aminomethyl) cyclohexanecarbonyl-D-Mrp-D-β-Nal-Phe-Lys-NH ".
7. A peptide according to claim 1, characterized in that it is selected from the group consisting of: Imidazo1aceti 1-D-Mrp-A1a-Trp-D-Phe-Lys-NH2 D-Ala-D-Mrp-Ala-Trp -D-Phe-Lys-NH2; D-Thr-D-Mrp-A 1 a-Trp-D-Phe-Lys-NH; His-D-Mrp-Ala-Trp-NH2; D-Thr-D - Mrp-Ala-Trp-NH,; His-D-Mrp-Ala-Phe-D-Trp-Lys-NH_; D-Thr-D-Mrp-A 1a-Trp-D-Phe-Lys-D-Thr-NH 2 D-Thr-H] s-D-Mrp-Ala-Trp-D-Phe-Lys-NH 2; His-D-Mrp-A1 -Trp-D-Phe-Lys-Thr-NH2 His-D-Mrp-A-Trp-D-Phe-Lys-D-Thr-NH2.
8. A peptide according to any of claims 6 or 7, characterized in that Mrp is 2-methyltriptofan.
9. The use of the peptides according to any of claims 1 to 8. for the manufacture of a medicament useful for promoting the release of growth hormone in an animal.
10. The use according to claim 9, characterized in that the medicament is useful in human medicine.
11. The use of the peptides according to claim 7, for the manufacture of a medicament useful for promoting the increase of the intracellular activity of adenyl cyclase.
12. Pharmaceutical compositions characterized in that they comprise an effective amount of at least one peptide according to any of claims 1 to 8 as an active ingredient, optionally mixed with carriers and excipients.
13. Compositions according to claim 12. in the form of compositions for parenteral, intranasal administration. oral, controlled release, subcutaneous implants.