HU201566B - Process for producing new analoguse of activatin factor of human growth hormon and pharmaceutical and veterinair compositions containing them - Google Patents

Abstract

The invention relates to a process for the preparation of novel peptides of the general formula (I) and salts thereof: HTyrR2AspAlaIlePheThrAsnSerTyr {peptides preparation; Secretion enhancement growth hormone; Drug}

Description

The present invention relates to novel analogs of human growth hormone secretagogue (hGRF), to human and veterinary pharmaceutical compositions containing the novel compounds, and to compositions for use in animal husbandry.

Hormones that stimulate growth hormone secretion, also known as Growth Hormone Releasing Factors (GRFs), and their analogues play an important role in both human and veterinary medicine and animal husbandry. They are used in human medicine to treat disorders associated with growth hormone deficiency or reduced secretion, and in veterinary medicine and animal husbandry, primarily to stimulate growth, improve food utilization, improve meat / fat ratios and increase milk production.

Following the isolation of native human GRF (hereafter "hGRF") (Natúré 300, 276 (1982); Science 218, 585 (1982)), studies to clarify the relationship between chemical structure and biological activity were soon begun. It has been found that the amino acid sequence of hGRF, a 44-membered peptide, is very similar to that of GRF isolated from several mammals (such as pigs, bovine animals, sheep). The active center of the molecules is generally shown in Figs. amino acid residues. Shorter peptide chains generally have lower biological activity than the native hormone, but experiments have shown that even hGRF (1-29) -NH2 (i.e., the native, human-derived peptide amide 1-29 from the N-terminus) ) also exhibits favorable activity over natural hGRF (1-44) -NH2 (Naturre 300, 276 (1982); J. Med. Chem. 30, 219 (1987); Peptides p. 481 (ed. D.) Theodoropoulos, Walter de Gruyter, Berlin, New York, 1988; Proceedings of the Ninth Am. Peptide Symposium, Publisher: Pierce Chem. Co., Rockford, Illinois, 1985/707, Biochem. Biophys.

Commun. 123, 854 (1984). Studies with hGRF- (1-29J-Nfa analogs) have shown that the N-terminal moiety is more bioactive than the C-terminal moiety and that the activity of the peptides is generally lower than that of the peptide amide (J. Med. Chem. 28, 181 (1985) It has also been found that the biological activity of peptides with a lower number of members than the native hormone can be positively influenced by replacing certain amino acids of the peptide chain with other amino acids (J, Med. Chem. 28, 181/1985).

It is also known from the foregoing publications that the native GRF (1-29) -NH2 has the same amino acid sequence in humans and pigs. For cattle, for example, they differ from these

28 (containing asparagine instead of serine), however, the addition of hGRF (1-44I-NH2 and hGRF (1-29) -NH2) has been shown to have a beneficial effect on bovine milk production, in both quantity and quality (J. Dairy Science 70, 2511 (1987) and 71, 92 (1988).

SUMMARY OF THE INVENTION It is an object of the present invention to provide hGRF analogs that at least achieve or exceed the activity of native hormone or its 1-29. amino acid sequence.

The present invention is based on the discovery that thirty amino acid analogues whose activity is significantly superior to that of native hGRF differ only in the amino acid sequence of native hGRFIL-30, -OH and hGRF (1-30) -NH2. the 27th position contains norleucine instead of methionine and the 30th position contains gamma-aminobutyric acid and gamma-aminobutyric acid amide instead of glutamine.

It is known from previously cited publications that hGRF (1-30) -NHz and hGRF (1-301-OH) have approximately half the activity of the native hormone, and that by replacing the methionine moiety at position 27 of the native peptide chain with a it is advantageous for chemical synthesis because the easily oxidizable methionine is replaced with the amino acid closest to it structurally, and the biological activity is not decreased, sometimes slightly increased. The 30th position of our preparation containing gamma-aminobutyric acid or gamma-aminobutyric acid amide however, the analogs showed surprisingly good biological activity due to the 30-position amino acid exchange.

In the specification, the three-letter abbreviation for amino acids is IUPAC-IVB Biochemical

Numenclature (J. Biol. Chem. 24 7, 977 (1972)), unless otherwise noted, the amino acids have the L-configuration. Further abbreviations are as follows:

Gaba

Boc

BHA

TFA

DIC

HOBt

HF

MPLC

HPLC

TLC hGRF hpGRF44

GH

STH release gamma-aminobutyric acid residue tert-butoxycarbonyl group benzhydrylamino group trifluoroacetic acid diisopropylcarbodiimide 1-hydroxybenzotriazole hydrogen fluoride medium pressure liquid chromatography high performance liquid chromatography (HLMS) 44-membered peptide) GRF isolated from human pancreatic tumor with the same amino acid composition as pituitary GRF growth hormone growth hormone secretion

HU 201566 Β

HGRF (1-30) -NH 2 has the formula: H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-NH2

The present invention relates to a process for the preparation of acid addition salts of novel peptides of formula (I)

H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu- (I )

-Gln-Asp-Ile-Nle-Ser-Arg-Gaba-Y - wherein Y is hydroxy or amino. According to the invention, the appropriately protected amino acids are coupled in the appropriate order by methods known in the art of peptide chemistry, the protecting groups are cleaved and, if desired, the peptide obtained as the acid addition salt is converted into another acid addition salt.

The compounds of formula (I) may be prepared by methods known in the art of peptide chemistry. The compounds may be prepared in the liquid phase by condensation of the appropriately protected amino acids or, more preferably, fragments of the appropriately protected amino acids, in a given sequence; however, the compounds of formula (I) are particularly preferably prepared by solid phase synthesis. In the latter case, the appropriately protected Gaba is first coupled to the resin and coupled to the appropriately protected amino acids or fragments thereof in the order of the sequence.

The novel compounds of the invention can be used in human medicine and animal husbandry to stimulate growth hormone secretion. For this purpose, pharmaceutical compositions and compositions useful in animal husbandry are prepared.

The invention thus also relates to a process for the manufacture of a medicament for promoting growth hormone release. According to the present invention, the compounds of formula (I) obtained in the form of a pharmaceutically acceptable acid addition salt prepared as above are admixed with conventional pharmaceutical excipients and converted into pharmaceutical compositions.

The present invention further relates to the preparation of compositions for use in animal husbandry, comprising mixing the compounds of formula (I) obtained as above in the form of an acid addition salt for animal husbandry with adjuvants for animal husbandry and converting them into animal husbandry compositions.

The acid addition salts of the compounds of formula I include, for example, hydrochlorides, hydrobromides, sulfates, phosphates, maleates, acetates or citrates.

The pharmaceutical and livestock compositions include, for example, intravenous, intramuscular, subcutaneous, intranasal and oral formulations.

Oral formulations include tablets which contain, in addition to the active ingredient, excipients such as carriers (such as calcium carbonate), binders (such as tragacanth, corn starch or gelatin), disintegrating agents (such as alginic acid) and lubricating agents (such as magnesium). contain. Flavoring agents may also be added to the composition for liquid administration. Oral formulations for use in animal husbandry may also be feed premixes.

Formulations for injection include, in addition to the active ingredient, a liquid carrier for injection, for example, isotonic saline or phosphate buffer.

The active compounds may also be formulated into sustained release formulations, for example, by incorporation of the active compounds into a known sustained release polymer.

The required daily dose of the active compounds will vary depending on a number of factors, including the species of the subject being treated, the route of administration and the effect desired, but usually a daily dose of 1-100 µg / kg body weight will be used.

The biological activity of the acid addition salts of the compounds of formula I was tested in vitro and in vivo by measuring GH secretion as a function of the concentration of compound administered. The data thus obtained were compared with the values obtained when native hpGRF or hGRF (1-29) -NH2 was added. The amount of GH selected was determined by radioimmunoassay with a 95% confidence limit.

In vitro, as determined by cell culture, approximately fifty fold activity was observed in superfusion assays relative to native hpGRF-44.

The results of cell culture assays are shown in Figure 1. The experiments were performed on rat pituitary gland. monolayer cell culture, and the results of 18 experiments are summarized in the diagram. The activity of hpGRF-44 was compared with that of Nle ^ '.gaba - ^ - hGRFd-SOJ-NHz acetate salt (Compound designated as SzB-4-179 in the figure). GH secretion is plotted against the concentration of peptides administered. The base corresponds to the natural GH secretion of the cells. It can be seen from the figure that about fifty times the concentration of hpGRF-44 is required to achieve the same STH release ng / culture relative to the concentration of the compound of the invention.

HU 201566 Β

The change in GH levels induced by the compounds of the present invention under in vivo conditions was as follows:

Sexually mature hira Wist 5 tar R Amsterdam rats weighing 250-300 g were anesthetized with 60 mg / kg pentobarbital (pentobarbital administered intraperitoneally). The peptides were dissolved in 0.9% w / w aqueous sodium chloride and administered 20 minutes after anesthesia by intravenous injection. Blood samples were taken from the jugular vein of animals prior to, and 5, 15 and 30 minutes after, drug administration, and blood GH levels were determined. By way of example, the results of the assay of the Nle 1 .gaba ³⁰ -hGRF (1-29) -NH 2 -acetate are reported. HGRF (1-29) -NH2 was used as a standard and the test sample and standard were administered intravenously at 2 pg / kg body weight. GH release was enhanced by both agents, but the GH levels of the animals treated with the compound of the invention were 164% after 5 minutes, 155% after 10 minutes and 204% after 10 minutes compared to standard treated animals.

The advantages of the novel compounds of the present invention can be summarized as follows:

- They show significantly higher activity than the native hormone.

- The metabolism of Gaba in a living organism (eg the nervous system), ie non-organismal, is clear. 35

- Due to Gaba's space filling, solid phase peptide synthesis is an excellent .spacer, thus providing a good synthesis efficiency when coupled to the peptide-bearing polymer as the first amino acid. 40

Wash 1 with CH 2 Cl 2

Cleavage 2 33% TFA / CfaCk

Cleavage 3 33% TFA / CH 2 Cl 2

Wash 4 with CH 2 Cl 2

5. wash with ethanol

6. Wash CHCh

Neutralization 7 with 10% Et3N / CHCl3

8. Wash CHCh

9. Wash CH2Cl2

10. Addition of boc-amino acid

Circuit 11 DIC

12. Wash with CH 2 Cl 2

13. Wash with ethanol

Substitution of Met with Nle at position 27 of the peptide is chemically advantageous for synthesis.

The following Examples illustrate the process of the invention in more detail. In the examples, the term "equivalent" refers to molar equivalent.

Example 1

Nle ^{: i7} , Preparation of Gaba ³⁰ -hGfiF (1-30) -NH 2 -acetate

The peptide was prepared on a benzhydrylamine resin (capacity: 0.65 milliequivalents / g) using a Beckman 990B peptide synthesizer. The Boc-Gaba-OH protected amino acid is used in an excess of three equivalents based on the resin capacity, with an equivalent amount of DIC condensing agent and an equivalent amount of HOBt catalyst. Reaction time for Boc-Gaba-OH activation is 12 hours. The resin - protected amino acid compound is then assayed for ninhydrin reaction to verify completeness of coupling to the resin. Boc-Gaba-0H resin coupling is usually perfect for first coupling; if the ninhydrin reaction may give a positive result (suggesting that the amino groups of the benzhydrylamine resin are not completely substituted), the coupling to the resin can be completed by a symmetric anhydride method. (Based on weight gain, the resin has a capacity of 75-80% of the capacity specified by the manufacturer.)

The Boc-Gaba-BHA resin is cleaved and neutralized in the usual manner, and peptide synthesis is then carried out step by step according to the following scheme:

three times 2 minutes once 2 minutes once 25 minutes three times 2 minutes three times 2 minutes three times 2 minutes twice 3 minutes twice 2 minutes three times 2 minutes once 120-300 minutes twice 2 minutes

When the Boc protecting group is cleaved, a mixture of 0.5% indole and 0.2% thioanisole or 1% anisole and 0.5% diethyl sulfide is used to prevent secondary reactions. 60

During the synthesis, 1 mmol of Boc-Gaba-BHA resin was used in sequence with 3 mmol of Boc-amino acid, 3 mmol of D1C coupling agent and 3 mmol of HOBt catalyst in dichloromethane solution. The Boc-65

For the coupling of -Arg (Tos) OH, a 1: 1 mixture of dimethylformamide and dichloromethane was used. Coupling of all protected amino acids was performed using the carbodynid method except for Boc-Gln and Boc-Asn, respectively, when the p-nitrophenyl ester of the corresponding protected amino acid was used in triplicate in the presence of an equivalent HOBt catalyst. In this case, the reaction is dimethyl5

HU 201566 Β

formamide / dichloromethane (1: 1) for 12-14 hours. The side chains of the protected amino acids are protected with Tyr, Ser and Thr with benzyl, Lys with 2-chlorobenzyloxycarbonyl (2Cl-Z), Arg 5 with tosyl (Tos), and Glu and Asp carboxyl groups with cyclohexyl (Chx).

The side-chain protecting groups and the peptide were removed from the resin using liquid HF so that the peptide-resin compound in the presence of 0.25 mmol peptide-BHA resin in the presence of 2.5 'ml of 10 wt. and kept in about 20-25 ml HF at 0 ° C for 1 hour. The HF was removed under reduced pressure, the residue was treated with absolute diethyl ether and the solid residue was dissolved in 15% acetic acid. The peptide present as the hydrogen fluoride salt was purified by gel puncturing on a Sephadex G-50 column in 15% by weight acetic acid. Subsequently, the peptide was subjected to MPLC method on a Cib reversed phase silica gel column (Syncroprep RP-4, 3u) using 0.25 M triethylamine phosphate (TEAP, pH 2.55) and 25 40 volumes of TEAP to 60 volumes of acetonitrile. purified. The fractions containing the peptide as the phosphate salt were identified by TLC and HPLC, and the fractions containing the desired product were combined and the solvent was evaporated under reduced pressure. The residue was repeatedly applied to a MPLC column for desalting and further purification and gradient elution from 10 wt.% Acetic acid solution and 10 wt. The purity and product content of the fractions containing the peptide as the acetate salt were determined as described above. Finally, the peptide acetate salt is isolated by freeze-drying as a solid. The thus prepared Nle ^ .gaba ³⁰ -hGRF (1-30) -NH2 is suitable for in vitro cell culture and superfusion assays, as well as for in vivo testing.

The physical characteristics of the product are as follows:

TLC: Rf = 0.54 (adsorbent: DC Alufolien Kieselgel 60, Cat. No. 5553), eluent: 15: 10: 3: 12 v / v n-butanol: 50: pyridine: acetic acid: water; 0.40 (eluent: 4: 1: 2 v / v n-butanol: acetic acid:

: water mixture).

HPLC: A Vydac 218 TP 546 column was used as the adsorbent and 30 volumes of acetonitrile 55 and 70 volumes of 0.25 M aqueous triethylammonium phosphate (pH 2.25) were used as eluent. UV detection at 215 nm with a sensitivity of 0.1 AuFS. ISCO (Model 60 2350) HPLC pumps were controlled by an IBM-XT computer according to ISCO ChemResearch. Isocratic elution was performed and the product was characterized by the quotient of retention time and breakthrough time (k '). k '= 4.37.

Example 2

Preparation of Na 2 O, GABA-hGRF (1-30) -OH-acetate

The peptide was prepared as described in Example 1 but prepared on Merrifield resin (chloromethyl resin). The resin (capacity: 0.5-0.9 milliequivalents / g) was reacted with triple excess Boc-Gaba cesium salt in dimethylformamide at 50 ° C for 20 hours. The reaction yields 0.4-0.6 mmol of Gaba / g substituted resin.

Peptide synthesis was performed as described in Example 1 and the resulting peptide was purified as in Example 1. The product was homogeneous by TLC and HPLC.

Claims (3)

PATENT CLAIMS A process for preparing acid addition salts of peptides of formula (I) H-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys- (I) -Leu-Leu-Gln-Asp-Ile-Nle-Ser-Arg-Gaba-Y wherein Y represents a hydroxyl group or an amino group, characterized in that the appropriately protected amino acids are linked in the appropriate order by methods known in the art of peptide chemistry, the protecting groups are cleaved and, if desired, the peptide obtained as acid addition salt is converted to another acid addition salt. 2. A process for the preparation of a growth hormone releasing pharmaceutical composition, wherein the compounds of formula I wherein Y is as defined in claim 1 are The pharmaceutically acceptable acid addition salts prepared according to claim 1 are mixed with the usual pharmaceutical excipients and converted into pharmaceutical compositions. 3. A process for the preparation of compositions for use in animal husbandry, comprising mixing the acid addition salts of the compounds of formula I, wherein Y is as defined in Claim 1, with the excipients suitable for use in animal husbandry, and in animal husbandry compositions. Published by the National Office for Inventions, Budapest Responsible: dr. Gabriella Swoboda Head of Department R-4967 - KJK