JPH06502618A - Novel synthetic GRF analogs - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Novel synthetic GRF analogs 1 Field of invention The present invention relates to novel peptides with therapeutic utility. More specifically, the present invention Growth hormone-releasing factor (growth hormone-r), a naturally occurring peptide eleasing factor: GRF).

The present invention also relates to therapeutic methods using these peptides. related.

2 Background of the invention Growth hormone (GH or somatotropin) is secreted by the anterior pituitary gland, 1 It is a peptide consisting of 91 amino acids. Growth hormone itself directly Although not truly growth promoting, many substances such as somatomedin produced by the liver It works by stimulating the production of one of the true growth factors. However, growth hormone The final effect of the operation is wide-ranging. At a global level, this hormone affects the skeletal, connective tissue, Affects muscles and internal organs. At the molecular level, growth hormone and somato Medin's metabolic effects include stimulation of nucleic acid and protein synthesis, induction of active nitrogen balance, and lipid production. Including stimulation of lipolysis and reduction of urea excretion.

Inappropriate levels of growth hormone in children can lead to problems with growth, endometrial development, and bone age. cause delay. It also causes retardation in the development of secondary sexual characteristics. growth hormone deficiency Additional effects of deficiency include impaired laryngeal development, delayed gonadal maturation, and hypoglycemia. Ru. All of these effects can be counteracted by normal levels of growth hormone. Ru.

Growth hormone production is both a releasing and inhibiting sensitizing factor located within the hypothalamus. is under the control of. -Second-release sensitizing factor is first released in the arcuate nucleus of the hypothalamus. growth hormone releasing factor (GRF or GHRH) produced and The offspring are transported to the pituitary gland by the portal circulation. However, other cells in the body, such as pancreatic tumor cells, However, they can also produce this hormone. human growth hormone releasing factor is a peptide consisting of 44 amino acids, of which the first 29 amino acids are all It has all kinds of biological activities. Also, similar peptides are found in cows, rats, and sheep. It has also been isolated from pigs and its sequence has been identified (Esch et al. , BiochemBiophys, Res, Collmun, 117: 772.1983).

Knowledge of GRF sequences provides a physiologically natural option for treating individuals with growth hormone deficiency. provided a means. Although subsequent efforts have been directed toward the development of synthetic GRF analogs, , this makes such analogs more effective for triggering the release of growth hormone This is because there was hope that it would be possible.

Efforts to create novel substitutions involving the N-terminus of molecules that have biological activity Salt points are matched to the moieties, especially residues 1 to 29. one or more Various substitutions in amino acid residues result in synthetic peptides compared to the corresponding native peptide. It has been shown to be effective in increasing the potency of putide. Peptide deza One of the recent approaches in incubation typically involves reinforcing the hydrophilic secondary structure of peptides. (De Grado et al, J, ^m, Che+n, Soc, 103:679-686, 1981; Mue et al, J, ^m, Chew, Soc, 105:4100-41 02.1983), certain peptide hormones, i.e. one side of the molecule contains hydrophobic residues. Peptide hormones that selectively have hydrophilic domains on the other side and that contain primarily hydrophilic domains The activity of these compounds can be enhanced by optimizing their hydrophilic properties. GRF is that- Binding with double bilayer membrane phospholipid vesicles and formation of monolayer at the air-water interface Based on observations, it has been described as having a substantial hydrophilic potential (Ka iser et, al,, 5science 223.249~255.19 84).

Many attempts to alter the secondary structure of GRF have been reported in the literature, e.g. Lance et al. reported that L-amino acids at positions 1 to 3 (Tyr', Ala2, Asp3) with the respective D-isomer in the N-terminal region of the GRF molecule. Attempts were made to restrict the conformations that occur (Biochem, Biophys, R es, Coma+, 119:265.1984) o D-analogs are original It was found that the activity was higher than that of peptides. In subsequent studies, this approach was extended to 4th, 5th, 6th, 7th and 8th place. 5th, 6th and 7th place Substitution of D-fi at position 4 substantially reduced activity, whereas substitution of D-fi at position 4 The activity was similar, and D-substitution at position 8 increased the activity (Coy et al. , J. Med. Chen, 28:181-185.1985).

Further attempts to alter the secondary structure of the native GRF molecule may include some other changes on the molecule. Attempts were focused on enhancing the planned hydrophilicity at points. For example, Tau focused on the predicted position of the α-helix on the GRF molecule (positions 1 to 29) and The specific amino acids in the region from position 3 to position 29 were changed from 6 to 29 where β-turn is expected to exist. Similar to the amino acid at position 10, substitution can modify a specific region of the GRF molecule. Ta. Replacement of Ser at position 9 with Ala (which is a “good α-helical former”) It was stated that the structure favors an extended helical conformation. Made with this pattern All released peptides showed enhanced activity compared to native GRF.

Substitution at position 8 of the original molecule by L-Ala or its equivalent amino acid residue is resulting in a huge increase in the growth hormone releasing activity of the peptides so modified A surprising finding was obtained. Other substitutions at position 8 have been suggested for some time. (e.g., in the US where D-ArgSSer and D-3er are substituted) Patent No. 4,518.586; Ser, ^sn, Thr or Gln U.S. Pat. No. 4,689.318 in which D-Arg or (see U.S. Pat. No. 4,626,523 in which D-Lys is substituted), No one intended to use Shiichi^1a in this position. In fact, Saito (S aito) et al. performed substitution with D-Ala at this position, resulting in active (7) real N (Biochem, Biophys, Res, Co mm, 149:531-537°1987). Therefore, in the special permutation of the present invention The observed enhancements are quite surprising.

3. Overview of the invention The present invention relates to a new group of peptides that are analogs of the native hormone GRF.

The new hormone converts Asn, which is normally located at position 8 in the original molecule, to form an α-helix. Characterized by substitution with inducible amino acids. In particular, the present invention provides: R1-R2-R3-Ala-11e-Phe-R7 for a peptide comprising the formula -RH-Rg-R16-^rg-R12-R13-Rl4415-G1n417 418-^1a-Arg-R21-Leu-R23-Rz<-R2s-Rzs- T? +7-Rzs-Rz.

However, in the formula, R1 is amino group-deficient Tyr (des-amino-Tyr) or A-R, where A is lower alkyl, lower cycloalkyl, benzyl or or lower acyl, and R1 has/has a C'Me or N'Me substituent. No Tyr, D-Tyr, Met.

PheSD-Phe, pcl-PheSLeu, His, or D-His and R2 is ^la, D-AlalD-NM^ or D-Arg; 3 is Asp or D-Asp; R7 is Thr, ^ib, NO2, Leu , Trp, β-Nal, or p-X-Phe, where X is H, FS Cl, Br1No2 or Me; R8 is ^la, ^1bSLeu, Tr p, β-Nal or p-X-Phe, where X is HlF, C1, B r, NO2 or Me; Ro is Ser, ^la, ^ib, Leu, Trp, β-Nal or p-X-Phe, where X is H, FlCl, Br, No2 or G; RIO is Tyr or D-Tyr.

R12 and R21 are represented by Lys, Arg or N'-B-Lys, in which case B is lower alkyl or cycloalkyl, even if R22 and RH are the same , may be different; R13 is He or Vat: R14 is Leu or D-Leu: R15 is cxy, ^la, Leu, ^sn, Gin or Aib; R17 is Leu or D-Leu :RI8 is Tyr or Ser; R23 is Leu or D-Leu; R24 is His or Gin; R25 is Glu, AspXD-Glu or D-Asp; R26 is Il e or Leu: R27 is MetSD-11etSAlaSNleSlie, Leu, Nva or Val; R211 is ^sn, Ser or missing (desR2g) and R29 is ^rg, D-Arg or deletion (desRu): and , or a pharmaceutically acceptable salt thereof.

In a preferred embodiment R, is Ala, and in a particularly preferred embodiment R8 and and R15 are Ala, most preferably R8, R8 and RI5 are Ala It is. Particularly effective peptides are those in which R8, R9 and R15 are Ala, and R It is formed when 2 is D-Ala.

Although the above formula contains 29 amino acid residues, one skilled in the art would understand that this is the molecule. It represents the end site of the sequence, and the addition of further amino acid residues up to the C-terminus of the sequence. It will be understood that does not affect its effectiveness. yields up to a total of 44 residues The addition will have no effect as expressed in the native GRF molecule.

These amino acids are not essential for activity, so their identity is Not important: they can be the same as in the original GRF sequence, or or an affordable equivalent. These alternative substitutions are described, for example, in U.S. Pat. No. 626.523 and No. 4.728.726.

The present invention also provides the above peptide/salt in combination with a pharmaceutically acceptable carrier. Compositions containing an effective amount to release growth hormone are also provided. Also, such treatment By administering the composition of the present invention to an individual in need of treatment, the release of growth hormone can be inhibited. A method of stimulation is also provided. Such methods benefit from the release of growth hormone is expected to be beneficial in the treatment of physiological conditions. Also, in normal animals Methods are also provided to stimulate its growth, in which the Also included is the administration of the compositions of the invention.

In one alternative embodiment, specific substitutions at positions 8, 9 and 15 In combination with D-Arg substitution at the R2 position, particularly effective G An RF receptor competitive inhibitor is produced. Such peptides inhibit innate GRF activity. inhibit sex and subsequently prevent the release of growth hormone. (And then again, this a pharmaceutically acceptable amount effective to competitively inhibit GRF of a peptide such as Also provided are therapeutic compositions comprising combinations with carriers that is administered to individuals in need of such treatment to stimulate the release of growth hormone. Methods for inhibiting are also provided.

4 Ming The following abbreviations are used throughout the specification and claims.

Ala Alanine Arg Arginine ^sn Asparagine Cys Cystine Gin Glutamine Phe Phenylalanine ＾ib　α-aminobutyric acid des-amino-Tyr [β-(p-hydroxyphenyl)propionyl Conolleucine Nle Norleucine Nva norvaline β-NMA D-alanine with α-amino group substituted with methyl Boc t-butyl oxycarbonyl Bzl Benzyl TosP-hluensulfonyl 2-CI-22-chlorobenzyloxycarbonyl 2-Br-22-bromoben Zyloxycarbonyl chx cyclohexyl In each case, isomeric forms of the amino acid exist, but they are heavier than the named residue. There are no essential ingredients, and one naturally occurring amino acid is intended. In addition, certain C-terminal Unless substitutions are noted, -○H (free acid) and -Nlh (amide) Both forms of the peptide are contemplated.

Similarly, as used herein, "lower alkyl", "lower acyl" and "lower cycloalkyl" The term "lower" used as "ru" refers to those with 3 to 4 carbon atoms. and "cycloalkyl" has 3 to 6 carbon atoms.

5. Detailed description of the invention The peptides of the present invention are analogs of native GRF and have a significant role in growth hormone release. It shows much better efficacy than traditional hormones. The potency enhancement of these peptides is G It is believed that this is due to the enhanced α-helical character of the N-terminal region of the RF molecule. . Enhancement of the hydrophilic α-helical properties of the C-terminal region enhances the potency of the peptide. It has been previously shown that how such modifications at the N-terminus affect the activity of the molecule. It was not known whether it would affect sea urchins. The results of the present invention are that G [at positions 1 to 29] At position 8 of RF, an amino acid residue with a tendency to form an α-helix It has been shown that substitutions of 1 to 10% of the total yield an unexpected enhancement of potency. inside amino acids Those that fall into this category are Ala, ^1bSI-euSTrpsβ-Nal, or p-X-Phe, where X is H, F, C1, Br, NO2 or is Me. The most preferred R8 is Ala. [1st to 29th place] GRF repair Analogues whose only decoration is the substitution of ^sn’ with Ala’ are naturally not present in vivo. (Table 1). ^la, L in 15th place Substitution with eu,^5nSGln or Aib, preferably Ala, It provides a significant potency enhancement, up to approximately 5 times that of the original GRF. further A preferred modification is in position 9 with one of the same preferred residues used in position 8. This is the replacement of These combinations of substitutions at positions 8,9 and 15 are especially When all of these are replaced by Ala, the potency is enhanced. Also, further G When lu” and Leu26.27 are combined, the activity is similar to that of the original GRF molecule. It will be strengthened up to 33 times. Substitution with D-Ala at position 2 [positions 1-29 ] It has been previously disclosed that it leads to an increase in GRF activity. Features of the present invention In a preferred embodiment, this substitution is one of the substitutions at positions 8.9 and 15. or more, in vivo, up to 49 times the native GRF. Create a high-power GRF analog with activity.

In order to enhance GRF competitive inhibitor activity, a peptide having Arg at the 2nd position is used. The planned substitutions at positions 8 and/or 9 and/or 15 were Particularly preferred. Use of Arg2 substitutions may result in GRF competitive inhibitor activity. Although previously reported, the planned combination is a more powerful D-Arg 2-position It produces a competitive inhibitor activity that is up to 10 times greater than the activity of conversion alone.

5.1. Preparation method Preparation of the peptides of the invention can be done by all known peptide synthesis methods. I can do it. A preferred method is by Merrifield Solid phase synthesis as described (J, Am, Chem, Soc, 85 :2149.1963), in this method the construction of the peptide starts from the C-terminus. ; An amino acid with a protected amino group is converted into a chloromethylated resin, α -aminodiphenylmethane resin or α-amino-(1,1-diphenyl)ethyl compounded into a suitable resin such as alcohol resin. Once there was a cutlet ring. After the deprotection of the terminal amino acids is carried out by techniques known to those skilled in the art. Noriyoshi Typical deprotection solvents include trichloroacetic acid alone or in combination with methylene chloride. It also contains The remaining protected amino acids of the desired sequence are linked in the appropriate order. It is subsequently added to the solid phase and coupled with the previous amino acid. normal coupling The cyanide solution is made of silanes such as dicyclohexyl cyanamide and diisopropyl cyanamide. It is an anamide derivative. Alternatively, some amino acids can be added before being added to the solid phase synthesizer. The amino acids may be coupled to each other.

Once the sequence is complete, all peptides are removed from the resin and subjected to HPLC or other can be purified by routine procedures of the technology, such as chromatographic techniques. Wear.

5.2. pharmaceutical composition Synthetic peptides often have pharmaceutically acceptable forms such as acid addition salts and metal complexes. It is used medicinally in the form of salts, and its acid addition salts include hydrochloride and hydrobromide. , sulfate, acetate, maleate, benzoate, citrate, tartrate, asco Rubates or phosphates are included, and metal complexes include iron or zinc.

The peptides are preferably administered parenterally, i.e. subcutaneously, intravenously, intramuscularly, intraperitoneally or intraperitoneally. Administered subcutaneously. For these modes of administration, peptides are typically administered in water or isotonic. Combined with a pharmaceutically acceptable carrier such as saline. Alternatively, the peptide is , administered orally in the form of tablets or capsules containing suitable binders, lubricants, etc. may be given.

The amount of peptide and/or unit dosage required per composition is currently Reference and comparison of known compositions and dosage forms used in It can be measured in advance by However, in general, the dosage range is individualized. The amount is 50 ng to 5 μg per kg of body weight. Dosage depends on the mode of administration and intended Depends on the results. However, such procedures are well within the capabilities of those skilled in the art. .

5.3. therapeutic use The synthetic peptides of the invention can be used in any situation where direct administration of growth hormone is desired. However, it is still useful. For veterinary use, e.g. cow, chicken, turkey, pig, goat, fish It can be administered to other livestock to promote growth, and can also be used to increase the protein (muscle) of these animals. This includes both changing or improving the ratio of meat (meat) to fat.

Peptides may also be useful in treating growth hormone deficiency-related disorders such as pituitary dwarfism. be. Growth hormone also plays a role in the metabolism and developmental processes of the stria, such as wound healing. affected by the GRF analogs of the present invention and therefore benefit from administration of the GRF analogs of the present invention. Can be done. Other uses for these peptides will be readily recognized by those skilled in the art. Dew.

One or more planned ^1 alil conversions are combined with Arg 2. Those peptides of the invention are competitive inhibitors of GRF and therefore inhibit growth. It can be used to treat conditions caused by hormone excess. the An example of such a condition is acromegaly, in which one of the symptoms is This causes the bones of the body to become abnormally large.

Benzylhydroylamine-polystyrene resin in chloride ion form (Advance (manufactured by edChemTech) (1.25g, 0.5mM) is Advanced It is placed in the reaction vessel of the ChemTech peptide synthesizer, which has the following: is programmed to carry out the reaction cycle: (a) methylene chloride: ( b) 33% trifluoroacetic acid in methylene chloride (twice 1 min and 15 min); (C) Methylene chloride: (d) Ethanol: (e) Methylene chloride: ket) Salt 10% diisopropylethylamine in methylene chloride.

The neutralized resin contains Boc-N'-tosyl-Arg and diisopropyl Stirred with carbodiimide (1.5mM each) in methylene chloride for 1 hour to obtain The removed amino acids are then subjected to a working cycle according to the cleaning program (a) to (f) above. are sent to each process. Next, add the following amino acids (1.5mM), Gin and Asn Coupled in the presence of 1.5mM 1-hydroxybenzotriazole. The steps are similar except that they are coupled sequentially: Boc-3et (Bzl), Boc-Met, Boa-Asp (chx), B oc-GinSBoc-LeuSBoc-Lys(2-CI|z), Boc-Arg (Tos), Boc-^1a, Boc-3er (Bzl), B oc-Leu, Boc-Gin, Boc-GlylBome|Leu.

Boc-ValSBoc-Lys (2-C1-z), Boc-Arg (Tos) , Boc-Tyr (2-Br-z), Boc-3er (Bzgeki, Boc-Ala, Boc-Thr (Bzl), Boa-Phe, Boc-1 1eSBoc-^1a, Boc-Asp, Boc-^1=A Boc- Tyr(2-Br-z). Remove the last t-butoxycarbonyl group, wash and The weight of the finished resin after drying was 2.5 g.

6, 1.2. A1a'GRF (1-29) NHz Previous section 6.1.1. Described in The resin (2.5g, 0.5mM) was mixed with p-cresol (5ml), dithiotre Itol (100 mg) and anhydrous hydrogen fluoride (35 ml) were mixed at 1°C. Stir for 45 minutes. Excess hydrogen fluoride is evaporated very quickly under a stream of dry nitrogen. The free peptide is precipitated and washed with ether.

The crude peptide was then dissolved in a minimal volume of 2M acetic acid and purified with sephade using the same solvent. Sephadex G-50 column (2.5 x 100 cm) will be released.

Next, fractions containing the main components by ultraviolet absorption and thin layer chromatography is collected and evaporated to a small volume, and PIDAC octadecylsilane Silica (Vydac octadecylsilane 5ilica) (1 0-15 μM) column (2.5×5 Qcm).

This is a linear gradient of 10-45% acetonitrile in 0.1% trifluoroacetic acid in water. It is separated using a Fractions were analyzed by thin layer chromatography and high performance analytical liquid Tested by chromatography and collected for maximum purity.

By repeatedly freeze-drying the solution from water, it becomes white and fluffy. A powdered product is obtained.

The product is found to be homogeneous by HPLC and TLC. acid ice Amino acid analysis of the solution confirms the octapeptide composition.

6.1.3. [^1a’1”]-GRF(1-29)-methyl norhydro Bennorhydroylamine-polystyrene resin in chloride ion form (manufactured by AdvancedChemTech) (1.25g, 5mM) is It is placed in the reaction vessel of the vanced ChemTech peptide synthesizer, but the The synthesizer is programmed to perform the following reaction cycle: (a) Chloride Methyl Thylene: (b) 33% trifluoroacetic acid in methylene chloride (1 min and 15 2 times); (C) Methylene chloride: (d) Ethanol: (e) Methylene chloride (f) 10% diisopropylethylamine in methylene chloride.

The neutralized resin is Boc-N'-tosyl-^rg and diisopropyl Stirred with carbodiimide (1.5mM each) in methylene chloride for 1 hour to obtain The removed amino acids are then subjected to a working cycle according to the cleaning program (a) to (f) above. are sent to each process. Next, the following amino acids (1.5mM), Gin and ^sn Coupled in the presence of 1.5mM 1-hydroxybenzotriazole. The steps are similar except that they are coupled sequentially: Boc-3er (Bzl), Boc-MetSBoc-^5p (chx), Bo c-GinSBoc-Leu, Boc-Lys(2-C1|z), Boc-^rg (Tos), Boc-A1. a, Boc-3er (Bzl), Boc-Leu, Boc-Gln, Boc-^1aSB Shield Me|Leu.

Boc-Val, Boc-Lys (2-C1-z), Boc-^rg (Tos) , Boc-Tyr (2-Br-z), Boa-3er (Bzgekij, Boc-Ala, Boc-Thr (Bzl), Boc-Phe, Boc-1 1e, Boc-AlaSBoc-AspSBoc-AlaB Boc-Tyr(2-Br-z). Remove the last t-butquine carbonyl group and wash. The weight of the finished resin after cleaning and drying was 26 g.

6, 1.4. Ala”+5-GRF (1-29) NH2 Previous section 6.1.3.1: Note The resin described (2.6 g, 0.5 mM) was prepared in 6.1.2. As already mentioned in section , cleavage using hydrogen fluoride and purification by column method.

By repeatedly freeze-drying the solution from water, a white, fluffy powder product is produced. is obtained.

The product is found to be homogeneous by HPLC and TLC. acid ice Amino acid analysis of the solution confirms the octapeptide composition.

The following peptides were also created using the same procedure: Aib", Ala"-G RF (1-29) NHz^1a8 IS, Glu25, Leu"t"-GRF ( 1-29) NFlzD-Ala2, Leu', Ala4, l5, Glu2 5, Leu" + 27-GRF (1-29) NH2O-^1a21.^1a8 ．． + 5, Glu”, Leu” 27-GRF (1 to 29) NH2AIB”+ 9. + s, Glu", Leu" 27-GRF (1-29) NH2O-A la", Ala'9"-GRF (1-29) NHzD-Ala21, Ala"t 　’　I”-GRF(1-29)NH2^La””　’-GRF(1-29) NH2Ala"+"+"-GRF(1-29)NH2O-^rg 2, Testing the utility of Ala”u’-GRF(1-29) for stimulating NH2 release It was done.

6, 2.1. Pituitary cell dispersion Weight 200-250g under controlled conditions (05:QO-19:00 hours open light conditions) The anterior pituitary gland from adult male rats reared in Han, Meth.

Enzymol, 103+249~257.1983; Hoefer et al,, Mo1. Ce11. EndocrinolA 35:229 ~235.1984) and the conventionally known trypsin/deoxyribonuclease Aase method (Heiman et al, Endocrinology 11 6:410-415.1985) using aseptic technique.

6, 2.2. cell culture The dispersed cells were grown in sterile-filtered Dulbecco's modified Eagle's medium. Earth [MEM　; Gibco Labrotories (GIBCO), G The medium contained 2.5% bovine fetus. Baby serum (GIBCO), 3% horse serum (GIBCO), (after collection stored on ice) (within 1 hour) 10% fresh pituitary donor rat serum, 1% MEM required Amino acids (GIBCO), gentamicin, 10 ng /ml: Sigma) and Nystatin, 10.0 00 U/ml; GIBCO) was added. Cells are hemocytometer (he macytometer) (appropriately 2.000 mm per pituitary gland). oaa cells), and wells (well, Co5tar cluster) 24; Rochester 5 scientific.

randomly distributed at a density of 200,000 cells per Rochester, NY). was placed. The placed cells were exposed to a supplement consisting of 95% air and 5% carbon dioxide. In preparation for the above hormone challenge in a humidified atmosphere, cells are and medium 199 (mcdium 199, GIBCO) to remove floating cells. Washed three times with (diluted in a silicone coated test tube) The application amount of the secretagogue was 1% BSA (fraction V; Total amount 1 including Sigma Chemical, St Louis, MO) Tested in 199 ml of medium. Keep the adjustment level within a narrow range and add maximal response to basal secretion levels without the addition of growth factors or glucocorticoids. To increase the ratio of levels stimulated to 0.1 nM somatos Pulsed in the presence of Tatin. 3 in an atmosphere of air/carbon dioxide (9515%) After 3 hours incubation at 7°C, the medium was removed and kept for 12 hours until assayed for hormone content. Stored at 0°C.

6, 2.4. GHRI A GH in plasma or media was determined by NIDDK and the University of Maryland School of Medicine. Ingredients widely available to the public through the National Program on Lumon and Pituitary. The standard double antibody RIA was used.

6, 2.5. result Efficacy was determined by the method described by Pugsley (Endocri, ey). Calculated by 4-point test according to Nology 39:161-176.1946) It was done. The results of these tests are shown in Table 1. seen in these data Thus, the combination of Ala at the 8th position and Ala at the 15th position is a type of only Ala15. It greatly increases the effectiveness of the peptide compared to its analogues. Similarly, ^la’ and Addition of D-Ala2 and/or D-Ala2 also enhances peptide GRF activity. on the other hand, Substitution with Ala at other positions in the N-terminal region, namely Ala at positions 7 and 10 When combined with the combination of Ala8, 9, and 15, the replacement by Has a hindrance effect.

Table 1 Efficacy of GRF (1-29) NH2 analogs in vivo ^1 a + Town 5 ± 1 (4) ^1a’ 4±1 (13) ^1a8,15 15±2(15) Aib’, ^1a+5 2±1(3) Ala4. + 5, Glu25, Leu26, 27 7 upper 2 (5) D-Al a2, Leu', Ala8. +5, Glu", Leu"+270.3±0 ．． 2 (5) D-Ala2, ^1a8, 15, Glu25, Lea"+ 27 7 ±1 (4) A1a8+ e, Is, Glu25, Leu"" 33±9 (6) D-Ala2, Ala"+" 27±10 (8) D-Ala2, D-^1a' + 9+ + 5 4g±14(8)^1at, g, o, +s 2±1(2)^ 1a'+ 9.10.15 1±1 (2) Efficacy was calculated by 4-point test. value is above the average value (sew, ). Values in parentheses are used to calculate average potency. The number of relevant experiments using multiple application rates of analogues. GRF (1~2 9) NHZ standard (potency = 1) was included in each assay. Further details Regarding Murphy and Coy's Peputid Please refer to e　Re5earch　1(1):36-41(1988) .

Submission of translation of written amendment (Article 184-8 of the Patent Law) November 4, 1992

Claims (1)

[Claims] 1. Peptides and pharmaceutically acceptable salts thereof containing the following sequences: ] [However, in the sequence, R1 is represented by amino group-deficient Tyr or A-R1, in which case A is lower alkyl, lower cycloalkyl, benzyl or lower acyl, and R1 is Tyr, D-Ty with or without C''Me or N''Me substituents r, Met, Phe, D-Phe, pCl-Phe, Lcu, His, or D -His; R2 is Ala, D-Ala, D-NMA or D-Arg. R3 is Asp or D-Asp; R7 is Thr, Aib, Leu, T rp, β-Nal, or p-X-Phe, where X is H, F, Cl, Br, NO2 or Me; R8 is Ala, Aib, Leu, Trp, β- Nal or p-X-Phe, where X is H, F, Cl, Br, NO2 or Me; R9 is Ser, Ala, Aib, Leu, Trp, β-Na 1 or p-X-Phe, where X is H, F, Cl, Br, NO2 or is Me; R10 is Tyr or D-Tyr; R12 and R21 are Lys, Arg or N'-B-Lys, where B is lower alkyl or is cycloalkyl, and R12 and R21 may be the same or different. Ku; R13 is ILe or Val; R14 is Leu or D-Leu; R15 is Gly, Ala, Leu, A sn, Gln or Aib; R17 is Leu or D-Leu; R 18 is Tyr or Ser; R23 is Leu or D-Leu; R24 is His or Gln; R25 is Glu, Asp, D-Glu or D-Asp; R26 is ILe or Leu; R27 is Met, D-Met, Ala, ILe, Leu, Nva, or Val R28 is Asn, Ser or deletion (desR28); R29 is Arg, D-Arg or deletion (desR29)]2. Said R8 is Ala The peptide according to claim 1, which is 3. 3. The peptide according to claim 2, wherein said R15 is Ala. 4. 4. The peptide according to claim 3, wherein said R9 is Ala. 5. The peptide according to any one of claims 1 to 4, wherein the R2 is D-Ala. 6. 5. The peptide according to claim 4, wherein said R25 is Glu. 7. Pep according to claim 6, wherein said R26 and R27 are each Leu. Chido. 8. 6. The peptide according to claim 5, wherein said R25 is Glu. 9. Pep according to claim 5, wherein said R26 and R27 are each Leu. Chido. 10. The peptide according to claim 1, containing Ala8-GRF (1-29). 11. The peptide according to claim 1, containing Ala8,15-GRF (1-29) Chido. 12. According to claim 1, containing Ala8,9,15-GRF(1-29) peptide. 13. Containing D-Ala2 and Ala8,15-GRF (1-29), The peptide according to claim 1. 14. Contains D-Ala2 and Ala8,9,15-GRF (1-29) , the peptide according to claim 1. 15. D-Ala2, Ala8,15, Glu25, Leu26,27-GRF The peptide according to claim 1, containing (1-29). 16. The peptide according to claim 1, wherein said R2 is D-Arg. 17. 17. The peptide according to claim 16, wherein said R2 is Ala. 18. 17. The peptide according to claim 16, wherein said R15 is Ala. 19. 18. The peptide according to claim 17, wherein said R15 is Ala. 20. 20. The peptide according to claim 19, wherein said R9 is Ala. 21. 21. The peptide according to claim 20, wherein said R25 is Glu. 22. 22. The peptide according to claim 21, wherein said R26 and R27 are Leu. . 23. Claim 16, comprising D-Arg2 and Ala8-GRF (1-29) Peptides described. 24. Claim comprising D-Arg2 and Ala8,15-GRF (1-29) 16. 25. Containing D-Arg2 and Ala8,9,15-GRF (1-29), 17. The peptide according to claim 16. 26. A peptide containing the following sequence in combination with a pharmaceutically acceptable carrier: and a pharmaceutical composition containing an effective amount of a pharmaceutically acceptable salt thereof: Masu】 [However, in the sequence, R1 is represented by amino group-deficient Tyr or A-R1, in which case A is lower alkyl, lower cycloalkyl, benzyl or lower acyl, and R1 is Tyr, D-Ty with or without C''Me or N''Me substituents r, Met, Phe, D-Phe, pCl-Phe, Leu, His, or D -His; R2 is Ala, D-Ala, D-NMA or D-Arg. R3 is Asp or D-Asp; R7 is Thr, Ala, Aib, L eu, Trp, β-Nal, or p-X-Phe, where X is H, F , Cl, Br, NO2 or Me; R8 is Ala, Aib, Leu, Tr p, β-Nal or p-X-Phe, where X is H, F, Cl, Br , NO2 or Me; R9 is Ser, Ala, Aib, Leu, Trp, β-Nal or p-X-Phe, where X is H, F, Cl, Br, N O2 or Me; R10 is Ala, Tyr or D-Tyr; R1 2 and R21 are represented by Lys, Arg or N''-B-Lys, in which case B is lower alkyl or cycloalkyl, even if R12 and R21 are the same , may be different; R13 is He or Val; R14 is Leu or D-Leu; R15 is Gly, Ala, Leu, A sn, Gln or Aib; R17 is Leu or D-Leu; R 18 is Tyr or Ser; R23 is Leu or D-Leu; R24 is His or Cln; R25 is Glu, Asp, D-Glu or D-Asp; R26 is He or Leu; R27 is Met, D-Met, Ala, Nle, He, Leu, Nva or Val; R28 is Asn, Ser or deletion (desR28); R 29 is Arg, D-Arg or deletion (desR29)] 27. Said R8 27. The composition of claim 26, wherein is Ala. 28. 28. The composition according to claim 27, wherein said R15 is Ala. 29. 30. The composition of claim 29, wherein R9 is Ala. 30. The composition according to any one of claims 26 to 29, wherein the R2 is D-Ala. thing. 31. 30. The composition of claim 29, wherein said R25 is Glu. 32. 30. The method of claim 29, wherein R26 and R27 are each Leu. Composition. 33. 31. The composition of claim 30, wherein said R25 is Glu. 34. according to claim 30, wherein said R26 and R27 are each Leu. Composition. 35. Claim 26, comprising a peptide containing Ala8-GRF (1-29). Compositions as described. 36. Claim 2 comprising a peptide containing Ala8,15-GRF (1-29) 6. The composition according to 6. 37. A claim containing a peptide containing Ala8,9,15-GRF(1-29) 27. The composition according to claim 26. 38. Peptides containing D-Ala2 and Ala8-GRF (1-29) 27. The composition of claim 26, comprising: 39. Pep containing D-Ala2 and Ala8,15-GRF (1-29) 27. The composition of claim 26, comprising tide. 40. D-Ala2, D-Ala2 and Ala8,9,15-GRF (1-2 27. The composition of claim 26, comprising a peptide containing 9). 41. D-Ala2, Ala8,15, Glu25, Leu26, ~27-GR 27. The composition of claim 26, comprising a peptide containing F(1-29). 42. 27. The composition of claim 26, wherein R2 is D-Arg. 43. 43. The composition of claim 42, wherein R8 is Ala. 44. 27. The composition according to claim 26, wherein said R15 is Ala. 45. 43. The composition of claim 42, wherein said R15 is Ala. 46. 46. The composition of claim 45, wherein R9 is Ala. 47. 47. The composition of claim 46, wherein said R25 is Glu. 48. 48. The set of claim 47, wherein said R26 and R27 are each Leu. A product. 49. Peptides containing D-Arg2 and Ala8-GRF (1-29) 43. The composition of claim 42, comprising: 50. Pep containing D-Arg2 and Ala8,15-GRF (1-29) 43. The composition of claim 42, comprising tide. 51. Contains D-Arg2 and Ala8,9,15-GRF (1-29) 43. The composition of claim 42, comprising a peptide. 52. A treatment comprising administering to an individual an effective amount of a peptide according to claim 1. A method of increasing growth hormone production in an individual in need of therapy. 53. 53. The method of claim 52, wherein the individual is a mammal. 54. 53. The method of claim 52, wherein the individual is a human. 55. Claims 52-54, wherein the unit dosage is from 5 μg to 5 μg per kg body weight. The method described in any of the above. 56. 53. The peptide according to claim 52, wherein the peptide is used for the treatment of pituitary dwarfism. Method. 57. Claim 52, wherein the peptide is used to enhance milk production in livestock. Method described. 58. 53. The peptide according to claim 52, wherein the peptide is used for promoting the growth of livestock. Method. 59. In order for the peptide to increase the ratio of protein to lipid in animals, 53. The method according to claim 52, wherein the method is used. 60. 61. The method according to any one of claims 58 to 60, wherein the livestock is a cow or a pig. . 61. comprising administering to an individual an effective amount of a peptide according to claim 16. A method of reducing growth hormone production in individuals in need of such treatment. 62. 62. The method of claim 61, wherein the individual is a human. 63. 61 or 62, wherein the unit dosage is from 5 μg to 5 μg per kg body weight. 62. 64. Any of claims 62 to 64, wherein the peptide is used for the treatment of acromegaly. The method described in